Arginine Residue 155 in the Second Intracellular Loop Plays a Critical Role in Rat Melanin-Concentrating Hormone Receptor 1 Activation
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内分泌学杂志 2005年第8期
Department of Pharmacology (Y.S., M.T., A.Y., K.M.), Saitama Medical School of Medicine, Iruma-gun, Saitama 350-0492; and Celestar Lexico-Sciences Inc. (S.S., K.I., H.D.), Makuhari, Chiba 261-8501, Japan
Address all correspondence and requests for reprints to: Yumiko Saito, Department of Pharmacology, Saitama Medical School, 38 Moro-Hongo, Moroyama-cho, Iruma-gun, Saitama 350-0492, Japan. E-mail: yumisait@saitama-med.ac.jp.
Abstract
Melanin-concentrating hormone (MCH) receptor 1 (MCH1R) is a class A G protein-coupled receptor. The MCH system has been linked to a variety of physiological functions, including the regulation of feeding and energy metabolism. We recently reported the importance of a dibasic motif in the membrane-proximal C-terminal region for MCH1R function. Here we reveal that an Arg residue in intracellular loop 2 of MCH1R plays a critical role in receptor function. We analyzed the roles of two distinct motifs, BBXXB and BXBB (in which B is a basic residue and X is a nonbasic residue), located in the three intracellular loops of MCH1R. Triple-substitution mutants of intracellular loops 1 and 3 could still activate calcium mobilization, albeit with lower efficacy or potency. However, mutations in intracellular loop 2 led to a complete loss of induction of signal transduction without changing the high affinity constant (Kd) value. By analyzing a series of single-substitution mutants, a point mutation of Arg155 in intracellular loop 2 was found to be responsible for the signaling pathway elicited by MCH. In addition, substitution at positions corresponding to Arg155 in human MCH receptor 2 and rat somatostatin receptor 2 also markedly abolished their ligand-induced signaling capacities, indicating that this Arg is a recognition determinant in several G protein-coupled receptors.
Introduction
MELANIN-CONCENTRATING HORMONE (MCH) is a cyclic neuropeptide that was first isolated from the pituitary gland of salmon (1). It is predominantly produced by neurons of the lateral hypothalamus in mammals (2) and plays key roles in the regulation of food-intake behavior and energy balance (3, 4). The orphan G protein-coupled receptor (GPCR) somatostatin-like receptor-1 (SLC-1) (5) is activated by MCH and belongs to the class A GPCRs (6, 7, 8, 9, 10). This receptor, now referred to as MCH receptor 1 (MCH1R), is widely expressed at high levels in the brain (11, 12). MCH1R is able to activate multiple signal transduction pathways, namely cAMP inhibition, calcium influx and MAPK, via Gi and Gq coupling (4, 5, 7, 13, 14). Although a second MCH receptor, MCH receptor 2 (MCH2R), was isolated in humans (15), it was found to be nonfunctional or to encode a nonfunctional pseudogene in nonhuman species, including rodents (16). Targeted disruption of the MCH1R gene in mice results in lean, hyperactive and hyperphagic mice that are resistant to diet-induced obesity (17, 18). Thus, MCH1R is regarded, at least in rodents, as an endogenous mediator of MCH for energy homeostasis. Moreover, recent studies have shown that selective MCH1R antagonists inhibit MCH-induced food intake in rats (19, 20, 21), and one of these antagonists also exhibited antidepressant and anxiolytic effects (20). Therefore, MCH1R has attracted considerable attention as a possible target for therapeutic intervention in obesity and some mental disorders. Recently two common single-nucleotide polymorphisms were identified in MCH1R, but neither was found to be associated with any obesity-related phenotypes in a population of 500 white subjects (22). Although other single-nucleotide polymorphisms have been identified in the MCH receptors, their associated phenotypic characteristics have not yet been analyzed (23). Further clarification is required in other populations of obese subjects and/or in genetically modified mice.
Several studies have reported structural information that underlies the activation of MCH1R. Biochemical analysis of MCH1R using a molecular model revealed that D123 in the third transmembrane domain is critical for ligand binding (24). We previously demonstrated that N23 in the extracellular N-terminal region mainly contributes to N-linked glycosylation of MCH1R and is necessary for cell surface expression (25). Furthermore, we recently demonstrated a key role for the C-terminal tail of MCH1R for receptor function. The membrane-proximal region of MCH1R is predicted to form an amphiphilic cytoplasmic helix 8, and a dibasic motif in this helix 8 is important for signal transduction (26). On the other hand, the distal portion of the C-terminal tail in MCH1R is necessary for the internalization process (27). However, in comparison with the adrenergic and muscarinic receptor subfamilies, relatively little is known about the structure/function relationships in MCH1R, and more extensive research is necessary to determine the factors that influence its G protein-interacting states.
Traditional mutagenesis approaches, including the use of hybrid receptors, have implicated a particular part of the transmembrane region and several intracellular portions in GPCR function (28, 29, 30, 31, 32, 33). In many GPCRs, intracellular loop 2 (i2) and the juxtamembrane portions of intracellular loop 3 (i3) are believed to alter the signal transduction capacity of the receptor by directly affecting G protein coupling or altering the packing of the transmembrane helices (28, 29, 30). Several reports have also shown that intracellular loop 1 (i1) is required for G protein activation (31). A series of single-substitution mutagenesis studies have attempted to identify the amino acids in different GPCRs that are responsible for the receptor activities. Some of these amino acid residues have been proposed to be involved in the effector coupling of several receptors (34, 35, 36). Furthermore, several conserved motifs were found to be involved in cell surface expression and effector coupling (26, 37, 38). Okamoto and Nishimoto (39) first proposed that a BBXB or BBXXB motif (in which B is a basic amino acid residue and X is a nonbasic residue) was required for Gi activation, as evaluated by a synthetic peptide study. Based on further structure-activity studies, a number of GPCRs were shown to contain these structural motifs in particular regions of their intracellular loops. In fact, such motifs have been found to affect the selectivity for receptor/G protein coupling or the intracellular signaling pathway in several GPCRs (40, 41, 42).
In the present study, we analyzed the functional roles of two distinct BBXXB and BXBB motifs located in the cytoplasmic loops of MCH1R. Although the basic motifs are generally present in i2, i3, and the C-terminal tail but absent from i1, in other GPCRs (39), mouse, rat, and human MCH1R commonly possess a BBXB motif in i1, a reversed BBXB in i2 and a BBXXB motif in i3 (5). Systematic examination revealed that the motifs in all three loops have profound functions in the receptor activity and that point mutation of R155 in i2 strongly affected the signaling, although no significant change was observed in the Kd value of radioligand binding assays. We further discuss the significance of the positions corresponding to R155 in some other GPCRs.
Materials and Methods
cDNA constructs for MCH1R, MCH2R, and somatostatin receptor (SSTR) 2 mutagenesis
Incorporation of a sequence encoding the Flag epitope tag before the first methionine in rat MCH1R was performed by PCR (25). The purified full-length cDNA of MCH1R (gi13929068) was subcloned into pcDNA3.1 (Invitrogen, Carlsbad, CA). Wild-type MCH1R and Flag-MCH1R have previously been shown to have similar EC50 values for MCH for calcium influx, indicating that the addition of the Flag tag does not affect the receptor function (25). Substitution mutants in the three cytoplasmic loops were produced by oligonucleotide-mediated site-directed mutagenesis using a Quickchange site-directed mutagenesis kit (Stratagene, La Jolla, CA). The substitution sites are shown in Fig. 1. First, to elucidate the cumulative effect, triple-substitution mutants of basic amino residues in each of the three intracellular loops, namely K67Q/K680Q/K70Q in i1 (i1/3Q), K153Q/R155Q/K156Q in i2 (i2/3Q), and K252Q/R253Q/R256Q in i3 (i3/3Q), of the full-length MCH1R were constructed. Next, a series of single-substitution mutants were constructed by mutating residues K67, K68, and K70 in i1; K153, R155, and K156 in i2; and K252, R253 and R256 in i3 to Q. For i2, R155 was mutated to K and D. All the mutations in the MCH1R cDNA sequence were confirmed by sequencing analysis. The mutated MCH1R cDNAs were excised by EcoR1 and XhoI digestion and inserted into the expression vector pcDNA3.1. The human MCH2R and SSTR2 cDNAs were a gift from Dr. O. Civelli (University of California, Irvine, CA). Substitution mutants of the MCH2R and SSTR2 cDNA sequences were created as described above.
FIG. 1. Schematic model of wild-type rat MCH1R and the single and triple BBXB or BBXXB substitution mutations. A BBXB motif in i1, a reversed BBXB motif in i2, and a BBXXB motif in i3 are denoted by the filled circles in square boxes in the putative arrangement of the rat MCH1R. wild-type MCH1R, and all the mutants were tagged with a Flag epitope at their N termini. The sequences of Flag-MCH1R and 14 mutants containing triple- or single-substitution mutations that exchanged basic residues for Q (i1: i1/3Q, K67Q, K68Q, and K70Q; i2: i2/3Q, K153Q, R155Q, and K156Q; i3: i3/3Q, K252Q, R253Q, and R256Q) and R155 in i2 by K or D (R155K and R155D, respectively) are shown. Each mutant was constructed and transfected into HEK293T cells. TM, Transmembrane.
Cell culture and transfection
DNA was mixed with LipofectAMINE PLUS transfection reagents (Invitrogen), and the mixture was then diluted with OptiMEM and added to 70–80% confluent human embryonic kidney (HEK)293T cells plated on 6-well plates. The transfected cells were cultured in DMEM containing 10% fetal bovine serum (FBS). At 48 h after the transfection, cell membranes were prepared from the cells for radioligand binding assays. For calcium influx assays, FACScan (Becton Dickinson Immunocytometer Systems Inc., Franklin Lakes, NJ) flow cytometric analysis and immunocytochemistry, the cells were plated on 96-well plates, 24-well plates, and coverslips, respectively, at 24 h after the transfection and then cultured for a further 24 h at 37 C. Flag-MCH1R, i1/3Q, i2/3Q, i3/3Q, and R155Q were also stably transfected into HEK293T cells. After 72 h, transfected cells were selected in the presence of zeocin at a final concentration of 0.4 μg/ml for 3 wk and used for measurement of the cAMP levels.
Western blotting and immunoprecipitation
Western blotting and immunoprecipitation were carried out essentially as described (23). To generate whole-cell extracts, HEK293T cells were lysed with ice-cold sodium dodecyl sulfate (SDS)-sample buffer [50 mM Tris-HCl (pH 6.8), 2% SDS, 50 mM ?-mercaptoethanol, and 10% glycerol] and then homogenized by sonication (Bioruptor; CosmoBio, Tokyo, Japan) at 4 C. For immunoprecipitation analyses, HEK293T cells were lysed with a rubber policeman in ice-cold solution A [50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.5% deoxycholate, and a protease inhibitor mixture (Roche, Indianapolis, IN)] for 20 min at 4 C and then cleared by centrifugation at 18,500 x g for 20 min at 4 C. For immunoprecipitation, aliquots of the cell lysates were precleared with protein-G-agarose on a rotator at 4 C for 30 min, and the protein-G-agarose was removed by centrifugation. Next, the lysates were incubated with 2 μg of an anti-Flag M2 antibody (Sigma, St. Louis, MO) and protein-G-agarose on a rotator for 15 h at 4 C. The immune complexes were washed three times with solution A and once with PBS and subsequently eluted from the protein-G-agarose by addition of SDS sample buffer. The proteins were separated in a 12.5% SDS-PAGE gel and then electrotransferred to a Hybond-P polyvinyl difluoride membrane (Amersham International, Little Chalfont, UK). After blocking with 5% skim milk dissolved in washing buffer (0.2% Tween 20 in Tris-HCl-buffered saline), Flag-MCH1R on the membrane was detected using the anti-Flag M2 antibody (2 μg/ml), followed by a horseradish peroxidase-conjugated goat antimouse IgG antibody. The reactive bands were visualized with enhanced chemiluminescence substrates (Amersham International) and analyzed using Scion Image (Scion Corp., Frederick, MD).
FACScan flow cytometric analysis of cell surface receptors
Flow cytometry was performed as described (23). Transfected HEK293T cells in 24-well plates were fixed with 1% paraformaldehyde and then incubated with 8 μg/ml anti-Flag M2 antibody in PBS containing 20% FBS and 0.05% sodium azide for 1 h. The cells were washed three times with PBS and then incubated with 10 μg/ml fluorescein isothiocyanate (FITC)-conjugated goat antimouse IgG secondary antibody for 1 h. The cells were collected from the wells with 5 mM EDTA and analyzed using a FACScan flow cytometer (Becton Dickinson Immunocytometer Systems). Cells were gated by light scatter or exclusion of propidium iodide, and 10,000 cells were acquired for each time point. The mean fluorescence of all the cells minus the mean cell fluorescence with the FITC-conjugated secondary antibody alone was used for the calculations.
Radioligand binding
Membrane preparations derived from transfected HEK293T cells were subjected to radioligand binding assays according to a previously described procedure (24). The membrane fractions (30 μg protein/assay) were incubated with increasing concentrations of [125I] (Phe13, Tyr19) MCH (Amersham International) from 0.01–6 nM in the presence or absence of 1 μM nonlabeled MCH (Peptide Institute, Osaka, Japan) in 500 μl of assay buffer [50 mM Tris-HCl (pH 7.4), 1 μM phosphoramidon, 0.5 mM phenylmethylsulfonylfluoride, and 0.2% BSA)] at room temperature for 2 h. The binding reaction was terminated by rapid filtration through GF/C glassfiber filter paper plates presoaked in 0.2% polyethylenimine, followed by three washes with 3 ml PBS. The radioactivity retained in the filter was determined using a -counter. Specific binding was defined as the difference between the total binding and the nonspecific binding.
Measurement of intracellular Ca2+
Transfected HEK293T cells or Chinese hamster ovary (CHO) cells seeded on black-walled 96-well plates (Becton Dickinson) were loaded with a nonwash calcium dye (calcium assay kit; Molecular Devices, Sunnyvale, CA) in Hanks’ balanced salt solution containing 20 mM HEPES (pH 7.5) for 1 h at 37 C. For each concentration of MCH or somatostatin 14, the level of intracellular Ca2+ ([Ca2+]i) was detected using a Flexstation imaging plate reader (Molecular Devices) over a 150-sec stimulation period. For pertussis toxin treatment, the transfected HEK293T cells were treated with 200 ng/ml pertussis toxin in DMEM containing 10% FBS for 18 h. Data were expressed as the fluorescence (arbitrary units) vs. time. The EC50 values for MCH or somatostatin 14 were obtained from sigmoidal fits using a nonlinear curve-fitting program (Prism version 3.0; GraphPad Software, San Diego, CA).
Measurement of cAMP production
Transfected HEK293T cells were seeded on 24-well plates and incubated for 24 h. The cells were preincubated with cAMP assay buffer [Hank’s balanced salt solution supplemented with 20 mM HEPES and 0.3 mM 3-isobutyl-1-methylxanthine (pH 7.5)] for 10 min. The cells were then incubated with forskolin (1 μM) and various concentrations of MCH for 15 min. Reactions were terminated with 0.3 N HCl, and the levels of extracted intracellular cAMP were measured using a RIA kit (Yamasa, Kyoto, Japan) following the manufacturer’s protocol.
GTPS binding assay
Aliquots (8 μg) of the membrane proteins isolated for radioligand binding as described above were incubated in 500 μl of 5'-O-(3-thiotriphosphate) (GTPS) binding buffer (20 mM HEPES-NaOH, pH 7.5, 100 mM NaCl, 5 mM MgCl2, 0.2% BSA, and 3 μM GDP) containing 0.2 nM [35S]GTPS and various concentrations of MCH for 30 min at 30 C. To determine the nonspecific binding, unlabeled GTPS was added to the binding mixtures to a final concentration of 100 μM. The bound [35S]GTPS was separated from free [35S]GTPS by rapid filtration through GF/C filters and washed three times with 3 ml of ice-cold binding buffer. The radioactivities of the filters were counted in 8 ml of the scintillation cocktail Emulsion-Scintillator Plus (Packard Bioscience, Groningen, The Netherlands) using a liquid scintillation counter.
Molecular modeling
We generated a homology model of rat MCH1R using a high-resolution (2.2A) x-ray structure of bovine rhodopsin [Protein Data Bank code 1U19] as a template. The first sequence alignment was carried out using the features of domains information in SWISS-PROT [MCR1_RAT (accession no. P94639) as rat MCH1R and OPSD_BOVIN (accession no. P02699) as bovine rhodopsin]. The packing of the transmembrane region of the seven-helix backbone in rhodopsin was used as a model structure for MCH1R, and the model structure of the extra- and intracellular loop regions was built by each template structure aligned above. Energy minimizations of i1, i2, and i3 were performed to relax the side chain geometries by the steepest descent and conjugate gradient minimization procedures with an Amber force field. Model structures of the MCH1R mutations in i2 were built from the model structure of wild-type MCH1R by exchanging side chain atoms and minimizing the energy. All model components were assembled using the biopolymer module of the SYBYL program package (TRIPOS Inc., St. Louis, MO).
Results
Effects of triple substitutions of highly conserved basic motifs in the intracellular loops of MCH1R on receptor activity
To elucidate the functional contributions of the highly conserved BXBB and BBXXB motifs in the intracellular loops of rat MCH1R, triple-substitution mutants were generated as shown in Fig. 1. Three triple-substitution mutant receptors were generated, in which the membrane-proximal-positive charges in i1, i2, or i3 were neutralized by substitution with Q. Flag-MCH1R and the mutant receptors were each transiently transfected into HEK293T cells to analyze their levels of protein expression and cell surface expression, radioligand saturation-binding, and signaling pathway. The levels of receptor expression were determined by immunoblotting analysis of the transfected cells using the anti-Flag M2 antibody (Fig. 2A, left panel). In cells transfected with wild-type Flag-MCH1R, the receptors migrated with the predicted molecular masses of 38, 44, 45, and 60 kDa. Our previous enzymatic deglycosylation study revealed that the 38-kDa band was the nonglycosylated form of MCH1R, whereas the three higher molecular mass bands (44, 45, and 60 kDa) were different N-linked glycosylated forms (25). Four immunoreactive bands were clearly detected in cells transfected with the i3 mutant (i3/3Q), and their levels of expression were nearly the same as those of Flag-MCH1R, as quantified by imaging analysis. However, for the i1 and i2 triple-substitution mutants (i1/3Q and i2/3Q, respectively), the 60-kDa receptor appeared to be reduced in intensity, compared with that for Flag-MCH1R. This finding was observed more clearly after immunoprecipitation (Fig. 2A, right panel). The intensities of the 60-kDa receptor were decreased by 67 and 61% in i1/3Q and i2/3Q, respectively, whereas the intensities of the other three bands in the mutants were nearly identical with those of Flag-MCH1R. The lower levels of expression of the 60-kDa receptor appeared to be due to a lack of appropriate glycosylation of the mutant receptors, as previously described in an N-linked glycosylation study and C-terminal truncated mutants of MCH1R (25, 26). These results may indicate that the highly conserved basic motifs in i1 and i2, but not that in i3, are responsible for correct intracellular trafficking and the extent of glycosylation.
FIG. 2. Analysis of the effects of triple-substitution mutations of the highly conserved basic motifs in the intracellular loops of MCH1R on receptor function. A, Protein expression of Flag-MCH1R and mutant receptors. Left, After lysis of transfected cells with SDS-sample buffer, 30 μg total protein were separated by 15% SDS-PAGE, transferred to a polyvinyl difluoride membrane, and immunoblotted with an anti-Flag M2 antibody. Four major immunoreactive bands of 38, 44, 45, and 60 kDa are present in Flag-MCH1R and the mutant receptors, although the intensity of the 60-kDa receptor (arrow) appears to be decreased in the i1 and i2 mutants. Right, After lysis of transfected cells and preclearing with protein-G-agarose, the lysates were subjected to immunoprecipitation with the anti-Flag M2 antibody. The 38-, 44-, 45-, and 60-kDa bands are clearly observed in Flag-MCH1R, but the intensity of the 60-kDa receptor (arrow) is decreased in the i1 and i2 mutants, similar to the results for the Western blotting. B, Saturation curves of Flag-MCH1R and the mutant receptors in radioligand binding. HEK293 cells transfected with Flag-MCH1R or the mutated receptors were harvested, and the membrane fractions were isolated. The membrane proteins (30 μg) were incubated with the indicated concentrations of [125I] (Phe13, Tyr19) MCH in the presence or absence of 1 μM nonlabeled MCH in 500 μl of assay buffer at room temperature for 2 h. Specific binding was defined as the difference between the total binding and the nonspecific binding. All experiments were independently performed three times in duplicate, and representative results are shown. C, Dose-response relationships of the MCH-stimulated calcium influx in HEK293T cells expressing Flag-MCH1R or the mutant receptors. Cells transfected with Flag-MCH1R or the triple-substitution mutant receptors were stimulated with the indicated concentrations of MCH, and the subsequent changes in the cytoplasmic-free Ca2+ levels were measured using a Flexstation. All experiments were independently performed at least three times in duplicate, and representative results are shown.
The level of cell surface expression was monitored by FACScan flow cytometry using the anti-Flag M2 antibody and a FITC-conjugated secondary antibody (Table 1). The mutated i1/3Q and i2/3Q receptors each showed a 60% reduction in the cell surface expression, whereas the level of i3/3Q was identical with that of Flag-MCH1R. Radioligand binding studies using cell membrane preparations demonstrated that the maximal binding capacity (Bmax) value for i3/3Q was similar to that for wild-type Flag-MCH1R, whereas those for i1/3Q and i2/3Q exhibited 75% decreases (Fig. 2B and Table 2). The maximal binding capacity values seemed to parallel those found by flow cytometry. In contrast, both i1/3Q and i2/3Q showed similar affinity constants (Kd) to that of Flag-MCH1R, whereas i3/3Q showed a 3-fold decrease (Table 2). Collectively, i1/3Q and i2/3Q revealed similar properties for their levels of protein expression and cell surface expression and similar parameters for radioligand binding.
TABLE 1. Cell surface expression and signaling of Flag-MCH1R and triple-substitution mutants of basic amino residues in the intracellular cytoplasmic loops of MCH1R after transfection into HEK293T cells
TABLE 2. Specific radioligand binding of Flag-MCH1R and mutant receptors
Next, we evaluated whether the highly basic regions of i1, i2, and i3 in MCH1R affect the MCH-stimulated calcium influx using a Flexstation. Representative curves are shown in Fig. 2C. Cells expressing i2/3Q completely lost their MCH-mediated calcium influx, whereas it was still observed in cells expressing i1/3Q or i3/3Q. Table 1 shows the EC50 values for Flag-MCH1R and the mutants. i1/3Q had a 20-fold higher EC50 value and a 50% reduction in the maximal response. In i3/3Q, the dose-response curve was shifted to the right and gave a 50-fold higher EC50 value with a similar maximal response to that of Flag-MCH1R (Fig. 2C). MCH1R has previously been shown to couple functionally to both Gq and Gi, which facilitate the calcium influx and inhibit cAMP accumulation (12, 13). Therefore, we characterized the phenotypes of the triple-substitution mutants by measuring the cAMP level after application of MCH to stably transfected cells (Table 3). MCH potently inhibited forskolin-stimulated accumulation of cAMP with an EC50 value of 1.2 nM and a 60% reduction in the maximal response of Flag-MCH1R. The EC50 values for i1/3Q and i3/3Q were 8- and 12-fold higher than the value for Flag-MCH1R, respectively. The effects of the MCH mutants on cAMP inhibition were smaller than those on the calcium influx, although direct comparisons of magnitude between different types of assay are not easy. However, cells expressing i2/3Q showed no inhibition of forskolin-stimulated accumulation of cAMP. The basal levels and forskolin-stimulated increases in the cAMP level in i1/3Q, i2/3Q and i3/3Q were identical with those induced in Flag-MCH1R-transfected cells. Thus, the triple-substitution mutations in the highly basic motifs of i1, i2, and i3 result in reductions in both Gq and Gi signaling.
TABLE 3. Forskolin-stimulated cAMP inhibition in HEK293T cells stably transfected with Flag-MCH1R, triple-substitution mutants of the intracellular cytoplasmic loops, or R155Q
Effects of individual substitutions in the i1 BBXB, i2 BBXB, and i3 BBXXB motifs on MCH1R function
The above results suggested that the basic motif in i2 may be a key determinant for signal transduction in Flag-MCH1R. To investigate the functional significance of individual basic residues in BBXB or BBXXB, a series of single-point mutants with substitution of B for Q were constructed as shown in Fig. 1. In cells expressing single-substitution mutants of i1, the cell surface expression of K68Q was identical with that of Flag-MCH1R, whereas 40% reductions were detected for K67Q and K70Q. However, K67Q elicited a calcium influx in a similar manner to Flag-MCH1R, whereas both K68Q and K70Q exhibited 2.5-fold higher EC50 values for calcium signaling without changing the high-affinity Kd value (Tables 2 and 4 and Fig. 3A). The cell surface expressions of all the single-substitution mutants of i3 were identical with that of Flag-MCH1R (Table 4). Regarding stimulation of a calcium influx, K252Q showed no significantly impaired function, whereas both R253Q and R256Q caused approximately 4.5-fold increases in the EC50 values with a 2-fold higher Kd value (Tables 2 and 4 and Fig. 3C). These results indicate that the last two amino acids in the context of the basic motifs in i1 and i3 affect receptor signaling with greater importance.
TABLE 4. Cell surface expression and signaling of Flag-MCH1R and single-substitution mutants of basic amino residues in the intracellular cytoplasmic loops of MCH1R after transfection into HEK293T cells
FIG. 3. Dose-response relationships of the MCH-stimulated calcium influx in HEK293T cells expressing Flag-MCH1R or single-substitution mutant receptors. Cells transfected with Flag-MCH1R or single-substitution mutant receptors of i1 (A), i2 (B), and i3 (C) were stimulated with the indicated concentrations of MCH as described in the legend for Fig. 2B. All experiments were independently performed at least three times in duplicate, and representative results are shown. A Western blotting analysis of the protein expressions of Flag-MCH1R and individual single-substitution mutant receptors of i2 is shown as an inset (B).
However, the single-substitution mutants in the conserved motif of i2 revealed distinct aspects from those of i1 and i3 for the receptor activity. Cells expressing R155Q showed a very small increase in the MCH-induced calcium influx with a 75-fold higher EC50 value and a 50% reduction in the maximal response, whereas mutations of the other two basic residues, K153Q and K156Q, had no significant effects on signaling (Table 4 and Fig. 3B). On the other hand, K153Q, R155Q, and K156Q all showed similar affinities (Kd) to Flag-MCH1R (Table 2) and similar characters in immunoblotting analyses of whole-cell extracts (Fig. 3B, inset). Furthermore, the levels of cell surface expression were nearly equivalent among the three mutants (Table 4). These data suggest that the profound impairment of the signaling function in R155Q is not secondary to the lack of glycosylation or low expression level at the cell surface. R155 was further changed into other amino acid residues to determine the specificity of this position in the signaling pathway of MCH1R (Table 4). Mutation of R155 to K produced a 50-fold higher EC50 value of MCH, compared with Flag-MCH1R, with a 50% reduction in the maximal response. Moreover, substitution of R155 with a negatively charged amino acid, D, completely abolished the ability of the receptor to stimulate calcium signaling with an 80% decrease in the level of cell surface expression. Next, we characterized the phenotype of R155Q by measuring the cAMP level in stably transfected cells. As observed for i2/3Q, R155Q had a drastic influence on the coupling to both the calcium response and adenylyl cyclase. In R155Q-expressing cells, MCH inhibited forskolin-stimulated accumulation of cAMP with a nearly 50-fold higher EC50 value, and the maximal inhibition of cAMP accumulation was reduced by nearly 50% (Table 3).
Because G proteins have been reported to directly affect the structure and ligand affinity of GPCRs, we determined whether the R155Q mutant was able to couple to G proteins and stimulate the exchange of GDP for GTP on G subunits after exposure to MCH. We quantified MCH-induced [35S]GTPS binding to G protein using membrane preparations from HEK293 cells transiently transfected with either Flag-MCH1R or R155Q. As shown in Fig. 4, Flag-MCH1R showed dose-dependent binding of [35S]GTPS with an EC50 value of 14.4 ± 0.80 nM. R155Q also stimulated GDP-GTP exchange on G subunits with a higher EC50 value of 201.9 ± 13.7 nM (Fig. 4). The maximal amounts of binding with 10 μM MCH were 142.0 and 132.5% in Flag-MCH1R- and R155Q-transfected cells, respectively. Thus, point mutations of R155 can drive MCH1R into a different affinity state for coupling to G protein, supporting the crucial role of this residue in the high efficiency activation process of the receptor.
FIG. 4. MCH-induced [35S]GTPS binding to Flag-MCH1R and R155Q. HEK293 cells were transfected with Flag-MCH1R or R155Q. After 48 h, the cells were harvested and the membrane fractions were recovered. The membrane proteins (8 μg) were subsequently incubated with 0.2 nM [35S]GTPS and 1 nM to 10 μM MCH in GTPS binding buffer for 30 min at 30 C. The amounts of radioactivity bound to the membrane preparations are shown as Flag-MCH1R (open circles) and R155Q (filled circles). All experiments were independently performed at least three times in triplicate, and representative results are shown.
Functional importance of corresponding arginine residues in i2 of other receptors
To further explore the key role of R155 in the basic motif of i2, we first examined whether an R was conserved at the equivalent position to R155 in i2 of MCH1R in orthologs, including fish. A sequence alignment search revealed that an R was evolutionally conserved at the position corresponding to R155 in rat MCH1R from mammals to zebrafish (43). We also searched whether the R position in i2 of the rat MCH1R is conserved in other GPCRs. Sequence alignment using a BLAST search clarified that human MCH2R, five SSTR subtypes, two opioid receptors, and an orphanin FQ/nociceptin receptor have a highly conserved R at the position corresponding to R155 of MCH1R, whereas rhodopsin does not. This R is the only amino acid residue that is well conserved in the C-terminal region of i2 (Table 5), whereas the junction of transmembrane 3 has a common motif of E/DRY among class A GPCRs.
TABLE 5. Amino acid sequence alignments of the c terminus of transmembrane 3 (TM3) and i2 of MCH1R and other GPCRs
This fact strongly suggests that the existence of an R at this position is critically important for receptor activity. Therefore, we generated single-substitution mutants at the corresponding residue R145 in human MCH2R and R154 in rat SSTR2 and examined the effects of these mutations on the receptor function. MCH2R has been shown to predominantly activate the Gq protein in HEK293 cells (15), and replacement of R145 in MCH2R with a neutrally charged amino acid, Q, led to pronounced changes in the calcium mobilization, with a 20-fold higher EC50 value for MCH and a 40% decrease in the maximal response compared with those of MCH2R (Table 6). On the other hand, all five somatostatin receptors have been shown to stimulate the activation of only Gi in both CHO cells and HEK293 cells (44). Therefore, we cotransfected SSTR2 with a Gq/i3 chimera that contained the five C-terminal residues of Gi3 and the rest of the Gq sequence (7). This chimera was designed to facilitate SSTR2 signaling in Gi3 to Gq activation because the five C-terminal residues of G are sufficient for receptor contact, whereas the remaining residues of G interact with the effector molecule. Receptor activity caused by somatostatin 14 in SSTR2 was recorded as a calcium influx in cotransfected cells. HEK293T cells expressing SSTR2 with the Gi3 chimera had an EC50 value of 0.0016 nM for somatostatin 14, whereas introduction of the point mutation R154Q produced an approximately 70-fold higher EC50 value with a 40% decrease in the maximal response, compared with those for SSTR2 (Table 6). Substitution at R154 also elicited decreased calcium mobilization in cotransfected CHO cells, as evidenced by the 30-fold higher EC50 value with a 45% decrease in the maximal response (Table 6). These results imply that, in some other GPCRs, the positions corresponding to R155 in i2 of MCH1R confer full activity for calcium influx and/or adenylyl cyclase caused by ligand stimulation.
TABLE 6. Signaling of human MCH2R, rat SSTR2 and their single-substitution mutant receptors after transfection into CHO and/or HEK293T cells
Discussion
Clarification of the amino acid sequences that influence the activation of G proteins in GPCRs is primarily important for understanding how each receptor forms critical contact points with G proteins. The amino acids of class A GPCRs have been extensively studied and various components in i2, i3, and helix 8 have been implicated as points of contact with G proteins or interacting molecules. In the current study, to determine the functional significance of the known structural motifs BBXXB and BXBB that exist in i1, i2, and i3 of MCH1R, we constructed a series of mutant receptors by replacing the basic amino acids with the noncharged amino acid Q. We started by generating triple-substitution mutants and then conducted individual mutation analyses. A single mutation at R155, but not at K153 or K156, in i2 caused very large reductions in the Gq- and Gi-mediated activities. Moreover, we showed the functional importance of the equivalent R in MCH2R and SSTR2 for Gq- and Gi-mediated activities, respectively. Our present data suggest that R155 in i2 represents a novel site with relevance to signal transduction, whereas the G protein coupling specificity seems to be governed by another receptor domain.
Importance of the BXBB and BBXXB motifs in MCH1R
Although the highly conserved basic motifs have been suggested to be functionally relevant (38, 39, 41), this could not be a universal rule for all GPCRs. One example is the m1 muscarinic acetylcholine receptor, in which the BBXXB motif in i3 is necessary for coupling to phosphoinositide hydrolysis, whereas the same motif in i2 is not required (40). However, our present data define the significance of the basic motifs in all three loops of MCH1R for the receptor activity. Interestingly, the contributions of each motif appeared to differ in terms of their ligand binding, protein expression, and ability to induce a calcium influx. The intensities of the positive bands in Western blotting and levels of cell surface expression were nearly identical for i1/3Q and i2/3Q, yet only the latter mutant resulted in a nonfunctional receptor for signaling. i3/3Q did not show any alterations in the intensity of protein expression or level of plasma membrane expression but did have a lower affinity than that of Flag-MCH1R. The mutant receptor i3/3Q therefore showed a right shift of the dose-response curve for the calcium influx, whereas retaining an identical maximal response to that of Flag-MCH1R. Reduced binding affinity was also observed in the R253Q and R256Q single-substitution mutants. Thus, the basic motif in i3 of MCH1R seems to be more responsible for agonist binding and G protein coupling than for trafficking of the receptor to the cell surface. The BBXXB motif in i3 may function in the packing of the transmembrane domains in MCH1R, resulting in either decreased accessibility of the G protein coupling site or inability of the receptor to form an active conformation upon MCH binding. In addition, all the triple-substitution mutants of the basic motifs caused reductions in the Gq- and Gi-mediated activities, suggesting that the G protein coupling specificity in MCH1R resides in regions other than the basic motifs.
The single-substitution analyses indicated that the role of each basic residue differed depending on its position within the basic motif. In terms of the receptor-mediated calcium influx mediated by MCH, the EC50 values for K68Q and K70Q were 2.5-fold higher than that for Flag-MCH1R. On the other hand, the values for K253Q and R256Q were 4.5-fold higher. K67Q and K252Q in i1 and i3, respectively, did not significantly alter the EC50 value, although K67Q showed a 30% reduction in the cell surface expression. These data demonstrate that the first basic residue of each motif plays a minor role, whereas the second and third residues are important for efficient activation of MCH1R. On the other hand, R155 in the BBXB motif of i2 was the only residue that caused a strong defect in signaling, despite the findings that K153Q and R155Q showed very similar levels of protein expression and cell surface expression. It should be noted that all the triple-substitution mutations of i1, i2, and i3 showed much stronger effects on the signaling and cell surface expression than any of the single-substitution mutations in each loop, implying that the individual basic residues work in mutually exclusive manners. Overall, these findings indicate that each basic residue in a motif has a different impact, but they work cooperatively for receptor activity. These findings are consistent with previous results for the m1 acetylcholine receptor and FSH receptor, in which individual residues function in a hierarchal and interdependent manner for receptor coupling (40) and cell surface expression (42), respectively.
Pivotal role of R155 in MCH1R for receptor signaling
A large number of experiments have identified conserved motifs in i2 as key determinants in various receptor functions. The E/DRY motif of transmembrane 3, which is the most conserved motif among class A GPCRs, is critical for the efficacy of G protein coupling and/or constitutive activation (38). In several GPCRs, it has been shown that the hydrophobic residue in the E/DRYXXV(I)XXPL motif plays a significant role in the G protein coupling specificity (37). In contrast, only a few papers have reported involvement of the BBXXB or BBXB motifs in i2 in receptor function. An example of these studies is the TSH receptor, in which an individual single mutation in BBXXB had only a modest effect on the signaling efficacy (45).
Apart from such basic motifs, several pieces of evidence have suggested that single or dibasic residues are critical for receptor function. For example, K in the proximal C-terminal region of i2 in the PTH/PTH-related protein receptor is a key determinant for Gq-selective coupling (46). In MCH1R, point mutation of R155 in i2 to the neutrally charged Q caused severe signaling impairment but had no effect on the G protein selectivity. Replacement of R155 by the negatively charged D resulted in a nonfunctional mutant with a 75% reduction in cell surface expression, accentuating the critical nature of R155, i.e. the importance of its positively charged nature. Interestingly, however, replacement of R155 with the moderately charged K also caused a profound reduction in signaling efficiency. This observation is in contrast to the case of a dibasic motif in the putative helix 8 in MCH1R, in which a moderate level of net charge is sufficient for activity (26). This fact may indicate that a sufficient level of positive charge at position 155 is strictly required for efficient signaling in MCH1R. To evaluate another possibility, we speculatively calculated the solvent accessible surface area (SASA) of the individual amino acids in i2 of rat MCH1R according to the method of Lee and Richards (47) with a 1.4A radius for the solvent probe. The extent of the solvent-exposed polar SASA of R155 appears to be greater than those of the other residues in the i2 region. On the other hand, the polar SASA of R155K is decreased to less than half that of the wild-type R155 (data not shown). The SASA histograms for R155Q and R155D showed very similar patterns to that of R155. These speculative analyses may indicate that the polarity of R155, as well as its positive charge, is also a key factor for maximizing information transfer from MCH-occupied receptors.
Interruption of the significance of R155 using three-dimensional modeling
Our findings highlight the fact that minor changes in the structure of i2 can lead to pronounced changes in the G protein coupling efficiency of MCH1R as suggested by the GTPS binding assays and probably also in some other GPCRs. How does amino acid substitution of R155 suppress the efficacy of the signaling pathway in MCH1R? The high resolution x-ray structure of bovine rhodopsin indicates that i2 has an L-like structure when viewed parallel to the membrane plane but lacks a regular secondary structure (48). Like the other intracellular loops, i2 does not fold over the area defined by the intracellular ends of transmembrane domains 1–7. To interpret a possible explanation for the role of R155, we generated a theoretical three-dimensional structure of the rat MCH1R by homology modeling, using the coordinates of the 2.2A crystal structure of bovine rhodopsin as a template (Fig. 5). At present, only rhodopsin can be used as a starting point, and therefore molecular modeling of the intracellular loops in several GPCRs has been reported based on the structure of rhodopsin (34, 45). However, we cannot state that our receptor model is representative of the actual structure of MCH1R. The arrangement of the transmembrane regions in the model may be quite reliable, but the homology of less than 40% between the i2 regions of rhodopsin and MCH1R (Table 5) may not favor molecular modeling. In the current study, the molecular analysis was used to locate the mutated residues and highlight possible relationships for receptor function.
FIG. 5. Model of the three-dimensional structure of the rat MCH1R. The depicted structure is based on the high-resolution x-ray structure of bovine rhodopsin (Protein Data Bank code 1U19; resolution = 2.2A). Energy minimization was applied to this model structure to refine the side chain positions. A, Model structure viewed from the direction of the cytoplasm to the extracellular region. Numbered arrows indicate the transmembrane regions. Transmembrane domains 3 and 4 are shown in yellow and purple, respectively. Loops i2 and i3 are shown in blue and orange, respectively. The highly conserved residues DRY (green) and R155 (violet) are shown as ball-and-stick models. In our model structure, the side chain of R155 is predicted to have no contact with any of the loops or transmembrane regions. B, Spatial orientations of K153, R155, and K156 in i2. The side chains are shown as ball-and-stick models, and the white arrow indicates the side chains in the DRY region.
The model predicts that R155 is located just at the N terminal of the bend of the L-like structure, which is a characteristic feature of i2 that is also seen in rhodopsin (48). The positively charged side chain of R155 is exposed and faces away from the interior of the structure and therefore cannot be important for cytoplasmic intracellular loop interactions. Furthermore, R155 does not interact with the nearby transmembrane domain. In addition, the side chains of D140(3.49), R141(3.50), and Y142(3.51) are speculatively oriented in the opposite direction to the side chain of R155. Therefore, it may be assumed that R155 is easily accessible to G proteins and that the switch in efficient signaling is most likely due to a molecular interaction. Figure 5B shows that the side chains of K153 and K156 are predicted to lie in the opposite direction to that of R155 and may therefore have less chance of contact with cytosolic molecules. This may explain why single substitutions of K153 and K156 did not significantly decrease the efficiency of signal transduction. Further studies are required to pinpoint the contact points between R155 in MCH1R and the G protein subunits directly involved in mediating responses in the target cells.
In summary, this study provides evidence for a crucial role of R155 in i2 as a recognition determinant in MCH1R. Our data further suggest the functional significance of the equivalent R in some other GPCRs. Although a final conclusion cannot be reached until the three-dimensional structure of the MCH1R-G protein complex is analyzed, the results of the present study suggest that the possible cytosolic orientation of the basic residue R155 is involved in a step of the G protein coupling. Therefore, we postulate that the mutant cannot properly signal the conformational changes to the downstream. Further investigations are also required to define the molecular structure and interacting molecules that determine the receptor-G protein specificity of MCH1R. These studies will allow additional understanding of the dual G protein coupling of MCH1R and particularly of the selectivity of activation.
Acknowledgments
We thank Dr. Y. Odagaki (Department of Psychiatry, Saitama Medical School, Saitama, Japan) for helpful advice regarding the GTP S binding assay. We also thank Drs. O. Civelli and R. Reinscheid (University of California, Irvine) for critically reviewing the manuscript.
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Address all correspondence and requests for reprints to: Yumiko Saito, Department of Pharmacology, Saitama Medical School, 38 Moro-Hongo, Moroyama-cho, Iruma-gun, Saitama 350-0492, Japan. E-mail: yumisait@saitama-med.ac.jp.
Abstract
Melanin-concentrating hormone (MCH) receptor 1 (MCH1R) is a class A G protein-coupled receptor. The MCH system has been linked to a variety of physiological functions, including the regulation of feeding and energy metabolism. We recently reported the importance of a dibasic motif in the membrane-proximal C-terminal region for MCH1R function. Here we reveal that an Arg residue in intracellular loop 2 of MCH1R plays a critical role in receptor function. We analyzed the roles of two distinct motifs, BBXXB and BXBB (in which B is a basic residue and X is a nonbasic residue), located in the three intracellular loops of MCH1R. Triple-substitution mutants of intracellular loops 1 and 3 could still activate calcium mobilization, albeit with lower efficacy or potency. However, mutations in intracellular loop 2 led to a complete loss of induction of signal transduction without changing the high affinity constant (Kd) value. By analyzing a series of single-substitution mutants, a point mutation of Arg155 in intracellular loop 2 was found to be responsible for the signaling pathway elicited by MCH. In addition, substitution at positions corresponding to Arg155 in human MCH receptor 2 and rat somatostatin receptor 2 also markedly abolished their ligand-induced signaling capacities, indicating that this Arg is a recognition determinant in several G protein-coupled receptors.
Introduction
MELANIN-CONCENTRATING HORMONE (MCH) is a cyclic neuropeptide that was first isolated from the pituitary gland of salmon (1). It is predominantly produced by neurons of the lateral hypothalamus in mammals (2) and plays key roles in the regulation of food-intake behavior and energy balance (3, 4). The orphan G protein-coupled receptor (GPCR) somatostatin-like receptor-1 (SLC-1) (5) is activated by MCH and belongs to the class A GPCRs (6, 7, 8, 9, 10). This receptor, now referred to as MCH receptor 1 (MCH1R), is widely expressed at high levels in the brain (11, 12). MCH1R is able to activate multiple signal transduction pathways, namely cAMP inhibition, calcium influx and MAPK, via Gi and Gq coupling (4, 5, 7, 13, 14). Although a second MCH receptor, MCH receptor 2 (MCH2R), was isolated in humans (15), it was found to be nonfunctional or to encode a nonfunctional pseudogene in nonhuman species, including rodents (16). Targeted disruption of the MCH1R gene in mice results in lean, hyperactive and hyperphagic mice that are resistant to diet-induced obesity (17, 18). Thus, MCH1R is regarded, at least in rodents, as an endogenous mediator of MCH for energy homeostasis. Moreover, recent studies have shown that selective MCH1R antagonists inhibit MCH-induced food intake in rats (19, 20, 21), and one of these antagonists also exhibited antidepressant and anxiolytic effects (20). Therefore, MCH1R has attracted considerable attention as a possible target for therapeutic intervention in obesity and some mental disorders. Recently two common single-nucleotide polymorphisms were identified in MCH1R, but neither was found to be associated with any obesity-related phenotypes in a population of 500 white subjects (22). Although other single-nucleotide polymorphisms have been identified in the MCH receptors, their associated phenotypic characteristics have not yet been analyzed (23). Further clarification is required in other populations of obese subjects and/or in genetically modified mice.
Several studies have reported structural information that underlies the activation of MCH1R. Biochemical analysis of MCH1R using a molecular model revealed that D123 in the third transmembrane domain is critical for ligand binding (24). We previously demonstrated that N23 in the extracellular N-terminal region mainly contributes to N-linked glycosylation of MCH1R and is necessary for cell surface expression (25). Furthermore, we recently demonstrated a key role for the C-terminal tail of MCH1R for receptor function. The membrane-proximal region of MCH1R is predicted to form an amphiphilic cytoplasmic helix 8, and a dibasic motif in this helix 8 is important for signal transduction (26). On the other hand, the distal portion of the C-terminal tail in MCH1R is necessary for the internalization process (27). However, in comparison with the adrenergic and muscarinic receptor subfamilies, relatively little is known about the structure/function relationships in MCH1R, and more extensive research is necessary to determine the factors that influence its G protein-interacting states.
Traditional mutagenesis approaches, including the use of hybrid receptors, have implicated a particular part of the transmembrane region and several intracellular portions in GPCR function (28, 29, 30, 31, 32, 33). In many GPCRs, intracellular loop 2 (i2) and the juxtamembrane portions of intracellular loop 3 (i3) are believed to alter the signal transduction capacity of the receptor by directly affecting G protein coupling or altering the packing of the transmembrane helices (28, 29, 30). Several reports have also shown that intracellular loop 1 (i1) is required for G protein activation (31). A series of single-substitution mutagenesis studies have attempted to identify the amino acids in different GPCRs that are responsible for the receptor activities. Some of these amino acid residues have been proposed to be involved in the effector coupling of several receptors (34, 35, 36). Furthermore, several conserved motifs were found to be involved in cell surface expression and effector coupling (26, 37, 38). Okamoto and Nishimoto (39) first proposed that a BBXB or BBXXB motif (in which B is a basic amino acid residue and X is a nonbasic residue) was required for Gi activation, as evaluated by a synthetic peptide study. Based on further structure-activity studies, a number of GPCRs were shown to contain these structural motifs in particular regions of their intracellular loops. In fact, such motifs have been found to affect the selectivity for receptor/G protein coupling or the intracellular signaling pathway in several GPCRs (40, 41, 42).
In the present study, we analyzed the functional roles of two distinct BBXXB and BXBB motifs located in the cytoplasmic loops of MCH1R. Although the basic motifs are generally present in i2, i3, and the C-terminal tail but absent from i1, in other GPCRs (39), mouse, rat, and human MCH1R commonly possess a BBXB motif in i1, a reversed BBXB in i2 and a BBXXB motif in i3 (5). Systematic examination revealed that the motifs in all three loops have profound functions in the receptor activity and that point mutation of R155 in i2 strongly affected the signaling, although no significant change was observed in the Kd value of radioligand binding assays. We further discuss the significance of the positions corresponding to R155 in some other GPCRs.
Materials and Methods
cDNA constructs for MCH1R, MCH2R, and somatostatin receptor (SSTR) 2 mutagenesis
Incorporation of a sequence encoding the Flag epitope tag before the first methionine in rat MCH1R was performed by PCR (25). The purified full-length cDNA of MCH1R (gi13929068) was subcloned into pcDNA3.1 (Invitrogen, Carlsbad, CA). Wild-type MCH1R and Flag-MCH1R have previously been shown to have similar EC50 values for MCH for calcium influx, indicating that the addition of the Flag tag does not affect the receptor function (25). Substitution mutants in the three cytoplasmic loops were produced by oligonucleotide-mediated site-directed mutagenesis using a Quickchange site-directed mutagenesis kit (Stratagene, La Jolla, CA). The substitution sites are shown in Fig. 1. First, to elucidate the cumulative effect, triple-substitution mutants of basic amino residues in each of the three intracellular loops, namely K67Q/K680Q/K70Q in i1 (i1/3Q), K153Q/R155Q/K156Q in i2 (i2/3Q), and K252Q/R253Q/R256Q in i3 (i3/3Q), of the full-length MCH1R were constructed. Next, a series of single-substitution mutants were constructed by mutating residues K67, K68, and K70 in i1; K153, R155, and K156 in i2; and K252, R253 and R256 in i3 to Q. For i2, R155 was mutated to K and D. All the mutations in the MCH1R cDNA sequence were confirmed by sequencing analysis. The mutated MCH1R cDNAs were excised by EcoR1 and XhoI digestion and inserted into the expression vector pcDNA3.1. The human MCH2R and SSTR2 cDNAs were a gift from Dr. O. Civelli (University of California, Irvine, CA). Substitution mutants of the MCH2R and SSTR2 cDNA sequences were created as described above.
FIG. 1. Schematic model of wild-type rat MCH1R and the single and triple BBXB or BBXXB substitution mutations. A BBXB motif in i1, a reversed BBXB motif in i2, and a BBXXB motif in i3 are denoted by the filled circles in square boxes in the putative arrangement of the rat MCH1R. wild-type MCH1R, and all the mutants were tagged with a Flag epitope at their N termini. The sequences of Flag-MCH1R and 14 mutants containing triple- or single-substitution mutations that exchanged basic residues for Q (i1: i1/3Q, K67Q, K68Q, and K70Q; i2: i2/3Q, K153Q, R155Q, and K156Q; i3: i3/3Q, K252Q, R253Q, and R256Q) and R155 in i2 by K or D (R155K and R155D, respectively) are shown. Each mutant was constructed and transfected into HEK293T cells. TM, Transmembrane.
Cell culture and transfection
DNA was mixed with LipofectAMINE PLUS transfection reagents (Invitrogen), and the mixture was then diluted with OptiMEM and added to 70–80% confluent human embryonic kidney (HEK)293T cells plated on 6-well plates. The transfected cells were cultured in DMEM containing 10% fetal bovine serum (FBS). At 48 h after the transfection, cell membranes were prepared from the cells for radioligand binding assays. For calcium influx assays, FACScan (Becton Dickinson Immunocytometer Systems Inc., Franklin Lakes, NJ) flow cytometric analysis and immunocytochemistry, the cells were plated on 96-well plates, 24-well plates, and coverslips, respectively, at 24 h after the transfection and then cultured for a further 24 h at 37 C. Flag-MCH1R, i1/3Q, i2/3Q, i3/3Q, and R155Q were also stably transfected into HEK293T cells. After 72 h, transfected cells were selected in the presence of zeocin at a final concentration of 0.4 μg/ml for 3 wk and used for measurement of the cAMP levels.
Western blotting and immunoprecipitation
Western blotting and immunoprecipitation were carried out essentially as described (23). To generate whole-cell extracts, HEK293T cells were lysed with ice-cold sodium dodecyl sulfate (SDS)-sample buffer [50 mM Tris-HCl (pH 6.8), 2% SDS, 50 mM ?-mercaptoethanol, and 10% glycerol] and then homogenized by sonication (Bioruptor; CosmoBio, Tokyo, Japan) at 4 C. For immunoprecipitation analyses, HEK293T cells were lysed with a rubber policeman in ice-cold solution A [50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.5% deoxycholate, and a protease inhibitor mixture (Roche, Indianapolis, IN)] for 20 min at 4 C and then cleared by centrifugation at 18,500 x g for 20 min at 4 C. For immunoprecipitation, aliquots of the cell lysates were precleared with protein-G-agarose on a rotator at 4 C for 30 min, and the protein-G-agarose was removed by centrifugation. Next, the lysates were incubated with 2 μg of an anti-Flag M2 antibody (Sigma, St. Louis, MO) and protein-G-agarose on a rotator for 15 h at 4 C. The immune complexes were washed three times with solution A and once with PBS and subsequently eluted from the protein-G-agarose by addition of SDS sample buffer. The proteins were separated in a 12.5% SDS-PAGE gel and then electrotransferred to a Hybond-P polyvinyl difluoride membrane (Amersham International, Little Chalfont, UK). After blocking with 5% skim milk dissolved in washing buffer (0.2% Tween 20 in Tris-HCl-buffered saline), Flag-MCH1R on the membrane was detected using the anti-Flag M2 antibody (2 μg/ml), followed by a horseradish peroxidase-conjugated goat antimouse IgG antibody. The reactive bands were visualized with enhanced chemiluminescence substrates (Amersham International) and analyzed using Scion Image (Scion Corp., Frederick, MD).
FACScan flow cytometric analysis of cell surface receptors
Flow cytometry was performed as described (23). Transfected HEK293T cells in 24-well plates were fixed with 1% paraformaldehyde and then incubated with 8 μg/ml anti-Flag M2 antibody in PBS containing 20% FBS and 0.05% sodium azide for 1 h. The cells were washed three times with PBS and then incubated with 10 μg/ml fluorescein isothiocyanate (FITC)-conjugated goat antimouse IgG secondary antibody for 1 h. The cells were collected from the wells with 5 mM EDTA and analyzed using a FACScan flow cytometer (Becton Dickinson Immunocytometer Systems). Cells were gated by light scatter or exclusion of propidium iodide, and 10,000 cells were acquired for each time point. The mean fluorescence of all the cells minus the mean cell fluorescence with the FITC-conjugated secondary antibody alone was used for the calculations.
Radioligand binding
Membrane preparations derived from transfected HEK293T cells were subjected to radioligand binding assays according to a previously described procedure (24). The membrane fractions (30 μg protein/assay) were incubated with increasing concentrations of [125I] (Phe13, Tyr19) MCH (Amersham International) from 0.01–6 nM in the presence or absence of 1 μM nonlabeled MCH (Peptide Institute, Osaka, Japan) in 500 μl of assay buffer [50 mM Tris-HCl (pH 7.4), 1 μM phosphoramidon, 0.5 mM phenylmethylsulfonylfluoride, and 0.2% BSA)] at room temperature for 2 h. The binding reaction was terminated by rapid filtration through GF/C glassfiber filter paper plates presoaked in 0.2% polyethylenimine, followed by three washes with 3 ml PBS. The radioactivity retained in the filter was determined using a -counter. Specific binding was defined as the difference between the total binding and the nonspecific binding.
Measurement of intracellular Ca2+
Transfected HEK293T cells or Chinese hamster ovary (CHO) cells seeded on black-walled 96-well plates (Becton Dickinson) were loaded with a nonwash calcium dye (calcium assay kit; Molecular Devices, Sunnyvale, CA) in Hanks’ balanced salt solution containing 20 mM HEPES (pH 7.5) for 1 h at 37 C. For each concentration of MCH or somatostatin 14, the level of intracellular Ca2+ ([Ca2+]i) was detected using a Flexstation imaging plate reader (Molecular Devices) over a 150-sec stimulation period. For pertussis toxin treatment, the transfected HEK293T cells were treated with 200 ng/ml pertussis toxin in DMEM containing 10% FBS for 18 h. Data were expressed as the fluorescence (arbitrary units) vs. time. The EC50 values for MCH or somatostatin 14 were obtained from sigmoidal fits using a nonlinear curve-fitting program (Prism version 3.0; GraphPad Software, San Diego, CA).
Measurement of cAMP production
Transfected HEK293T cells were seeded on 24-well plates and incubated for 24 h. The cells were preincubated with cAMP assay buffer [Hank’s balanced salt solution supplemented with 20 mM HEPES and 0.3 mM 3-isobutyl-1-methylxanthine (pH 7.5)] for 10 min. The cells were then incubated with forskolin (1 μM) and various concentrations of MCH for 15 min. Reactions were terminated with 0.3 N HCl, and the levels of extracted intracellular cAMP were measured using a RIA kit (Yamasa, Kyoto, Japan) following the manufacturer’s protocol.
GTPS binding assay
Aliquots (8 μg) of the membrane proteins isolated for radioligand binding as described above were incubated in 500 μl of 5'-O-(3-thiotriphosphate) (GTPS) binding buffer (20 mM HEPES-NaOH, pH 7.5, 100 mM NaCl, 5 mM MgCl2, 0.2% BSA, and 3 μM GDP) containing 0.2 nM [35S]GTPS and various concentrations of MCH for 30 min at 30 C. To determine the nonspecific binding, unlabeled GTPS was added to the binding mixtures to a final concentration of 100 μM. The bound [35S]GTPS was separated from free [35S]GTPS by rapid filtration through GF/C filters and washed three times with 3 ml of ice-cold binding buffer. The radioactivities of the filters were counted in 8 ml of the scintillation cocktail Emulsion-Scintillator Plus (Packard Bioscience, Groningen, The Netherlands) using a liquid scintillation counter.
Molecular modeling
We generated a homology model of rat MCH1R using a high-resolution (2.2A) x-ray structure of bovine rhodopsin [Protein Data Bank code 1U19] as a template. The first sequence alignment was carried out using the features of domains information in SWISS-PROT [MCR1_RAT (accession no. P94639) as rat MCH1R and OPSD_BOVIN (accession no. P02699) as bovine rhodopsin]. The packing of the transmembrane region of the seven-helix backbone in rhodopsin was used as a model structure for MCH1R, and the model structure of the extra- and intracellular loop regions was built by each template structure aligned above. Energy minimizations of i1, i2, and i3 were performed to relax the side chain geometries by the steepest descent and conjugate gradient minimization procedures with an Amber force field. Model structures of the MCH1R mutations in i2 were built from the model structure of wild-type MCH1R by exchanging side chain atoms and minimizing the energy. All model components were assembled using the biopolymer module of the SYBYL program package (TRIPOS Inc., St. Louis, MO).
Results
Effects of triple substitutions of highly conserved basic motifs in the intracellular loops of MCH1R on receptor activity
To elucidate the functional contributions of the highly conserved BXBB and BBXXB motifs in the intracellular loops of rat MCH1R, triple-substitution mutants were generated as shown in Fig. 1. Three triple-substitution mutant receptors were generated, in which the membrane-proximal-positive charges in i1, i2, or i3 were neutralized by substitution with Q. Flag-MCH1R and the mutant receptors were each transiently transfected into HEK293T cells to analyze their levels of protein expression and cell surface expression, radioligand saturation-binding, and signaling pathway. The levels of receptor expression were determined by immunoblotting analysis of the transfected cells using the anti-Flag M2 antibody (Fig. 2A, left panel). In cells transfected with wild-type Flag-MCH1R, the receptors migrated with the predicted molecular masses of 38, 44, 45, and 60 kDa. Our previous enzymatic deglycosylation study revealed that the 38-kDa band was the nonglycosylated form of MCH1R, whereas the three higher molecular mass bands (44, 45, and 60 kDa) were different N-linked glycosylated forms (25). Four immunoreactive bands were clearly detected in cells transfected with the i3 mutant (i3/3Q), and their levels of expression were nearly the same as those of Flag-MCH1R, as quantified by imaging analysis. However, for the i1 and i2 triple-substitution mutants (i1/3Q and i2/3Q, respectively), the 60-kDa receptor appeared to be reduced in intensity, compared with that for Flag-MCH1R. This finding was observed more clearly after immunoprecipitation (Fig. 2A, right panel). The intensities of the 60-kDa receptor were decreased by 67 and 61% in i1/3Q and i2/3Q, respectively, whereas the intensities of the other three bands in the mutants were nearly identical with those of Flag-MCH1R. The lower levels of expression of the 60-kDa receptor appeared to be due to a lack of appropriate glycosylation of the mutant receptors, as previously described in an N-linked glycosylation study and C-terminal truncated mutants of MCH1R (25, 26). These results may indicate that the highly conserved basic motifs in i1 and i2, but not that in i3, are responsible for correct intracellular trafficking and the extent of glycosylation.
FIG. 2. Analysis of the effects of triple-substitution mutations of the highly conserved basic motifs in the intracellular loops of MCH1R on receptor function. A, Protein expression of Flag-MCH1R and mutant receptors. Left, After lysis of transfected cells with SDS-sample buffer, 30 μg total protein were separated by 15% SDS-PAGE, transferred to a polyvinyl difluoride membrane, and immunoblotted with an anti-Flag M2 antibody. Four major immunoreactive bands of 38, 44, 45, and 60 kDa are present in Flag-MCH1R and the mutant receptors, although the intensity of the 60-kDa receptor (arrow) appears to be decreased in the i1 and i2 mutants. Right, After lysis of transfected cells and preclearing with protein-G-agarose, the lysates were subjected to immunoprecipitation with the anti-Flag M2 antibody. The 38-, 44-, 45-, and 60-kDa bands are clearly observed in Flag-MCH1R, but the intensity of the 60-kDa receptor (arrow) is decreased in the i1 and i2 mutants, similar to the results for the Western blotting. B, Saturation curves of Flag-MCH1R and the mutant receptors in radioligand binding. HEK293 cells transfected with Flag-MCH1R or the mutated receptors were harvested, and the membrane fractions were isolated. The membrane proteins (30 μg) were incubated with the indicated concentrations of [125I] (Phe13, Tyr19) MCH in the presence or absence of 1 μM nonlabeled MCH in 500 μl of assay buffer at room temperature for 2 h. Specific binding was defined as the difference between the total binding and the nonspecific binding. All experiments were independently performed three times in duplicate, and representative results are shown. C, Dose-response relationships of the MCH-stimulated calcium influx in HEK293T cells expressing Flag-MCH1R or the mutant receptors. Cells transfected with Flag-MCH1R or the triple-substitution mutant receptors were stimulated with the indicated concentrations of MCH, and the subsequent changes in the cytoplasmic-free Ca2+ levels were measured using a Flexstation. All experiments were independently performed at least three times in duplicate, and representative results are shown.
The level of cell surface expression was monitored by FACScan flow cytometry using the anti-Flag M2 antibody and a FITC-conjugated secondary antibody (Table 1). The mutated i1/3Q and i2/3Q receptors each showed a 60% reduction in the cell surface expression, whereas the level of i3/3Q was identical with that of Flag-MCH1R. Radioligand binding studies using cell membrane preparations demonstrated that the maximal binding capacity (Bmax) value for i3/3Q was similar to that for wild-type Flag-MCH1R, whereas those for i1/3Q and i2/3Q exhibited 75% decreases (Fig. 2B and Table 2). The maximal binding capacity values seemed to parallel those found by flow cytometry. In contrast, both i1/3Q and i2/3Q showed similar affinity constants (Kd) to that of Flag-MCH1R, whereas i3/3Q showed a 3-fold decrease (Table 2). Collectively, i1/3Q and i2/3Q revealed similar properties for their levels of protein expression and cell surface expression and similar parameters for radioligand binding.
TABLE 1. Cell surface expression and signaling of Flag-MCH1R and triple-substitution mutants of basic amino residues in the intracellular cytoplasmic loops of MCH1R after transfection into HEK293T cells
TABLE 2. Specific radioligand binding of Flag-MCH1R and mutant receptors
Next, we evaluated whether the highly basic regions of i1, i2, and i3 in MCH1R affect the MCH-stimulated calcium influx using a Flexstation. Representative curves are shown in Fig. 2C. Cells expressing i2/3Q completely lost their MCH-mediated calcium influx, whereas it was still observed in cells expressing i1/3Q or i3/3Q. Table 1 shows the EC50 values for Flag-MCH1R and the mutants. i1/3Q had a 20-fold higher EC50 value and a 50% reduction in the maximal response. In i3/3Q, the dose-response curve was shifted to the right and gave a 50-fold higher EC50 value with a similar maximal response to that of Flag-MCH1R (Fig. 2C). MCH1R has previously been shown to couple functionally to both Gq and Gi, which facilitate the calcium influx and inhibit cAMP accumulation (12, 13). Therefore, we characterized the phenotypes of the triple-substitution mutants by measuring the cAMP level after application of MCH to stably transfected cells (Table 3). MCH potently inhibited forskolin-stimulated accumulation of cAMP with an EC50 value of 1.2 nM and a 60% reduction in the maximal response of Flag-MCH1R. The EC50 values for i1/3Q and i3/3Q were 8- and 12-fold higher than the value for Flag-MCH1R, respectively. The effects of the MCH mutants on cAMP inhibition were smaller than those on the calcium influx, although direct comparisons of magnitude between different types of assay are not easy. However, cells expressing i2/3Q showed no inhibition of forskolin-stimulated accumulation of cAMP. The basal levels and forskolin-stimulated increases in the cAMP level in i1/3Q, i2/3Q and i3/3Q were identical with those induced in Flag-MCH1R-transfected cells. Thus, the triple-substitution mutations in the highly basic motifs of i1, i2, and i3 result in reductions in both Gq and Gi signaling.
TABLE 3. Forskolin-stimulated cAMP inhibition in HEK293T cells stably transfected with Flag-MCH1R, triple-substitution mutants of the intracellular cytoplasmic loops, or R155Q
Effects of individual substitutions in the i1 BBXB, i2 BBXB, and i3 BBXXB motifs on MCH1R function
The above results suggested that the basic motif in i2 may be a key determinant for signal transduction in Flag-MCH1R. To investigate the functional significance of individual basic residues in BBXB or BBXXB, a series of single-point mutants with substitution of B for Q were constructed as shown in Fig. 1. In cells expressing single-substitution mutants of i1, the cell surface expression of K68Q was identical with that of Flag-MCH1R, whereas 40% reductions were detected for K67Q and K70Q. However, K67Q elicited a calcium influx in a similar manner to Flag-MCH1R, whereas both K68Q and K70Q exhibited 2.5-fold higher EC50 values for calcium signaling without changing the high-affinity Kd value (Tables 2 and 4 and Fig. 3A). The cell surface expressions of all the single-substitution mutants of i3 were identical with that of Flag-MCH1R (Table 4). Regarding stimulation of a calcium influx, K252Q showed no significantly impaired function, whereas both R253Q and R256Q caused approximately 4.5-fold increases in the EC50 values with a 2-fold higher Kd value (Tables 2 and 4 and Fig. 3C). These results indicate that the last two amino acids in the context of the basic motifs in i1 and i3 affect receptor signaling with greater importance.
TABLE 4. Cell surface expression and signaling of Flag-MCH1R and single-substitution mutants of basic amino residues in the intracellular cytoplasmic loops of MCH1R after transfection into HEK293T cells
FIG. 3. Dose-response relationships of the MCH-stimulated calcium influx in HEK293T cells expressing Flag-MCH1R or single-substitution mutant receptors. Cells transfected with Flag-MCH1R or single-substitution mutant receptors of i1 (A), i2 (B), and i3 (C) were stimulated with the indicated concentrations of MCH as described in the legend for Fig. 2B. All experiments were independently performed at least three times in duplicate, and representative results are shown. A Western blotting analysis of the protein expressions of Flag-MCH1R and individual single-substitution mutant receptors of i2 is shown as an inset (B).
However, the single-substitution mutants in the conserved motif of i2 revealed distinct aspects from those of i1 and i3 for the receptor activity. Cells expressing R155Q showed a very small increase in the MCH-induced calcium influx with a 75-fold higher EC50 value and a 50% reduction in the maximal response, whereas mutations of the other two basic residues, K153Q and K156Q, had no significant effects on signaling (Table 4 and Fig. 3B). On the other hand, K153Q, R155Q, and K156Q all showed similar affinities (Kd) to Flag-MCH1R (Table 2) and similar characters in immunoblotting analyses of whole-cell extracts (Fig. 3B, inset). Furthermore, the levels of cell surface expression were nearly equivalent among the three mutants (Table 4). These data suggest that the profound impairment of the signaling function in R155Q is not secondary to the lack of glycosylation or low expression level at the cell surface. R155 was further changed into other amino acid residues to determine the specificity of this position in the signaling pathway of MCH1R (Table 4). Mutation of R155 to K produced a 50-fold higher EC50 value of MCH, compared with Flag-MCH1R, with a 50% reduction in the maximal response. Moreover, substitution of R155 with a negatively charged amino acid, D, completely abolished the ability of the receptor to stimulate calcium signaling with an 80% decrease in the level of cell surface expression. Next, we characterized the phenotype of R155Q by measuring the cAMP level in stably transfected cells. As observed for i2/3Q, R155Q had a drastic influence on the coupling to both the calcium response and adenylyl cyclase. In R155Q-expressing cells, MCH inhibited forskolin-stimulated accumulation of cAMP with a nearly 50-fold higher EC50 value, and the maximal inhibition of cAMP accumulation was reduced by nearly 50% (Table 3).
Because G proteins have been reported to directly affect the structure and ligand affinity of GPCRs, we determined whether the R155Q mutant was able to couple to G proteins and stimulate the exchange of GDP for GTP on G subunits after exposure to MCH. We quantified MCH-induced [35S]GTPS binding to G protein using membrane preparations from HEK293 cells transiently transfected with either Flag-MCH1R or R155Q. As shown in Fig. 4, Flag-MCH1R showed dose-dependent binding of [35S]GTPS with an EC50 value of 14.4 ± 0.80 nM. R155Q also stimulated GDP-GTP exchange on G subunits with a higher EC50 value of 201.9 ± 13.7 nM (Fig. 4). The maximal amounts of binding with 10 μM MCH were 142.0 and 132.5% in Flag-MCH1R- and R155Q-transfected cells, respectively. Thus, point mutations of R155 can drive MCH1R into a different affinity state for coupling to G protein, supporting the crucial role of this residue in the high efficiency activation process of the receptor.
FIG. 4. MCH-induced [35S]GTPS binding to Flag-MCH1R and R155Q. HEK293 cells were transfected with Flag-MCH1R or R155Q. After 48 h, the cells were harvested and the membrane fractions were recovered. The membrane proteins (8 μg) were subsequently incubated with 0.2 nM [35S]GTPS and 1 nM to 10 μM MCH in GTPS binding buffer for 30 min at 30 C. The amounts of radioactivity bound to the membrane preparations are shown as Flag-MCH1R (open circles) and R155Q (filled circles). All experiments were independently performed at least three times in triplicate, and representative results are shown.
Functional importance of corresponding arginine residues in i2 of other receptors
To further explore the key role of R155 in the basic motif of i2, we first examined whether an R was conserved at the equivalent position to R155 in i2 of MCH1R in orthologs, including fish. A sequence alignment search revealed that an R was evolutionally conserved at the position corresponding to R155 in rat MCH1R from mammals to zebrafish (43). We also searched whether the R position in i2 of the rat MCH1R is conserved in other GPCRs. Sequence alignment using a BLAST search clarified that human MCH2R, five SSTR subtypes, two opioid receptors, and an orphanin FQ/nociceptin receptor have a highly conserved R at the position corresponding to R155 of MCH1R, whereas rhodopsin does not. This R is the only amino acid residue that is well conserved in the C-terminal region of i2 (Table 5), whereas the junction of transmembrane 3 has a common motif of E/DRY among class A GPCRs.
TABLE 5. Amino acid sequence alignments of the c terminus of transmembrane 3 (TM3) and i2 of MCH1R and other GPCRs
This fact strongly suggests that the existence of an R at this position is critically important for receptor activity. Therefore, we generated single-substitution mutants at the corresponding residue R145 in human MCH2R and R154 in rat SSTR2 and examined the effects of these mutations on the receptor function. MCH2R has been shown to predominantly activate the Gq protein in HEK293 cells (15), and replacement of R145 in MCH2R with a neutrally charged amino acid, Q, led to pronounced changes in the calcium mobilization, with a 20-fold higher EC50 value for MCH and a 40% decrease in the maximal response compared with those of MCH2R (Table 6). On the other hand, all five somatostatin receptors have been shown to stimulate the activation of only Gi in both CHO cells and HEK293 cells (44). Therefore, we cotransfected SSTR2 with a Gq/i3 chimera that contained the five C-terminal residues of Gi3 and the rest of the Gq sequence (7). This chimera was designed to facilitate SSTR2 signaling in Gi3 to Gq activation because the five C-terminal residues of G are sufficient for receptor contact, whereas the remaining residues of G interact with the effector molecule. Receptor activity caused by somatostatin 14 in SSTR2 was recorded as a calcium influx in cotransfected cells. HEK293T cells expressing SSTR2 with the Gi3 chimera had an EC50 value of 0.0016 nM for somatostatin 14, whereas introduction of the point mutation R154Q produced an approximately 70-fold higher EC50 value with a 40% decrease in the maximal response, compared with those for SSTR2 (Table 6). Substitution at R154 also elicited decreased calcium mobilization in cotransfected CHO cells, as evidenced by the 30-fold higher EC50 value with a 45% decrease in the maximal response (Table 6). These results imply that, in some other GPCRs, the positions corresponding to R155 in i2 of MCH1R confer full activity for calcium influx and/or adenylyl cyclase caused by ligand stimulation.
TABLE 6. Signaling of human MCH2R, rat SSTR2 and their single-substitution mutant receptors after transfection into CHO and/or HEK293T cells
Discussion
Clarification of the amino acid sequences that influence the activation of G proteins in GPCRs is primarily important for understanding how each receptor forms critical contact points with G proteins. The amino acids of class A GPCRs have been extensively studied and various components in i2, i3, and helix 8 have been implicated as points of contact with G proteins or interacting molecules. In the current study, to determine the functional significance of the known structural motifs BBXXB and BXBB that exist in i1, i2, and i3 of MCH1R, we constructed a series of mutant receptors by replacing the basic amino acids with the noncharged amino acid Q. We started by generating triple-substitution mutants and then conducted individual mutation analyses. A single mutation at R155, but not at K153 or K156, in i2 caused very large reductions in the Gq- and Gi-mediated activities. Moreover, we showed the functional importance of the equivalent R in MCH2R and SSTR2 for Gq- and Gi-mediated activities, respectively. Our present data suggest that R155 in i2 represents a novel site with relevance to signal transduction, whereas the G protein coupling specificity seems to be governed by another receptor domain.
Importance of the BXBB and BBXXB motifs in MCH1R
Although the highly conserved basic motifs have been suggested to be functionally relevant (38, 39, 41), this could not be a universal rule for all GPCRs. One example is the m1 muscarinic acetylcholine receptor, in which the BBXXB motif in i3 is necessary for coupling to phosphoinositide hydrolysis, whereas the same motif in i2 is not required (40). However, our present data define the significance of the basic motifs in all three loops of MCH1R for the receptor activity. Interestingly, the contributions of each motif appeared to differ in terms of their ligand binding, protein expression, and ability to induce a calcium influx. The intensities of the positive bands in Western blotting and levels of cell surface expression were nearly identical for i1/3Q and i2/3Q, yet only the latter mutant resulted in a nonfunctional receptor for signaling. i3/3Q did not show any alterations in the intensity of protein expression or level of plasma membrane expression but did have a lower affinity than that of Flag-MCH1R. The mutant receptor i3/3Q therefore showed a right shift of the dose-response curve for the calcium influx, whereas retaining an identical maximal response to that of Flag-MCH1R. Reduced binding affinity was also observed in the R253Q and R256Q single-substitution mutants. Thus, the basic motif in i3 of MCH1R seems to be more responsible for agonist binding and G protein coupling than for trafficking of the receptor to the cell surface. The BBXXB motif in i3 may function in the packing of the transmembrane domains in MCH1R, resulting in either decreased accessibility of the G protein coupling site or inability of the receptor to form an active conformation upon MCH binding. In addition, all the triple-substitution mutants of the basic motifs caused reductions in the Gq- and Gi-mediated activities, suggesting that the G protein coupling specificity in MCH1R resides in regions other than the basic motifs.
The single-substitution analyses indicated that the role of each basic residue differed depending on its position within the basic motif. In terms of the receptor-mediated calcium influx mediated by MCH, the EC50 values for K68Q and K70Q were 2.5-fold higher than that for Flag-MCH1R. On the other hand, the values for K253Q and R256Q were 4.5-fold higher. K67Q and K252Q in i1 and i3, respectively, did not significantly alter the EC50 value, although K67Q showed a 30% reduction in the cell surface expression. These data demonstrate that the first basic residue of each motif plays a minor role, whereas the second and third residues are important for efficient activation of MCH1R. On the other hand, R155 in the BBXB motif of i2 was the only residue that caused a strong defect in signaling, despite the findings that K153Q and R155Q showed very similar levels of protein expression and cell surface expression. It should be noted that all the triple-substitution mutations of i1, i2, and i3 showed much stronger effects on the signaling and cell surface expression than any of the single-substitution mutations in each loop, implying that the individual basic residues work in mutually exclusive manners. Overall, these findings indicate that each basic residue in a motif has a different impact, but they work cooperatively for receptor activity. These findings are consistent with previous results for the m1 acetylcholine receptor and FSH receptor, in which individual residues function in a hierarchal and interdependent manner for receptor coupling (40) and cell surface expression (42), respectively.
Pivotal role of R155 in MCH1R for receptor signaling
A large number of experiments have identified conserved motifs in i2 as key determinants in various receptor functions. The E/DRY motif of transmembrane 3, which is the most conserved motif among class A GPCRs, is critical for the efficacy of G protein coupling and/or constitutive activation (38). In several GPCRs, it has been shown that the hydrophobic residue in the E/DRYXXV(I)XXPL motif plays a significant role in the G protein coupling specificity (37). In contrast, only a few papers have reported involvement of the BBXXB or BBXB motifs in i2 in receptor function. An example of these studies is the TSH receptor, in which an individual single mutation in BBXXB had only a modest effect on the signaling efficacy (45).
Apart from such basic motifs, several pieces of evidence have suggested that single or dibasic residues are critical for receptor function. For example, K in the proximal C-terminal region of i2 in the PTH/PTH-related protein receptor is a key determinant for Gq-selective coupling (46). In MCH1R, point mutation of R155 in i2 to the neutrally charged Q caused severe signaling impairment but had no effect on the G protein selectivity. Replacement of R155 by the negatively charged D resulted in a nonfunctional mutant with a 75% reduction in cell surface expression, accentuating the critical nature of R155, i.e. the importance of its positively charged nature. Interestingly, however, replacement of R155 with the moderately charged K also caused a profound reduction in signaling efficiency. This observation is in contrast to the case of a dibasic motif in the putative helix 8 in MCH1R, in which a moderate level of net charge is sufficient for activity (26). This fact may indicate that a sufficient level of positive charge at position 155 is strictly required for efficient signaling in MCH1R. To evaluate another possibility, we speculatively calculated the solvent accessible surface area (SASA) of the individual amino acids in i2 of rat MCH1R according to the method of Lee and Richards (47) with a 1.4A radius for the solvent probe. The extent of the solvent-exposed polar SASA of R155 appears to be greater than those of the other residues in the i2 region. On the other hand, the polar SASA of R155K is decreased to less than half that of the wild-type R155 (data not shown). The SASA histograms for R155Q and R155D showed very similar patterns to that of R155. These speculative analyses may indicate that the polarity of R155, as well as its positive charge, is also a key factor for maximizing information transfer from MCH-occupied receptors.
Interruption of the significance of R155 using three-dimensional modeling
Our findings highlight the fact that minor changes in the structure of i2 can lead to pronounced changes in the G protein coupling efficiency of MCH1R as suggested by the GTPS binding assays and probably also in some other GPCRs. How does amino acid substitution of R155 suppress the efficacy of the signaling pathway in MCH1R? The high resolution x-ray structure of bovine rhodopsin indicates that i2 has an L-like structure when viewed parallel to the membrane plane but lacks a regular secondary structure (48). Like the other intracellular loops, i2 does not fold over the area defined by the intracellular ends of transmembrane domains 1–7. To interpret a possible explanation for the role of R155, we generated a theoretical three-dimensional structure of the rat MCH1R by homology modeling, using the coordinates of the 2.2A crystal structure of bovine rhodopsin as a template (Fig. 5). At present, only rhodopsin can be used as a starting point, and therefore molecular modeling of the intracellular loops in several GPCRs has been reported based on the structure of rhodopsin (34, 45). However, we cannot state that our receptor model is representative of the actual structure of MCH1R. The arrangement of the transmembrane regions in the model may be quite reliable, but the homology of less than 40% between the i2 regions of rhodopsin and MCH1R (Table 5) may not favor molecular modeling. In the current study, the molecular analysis was used to locate the mutated residues and highlight possible relationships for receptor function.
FIG. 5. Model of the three-dimensional structure of the rat MCH1R. The depicted structure is based on the high-resolution x-ray structure of bovine rhodopsin (Protein Data Bank code 1U19; resolution = 2.2A). Energy minimization was applied to this model structure to refine the side chain positions. A, Model structure viewed from the direction of the cytoplasm to the extracellular region. Numbered arrows indicate the transmembrane regions. Transmembrane domains 3 and 4 are shown in yellow and purple, respectively. Loops i2 and i3 are shown in blue and orange, respectively. The highly conserved residues DRY (green) and R155 (violet) are shown as ball-and-stick models. In our model structure, the side chain of R155 is predicted to have no contact with any of the loops or transmembrane regions. B, Spatial orientations of K153, R155, and K156 in i2. The side chains are shown as ball-and-stick models, and the white arrow indicates the side chains in the DRY region.
The model predicts that R155 is located just at the N terminal of the bend of the L-like structure, which is a characteristic feature of i2 that is also seen in rhodopsin (48). The positively charged side chain of R155 is exposed and faces away from the interior of the structure and therefore cannot be important for cytoplasmic intracellular loop interactions. Furthermore, R155 does not interact with the nearby transmembrane domain. In addition, the side chains of D140(3.49), R141(3.50), and Y142(3.51) are speculatively oriented in the opposite direction to the side chain of R155. Therefore, it may be assumed that R155 is easily accessible to G proteins and that the switch in efficient signaling is most likely due to a molecular interaction. Figure 5B shows that the side chains of K153 and K156 are predicted to lie in the opposite direction to that of R155 and may therefore have less chance of contact with cytosolic molecules. This may explain why single substitutions of K153 and K156 did not significantly decrease the efficiency of signal transduction. Further studies are required to pinpoint the contact points between R155 in MCH1R and the G protein subunits directly involved in mediating responses in the target cells.
In summary, this study provides evidence for a crucial role of R155 in i2 as a recognition determinant in MCH1R. Our data further suggest the functional significance of the equivalent R in some other GPCRs. Although a final conclusion cannot be reached until the three-dimensional structure of the MCH1R-G protein complex is analyzed, the results of the present study suggest that the possible cytosolic orientation of the basic residue R155 is involved in a step of the G protein coupling. Therefore, we postulate that the mutant cannot properly signal the conformational changes to the downstream. Further investigations are also required to define the molecular structure and interacting molecules that determine the receptor-G protein specificity of MCH1R. These studies will allow additional understanding of the dual G protein coupling of MCH1R and particularly of the selectivity of activation.
Acknowledgments
We thank Dr. Y. Odagaki (Department of Psychiatry, Saitama Medical School, Saitama, Japan) for helpful advice regarding the GTP S binding assay. We also thank Drs. O. Civelli and R. Reinscheid (University of California, Irvine) for critically reviewing the manuscript.
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