Arrestin-Independent Internalization and Recycling of the Urotensin Receptor Contribute to Long-Lasting Urotensin IIeCMediated Vasoconstrict
http://www.100md.com
循环研究杂志 2005年第9期
the Med. Klinik IV-Nephrologie, Charitee, Campus Benjamin Franklin, Berlin (G.G., M.T., W.Z., M.v.d.G.)
Institut fe Pharmakologie, Charitee, Campus Benjamin Franklin, Berlin (J.J., A.O.)
Forschungsinstitut fe Molekulare Pharmakologie, Berlin (J.E., J.F., M.B., W.R.)
Universitt Potsdam, Institut fe Ernhrungswissenschaft, Nuthetal (F.N.-R.), Germany.
Abstract
Urotensin II (UII), which acts on the G protein-coupled urotensin (UT) receptor, elicits long-lasting vasoconstriction. The role of UT receptor internalization and intracellular trafficking in vasoconstriction has yet not been analyzed. Therefore, UII-mediated contractile responses of aortic ring preparations in wire myography and rat UT (rUT) receptor internalization and intracellular trafficking in binding and imaging analyses were compared. UII elicited a concentration-dependent vasoconstriction of rat aorta (eClog EC50, mol/L:9.0±0.1). A second application of UII after 30 minutes elicited a reduced contraction (36±4% of the initial response), but when applied after 60 minutes elicited a full contraction. In internalization experiments with radioactive labeled VII (125I-UII), 70% of rUT receptors expressed on the cell surface of human embryonic kidney 293 cells were sequestered within 30 minutes (half life [th]: 5.6±0.2 minutes), but recycled quantitatively within 60 minutes (th 31.9±2.6 minutes). UII-bound rUT receptors were sorted to early and recycling endosomes, as evidenced by colocalization of rUT receptors with the early endosomal antigen and the transferrin receptor. Real-time imaging with a newly developed fluorescent UII (Cy3-UII) revealed that rUT receptors recruited arrestin3 green fluorescent protein to the plasma membrane. Arrestin3 was not required for the endocytosis of the rUT receptor, however, as internalization of Cy3-UII was not altered in mouse embryonic fibroblasts lacking endogenous arrestin2/arrestin3 expression. The data demonstrate that the rUT receptor internalizes arrestin independently and recycles quantitatively. The continuous externalization of rUT receptors provides the basis for repetitive and lasting UII-mediated vasoconstriction.
Key Words: urotensin II vascular tone urotensin receptor recycling internalization
Introduction
Urotensin II (UII) is among the most potent mammalian vasoconstrictors identified so far.1 UII was characterized as being 1 to 2 orders of magnitude more potent than endothelin-1 (ET-1)2and acts on the urotensin (UT) receptor, formerly known as GPR14.3 Stimulation of the UT receptor results in a phospholipase C-mediated increase in cytosolic Ca2+ and the activation of rho-kinase.4,5 Both signaling pathways promote an increase in MLCK phosphorylation, resulting in a strong contraction of vascular smooth muscle cells.6 In addition, stimulation of the UT receptor also results in an activation of tyrosine kinases, the mitogen-activated protein kinase (MAPK) p38, and extracellular signal regulated kinases (ERK)1/2.6,7
UII-mediated vasoactive effects are variable and depend on the species and the disease state investigated.8,9 In humans, UII peptide and human UT (hUT) receptor mRNAs are found in the heart (atrial and ventricular myocytes, fibroblasts)10 and kidney (epithelia of tubules and ducts, renal capillary and glomerular endothelial cells).11 Low levels of UII binding sites have been identified in the coronary arteries, kidney, left ventricle, and skeletal muscle.12 A dose-dependent reduction of forearm blood flow in healthy subjects and vasoconstrictor response in coronary arteries have been described,12,13 whereas vasodilatory responses have been observed in pulmonary and abdominal resistance arteries.14 Interestingly, differential effects of UII have been demonstrated between healthy subjects and patients with heart failure. Iontophoretic application of UII decreased the microvascular tone in the skin of healthy subjects, but increased it in patients with heart failure.15 In contrast, in 2 other studies, small or no effects on blood flow, heart rate, cardiac output, and mean arterial pressure have been reported,16,17 and in a further study with isolated human arteries of different sizes and vascular beds, no UII-mediated vasoconstriction has been observed.18 In disease states, the expression of UII and hUT receptors is upregulated. For example, plasma and/or urinary UII are increased in essential and portal hypertension, liver cirrhosis, end-stage heart failure and renal disease.2,19 Clozel and coworkers20 have demonstrated in a rat model of renal ischemia that the non-peptide UII receptor antagonist ACT-058362 prevents the no-reflow phenomenon, which leads to postischemic renal vasoconstriction and acute renal failure. These findings point to a role of the UT receptor in pathological states.
UII-induced vasoconstriction of rat aorta is very similar to that caused by ET-1, as both peptides elicit vasoconstriction with a slow onset but long duration.7,21 For ET-1, it has been shown that contractile responses in rat aorta correlate with the extent of endothelin A receptor externalization.22 The mechanisms involved in the regulation of cell surface expression of the UT receptor remain elusive. For example, the mode of internalization and the intracellular trafficking routes of the UT receptor, eg, whether it recycles or is down regulated, are unknown.
UII-induced vasoconstriction and resensitization of UII-mediated responses in rat aorta were studied. The functional effects of UII on contractile responses of the rat aorta were compared with data obtained from internalization and recycling experiments with radioactive labeled UII (125I-UII). In addition, the intracellular trafficking of the rat UT (rUT) receptor was studied in real-time imaging analysis using a newly developed fluorescent analogue of UII in conjunction with a fluorescent rUT receptor fusion protein or fluorescent arrestin isoforms.
Materials and Methods
Detailed methods are described in the expanded Materials and Methods in the Data Supplement, available online at http://circres.ahajournals.org.
Peptide Synthesis and Fluorescence Labeling
Synthesis of UII by the solid phase method using standard 9-fluorenylmethoxy-carbonyl chemistry and labeling of UII with Cy3 NHS ester were performed as described recently.23
Reverse Transcription Polymerase Chain Reaction and Generation of Expression Plasmids Encoding the Rat UT Receptor
The rUT receptor (Accession no. U32673) was amplified from a rat aorta cDNA library. The polymerase chain reaction fragment was cloned into a pcDNA3 vector, resulting in the plasmid pcDNA3.rUT. For generation of fusion proteins, UT-receptor polymerase chain reaction fragment was cloned into the pEGFP.N1 vector, resulting in the plasmid pEGFP.rUT.
125I-UII Binding Analysis With Membrane Preparations and Intact Cells
Membranes of human embryonic kidney (HEK) cells expressing the rUT receptor or the rUT·green fluorescent protein (GFP) receptor were prepared as described recently.24 Saturation and displacement binding experiments were performed as described recently.23 Maximum specific binding volume (Bmax) values in the different assays systems ranged from 2.3 to 6.4 pmol/mg protein for HEK293 cells and between 4.5 to 7.3 pmol/mg protein for COS.M6 cells.
Inositol Phosphate Assay
Determination of UII-induced inositol phosphate formation in COS-M6 cells expressing rUT receptor or rUT.GFP receptor was performed as described.24
Calcium Measurements
HEK293 cells expressing rUT receptor of rUT·GFP were loaded with 4 eol/L Fluo4-AM and analyzed in a fluorescent image plate reader (FLIPR) in the absence or presence of increasing amounts of UII (Molecular Devices) as described previously.25
Fluorescence Microscopy and Image Analysis
Fixed and living cells were examined with a Zeiss LSM510 Meta system, equipped with an Axiovert 135 microscope and 63x/1.4 oil and 100x/1.4 oil objectives. For immunofluorescence studies, cells were processed as described.26 Real-time imaging of rUT receptor internalization were performed in a temperable insert described recently27 for continuous incubation of the sample at 37°C.
Wire Myography
Rat thoracic aorta of 4-month-old male Wistar-Kyoto rats (Charles River; Sulzfeld, Germany) were prepared as described previously and the force and wall tension of the vasculature were measured using established methodology.28
Statistics
Data are expressed as mean±SEM. Data were statistically analyzed using the Mann-Whitney test for unpaired data. All statistical analysis were done using Graphpad Prism 4.02. EC50 values in vessel experiments were calculated using curve fitting with sigmoidal dose response curve.
Results
Cumulative or Repeated UII Application Elicits Lasting Vasoconstriction of Rat Aortic Rings
Cumulative application of UII leads to a concentration-dependent vasoconstriction (eClog EC50 [mol/L]:9.0±0.1; Figure 1A and 1B). In the continuous presence of UII (10 nmol/L), a sustained vasoconstriction for at least 12 minutes was observed, which reached 94±4% of the maximal UII-induced vasoconstriction. The UII-mediated vasoconstriction could be reversed to baseline values by repeated washing (Figure 1C). When aortic rings were rechallenged with UII (10 nmol/L) 30 minutes after washing, a reduced vasoconstriction was observed (36±4% of initial response, P<0.05). In contrast, a complete vasoconstriction was observed when aortic rings were rechallenged after 60 minutes (Figure 1D).
rUT Receptors Show Rapid Internalization and Recycling
To gain more insight into the trafficking properties of the rUT receptor, we analyzed the internalization of rUT receptors in transfected HEK293 cells using 125I-UII as the radioligand. To induce internalization, cells were incubated with saturating levels of UII (100 nmol/L) for up to 60 minutes at 37°C. The cells were then placed on ice to prevent further internalization and washed twice with an ice cold acidic buffer (pH 5.0) to remove free and cell-bound UII. The amount of cell surface receptors was then determined in 125I-UII binding studies. After 60 minutes of UII application, 64±6% of the receptors were sequestered, with a half time for internalization of 5.6±0.2 minutes (Figure 2A).
Further, it was analyzed whether internalized rUT receptors can recycle to the cell surface. To this end, cells expressing the rUT receptor were incubated with UII (100 nmol/L) for 10 minutes at 37°C to promote receptor sequestration. The cells were then placed on ice and UII was removed using an acidic buffer (pH 5.0) and further incubated with 125I-UII for up to 60 minutes at 37°C. Figure 2B shows that rUT receptors reappeared at the cell surface within 60 minutes after the initial UII challenge. The half time for the reappearance of the binding site was 31.9±2.6 minutes (see Figure 2B). Similar results were obtained with cells incubated in the presence of cycloheximide. The results suggest that rUT receptors are rapidly internalized after agonist stimulation and recycle to the cell surface quantitatively within 60 minutes.
Characterization of Fluorescent rUT Receptors and UII Peptides
To demonstrate internalization and recycling of the rUT receptor in real-time imaging, a plasmid encoding a fusion protein (rUT·GFP) consisting of the rUT receptor and the GFP fused to the C terminus of the receptor was generated. Before imaging, the properties of the native rUT receptor and the rUT·GFP receptor were compared. In saturation binding analyses, no significant differences were observed (Kd values for the native rUT-receptor and the rUT·GFP receptor were 2.7±0.1 nmol/L and 2.9±0.1 nmol/L, respectively; n=3). The ability of the native rUT receptor and the rUT·GFP receptor to stimulate inositol phosphate formation was studied. On stimulation of rUT receptors in HEK cells, only small increases in inositol phosphate formation were observed. Thus, COS.M6 cells were used, which yielded robust increases in inositol phosphate formation. COS.M6 cells expressing the rUT receptor or the rUT·GFP receptor revealed very similar EC50 values for inositol phosphate formation (EC50 [eClog mol/L] values for the native and the rUT·GFP receptor were 8.1±0.5 and 8.4±0.2, respectively; n=3) (see Figure 3A). Similarily, HEK293 cells expressing rUT receptor or rUT·GFP and analyzed an increase in cytosolic calcium showed no gross differences in their EC50 values ([eClog mol/L], rUT: 7.3±0.1 and rUT·GFP: 7.6±0.1) (Figure 3B). These data demonstrate that the GFP moiety at the very C terminus of the rUT receptor does not alter the receptor’s affinity for the natural agonist, nor does it impair its ability to activate G proteins.
To study rUT receptor trafficking of the native rUT receptor, fluorescent UII derivatives using a monoreactive Cy3-NHS ester was generated. Depending on the pH of the reaction buffer, incorporation of Cy3 occurred either at the -amino group of the N-terminal glutamate residue (E1; at neutral pH) or at the -amino group of lysine residue 8 (K8; at alkaline pH). UII, Cy3-UII (E1), and Cy3-UII (K8) were compared in displacement experiments, with membrane preparations from HEK cells expressing the rUT receptor. Here, Ki values for native UII and Cy3-UII (E1) were very similar (Ki values for native and Cy3-UII [E1] were 5.3±0.6 and 4.7±0.5 nmol/L, respectively; n=3). In contrast, no significant binding of Cy3-UII (K8) was observed. The data show that the cyanin moiety at the -amino group of E1 does not alter the receptor’s affinity for the ligand, whereas the presence of the fluorescent moiety at K8 greatly reduces the receptor’s affinity. The findings are in agreement with results obtained from structure-activity studies of UII: The deletion or alanine replacement of E1 did not affect biological activity, but alanine replacement of K8 reduced biological activity >10 000-fold.29,30
Live Cell Imaging of rUT Receptor and UII Internalization
HEK cells expressing the rUT·GFP receptor were analyzed at 37°C with a confocal laser scanning microscope equipped with a temperable insert. In the absence of ligand, the rUT·GFP receptor was predominantly expressed at the plasma membrane. In addition, rUT·GFP receptor was also found in the cells’ interior. This pool could represent newly synthesized protein being transported to the plasma membrane or constitutively internalized receptors in a perinuclear compartment, eg, the pericentriolar recycling center. Within 3 minutes after application of Cy3-UII (50 nmol/L), both the rUT receptor and UII colocalized at the plasma membrane (yellow signals in the overlay, Figure 4). In addition, UII/rUT receptors within the cells were observed, indicating rapid internalization (see Figure 4, arrows). On further incubation (10 minutes), internalization of Cy3-rUII/UT·GFP complexes increased, resulting in prominent colocalization within endosomal compartments.
Internalized rUT Receptor Is Found in Early and Recycling Endosomes
To define the endocytic route of the agonist-bound rUT receptor, further immunofluorescence studies were performed (Figure 5). Because the radioactive binding experiments provided evidence that the rUT receptor undergoes recycling, the potential colocalization of agonist-treated rUT receptors with markers of the recycling pathway (transferrin receptor, early endosomal antigen; EEA1) was investigated. To this end, HEK cells expressing the rUT·GFP receptor were incubated with UII (100 nmol/L) for 60 minutes at 18°C, so that rUT receptors quantitatively bound UII but could not internalize until cells were incubated at 37°C. In cells incubated at 18°C only, staining with an EEA1 antibody revealed endosomal structures in the cells’ interiors, which were not colocalized with rUT·GFP. The latter was predominately localized in the plasma membrane. In cells further incubated at 37°C (15 minutes), rUT·GFP colocalized with almost all EEA1-positive endosomes. In addition, rUT·GFP was found in endosomal structures that were not labeled with EEA1. Because the transferrin receptor is found in EEA1-positive early endosomes and also in EEA1-negative recycling endosomes, it was analyzed whether internalized rUT·GFP showed an intense colocalization with the transferrin receptor. In cells incubated at 18°C, a distinct colocalization of rUT·GFP with the transferrin receptor (close to the nucleus) was observed. This compartment, which was observed in 30% of cells, could represent the pericentriolar recycling center. When cells were further incubated for 15 minutes at 37°C, an extensive colocalization of rUT·GFP with the transferrin receptor in endosomal compartments was observed. No colocalization was found for rUT·GFP with late endosomal (Rab7; data not shown) or lysosomal markers (Rab9, Lamp1; Figure 5). The results suggest that rUT receptors recycle quantitatively and are not sorted to the late endosomal/lysosomal pathway.
UII-Stimulated rUT Receptors Recruit Arrestin3 to the Plasma Membrane, but Reveal Arrestin-Independent Internalization
Arrestin2 and arrestin3 are involved in the desensitization and internalization of many G protein-coupled receptors.31 To identify the isoforms involved in the desensitization and/or internalization of the rUT receptor, live cell imaging of HEK293 cells coexpressing the rUT receptor and arrestin3·GFP was performed. In the resting state at 37°C, arrestin3·GFP reveals a diffuse cytoplasmic distribution. Within 3 minutes after addition of Cy3-UII, however, arrestin3·GFP was recruited to the plasma membrane, where it partially colocalizes with the fluorescent agonist. Further incubation (10 minutes) resulted in a more intensive recruitment of arrestin3·GFP to the plasma membrane, although most of Cy3-UII is now found intracellulary (Figure 6, upper panel). In cells coexpressing the rUT receptor, arrestin3·GFP and a dominant-negative mutant of dynamin, K44A·dynamin, the recruitment of arrestin3·GFP by Cy3-UII-bound rUT receptor was even more prominent because K44A·dynamin inhibited internalization (Figure 6, middle panel). In cells coexpressing the rUT receptor and arrestin2·GFP, no recruitment of arrestin2 to the plasma membrane was observed (Figure 6, lower panel). The data show that rUT receptors recruit arrestin3 but not arrestin2 to the plasma membrane. During the endocytic transport of rUT receptors, however, no association of arrestin3 was observed. To explore the role of arrestin3 for rUT receptor function in more detail, we analyzed whether overexpression of arrestin3 or the mutant V54D.arrestin3 alters the UII-induced internalization rate of rUT receptors. The V54D.arrestin3 mutant is known to interfere with proper targeting of several G protein-coupled receptors to clathrin-coated pits and subsequently inhibit internalization.32 In binding experiments with 125I-UII as radioligand, however, no significant change in the internalization rate of the rUT receptor was observed (Figure 7A). The data suggest that arrestin3 is not required for proper receptor internalization. To prove more vigorously the requirement of arrestins for the endocytosis, rUT receptor internalization in mouse embryonic fibroblasts (MEF) derived from mice with a targeted deletion of arrestin2/arrestin3 genes (arr2eC/eC/arr3eC/eC) was analyzed. In the resting state, rUT receptors were mainly expressed in the plasma membrane of arr2eC/ eC/arr3eC/ eC MEF cells. On incubation with Cy3-UII for 25 minutes at 37°C, complexes of Cy3-UII/rUT·GFP receptors were observed in endosomal compartments (see Figure 7B). The data provide strong evidence that the UII-bound rUT receptor internalizes independently of arrestin2 and arrestin3.
Discussion
Our data demonstrate that the UII-bound rUT receptor is rapidly internalized in a dynamin-dependent but arrestin-independent manner. In the absence of the agonist, the receptor recycles back to the plasma membrane. Because the speed of internalization is 6 times faster than the externalization of rUT receptors, saturating levels of UII result in transient downregulation of rUT receptors. On removal of the agonist, the level of rUT receptors expressed at the cell surface recover to that before UII stimulation, indicating quantitative recycling. The time course of recovery of UII-mediated vasoconstriction after an initial UII application was very similar to that of rUT receptor externalization (compare Figure 1D and Figure 2B). The data suggest that the crucial determinant in long-lasting and repetitive UII-induced vasoconstriction is the rate of rUT receptor externalization.
UII and ET-1 are unique in their action on the vascular system. Both peptides elicit a strong and lasting vasoconstriction.21 Similar to the finding for UII and the rUT receptor, ET-1eCmediated contractile responses depend on the externalization rate of ETA receptors.22 Angiotensin II, which elicits only a transient vasoconstriction,21 shows only delayed recycling. Interestingly, AT1 receptors form stable complexes with arrestin2 and/or arrestin3 that are involved in the sorting of AT1 receptors to deep endosomes. This kind of receptor trafficking also applies to the vasopressin V2 receptor, the substance P receptor, and the thyrotropin-releasing hormone receptor.33 Although ETA33 and rUT receptors associate with arrestin3 at the plasma membrane, they do not traffic with arrestin3 to endocytic compartments. Interestingly, coexpression of rUT receptors with the mutant V54D.arrestin3, or rUT receptor expression in MEF cells, which lack endogenous expression of arrestin2 and arrestin3 (arr2eC/ eC/arr3eC/ eC), did not inhibit UII-induced internalization. Thus, the rUT receptor does not require arrestin3 for internalization. It is likely, however, that arrestin3 is involved in the desensitization of activated rUT receptors. Similar findings have been reported for the m2 muscarinic cholinergic receptor, the thrombin receptor (PAR1), the prostacyclin receptor, and the 5-HT2A receptor.34eC36 In agreement with our studies, a weak and transient arrestin recruitment was also reported by Onan in her thesis.37 In contrast to the presented results and Onan’s findings, Proulx and coworkers38 recently reported that rUT receptors form a stable complex with arrestin2 or arrestin3. The reason for this discrepancy is not clear. In the study by Proulx et al, however, arrestin/rUT receptor interaction was analyzed by the combined use of fluorescent UT·GFP receptor and arrestin2·YFP or arrestin3·YFP fusions proteins. Because GFP has a tendency to form dimers at higher concentrations, it cannot be certain whether recruitment of arrestin to the rUT receptor could result in a persistent arrestineCreceptor interaction due to the dimerization of GFP/YFP moieties.
Currently, it is not known whether the data presented for the rUT receptor are also applicable to UT receptors of other species. Considerable variations in the amino acid sequence of the different species are found, in particular within the C terminus. Because interactions of the C terminus with G protein-coupled receptor interacting proteins determine trafficking of G protein-coupled receptors,39 variations in the amino acid sequence could account for altered receptor trafficking by different G protein-coupled receptor interacting proteins interactions. However, several consensus motifs for protein kinases are conserved among UT receptors of rats, mice, humans, and monkeys. Notably, a serine cluster in the C terminus, displaying consensus motifs for protein kinase C and casein kinase I phosphorylation, was demonstrated to be important for the internalization of rUT.38 Interestingly, for the feline UT receptor, variations in this serine cluster are found that lacked the protein kinase C site and harbored only 1 of 2 casein kinase I sites.40 Whether species-dependent differences in this motif result in altered UT receptor trafficking awaits further characterization.
Under normal physiological conditions, UT receptor expression is low in human arteries, ie, coronary vessels and aortae,12 and is consistently observed in the thoracic aortae of rodents.12,41 In rat thoracic aortae, UII-induced vasoconstriction falls off with an increasing distance to the carotid bifurcation.12 Similarly, in rodents, UII-mediated contractile responses are restricted to the thoracic aorta.21
In humans, vasoconstrictive effects of UII have been observed in coronary, mammary, and radial arteries and in the saphenous and umbilical vein.12 Interestingly, in disease states, eg, in portal hypertension, liver cirrhosis, end-stage heart failure, and renal disease, an up regulation of UII and UT receptor expression was observed.2,19 This may also explain differential effects of UII in healthy and diseased patients on microvascular tone in skin. Lim and coworkers15 demonstrated that patients with coronary heart failure show a dose-dependent UII-induced constriction of microvascular skin vessels, whereas healthy subjects show a dose-dependent vasodilation. Therefore, the UT receptor may be classified as a prototypical member of G protein-coupled receptors that does contribute at low level to the regulation of organ function in healthy states, but that plays a significant role in disease states. Once upregulation of UT receptor expression has been initiated in disease, the intracellular trafficking properties of the receptor allow a continuous receptor externalization, thereby providing the molecular basis for long-lasting UII-mediated responses.
Acknowledgments
The work was supported by the Deutsche Forschungsgemeinschaft (FG 341), the Sonnenfeld-Stiftung, and the Fonds der Chemischen Industrie. We thank for Dr Dietmar Krutwurst for help in calcium measurements, Dr Tim Plant for critical reading, and Monika Bigalke for excellent technical assistance.
Both authors contributed equally to this study.
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Oakley RH, Laporte SA, Holt JA, Caron MG, Barak LS. Differential affinities of visual arrestin, beta arrestin1, and beta arrestin2 for G protein-coupled receptors delineate two major classes of receptors. J Biol Chem. 2000; 275: 17201eC17210.
Lee KB, Pals-Rylaarsdam R, Benovic JL, Hosey MM. Arrestin-independent internalization of the m1, m3, and m4 subtypes of muscarinic cholinergic receptors. J Biol Chem. 1998; 273: 12967eC12972.
Smyth EM, Austin SC, Reilly MP, FitzGerald GA. Internalization and sequestration of the human prostacyclin receptor. J Biol Chem. 2000; 275: 32037eC32045.
Paing MM, Stutts AB, Kohout TA, Lefkowitz RJ, Trejo J. Beta-arrestins regulate protease-activated receptor-1 desensitization but not internalization or down-regulation. J Biol Chem. 2002; 277: 1292eC1300.
Onan D. Cardiovascular and Regulatory Aspects of the Urotensin-II Receptor [thesis]. Prahran, Australia: Molecular Endocrinology Laboratory, Baker Heart Research Institute, Monash University; 2004.
Proulx CD, Simaan M, Escher E, Laporte SA, Guillemette G, Leduc R. Involvement of a cytoplasmic-tail serine cluster in urotensin II receptor internalization. Biochem J. 2005; 385: 115eC123.
Bockaert J, Fagni L, Dumuis A, Marin P. GPCR interacting proteins (GIP). Pharmacol Ther. 2004; 103: 203eC221.
Aiyar N, Johns DG, Ao Z, Disa J, Behm DJ, Foley JJ, Buckley PT, Sarau HM, van-der-Keyl HK, Elshourbagy NA, Douglas SA. Cloning and pharmacological characterization of the cat urotensin-II receptor (UT). Biochem Pharmacol. 2005; 69: 1069eC1079.
Nothacker HP, Wang Z, McNeill AM, Saito Y, Merten S, O’Dowd B, Duckles SP, Civelli O. Identification of the natural ligand of an orphan G-protein-coupled receptor involved in the regulation of vasoconstriction. Nat Cell Biol. 1999; 1: 383eC385.(Geter Giebing, Markus Tll)
Institut fe Pharmakologie, Charitee, Campus Benjamin Franklin, Berlin (J.J., A.O.)
Forschungsinstitut fe Molekulare Pharmakologie, Berlin (J.E., J.F., M.B., W.R.)
Universitt Potsdam, Institut fe Ernhrungswissenschaft, Nuthetal (F.N.-R.), Germany.
Abstract
Urotensin II (UII), which acts on the G protein-coupled urotensin (UT) receptor, elicits long-lasting vasoconstriction. The role of UT receptor internalization and intracellular trafficking in vasoconstriction has yet not been analyzed. Therefore, UII-mediated contractile responses of aortic ring preparations in wire myography and rat UT (rUT) receptor internalization and intracellular trafficking in binding and imaging analyses were compared. UII elicited a concentration-dependent vasoconstriction of rat aorta (eClog EC50, mol/L:9.0±0.1). A second application of UII after 30 minutes elicited a reduced contraction (36±4% of the initial response), but when applied after 60 minutes elicited a full contraction. In internalization experiments with radioactive labeled VII (125I-UII), 70% of rUT receptors expressed on the cell surface of human embryonic kidney 293 cells were sequestered within 30 minutes (half life [th]: 5.6±0.2 minutes), but recycled quantitatively within 60 minutes (th 31.9±2.6 minutes). UII-bound rUT receptors were sorted to early and recycling endosomes, as evidenced by colocalization of rUT receptors with the early endosomal antigen and the transferrin receptor. Real-time imaging with a newly developed fluorescent UII (Cy3-UII) revealed that rUT receptors recruited arrestin3 green fluorescent protein to the plasma membrane. Arrestin3 was not required for the endocytosis of the rUT receptor, however, as internalization of Cy3-UII was not altered in mouse embryonic fibroblasts lacking endogenous arrestin2/arrestin3 expression. The data demonstrate that the rUT receptor internalizes arrestin independently and recycles quantitatively. The continuous externalization of rUT receptors provides the basis for repetitive and lasting UII-mediated vasoconstriction.
Key Words: urotensin II vascular tone urotensin receptor recycling internalization
Introduction
Urotensin II (UII) is among the most potent mammalian vasoconstrictors identified so far.1 UII was characterized as being 1 to 2 orders of magnitude more potent than endothelin-1 (ET-1)2and acts on the urotensin (UT) receptor, formerly known as GPR14.3 Stimulation of the UT receptor results in a phospholipase C-mediated increase in cytosolic Ca2+ and the activation of rho-kinase.4,5 Both signaling pathways promote an increase in MLCK phosphorylation, resulting in a strong contraction of vascular smooth muscle cells.6 In addition, stimulation of the UT receptor also results in an activation of tyrosine kinases, the mitogen-activated protein kinase (MAPK) p38, and extracellular signal regulated kinases (ERK)1/2.6,7
UII-mediated vasoactive effects are variable and depend on the species and the disease state investigated.8,9 In humans, UII peptide and human UT (hUT) receptor mRNAs are found in the heart (atrial and ventricular myocytes, fibroblasts)10 and kidney (epithelia of tubules and ducts, renal capillary and glomerular endothelial cells).11 Low levels of UII binding sites have been identified in the coronary arteries, kidney, left ventricle, and skeletal muscle.12 A dose-dependent reduction of forearm blood flow in healthy subjects and vasoconstrictor response in coronary arteries have been described,12,13 whereas vasodilatory responses have been observed in pulmonary and abdominal resistance arteries.14 Interestingly, differential effects of UII have been demonstrated between healthy subjects and patients with heart failure. Iontophoretic application of UII decreased the microvascular tone in the skin of healthy subjects, but increased it in patients with heart failure.15 In contrast, in 2 other studies, small or no effects on blood flow, heart rate, cardiac output, and mean arterial pressure have been reported,16,17 and in a further study with isolated human arteries of different sizes and vascular beds, no UII-mediated vasoconstriction has been observed.18 In disease states, the expression of UII and hUT receptors is upregulated. For example, plasma and/or urinary UII are increased in essential and portal hypertension, liver cirrhosis, end-stage heart failure and renal disease.2,19 Clozel and coworkers20 have demonstrated in a rat model of renal ischemia that the non-peptide UII receptor antagonist ACT-058362 prevents the no-reflow phenomenon, which leads to postischemic renal vasoconstriction and acute renal failure. These findings point to a role of the UT receptor in pathological states.
UII-induced vasoconstriction of rat aorta is very similar to that caused by ET-1, as both peptides elicit vasoconstriction with a slow onset but long duration.7,21 For ET-1, it has been shown that contractile responses in rat aorta correlate with the extent of endothelin A receptor externalization.22 The mechanisms involved in the regulation of cell surface expression of the UT receptor remain elusive. For example, the mode of internalization and the intracellular trafficking routes of the UT receptor, eg, whether it recycles or is down regulated, are unknown.
UII-induced vasoconstriction and resensitization of UII-mediated responses in rat aorta were studied. The functional effects of UII on contractile responses of the rat aorta were compared with data obtained from internalization and recycling experiments with radioactive labeled UII (125I-UII). In addition, the intracellular trafficking of the rat UT (rUT) receptor was studied in real-time imaging analysis using a newly developed fluorescent analogue of UII in conjunction with a fluorescent rUT receptor fusion protein or fluorescent arrestin isoforms.
Materials and Methods
Detailed methods are described in the expanded Materials and Methods in the Data Supplement, available online at http://circres.ahajournals.org.
Peptide Synthesis and Fluorescence Labeling
Synthesis of UII by the solid phase method using standard 9-fluorenylmethoxy-carbonyl chemistry and labeling of UII with Cy3 NHS ester were performed as described recently.23
Reverse Transcription Polymerase Chain Reaction and Generation of Expression Plasmids Encoding the Rat UT Receptor
The rUT receptor (Accession no. U32673) was amplified from a rat aorta cDNA library. The polymerase chain reaction fragment was cloned into a pcDNA3 vector, resulting in the plasmid pcDNA3.rUT. For generation of fusion proteins, UT-receptor polymerase chain reaction fragment was cloned into the pEGFP.N1 vector, resulting in the plasmid pEGFP.rUT.
125I-UII Binding Analysis With Membrane Preparations and Intact Cells
Membranes of human embryonic kidney (HEK) cells expressing the rUT receptor or the rUT·green fluorescent protein (GFP) receptor were prepared as described recently.24 Saturation and displacement binding experiments were performed as described recently.23 Maximum specific binding volume (Bmax) values in the different assays systems ranged from 2.3 to 6.4 pmol/mg protein for HEK293 cells and between 4.5 to 7.3 pmol/mg protein for COS.M6 cells.
Inositol Phosphate Assay
Determination of UII-induced inositol phosphate formation in COS-M6 cells expressing rUT receptor or rUT.GFP receptor was performed as described.24
Calcium Measurements
HEK293 cells expressing rUT receptor of rUT·GFP were loaded with 4 eol/L Fluo4-AM and analyzed in a fluorescent image plate reader (FLIPR) in the absence or presence of increasing amounts of UII (Molecular Devices) as described previously.25
Fluorescence Microscopy and Image Analysis
Fixed and living cells were examined with a Zeiss LSM510 Meta system, equipped with an Axiovert 135 microscope and 63x/1.4 oil and 100x/1.4 oil objectives. For immunofluorescence studies, cells were processed as described.26 Real-time imaging of rUT receptor internalization were performed in a temperable insert described recently27 for continuous incubation of the sample at 37°C.
Wire Myography
Rat thoracic aorta of 4-month-old male Wistar-Kyoto rats (Charles River; Sulzfeld, Germany) were prepared as described previously and the force and wall tension of the vasculature were measured using established methodology.28
Statistics
Data are expressed as mean±SEM. Data were statistically analyzed using the Mann-Whitney test for unpaired data. All statistical analysis were done using Graphpad Prism 4.02. EC50 values in vessel experiments were calculated using curve fitting with sigmoidal dose response curve.
Results
Cumulative or Repeated UII Application Elicits Lasting Vasoconstriction of Rat Aortic Rings
Cumulative application of UII leads to a concentration-dependent vasoconstriction (eClog EC50 [mol/L]:9.0±0.1; Figure 1A and 1B). In the continuous presence of UII (10 nmol/L), a sustained vasoconstriction for at least 12 minutes was observed, which reached 94±4% of the maximal UII-induced vasoconstriction. The UII-mediated vasoconstriction could be reversed to baseline values by repeated washing (Figure 1C). When aortic rings were rechallenged with UII (10 nmol/L) 30 minutes after washing, a reduced vasoconstriction was observed (36±4% of initial response, P<0.05). In contrast, a complete vasoconstriction was observed when aortic rings were rechallenged after 60 minutes (Figure 1D).
rUT Receptors Show Rapid Internalization and Recycling
To gain more insight into the trafficking properties of the rUT receptor, we analyzed the internalization of rUT receptors in transfected HEK293 cells using 125I-UII as the radioligand. To induce internalization, cells were incubated with saturating levels of UII (100 nmol/L) for up to 60 minutes at 37°C. The cells were then placed on ice to prevent further internalization and washed twice with an ice cold acidic buffer (pH 5.0) to remove free and cell-bound UII. The amount of cell surface receptors was then determined in 125I-UII binding studies. After 60 minutes of UII application, 64±6% of the receptors were sequestered, with a half time for internalization of 5.6±0.2 minutes (Figure 2A).
Further, it was analyzed whether internalized rUT receptors can recycle to the cell surface. To this end, cells expressing the rUT receptor were incubated with UII (100 nmol/L) for 10 minutes at 37°C to promote receptor sequestration. The cells were then placed on ice and UII was removed using an acidic buffer (pH 5.0) and further incubated with 125I-UII for up to 60 minutes at 37°C. Figure 2B shows that rUT receptors reappeared at the cell surface within 60 minutes after the initial UII challenge. The half time for the reappearance of the binding site was 31.9±2.6 minutes (see Figure 2B). Similar results were obtained with cells incubated in the presence of cycloheximide. The results suggest that rUT receptors are rapidly internalized after agonist stimulation and recycle to the cell surface quantitatively within 60 minutes.
Characterization of Fluorescent rUT Receptors and UII Peptides
To demonstrate internalization and recycling of the rUT receptor in real-time imaging, a plasmid encoding a fusion protein (rUT·GFP) consisting of the rUT receptor and the GFP fused to the C terminus of the receptor was generated. Before imaging, the properties of the native rUT receptor and the rUT·GFP receptor were compared. In saturation binding analyses, no significant differences were observed (Kd values for the native rUT-receptor and the rUT·GFP receptor were 2.7±0.1 nmol/L and 2.9±0.1 nmol/L, respectively; n=3). The ability of the native rUT receptor and the rUT·GFP receptor to stimulate inositol phosphate formation was studied. On stimulation of rUT receptors in HEK cells, only small increases in inositol phosphate formation were observed. Thus, COS.M6 cells were used, which yielded robust increases in inositol phosphate formation. COS.M6 cells expressing the rUT receptor or the rUT·GFP receptor revealed very similar EC50 values for inositol phosphate formation (EC50 [eClog mol/L] values for the native and the rUT·GFP receptor were 8.1±0.5 and 8.4±0.2, respectively; n=3) (see Figure 3A). Similarily, HEK293 cells expressing rUT receptor or rUT·GFP and analyzed an increase in cytosolic calcium showed no gross differences in their EC50 values ([eClog mol/L], rUT: 7.3±0.1 and rUT·GFP: 7.6±0.1) (Figure 3B). These data demonstrate that the GFP moiety at the very C terminus of the rUT receptor does not alter the receptor’s affinity for the natural agonist, nor does it impair its ability to activate G proteins.
To study rUT receptor trafficking of the native rUT receptor, fluorescent UII derivatives using a monoreactive Cy3-NHS ester was generated. Depending on the pH of the reaction buffer, incorporation of Cy3 occurred either at the -amino group of the N-terminal glutamate residue (E1; at neutral pH) or at the -amino group of lysine residue 8 (K8; at alkaline pH). UII, Cy3-UII (E1), and Cy3-UII (K8) were compared in displacement experiments, with membrane preparations from HEK cells expressing the rUT receptor. Here, Ki values for native UII and Cy3-UII (E1) were very similar (Ki values for native and Cy3-UII [E1] were 5.3±0.6 and 4.7±0.5 nmol/L, respectively; n=3). In contrast, no significant binding of Cy3-UII (K8) was observed. The data show that the cyanin moiety at the -amino group of E1 does not alter the receptor’s affinity for the ligand, whereas the presence of the fluorescent moiety at K8 greatly reduces the receptor’s affinity. The findings are in agreement with results obtained from structure-activity studies of UII: The deletion or alanine replacement of E1 did not affect biological activity, but alanine replacement of K8 reduced biological activity >10 000-fold.29,30
Live Cell Imaging of rUT Receptor and UII Internalization
HEK cells expressing the rUT·GFP receptor were analyzed at 37°C with a confocal laser scanning microscope equipped with a temperable insert. In the absence of ligand, the rUT·GFP receptor was predominantly expressed at the plasma membrane. In addition, rUT·GFP receptor was also found in the cells’ interior. This pool could represent newly synthesized protein being transported to the plasma membrane or constitutively internalized receptors in a perinuclear compartment, eg, the pericentriolar recycling center. Within 3 minutes after application of Cy3-UII (50 nmol/L), both the rUT receptor and UII colocalized at the plasma membrane (yellow signals in the overlay, Figure 4). In addition, UII/rUT receptors within the cells were observed, indicating rapid internalization (see Figure 4, arrows). On further incubation (10 minutes), internalization of Cy3-rUII/UT·GFP complexes increased, resulting in prominent colocalization within endosomal compartments.
Internalized rUT Receptor Is Found in Early and Recycling Endosomes
To define the endocytic route of the agonist-bound rUT receptor, further immunofluorescence studies were performed (Figure 5). Because the radioactive binding experiments provided evidence that the rUT receptor undergoes recycling, the potential colocalization of agonist-treated rUT receptors with markers of the recycling pathway (transferrin receptor, early endosomal antigen; EEA1) was investigated. To this end, HEK cells expressing the rUT·GFP receptor were incubated with UII (100 nmol/L) for 60 minutes at 18°C, so that rUT receptors quantitatively bound UII but could not internalize until cells were incubated at 37°C. In cells incubated at 18°C only, staining with an EEA1 antibody revealed endosomal structures in the cells’ interiors, which were not colocalized with rUT·GFP. The latter was predominately localized in the plasma membrane. In cells further incubated at 37°C (15 minutes), rUT·GFP colocalized with almost all EEA1-positive endosomes. In addition, rUT·GFP was found in endosomal structures that were not labeled with EEA1. Because the transferrin receptor is found in EEA1-positive early endosomes and also in EEA1-negative recycling endosomes, it was analyzed whether internalized rUT·GFP showed an intense colocalization with the transferrin receptor. In cells incubated at 18°C, a distinct colocalization of rUT·GFP with the transferrin receptor (close to the nucleus) was observed. This compartment, which was observed in 30% of cells, could represent the pericentriolar recycling center. When cells were further incubated for 15 minutes at 37°C, an extensive colocalization of rUT·GFP with the transferrin receptor in endosomal compartments was observed. No colocalization was found for rUT·GFP with late endosomal (Rab7; data not shown) or lysosomal markers (Rab9, Lamp1; Figure 5). The results suggest that rUT receptors recycle quantitatively and are not sorted to the late endosomal/lysosomal pathway.
UII-Stimulated rUT Receptors Recruit Arrestin3 to the Plasma Membrane, but Reveal Arrestin-Independent Internalization
Arrestin2 and arrestin3 are involved in the desensitization and internalization of many G protein-coupled receptors.31 To identify the isoforms involved in the desensitization and/or internalization of the rUT receptor, live cell imaging of HEK293 cells coexpressing the rUT receptor and arrestin3·GFP was performed. In the resting state at 37°C, arrestin3·GFP reveals a diffuse cytoplasmic distribution. Within 3 minutes after addition of Cy3-UII, however, arrestin3·GFP was recruited to the plasma membrane, where it partially colocalizes with the fluorescent agonist. Further incubation (10 minutes) resulted in a more intensive recruitment of arrestin3·GFP to the plasma membrane, although most of Cy3-UII is now found intracellulary (Figure 6, upper panel). In cells coexpressing the rUT receptor, arrestin3·GFP and a dominant-negative mutant of dynamin, K44A·dynamin, the recruitment of arrestin3·GFP by Cy3-UII-bound rUT receptor was even more prominent because K44A·dynamin inhibited internalization (Figure 6, middle panel). In cells coexpressing the rUT receptor and arrestin2·GFP, no recruitment of arrestin2 to the plasma membrane was observed (Figure 6, lower panel). The data show that rUT receptors recruit arrestin3 but not arrestin2 to the plasma membrane. During the endocytic transport of rUT receptors, however, no association of arrestin3 was observed. To explore the role of arrestin3 for rUT receptor function in more detail, we analyzed whether overexpression of arrestin3 or the mutant V54D.arrestin3 alters the UII-induced internalization rate of rUT receptors. The V54D.arrestin3 mutant is known to interfere with proper targeting of several G protein-coupled receptors to clathrin-coated pits and subsequently inhibit internalization.32 In binding experiments with 125I-UII as radioligand, however, no significant change in the internalization rate of the rUT receptor was observed (Figure 7A). The data suggest that arrestin3 is not required for proper receptor internalization. To prove more vigorously the requirement of arrestins for the endocytosis, rUT receptor internalization in mouse embryonic fibroblasts (MEF) derived from mice with a targeted deletion of arrestin2/arrestin3 genes (arr2eC/eC/arr3eC/eC) was analyzed. In the resting state, rUT receptors were mainly expressed in the plasma membrane of arr2eC/ eC/arr3eC/ eC MEF cells. On incubation with Cy3-UII for 25 minutes at 37°C, complexes of Cy3-UII/rUT·GFP receptors were observed in endosomal compartments (see Figure 7B). The data provide strong evidence that the UII-bound rUT receptor internalizes independently of arrestin2 and arrestin3.
Discussion
Our data demonstrate that the UII-bound rUT receptor is rapidly internalized in a dynamin-dependent but arrestin-independent manner. In the absence of the agonist, the receptor recycles back to the plasma membrane. Because the speed of internalization is 6 times faster than the externalization of rUT receptors, saturating levels of UII result in transient downregulation of rUT receptors. On removal of the agonist, the level of rUT receptors expressed at the cell surface recover to that before UII stimulation, indicating quantitative recycling. The time course of recovery of UII-mediated vasoconstriction after an initial UII application was very similar to that of rUT receptor externalization (compare Figure 1D and Figure 2B). The data suggest that the crucial determinant in long-lasting and repetitive UII-induced vasoconstriction is the rate of rUT receptor externalization.
UII and ET-1 are unique in their action on the vascular system. Both peptides elicit a strong and lasting vasoconstriction.21 Similar to the finding for UII and the rUT receptor, ET-1eCmediated contractile responses depend on the externalization rate of ETA receptors.22 Angiotensin II, which elicits only a transient vasoconstriction,21 shows only delayed recycling. Interestingly, AT1 receptors form stable complexes with arrestin2 and/or arrestin3 that are involved in the sorting of AT1 receptors to deep endosomes. This kind of receptor trafficking also applies to the vasopressin V2 receptor, the substance P receptor, and the thyrotropin-releasing hormone receptor.33 Although ETA33 and rUT receptors associate with arrestin3 at the plasma membrane, they do not traffic with arrestin3 to endocytic compartments. Interestingly, coexpression of rUT receptors with the mutant V54D.arrestin3, or rUT receptor expression in MEF cells, which lack endogenous expression of arrestin2 and arrestin3 (arr2eC/ eC/arr3eC/ eC), did not inhibit UII-induced internalization. Thus, the rUT receptor does not require arrestin3 for internalization. It is likely, however, that arrestin3 is involved in the desensitization of activated rUT receptors. Similar findings have been reported for the m2 muscarinic cholinergic receptor, the thrombin receptor (PAR1), the prostacyclin receptor, and the 5-HT2A receptor.34eC36 In agreement with our studies, a weak and transient arrestin recruitment was also reported by Onan in her thesis.37 In contrast to the presented results and Onan’s findings, Proulx and coworkers38 recently reported that rUT receptors form a stable complex with arrestin2 or arrestin3. The reason for this discrepancy is not clear. In the study by Proulx et al, however, arrestin/rUT receptor interaction was analyzed by the combined use of fluorescent UT·GFP receptor and arrestin2·YFP or arrestin3·YFP fusions proteins. Because GFP has a tendency to form dimers at higher concentrations, it cannot be certain whether recruitment of arrestin to the rUT receptor could result in a persistent arrestineCreceptor interaction due to the dimerization of GFP/YFP moieties.
Currently, it is not known whether the data presented for the rUT receptor are also applicable to UT receptors of other species. Considerable variations in the amino acid sequence of the different species are found, in particular within the C terminus. Because interactions of the C terminus with G protein-coupled receptor interacting proteins determine trafficking of G protein-coupled receptors,39 variations in the amino acid sequence could account for altered receptor trafficking by different G protein-coupled receptor interacting proteins interactions. However, several consensus motifs for protein kinases are conserved among UT receptors of rats, mice, humans, and monkeys. Notably, a serine cluster in the C terminus, displaying consensus motifs for protein kinase C and casein kinase I phosphorylation, was demonstrated to be important for the internalization of rUT.38 Interestingly, for the feline UT receptor, variations in this serine cluster are found that lacked the protein kinase C site and harbored only 1 of 2 casein kinase I sites.40 Whether species-dependent differences in this motif result in altered UT receptor trafficking awaits further characterization.
Under normal physiological conditions, UT receptor expression is low in human arteries, ie, coronary vessels and aortae,12 and is consistently observed in the thoracic aortae of rodents.12,41 In rat thoracic aortae, UII-induced vasoconstriction falls off with an increasing distance to the carotid bifurcation.12 Similarly, in rodents, UII-mediated contractile responses are restricted to the thoracic aorta.21
In humans, vasoconstrictive effects of UII have been observed in coronary, mammary, and radial arteries and in the saphenous and umbilical vein.12 Interestingly, in disease states, eg, in portal hypertension, liver cirrhosis, end-stage heart failure, and renal disease, an up regulation of UII and UT receptor expression was observed.2,19 This may also explain differential effects of UII in healthy and diseased patients on microvascular tone in skin. Lim and coworkers15 demonstrated that patients with coronary heart failure show a dose-dependent UII-induced constriction of microvascular skin vessels, whereas healthy subjects show a dose-dependent vasodilation. Therefore, the UT receptor may be classified as a prototypical member of G protein-coupled receptors that does contribute at low level to the regulation of organ function in healthy states, but that plays a significant role in disease states. Once upregulation of UT receptor expression has been initiated in disease, the intracellular trafficking properties of the receptor allow a continuous receptor externalization, thereby providing the molecular basis for long-lasting UII-mediated responses.
Acknowledgments
The work was supported by the Deutsche Forschungsgemeinschaft (FG 341), the Sonnenfeld-Stiftung, and the Fonds der Chemischen Industrie. We thank for Dr Dietmar Krutwurst for help in calcium measurements, Dr Tim Plant for critical reading, and Monika Bigalke for excellent technical assistance.
Both authors contributed equally to this study.
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