Multi-Tasking RGS Proteins in the Heart
http://www.100md.com
循环研究杂志 2005年第3期
the Department of Pharmacology (E.L.R., P.A.I.), University of California San Diego, La Jolla
the Department of Physiology (R.A.S., M.B.), University of Maryland School of Medicine, Baltimore
the Department of Molecular Cardiology (M.B.), Lerner Research Institute, The Cleveland Clinic Foundation, Ohio.
Abstract
Regulator of G-proteineCsignaling (RGS) proteins play a key role in the regulation of G-proteineCcoupled receptor (GPCR) signaling. The characteristic hallmark of RGS proteins is a conserved 120-aa RGS region that confers on these proteins the ability to serve as GTPase-activating proteins (GAPs) for G proteins. Most RGS proteins can serve as GAPs for multiple isoforms of G and therefore have the potential to influence many cellular signaling pathways. However, RGS proteins can be highly regulated and can demonstrate extreme specificity for a particular signaling pathway. RGS proteins can be regulated by altering their GAP activity or subcellular localization; such regulation is achieved by phosphorylation, palmitoylation, and interaction with protein and lipid-binding partners. Many RGS proteins have GAP-independent functions that influence GPCR and non-GPCReCmediated signaling, such as effector regulation or action as an effector. Hence, RGS proteins should be considered multifunctional signaling regulators. GPCR-mediated signaling is critical for normal function in the cardiovascular system and is currently the primary target for the pharmacological treatment of disease. Alterations in RGS protein levels, in particular RGS2 and RGS4, produce cardiovascular phenotypes. Thus, because of the importance of GPCR-signaling pathways and the profound influence of RGS proteins on these pathways, RGS proteins are regulators of cardiovascular physiology and potentially novel drug targets as well.
Key Words: RGS protein regulator of G-protein signaling GPCRheart
Introduction
G-ProteineCcoupled receptors (GPCRs) are found ubiquitously through the body and are involved in virtually every physiological process. Cardiovascular cells possess >100 different GPCRs.1 GPCRs bind a heterotrimeric G-protein complex of -, -, and -subunits. G-Proteins are divided into four families (eg, Gs, Gi, Gq, and G12) based on similarity of -subunits among individual family members;2 at least 20 -subunits are found in mammalian cells.3 Agonist binding of GPCRs promotes G-protein activation. This activation is achieved by catalyzing GDPeCGTP exchange on the -subunit. A conformational change in the GTP-bound -subunit leads to a dissociation of G from the -subunits. GTP-bound G-subunits and dissociated -dimers regulate downstream effectors. These signaling events are terminated as a consequence of intrinsic GTPase activity of the G-subunit, which hydrolyzes bound GTP to GDP, resulting in a reassociation of the G-protein heterotrimer. The intrinsic GTPase activity of -subunits is generally insufficient to correlate with physiological rates of G-protein inactivation, but this activity can be accelerated by the presence of GTPase-activating proteins (GAPs), such as regulator of G-proteineCsignaling (RGS) proteins.
The discovery of RGS proteins suggested the possibility of a specific RGS protein for each G subtype. However, this idea was discarded when it was discovered that the number of RGS proteins (now >30) was greater than that of G-subunits, along with the recognition that individual RGS proteins could act as GAPs for multiple families of G. However, as understanding of RGS function increases, evidence has emerged for G specificity of certain RGS proteins, especially in the in vivo setting, as well as their modulation of distinct GPCR-mediated signaling pathways. Although a substantial amount is known regarding identity and expression of RGS proteins, much remains to be elucidated regarding their function, localization, and regulation. Their GAP activity is a primary characteristic, but RGS proteins use a variety of mechanisms to regulate signaling. Several excellent reviews have provided insight on the structure and function of RGS proteins.4eC7 However, numerous recent developments related to RGS proteins have important implications, especially for cardiovascular physiology and pathophysiology. In this review, we first provide an overview of RGS structure, emphasizing non-RGS domains. Second, we discuss mechanisms for the regulation of RGS proteins. Finally, we bring the reader up to date on current knowledge of the importance of these "multitasking" RGS proteins in the cardiovascular system.
RGS Protein Structure
Figure 1 summarizes key features of RGS genes and proteins. Many RGS genes produce different isoforms (eg, rgs6 encodes 36 distinct transcripts8). A description of each isoform is beyond the scope of this review. RGS proteins are diverse, ranging in size from 152 (RGS21) to 1376 (RGS12) amino acids. Many, indeed most, RGS proteins identified to date exhibit GAP activity toward the Gi and Gq families of G-proteins. However, it is important to note that even though an RGS protein may be able to serve as a GAP for a particular G-subunit, particular GPCR signaling pathways involving the identical G-subunit can show receptor-selective regulation by RGS proteins.9
On the basis of sequence similarity of the RGS domain, RGS proteins can be classified into one of five subfamilies: R4, R7, R12, RZ, and RL (Figure 1). Although some RGS proteins consist almost exclusively of the RGS domain, a conserved 120-aa region, others contain additional domains. The RGS domain is necessary and sufficient to confer GAP activity;10 however, other domains, to be discussed subsequently, can increase specificity, determine cellular localization, and provide additional activities.
PSD-95 Disk-Large ZO-1 Domain
PSD-95 disk-large ZO-1 (PDZ) domains, 90-aa regions with a highly conserved four-residue GLGF sequence, are involved in the clustering of multiprotein signaling complexes.11 RGS12 and a variant of RGS3, PDZ-RGS3, contain PDZ domains. The PDZ domain of PDZ-RGS3 binds the non-GPCR B-ephrin receptor, thereby providing a possible link between GPCR signaling and B-ephrin signaling.12 In addition, PDZ-RGS3 could be important for B-ephrineCregulated cardiovascular development.13
The PDZ domain of RGS12 binds selectively to the interleukin-8 receptor B (CXCR2), a Gi-coupled GPCR.14 Although RGS12 has GAP activity toward Gi, it remains to be determined whether RGS12 alters CXCR2-mediated G-protein signaling. The interaction of RGS12 with CXCR2 has the potential to influence myocardial viability during ischemia reperfusion.15
G-Protein SubuniteCLike Domain
RGS6, RGS7, RGS9, and RGS11 contain G-protein subuniteClike (GGL) domains, a 64-aa region with a high level of similarity to the G-subunit, can form dimers with particular G-protein subunits (ie, G5) but not others (eg, G1 to G3).16 This RGS/G5 interaction appears to influence RGS and G5 protein stability,17,18 GAP activity of RGS proteins,16,19 and subcellular localization of RGS and G5.8,20
GoLoco Domain
RGS12 and RGS14 contain GoLoco domains, a 19-aa Gi binding motif that acts as a guanine nucleotide dissociation inhibitor (GDI) by binding and stabilizing GDP-bound Gi, inhibiting the rate of exchange of GDP for GTP in a GAP-independent manner.21 The RGS and GoLoco domains of RGS14 can independently inhibit Gi signaling, but both domains are necessary for maximal inhibition.22 The GoLoco domain of RGS14 exhibits GDI activity toward Gi1 and Gi3 but not Gi2 or Go even though the GAP activity is not G-protein subtype selective.23
PX Domain
RGS-PX1, the only RGS protein thus far identified with GAP activity for Gs, contains a PX domain, an 120-aa phosphoinositide-binding domain involved in membrane targeting.24,25 RGS-PX1 delays lysosomal degradation of the EGF receptor, most likely as a result of the sorting nexin function of the PX domain.24 The relationship between RGS-PX1 and the EGF receptor suggests a possible role for RGS-PX1 in angiotensin II (Ang II)eCinduced cardiac hypertrophy.26
Disheveled EGL-10 Pleckstrin Domain
The 70-aa disheveled EGL-10 pleckstrin (DEP) domains are present in various signaling proteins, including RGS6, RGS7, RGS9, and RGS11, but little is known about their function. The DEP domain of RGS9 appears to be critical for interaction with R9AP (RGS9 anchoring protein) and subcellular targeting of RGS9 to the rod outer segment.27 The DEP domain of RGS7 can bind snapin, a protein that interacts with synaptosomal-associated protein of 25 kDa, a component of the soluble N-ethylmaleimideeCsensitive factor attachment protein receptor complex, suggesting a role for RGS7 in synaptic vesicle exocytosis.28 Interestingly, snapin was discovered recently to bind adenylyl cyclase type 6 (AC6),29 a highly expressed isoform of adenylyl cyclase in the heart,30 providing a possible link for RGS7 in the regulation of cAMP levels.
Regulation of RGS Proteins
RGS proteins have the potential to decrease or stop GPCR-mediated signaling. This important action implies that activity and expression of RGS proteins must be regulated. Indeed, RGS proteins are highly regulated through various mechanisms such as alterations in expression levels, subcellular localization, post-translational modifications, and binding partners. In addition to direct regulation of RGS proteins, the effects of RGS proteins can be regulated by post-translational modification of the G-protein -subunit.
Regulation of RGS Expression
Increases or decreases in cellular levels of RGS proteins have the potential to be critical for RGS-induced regulation of G-protein signaling. Increasing evidence demonstrates that RGS protein and mRNA levels are dynamically altered by various drugs, second messengers, and disease states. Although numerous RGS proteins show dynamic expression, RGS2 provides an excellent example of a highly regulated RGS protein. Dopamine D1 receptor agonists increase RGS2 mRNA, whereas a decrease in RGS2 mRNA occurs with agonism of the dopamine D2 receptor.31 Increases in cAMP levels by forskolin appear to increase RGS2 protein levels.32 Alterations in RGS2 levels show pathophysiological relevance because an overabundance of RGS2 protein is seen in individuals with Bartter’s/Gitelman’s Syndrome,33 and RGS2 knockout mice exhibit a severe cardiovascular phenotype,34 as is discussed subsequently. Chronic pharmacological therapy, as commonly used in the treatment of cardiovascular disease, likely alters RGS expression. The impact of such alterations on signal transduction has not been well studied but could lead to a myriad of effects, including sensitization or desensitization of signaling pathways, side effects, tolerance, dependence, etc.
Subcellular Localization
In order for an RGS protein to actively serve as a GAP, it must be localized in a region in the cell where it can bind its target G-subunit. Localization of RGS proteins in a particular subcellular compartment could increase the specificity of an RGS protein for particular G-proteins and GPCRs or pathway even though the GAP activity of an RGS protein may have multiple potential G targets.
On G-protein activation, RGS3 is translocated from the cytosol to the plasma membrane.35 In addition, the phosphorylation of RGS10 promotes its translocation from the plasma membrane and cytosol to the nucleus.36 The subcellular localization of RGS2 and RGS4 are dependent on specific G-proteins and GPCRs.37 In human embryonic kidney 293 cells, transfected RGS2 and RGS4 localize to the nucleus and cytosol, respectively. Whereas RGS2 translocates to the plasma membrane when cotransfected with Gq or Gs but not Gi2 RGS4 translocates to the plasma membrane when cotransfected with Gi2 but not Gq or Gs. A similar translocation profile is observed when GPCRs have been expressed that preferentially interact with specific G-protein family members. For example, the Ang II receptor (Gq-coupled) and 2-adrenergic receptor (Gs-coupled) promoted plasmalemmal translocation of RGS2, whereas RGS4 was only translocated by expression of the M2 muscarinic cholinergic receptor (mAChR; Gi-coupled). Unlike RGS3, the translocation of RGS2 and RGS4 does not appear to depend on G-protein activation.
Colocalization of proteins in a signaling pathway may be critical for signaling. Membrane microdomains, such as lipid rafts and caveolae, allow for the clustering and compartmentation of signaling molecules and are likely important for integrating GPCR-mediated signaling pathways.38 Many GPCRs move into or out of lipid rafts on agonist stimulation,39,40 an effect that could move the GPCR closer to or away from an RGS protein. Little work has been performed to determine whether RGS proteins are found in membrane microdomains. However, RGS16 localizes to lipid rafts on palmitoylation,41,42 which may be important for it to exert GAP activity for a particular signaling pathway.
Phosphorylation and Palmitoylation
Phosphorylation and palmitoylation, which reportedly can occur for multiple RGS proteins, produce a variety of effects, including alterations in subcellular localization, protein stability, and alterations in GAP activity. However, the physiological importance of these modifications remains to be determined. Tables 1 and 2 provide a summary of RGS proteins known to be influenced by phosphorylation and palmitoylation, respectively. It is likely that most, if not all, RGS proteins are regulated by phosphorylation or palmitoylation; however, much work needs to be done in this area.43 Phosphorylation and palmitoylation of G-subunits can affect RGS GAP activity, in particular, decreasing such activity.44eC46
Protein and Lipid-Binding Partners
Several RGS protein-binding partners have been identified. Of particular interest are 14-3-3 proteins, which bind target proteins at phosphorylated residues.47 14-3-3 binds RGS7 on phosphoserine 434 within the RGS domain, thereby decreasing RGS7 GAP activity;48 this binding is inhibited by tumor necrosis factor-, which can decrease serine 434 phosphorylation.49 In contrast, 14-3-3 binds RGS3 at phosphoserine 264 in a region outside the RGS domain but still reduces the potency of RGS3 in the inhibition of G-protein signaling.50 Only RGS3 and RGS7 have thus far been reported to bind 14-3-3, although other RGS proteins possess putative binding sites.48,50
The GAP activity of many RGS proteins is inhibited by phosphatidylinositol-3,4,5,-trisphosphate (PIP3), phosphatidic acid (PA), and lysophosphatidic acid, effects reversed by Ca2+/calmodulin.51eC55 PIP3 and PA can bind RGS4 and additively inhibit GAP activity, which suggests multiple binding sites. The binding of Ca2+/calmodulin can occur on two sites of RGS4 to reverse PA- and PIP3-mediated GAP inhibition without affecting GAP activity alone.55 The physiological significance for PIP3-mediated inhibition of RGS proteins includes regulation of G-proteineCgated K+ channels in cardiac myocytes, as is discussed subsequently.
Receptor Selectivity
As noted above, some RGS proteins can be very "promiscuous" in that they have GAP activity in vitro for many subtypes of G-subunits. However, it has become apparent that RGS proteins are highly regulated and can be extremely specific in modulating distinct signal transduction pathways. In addition to the various methods of RGS regulation discussed above, RGS proteins may selectively alter GPCR signaling in a receptor-specific manner, as summarized in Table 3.
Many of the methods used to determine receptor selectivity involve overexpression of GPCRs or RGS proteins. Overexpression can lead to artifactual results by producing "un-natural" protein levels. Alternatively, specific knockdown of proteins with ribozymes, antisense oligonucleotides, RNA interference, etc, avoids this problem, albeit introducing reagents with other potential sets of actions. Even so, one imagines that "knockdown strategies" can facilitate study of RGS/GPCR interactions in a setting with less potential for nonspecific perturbation than overexpression. Wang et al56 have demonstrated that knockdown of RGS3 and RGS5 alters Gq signaling in a receptor-specific manner (ie, of muscarinic M3 and Ang II type 1 [AT1] receptor signaling, respectively).56
It is clear that the ability of a RGS protein to show GAP activity for a specific G-subunit in vitro does not allow for the prediction of the precise signaling pathways that the RGS protein regulates in vivo. Because there are many factors that influence RGS activity or subcellular localization, it is not unreasonable to imagine cell-specific effects where a particular GPCR expressed in different cell types is differentially regulated by RGS proteins.
In addition to RGS selectivity for receptor signaling, Bernstein et al57 have recently demonstrated differential physical binding of RGS proteins to mAChRs. RGS1, RGS2, RGS4, and RGS16 were tested for interaction with the third intracellular loops of each of the five mAChR subtypes. RGS2, but not RGS1 or RGS16, bound the M1 mAChR, whereas none bound the M2 mAChR. Moreover, RGS2, but not RGS16, colocalized with the M1 mAChR at the plasma membrane and inhibited M1 mAChR-induced phosphoinositide hydrolysis.
RGS and G-Protein Selectivity
As shown in Figure 1, most RGS proteins are GAPs for the Gi and Gq families of G-proteins. However, many RGS proteins show G subtype-selective activity, as demonstrated by the following. (1) RGS19 interacts with Gi1, Gi3, and G0 but not Gi2;58,59 (2) RGS4 shows selectivity for Gi2 and Go1 over Gi1 and Gi3;60 (3) GPCR kinase 2 (GRK2) binds Gq, G11, and G14 but not G16;61 (4) RGS14 inhibits guanine nucleotide exchange on Gi1 and Gi3 but not Gi2;23 and (5) A splice variant of RGS20, RGSZ1, is 100-fold selective for GZ over Gi.45
Only one RGS protein, RGS-PX1, has thus far been found to exhibit GAP activity for the Gs family.24 Even though other RGS proteins do not regulate Gs-mediated signaling pathways via GAP activity, GAP-independent regulation occurs. Initial evidence demonstrated that RGS2 and a truncated form of RGS3, RGS3T, inhibited cAMP production.62,63 Later, Sinnarajah et al64 showed that RGS1, RGS2, and RGS3, but not RGS4 or RGS5, decreased odorant-induced cAMP production in olfactory membranes. Those workers also demonstrated that RGS2 inhibits cAMP production from AC3, AC5, and AC6, but not AC1 or AC2. This inhibition is achieved by the N terminal of RGS2 directly binding the C(1) domain of AC5.64,65
p115RhoeCguanine nucleotide exchange factor (GEF) is an RGS protein that shows selective GAP activity for members of the G12 family of G-proteins, G12 and G13, but also has an additional functional role.66 Activated G13 stimulates p115Rho-GEF and enhances its GEF activity for the monomeric G-protein Rho.67 Rho is involved in a variety of cellular responses, such as actin stress fiber formation, gene transcription and transformation. Of particular importance to the cardiovascular system, Rho induces hypertrophic responses in isolated cardiac myocytes,68 and cardiac overexpression of Rho in mice results in the development of ventricular failure.69 Because p115Rho-GEF activates (via GEF activity on Rho) and prevents activation (via GAP activity on G13) of Rho, it has a unique dual role in Rho signaling that may be critical for cardiovascular function. Although no other RGS proteins have been reported to be GAPs for G13, RGS16 is involved in the inhibition of G13 signaling via a GAP-independent mechanism. RGS16 binds G13 and translocates it to detergent-resistant membranes, presumably lipid rafts, preventing effector interaction and therefore inhibiting G13 signaling.70
RGS Proteins and the Cardiovascular System
GPCRs, and in turn, RGS proteins, are important regulators of cardiovascular signaling. Figure 1 demonstrates that the gene expression of virtually all known RGS proteins has been detected in the mammalian heart. Emerging evidence demonstrates that RGS proteins are needed for normal cardiovascular function and are altered in various cardiovascular disease states.
RGS2
Many GPCRs in the cardiovascular system responsible for vasoconstriction are coupled to Gq. More than half of the currently known RGS proteins exhibit GAP activity toward Gq and therefore have the potential to influence vascular tone. RGS2 shows some selectivity toward Gq and may be its most potent GAP.34,71 RGS2 has emerged as a potentially critical regulator of cardiovascular function because its GAP activity for Gq antagonizes Gq-mediated vasoconstriction. Although RGS2 can regulate Gi and Gs signaling (through GAP and non-GAP mechanisms), its potent regulation of Gq71,72 signaling appears to produce the most significant physiological effects.
Ang II is a vasoconstrictor, the effects of which are predominantly mediated through AT1, a GPCR coupled to Gq. In cultured vascular smooth muscle cells, Ang II stimulates the gene expression of RGS2,73 a response that may serve as negative feedback regulation, because RGS2 could inactivate this pathway via its GAP activity on Gq. In mice deficient for the RGS2 gene, a strong hypertensive phenotype is observed.34 In anesthetized mice, this phenotype appears to be attributable exclusively to the Ang II signaling pathway because it can be reversed with an AT1 antagonist or blockade of Ang II production with an angiotensin-converting enzyme inhibitor. Interestingly, rgs2+/eC and rgs2eC/eC mice exhibit a similar hypertensive phenotype, demonstrating that both copies of the gene are essential for normal cardiovascular function.
RGS2 is also regulated through a GPCR-independent pathway. NO is a potent vasodilator that induces the activation of cyclic GMPeCdependent protein kinases (PKGs). PKGs promote vascular relaxation through a variety of mechanisms including activation of RGS2.74 In particular, cGMP-dependent protein kinase I- binds directly to and phosphorylates RGS2, which increases its GAP activity on Gq and results in vasodilation. In RGS2 knockout mice, this pathway is disrupted. Aortas from RGS2-deficient mice show increased vasoconstriction in vitro in response to Gq-coupled agonists and decreased relaxation in response to cyclic GMP.34
RGS4
RGS4 is a key regulator of the G-proteineCgated K+ (KG) channels. Although it was discovered >80 years ago that acetylcholine (ACh) released from stimulation of the vagus nerve causes bradycardia,75 it was not until the recent discovery and characterization of RGS proteins that the kinetic mechanisms behind ACh-induced heart deceleration could be explained more fully (Figure 2). KG channels found in sino-atrial node cells decrease heart rate when activated by G. At a resting diastolic state (with low intracellular Ca2+) in the cardiac myocyte, PIP3 binds RGS4 within its RGS domain, preventing GAP activity.51eC54 Because GAP activity is prevented, an ACh-bound M2 mAChR causes the heterotrimeric G-protein complex to dissociate, allowing the G-subunit to bind and activate KG channels, which leads to K+ efflux and cellular hyperpolarization.76 On depolarization and subsequent Ca2+ influx, the Ca2+/calmodulin complex binds RGS4, relieving the PIP3 inhibition. This allows RGS4 to accelerate the GTPase activity of the -subunit, promoting reassociation of the heterotrimeric complex and inactivation of the KG channel. Because intracellular Ca2+ decreases, Ca2+/calmodulin dissociates from RGS4, allowing PIP3 to again bind and inhibit RGS4. RGS proteins thus appear to speed the activation and deactivation of KG channels.77
In order for the kinetics of KG channel activation and deactivation to approximate native conditions, RGS proteins must be present; however, it is still a subject of controversy whether physiological appropriate rates can be obtained,78 perhaps as a consequence of assembly of different tetrameric KG channels. Increased deactivation can be explained by RGS GAP activity promoting reassociation of the heterotrimeric G-protein complex. RGS-induced increases in channel activation are more difficult to explain with no conclusive data available thus far.
Muslin et al79eC81 have characterized an additional role for RGS4 in influencing cardiac hypertrophy. Reversible exercise-induced cardiac hypertrophy is not detrimental, whereas chronic hypertrophy is associated with cardiac arrhythmias, congestive heart failure, and death.82eC84 Initially, the overexpression of RGS4 in neonatal cardiac myocytes was observed to inhibit phenylephrine- and endothelin-1eCinduced hypertrophy.80 A second study used transgenic mice overexpressing RGS4 in ventricular tissue to study cardiac physiology.79 Overexpression of RGS4 did not affect basal cardiac function but significantly reduced the ability of the heart to adapt to an increase in cardiac afterload induced by transverse aortic constriction. Compared with littermate controls, the transgenic mice exhibited reduced ventricular hypertrophy, left ventricular dilation, depressed systolic function, and increased mortality in response to transverse aortic constriction. Thus, ventricular RGS4 overexpression appears to block beneficial compensatory hypertrophic mechanisms of the heart in response to an acute increase in cardiac afterload. Such results possibly suggest that increased cardiac RGS4 expression/activity would be unfavorable. However, a third study demonstrated favorable effects of RGS4 overexpression in mice that co-overexpress Gq.79 Transgenic mice overexpressing Gq in the heart exhibit a phenotype similar to human cardiac hypertrophy,85,86 but co-overexpression of RGS4 and Gq delays the Gq-mediated onset of cardiac hypertrophy.79
GRK2
GRKs decrease -adrenergic receptor signaling via phosphorylation of the activated receptor and by antagonizing G-protein signaling. In addition to its kinase activity, GRK2 (also known as ARK1) is an RGS protein that shows weak GAP activity for Gq.87 However, GRK2-mediated inhibition of G signaling is likely attributable to its binding and sequestration of activated G instead of the weak GAP activity.87 GRK2 activity and expression are increased in human hypertension and heart disease, including heart failure.88eC91 GRK2 inhibition can help prevent and blunt heart failure in animal models.92,93 In addition, the RGS domain of a kinase-inactivated mutant of GRK2 decreases endothelin-1 and Ang II signaling.94 Such data suggest that GRK2 has a dual role, perhaps in serving as a negative RGS (via its GAP activity and its sequestration of G) and in addition, as a receptor-desensitizing kinase for GPCRs. The ability of GRK2 to interact with caveolin may also contribute to its localization and actions, including its RGS activity.95
Human Heart Failure
Although variable results have been obtained, the expression profile of RGS2, RGS3, and RGS4 are altered in failing human hearts. Mittmann et al96 observed an increase in RGS4 mRNA but no change in RGS2 or RGS3 in such hearts, whereas Owen et al97 found an apparent upregulation of RGS3 and RGS4 protein and mRNA in human heart failure, and Takeishi et al98 identified an apparent decrease in RGS2 protein. These alterations in RGS expression have the potential to significantly alter cardiac physiology, as demonstrated in animal studies that show cardiovascular abnormalities in mice overexpressing RGS479 or deficient for RGS2.34
RGS Proteins as Drug Targets
Approximately 50% of all drugs target GPCRs,99,100 a fact that underscores the importance of GPCR signaling in the treatment of disease. However, there are many cases in which the current drugs targeted to GPCRs are inadequate. A good example is targeting of -adrenergic receptors. 2-Adrenergic receptor agonists are widely used for the treatment of bronchospasm, but these drugs can stimulate cardiac -adrenergic receptors as well. -Adrenergic receptor antagonists are commonly used for the treatment of various cardiovascular disorders. Difficulties can arise when an individual requires a -agonist for bronchospasm but has associated cardiovascular disease.101 Conversely, nonselective -blockers can be contraindicated in asthma and chronic obstructive pulmonary disease because of induced bronchoconstriction, and even 1-selective antagonists can cause problems in some patients.102 At higher doses, the selectivity of the "cardioselective" -blockers is lost and an increase in pulmonary side effects are seen. In the treatment of asthma, the use of 2-agonists leads to an increase in heart rate and is associated with an increased risk of adverse cardiovascular events.101 Thus, although -adrenergic receptors are important targets for the treatment of bronchospasm and cardiovascular disorders, treatment of one disease can precipitate dangerous side effects in the other organ system, thereby complicating therapy directed to the GPCR. RGS proteins, as potent regulators of GPCR signaling, potentially provide a new target for the regulation of GPCR signaling in such settings. A possible target could be RGS1, which shows strong mRNA expression in the lung but not the heart.103 In addition, RGS4 is found at moderate levels in the heart but low levels in the lung.103,104
In some reconstituted and in vitro systems, RGS proteins can exhibit GAP activity toward a variety of G-subunits. However, data in more physiological systems suggest that RGS proteins act with much more specificity. As the unique specificity profile of each RGS protein is determined, in particular in the in vivo setting, pharmacological manipulation of RGS proteins seem likely to become more enticing. Pharmacological regulation of RGS proteins could: (1) potentiate or attenuate the actions of an endogenous agonist; (2) complement a GPCR pharmacological agonist or antagonist, thereby reducing the dose needed; or 3) combat the drug-induced side effects produced by GPCR agonism or antagonism.
The mechanistic effects of pharmacological manipulation of RGS proteins can be grouped into two categories: (1) alteration of RGS activity; and 2) addition or removal of an RGS protein from a particular pathway, perhaps by altering subcellular localization. Drugs developed to alter RGS activity may be preferred to drugs that alter localization because drugs with the latter action have the potential to promote RGS protein interaction with other signaling pathways.
RGS proteins of the R4 subfamily are the most studied because of their "simple" structure. However, these "simple" proteins exert profound physiological effects and, perhaps as a consequence, are highly regulated via a variety of mechanisms. For example, RGS4 is regulated by phosphorylation, palmitoylation, PIP3, Ca2+/calmodulin, G-proteins, and GPCRs. Other RGS proteins with a more complex structure would presumably be subject to an even larger number of regulatory factors. The complexity of RGS protein structure or regulation may present problems in developing drugs that selectively alter a specific pathway without influencing other pathways. However, from another perspective, the numerous methods of regulation could provide additional drug targets for the manipulation of RGS activity.
One could argue that every physiological process uses a unique subset of signaling events. The challenge in developing drugs to treat disease is in finding unique targets within a specific signaling pathway that would yield efficacy without toxicity. Drugs targeted to GPCRs have proven to be extremely important for the treatment of many diseases, in part as a consequence of unique patterns of tissue expression and accessibility on the cell surface. RGS proteins appear to offer an alternative target to alter GPCR and G-proteineCmediated signaling. With a tissue distribution different from that of GPCRs, RGS proteins may prove to be useful targets. Work is already under way to develop agents that alter RGS function. Mosberg et al105 have begun developing RGS4 inhibitors that could be useful in treatment of cardiovascular disorders.79 However, RGS proteins may not prove immune to specificity problems, and accessibility of drugs to key sites on the proteins will need to be achieved. In the case of RGS4, it would be important that RGS4 activity in the brain is not altered because it may lead to schizophrenic symptoms.106
The "gaps" in current understanding of the unique mechanisms by which each RGS protein alters signaling make it challenging to predict the impact that drugs targeted to RGS proteins would have on human cardiovascular physiology. However, in our view, the significance of RGS proteins in the regulation of cell signaling warrants drug discovery efforts.
Acknowledgments
This work was supported by National Institutes of Health (NIH) grants HL007261 (to E.L.R.), and NIH AG16613 and NIH HL56256 (to M.B.), American Heart Association postdoctoral fellowship (to R.S.), and NIH grants NIH HL69758, HL66941, HL58120, and HL53773 (to P.A.I.).
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the Department of Physiology (R.A.S., M.B.), University of Maryland School of Medicine, Baltimore
the Department of Molecular Cardiology (M.B.), Lerner Research Institute, The Cleveland Clinic Foundation, Ohio.
Abstract
Regulator of G-proteineCsignaling (RGS) proteins play a key role in the regulation of G-proteineCcoupled receptor (GPCR) signaling. The characteristic hallmark of RGS proteins is a conserved 120-aa RGS region that confers on these proteins the ability to serve as GTPase-activating proteins (GAPs) for G proteins. Most RGS proteins can serve as GAPs for multiple isoforms of G and therefore have the potential to influence many cellular signaling pathways. However, RGS proteins can be highly regulated and can demonstrate extreme specificity for a particular signaling pathway. RGS proteins can be regulated by altering their GAP activity or subcellular localization; such regulation is achieved by phosphorylation, palmitoylation, and interaction with protein and lipid-binding partners. Many RGS proteins have GAP-independent functions that influence GPCR and non-GPCReCmediated signaling, such as effector regulation or action as an effector. Hence, RGS proteins should be considered multifunctional signaling regulators. GPCR-mediated signaling is critical for normal function in the cardiovascular system and is currently the primary target for the pharmacological treatment of disease. Alterations in RGS protein levels, in particular RGS2 and RGS4, produce cardiovascular phenotypes. Thus, because of the importance of GPCR-signaling pathways and the profound influence of RGS proteins on these pathways, RGS proteins are regulators of cardiovascular physiology and potentially novel drug targets as well.
Key Words: RGS protein regulator of G-protein signaling GPCRheart
Introduction
G-ProteineCcoupled receptors (GPCRs) are found ubiquitously through the body and are involved in virtually every physiological process. Cardiovascular cells possess >100 different GPCRs.1 GPCRs bind a heterotrimeric G-protein complex of -, -, and -subunits. G-Proteins are divided into four families (eg, Gs, Gi, Gq, and G12) based on similarity of -subunits among individual family members;2 at least 20 -subunits are found in mammalian cells.3 Agonist binding of GPCRs promotes G-protein activation. This activation is achieved by catalyzing GDPeCGTP exchange on the -subunit. A conformational change in the GTP-bound -subunit leads to a dissociation of G from the -subunits. GTP-bound G-subunits and dissociated -dimers regulate downstream effectors. These signaling events are terminated as a consequence of intrinsic GTPase activity of the G-subunit, which hydrolyzes bound GTP to GDP, resulting in a reassociation of the G-protein heterotrimer. The intrinsic GTPase activity of -subunits is generally insufficient to correlate with physiological rates of G-protein inactivation, but this activity can be accelerated by the presence of GTPase-activating proteins (GAPs), such as regulator of G-proteineCsignaling (RGS) proteins.
The discovery of RGS proteins suggested the possibility of a specific RGS protein for each G subtype. However, this idea was discarded when it was discovered that the number of RGS proteins (now >30) was greater than that of G-subunits, along with the recognition that individual RGS proteins could act as GAPs for multiple families of G. However, as understanding of RGS function increases, evidence has emerged for G specificity of certain RGS proteins, especially in the in vivo setting, as well as their modulation of distinct GPCR-mediated signaling pathways. Although a substantial amount is known regarding identity and expression of RGS proteins, much remains to be elucidated regarding their function, localization, and regulation. Their GAP activity is a primary characteristic, but RGS proteins use a variety of mechanisms to regulate signaling. Several excellent reviews have provided insight on the structure and function of RGS proteins.4eC7 However, numerous recent developments related to RGS proteins have important implications, especially for cardiovascular physiology and pathophysiology. In this review, we first provide an overview of RGS structure, emphasizing non-RGS domains. Second, we discuss mechanisms for the regulation of RGS proteins. Finally, we bring the reader up to date on current knowledge of the importance of these "multitasking" RGS proteins in the cardiovascular system.
RGS Protein Structure
Figure 1 summarizes key features of RGS genes and proteins. Many RGS genes produce different isoforms (eg, rgs6 encodes 36 distinct transcripts8). A description of each isoform is beyond the scope of this review. RGS proteins are diverse, ranging in size from 152 (RGS21) to 1376 (RGS12) amino acids. Many, indeed most, RGS proteins identified to date exhibit GAP activity toward the Gi and Gq families of G-proteins. However, it is important to note that even though an RGS protein may be able to serve as a GAP for a particular G-subunit, particular GPCR signaling pathways involving the identical G-subunit can show receptor-selective regulation by RGS proteins.9
On the basis of sequence similarity of the RGS domain, RGS proteins can be classified into one of five subfamilies: R4, R7, R12, RZ, and RL (Figure 1). Although some RGS proteins consist almost exclusively of the RGS domain, a conserved 120-aa region, others contain additional domains. The RGS domain is necessary and sufficient to confer GAP activity;10 however, other domains, to be discussed subsequently, can increase specificity, determine cellular localization, and provide additional activities.
PSD-95 Disk-Large ZO-1 Domain
PSD-95 disk-large ZO-1 (PDZ) domains, 90-aa regions with a highly conserved four-residue GLGF sequence, are involved in the clustering of multiprotein signaling complexes.11 RGS12 and a variant of RGS3, PDZ-RGS3, contain PDZ domains. The PDZ domain of PDZ-RGS3 binds the non-GPCR B-ephrin receptor, thereby providing a possible link between GPCR signaling and B-ephrin signaling.12 In addition, PDZ-RGS3 could be important for B-ephrineCregulated cardiovascular development.13
The PDZ domain of RGS12 binds selectively to the interleukin-8 receptor B (CXCR2), a Gi-coupled GPCR.14 Although RGS12 has GAP activity toward Gi, it remains to be determined whether RGS12 alters CXCR2-mediated G-protein signaling. The interaction of RGS12 with CXCR2 has the potential to influence myocardial viability during ischemia reperfusion.15
G-Protein SubuniteCLike Domain
RGS6, RGS7, RGS9, and RGS11 contain G-protein subuniteClike (GGL) domains, a 64-aa region with a high level of similarity to the G-subunit, can form dimers with particular G-protein subunits (ie, G5) but not others (eg, G1 to G3).16 This RGS/G5 interaction appears to influence RGS and G5 protein stability,17,18 GAP activity of RGS proteins,16,19 and subcellular localization of RGS and G5.8,20
GoLoco Domain
RGS12 and RGS14 contain GoLoco domains, a 19-aa Gi binding motif that acts as a guanine nucleotide dissociation inhibitor (GDI) by binding and stabilizing GDP-bound Gi, inhibiting the rate of exchange of GDP for GTP in a GAP-independent manner.21 The RGS and GoLoco domains of RGS14 can independently inhibit Gi signaling, but both domains are necessary for maximal inhibition.22 The GoLoco domain of RGS14 exhibits GDI activity toward Gi1 and Gi3 but not Gi2 or Go even though the GAP activity is not G-protein subtype selective.23
PX Domain
RGS-PX1, the only RGS protein thus far identified with GAP activity for Gs, contains a PX domain, an 120-aa phosphoinositide-binding domain involved in membrane targeting.24,25 RGS-PX1 delays lysosomal degradation of the EGF receptor, most likely as a result of the sorting nexin function of the PX domain.24 The relationship between RGS-PX1 and the EGF receptor suggests a possible role for RGS-PX1 in angiotensin II (Ang II)eCinduced cardiac hypertrophy.26
Disheveled EGL-10 Pleckstrin Domain
The 70-aa disheveled EGL-10 pleckstrin (DEP) domains are present in various signaling proteins, including RGS6, RGS7, RGS9, and RGS11, but little is known about their function. The DEP domain of RGS9 appears to be critical for interaction with R9AP (RGS9 anchoring protein) and subcellular targeting of RGS9 to the rod outer segment.27 The DEP domain of RGS7 can bind snapin, a protein that interacts with synaptosomal-associated protein of 25 kDa, a component of the soluble N-ethylmaleimideeCsensitive factor attachment protein receptor complex, suggesting a role for RGS7 in synaptic vesicle exocytosis.28 Interestingly, snapin was discovered recently to bind adenylyl cyclase type 6 (AC6),29 a highly expressed isoform of adenylyl cyclase in the heart,30 providing a possible link for RGS7 in the regulation of cAMP levels.
Regulation of RGS Proteins
RGS proteins have the potential to decrease or stop GPCR-mediated signaling. This important action implies that activity and expression of RGS proteins must be regulated. Indeed, RGS proteins are highly regulated through various mechanisms such as alterations in expression levels, subcellular localization, post-translational modifications, and binding partners. In addition to direct regulation of RGS proteins, the effects of RGS proteins can be regulated by post-translational modification of the G-protein -subunit.
Regulation of RGS Expression
Increases or decreases in cellular levels of RGS proteins have the potential to be critical for RGS-induced regulation of G-protein signaling. Increasing evidence demonstrates that RGS protein and mRNA levels are dynamically altered by various drugs, second messengers, and disease states. Although numerous RGS proteins show dynamic expression, RGS2 provides an excellent example of a highly regulated RGS protein. Dopamine D1 receptor agonists increase RGS2 mRNA, whereas a decrease in RGS2 mRNA occurs with agonism of the dopamine D2 receptor.31 Increases in cAMP levels by forskolin appear to increase RGS2 protein levels.32 Alterations in RGS2 levels show pathophysiological relevance because an overabundance of RGS2 protein is seen in individuals with Bartter’s/Gitelman’s Syndrome,33 and RGS2 knockout mice exhibit a severe cardiovascular phenotype,34 as is discussed subsequently. Chronic pharmacological therapy, as commonly used in the treatment of cardiovascular disease, likely alters RGS expression. The impact of such alterations on signal transduction has not been well studied but could lead to a myriad of effects, including sensitization or desensitization of signaling pathways, side effects, tolerance, dependence, etc.
Subcellular Localization
In order for an RGS protein to actively serve as a GAP, it must be localized in a region in the cell where it can bind its target G-subunit. Localization of RGS proteins in a particular subcellular compartment could increase the specificity of an RGS protein for particular G-proteins and GPCRs or pathway even though the GAP activity of an RGS protein may have multiple potential G targets.
On G-protein activation, RGS3 is translocated from the cytosol to the plasma membrane.35 In addition, the phosphorylation of RGS10 promotes its translocation from the plasma membrane and cytosol to the nucleus.36 The subcellular localization of RGS2 and RGS4 are dependent on specific G-proteins and GPCRs.37 In human embryonic kidney 293 cells, transfected RGS2 and RGS4 localize to the nucleus and cytosol, respectively. Whereas RGS2 translocates to the plasma membrane when cotransfected with Gq or Gs but not Gi2 RGS4 translocates to the plasma membrane when cotransfected with Gi2 but not Gq or Gs. A similar translocation profile is observed when GPCRs have been expressed that preferentially interact with specific G-protein family members. For example, the Ang II receptor (Gq-coupled) and 2-adrenergic receptor (Gs-coupled) promoted plasmalemmal translocation of RGS2, whereas RGS4 was only translocated by expression of the M2 muscarinic cholinergic receptor (mAChR; Gi-coupled). Unlike RGS3, the translocation of RGS2 and RGS4 does not appear to depend on G-protein activation.
Colocalization of proteins in a signaling pathway may be critical for signaling. Membrane microdomains, such as lipid rafts and caveolae, allow for the clustering and compartmentation of signaling molecules and are likely important for integrating GPCR-mediated signaling pathways.38 Many GPCRs move into or out of lipid rafts on agonist stimulation,39,40 an effect that could move the GPCR closer to or away from an RGS protein. Little work has been performed to determine whether RGS proteins are found in membrane microdomains. However, RGS16 localizes to lipid rafts on palmitoylation,41,42 which may be important for it to exert GAP activity for a particular signaling pathway.
Phosphorylation and Palmitoylation
Phosphorylation and palmitoylation, which reportedly can occur for multiple RGS proteins, produce a variety of effects, including alterations in subcellular localization, protein stability, and alterations in GAP activity. However, the physiological importance of these modifications remains to be determined. Tables 1 and 2 provide a summary of RGS proteins known to be influenced by phosphorylation and palmitoylation, respectively. It is likely that most, if not all, RGS proteins are regulated by phosphorylation or palmitoylation; however, much work needs to be done in this area.43 Phosphorylation and palmitoylation of G-subunits can affect RGS GAP activity, in particular, decreasing such activity.44eC46
Protein and Lipid-Binding Partners
Several RGS protein-binding partners have been identified. Of particular interest are 14-3-3 proteins, which bind target proteins at phosphorylated residues.47 14-3-3 binds RGS7 on phosphoserine 434 within the RGS domain, thereby decreasing RGS7 GAP activity;48 this binding is inhibited by tumor necrosis factor-, which can decrease serine 434 phosphorylation.49 In contrast, 14-3-3 binds RGS3 at phosphoserine 264 in a region outside the RGS domain but still reduces the potency of RGS3 in the inhibition of G-protein signaling.50 Only RGS3 and RGS7 have thus far been reported to bind 14-3-3, although other RGS proteins possess putative binding sites.48,50
The GAP activity of many RGS proteins is inhibited by phosphatidylinositol-3,4,5,-trisphosphate (PIP3), phosphatidic acid (PA), and lysophosphatidic acid, effects reversed by Ca2+/calmodulin.51eC55 PIP3 and PA can bind RGS4 and additively inhibit GAP activity, which suggests multiple binding sites. The binding of Ca2+/calmodulin can occur on two sites of RGS4 to reverse PA- and PIP3-mediated GAP inhibition without affecting GAP activity alone.55 The physiological significance for PIP3-mediated inhibition of RGS proteins includes regulation of G-proteineCgated K+ channels in cardiac myocytes, as is discussed subsequently.
Receptor Selectivity
As noted above, some RGS proteins can be very "promiscuous" in that they have GAP activity in vitro for many subtypes of G-subunits. However, it has become apparent that RGS proteins are highly regulated and can be extremely specific in modulating distinct signal transduction pathways. In addition to the various methods of RGS regulation discussed above, RGS proteins may selectively alter GPCR signaling in a receptor-specific manner, as summarized in Table 3.
Many of the methods used to determine receptor selectivity involve overexpression of GPCRs or RGS proteins. Overexpression can lead to artifactual results by producing "un-natural" protein levels. Alternatively, specific knockdown of proteins with ribozymes, antisense oligonucleotides, RNA interference, etc, avoids this problem, albeit introducing reagents with other potential sets of actions. Even so, one imagines that "knockdown strategies" can facilitate study of RGS/GPCR interactions in a setting with less potential for nonspecific perturbation than overexpression. Wang et al56 have demonstrated that knockdown of RGS3 and RGS5 alters Gq signaling in a receptor-specific manner (ie, of muscarinic M3 and Ang II type 1 [AT1] receptor signaling, respectively).56
It is clear that the ability of a RGS protein to show GAP activity for a specific G-subunit in vitro does not allow for the prediction of the precise signaling pathways that the RGS protein regulates in vivo. Because there are many factors that influence RGS activity or subcellular localization, it is not unreasonable to imagine cell-specific effects where a particular GPCR expressed in different cell types is differentially regulated by RGS proteins.
In addition to RGS selectivity for receptor signaling, Bernstein et al57 have recently demonstrated differential physical binding of RGS proteins to mAChRs. RGS1, RGS2, RGS4, and RGS16 were tested for interaction with the third intracellular loops of each of the five mAChR subtypes. RGS2, but not RGS1 or RGS16, bound the M1 mAChR, whereas none bound the M2 mAChR. Moreover, RGS2, but not RGS16, colocalized with the M1 mAChR at the plasma membrane and inhibited M1 mAChR-induced phosphoinositide hydrolysis.
RGS and G-Protein Selectivity
As shown in Figure 1, most RGS proteins are GAPs for the Gi and Gq families of G-proteins. However, many RGS proteins show G subtype-selective activity, as demonstrated by the following. (1) RGS19 interacts with Gi1, Gi3, and G0 but not Gi2;58,59 (2) RGS4 shows selectivity for Gi2 and Go1 over Gi1 and Gi3;60 (3) GPCR kinase 2 (GRK2) binds Gq, G11, and G14 but not G16;61 (4) RGS14 inhibits guanine nucleotide exchange on Gi1 and Gi3 but not Gi2;23 and (5) A splice variant of RGS20, RGSZ1, is 100-fold selective for GZ over Gi.45
Only one RGS protein, RGS-PX1, has thus far been found to exhibit GAP activity for the Gs family.24 Even though other RGS proteins do not regulate Gs-mediated signaling pathways via GAP activity, GAP-independent regulation occurs. Initial evidence demonstrated that RGS2 and a truncated form of RGS3, RGS3T, inhibited cAMP production.62,63 Later, Sinnarajah et al64 showed that RGS1, RGS2, and RGS3, but not RGS4 or RGS5, decreased odorant-induced cAMP production in olfactory membranes. Those workers also demonstrated that RGS2 inhibits cAMP production from AC3, AC5, and AC6, but not AC1 or AC2. This inhibition is achieved by the N terminal of RGS2 directly binding the C(1) domain of AC5.64,65
p115RhoeCguanine nucleotide exchange factor (GEF) is an RGS protein that shows selective GAP activity for members of the G12 family of G-proteins, G12 and G13, but also has an additional functional role.66 Activated G13 stimulates p115Rho-GEF and enhances its GEF activity for the monomeric G-protein Rho.67 Rho is involved in a variety of cellular responses, such as actin stress fiber formation, gene transcription and transformation. Of particular importance to the cardiovascular system, Rho induces hypertrophic responses in isolated cardiac myocytes,68 and cardiac overexpression of Rho in mice results in the development of ventricular failure.69 Because p115Rho-GEF activates (via GEF activity on Rho) and prevents activation (via GAP activity on G13) of Rho, it has a unique dual role in Rho signaling that may be critical for cardiovascular function. Although no other RGS proteins have been reported to be GAPs for G13, RGS16 is involved in the inhibition of G13 signaling via a GAP-independent mechanism. RGS16 binds G13 and translocates it to detergent-resistant membranes, presumably lipid rafts, preventing effector interaction and therefore inhibiting G13 signaling.70
RGS Proteins and the Cardiovascular System
GPCRs, and in turn, RGS proteins, are important regulators of cardiovascular signaling. Figure 1 demonstrates that the gene expression of virtually all known RGS proteins has been detected in the mammalian heart. Emerging evidence demonstrates that RGS proteins are needed for normal cardiovascular function and are altered in various cardiovascular disease states.
RGS2
Many GPCRs in the cardiovascular system responsible for vasoconstriction are coupled to Gq. More than half of the currently known RGS proteins exhibit GAP activity toward Gq and therefore have the potential to influence vascular tone. RGS2 shows some selectivity toward Gq and may be its most potent GAP.34,71 RGS2 has emerged as a potentially critical regulator of cardiovascular function because its GAP activity for Gq antagonizes Gq-mediated vasoconstriction. Although RGS2 can regulate Gi and Gs signaling (through GAP and non-GAP mechanisms), its potent regulation of Gq71,72 signaling appears to produce the most significant physiological effects.
Ang II is a vasoconstrictor, the effects of which are predominantly mediated through AT1, a GPCR coupled to Gq. In cultured vascular smooth muscle cells, Ang II stimulates the gene expression of RGS2,73 a response that may serve as negative feedback regulation, because RGS2 could inactivate this pathway via its GAP activity on Gq. In mice deficient for the RGS2 gene, a strong hypertensive phenotype is observed.34 In anesthetized mice, this phenotype appears to be attributable exclusively to the Ang II signaling pathway because it can be reversed with an AT1 antagonist or blockade of Ang II production with an angiotensin-converting enzyme inhibitor. Interestingly, rgs2+/eC and rgs2eC/eC mice exhibit a similar hypertensive phenotype, demonstrating that both copies of the gene are essential for normal cardiovascular function.
RGS2 is also regulated through a GPCR-independent pathway. NO is a potent vasodilator that induces the activation of cyclic GMPeCdependent protein kinases (PKGs). PKGs promote vascular relaxation through a variety of mechanisms including activation of RGS2.74 In particular, cGMP-dependent protein kinase I- binds directly to and phosphorylates RGS2, which increases its GAP activity on Gq and results in vasodilation. In RGS2 knockout mice, this pathway is disrupted. Aortas from RGS2-deficient mice show increased vasoconstriction in vitro in response to Gq-coupled agonists and decreased relaxation in response to cyclic GMP.34
RGS4
RGS4 is a key regulator of the G-proteineCgated K+ (KG) channels. Although it was discovered >80 years ago that acetylcholine (ACh) released from stimulation of the vagus nerve causes bradycardia,75 it was not until the recent discovery and characterization of RGS proteins that the kinetic mechanisms behind ACh-induced heart deceleration could be explained more fully (Figure 2). KG channels found in sino-atrial node cells decrease heart rate when activated by G. At a resting diastolic state (with low intracellular Ca2+) in the cardiac myocyte, PIP3 binds RGS4 within its RGS domain, preventing GAP activity.51eC54 Because GAP activity is prevented, an ACh-bound M2 mAChR causes the heterotrimeric G-protein complex to dissociate, allowing the G-subunit to bind and activate KG channels, which leads to K+ efflux and cellular hyperpolarization.76 On depolarization and subsequent Ca2+ influx, the Ca2+/calmodulin complex binds RGS4, relieving the PIP3 inhibition. This allows RGS4 to accelerate the GTPase activity of the -subunit, promoting reassociation of the heterotrimeric complex and inactivation of the KG channel. Because intracellular Ca2+ decreases, Ca2+/calmodulin dissociates from RGS4, allowing PIP3 to again bind and inhibit RGS4. RGS proteins thus appear to speed the activation and deactivation of KG channels.77
In order for the kinetics of KG channel activation and deactivation to approximate native conditions, RGS proteins must be present; however, it is still a subject of controversy whether physiological appropriate rates can be obtained,78 perhaps as a consequence of assembly of different tetrameric KG channels. Increased deactivation can be explained by RGS GAP activity promoting reassociation of the heterotrimeric G-protein complex. RGS-induced increases in channel activation are more difficult to explain with no conclusive data available thus far.
Muslin et al79eC81 have characterized an additional role for RGS4 in influencing cardiac hypertrophy. Reversible exercise-induced cardiac hypertrophy is not detrimental, whereas chronic hypertrophy is associated with cardiac arrhythmias, congestive heart failure, and death.82eC84 Initially, the overexpression of RGS4 in neonatal cardiac myocytes was observed to inhibit phenylephrine- and endothelin-1eCinduced hypertrophy.80 A second study used transgenic mice overexpressing RGS4 in ventricular tissue to study cardiac physiology.79 Overexpression of RGS4 did not affect basal cardiac function but significantly reduced the ability of the heart to adapt to an increase in cardiac afterload induced by transverse aortic constriction. Compared with littermate controls, the transgenic mice exhibited reduced ventricular hypertrophy, left ventricular dilation, depressed systolic function, and increased mortality in response to transverse aortic constriction. Thus, ventricular RGS4 overexpression appears to block beneficial compensatory hypertrophic mechanisms of the heart in response to an acute increase in cardiac afterload. Such results possibly suggest that increased cardiac RGS4 expression/activity would be unfavorable. However, a third study demonstrated favorable effects of RGS4 overexpression in mice that co-overexpress Gq.79 Transgenic mice overexpressing Gq in the heart exhibit a phenotype similar to human cardiac hypertrophy,85,86 but co-overexpression of RGS4 and Gq delays the Gq-mediated onset of cardiac hypertrophy.79
GRK2
GRKs decrease -adrenergic receptor signaling via phosphorylation of the activated receptor and by antagonizing G-protein signaling. In addition to its kinase activity, GRK2 (also known as ARK1) is an RGS protein that shows weak GAP activity for Gq.87 However, GRK2-mediated inhibition of G signaling is likely attributable to its binding and sequestration of activated G instead of the weak GAP activity.87 GRK2 activity and expression are increased in human hypertension and heart disease, including heart failure.88eC91 GRK2 inhibition can help prevent and blunt heart failure in animal models.92,93 In addition, the RGS domain of a kinase-inactivated mutant of GRK2 decreases endothelin-1 and Ang II signaling.94 Such data suggest that GRK2 has a dual role, perhaps in serving as a negative RGS (via its GAP activity and its sequestration of G) and in addition, as a receptor-desensitizing kinase for GPCRs. The ability of GRK2 to interact with caveolin may also contribute to its localization and actions, including its RGS activity.95
Human Heart Failure
Although variable results have been obtained, the expression profile of RGS2, RGS3, and RGS4 are altered in failing human hearts. Mittmann et al96 observed an increase in RGS4 mRNA but no change in RGS2 or RGS3 in such hearts, whereas Owen et al97 found an apparent upregulation of RGS3 and RGS4 protein and mRNA in human heart failure, and Takeishi et al98 identified an apparent decrease in RGS2 protein. These alterations in RGS expression have the potential to significantly alter cardiac physiology, as demonstrated in animal studies that show cardiovascular abnormalities in mice overexpressing RGS479 or deficient for RGS2.34
RGS Proteins as Drug Targets
Approximately 50% of all drugs target GPCRs,99,100 a fact that underscores the importance of GPCR signaling in the treatment of disease. However, there are many cases in which the current drugs targeted to GPCRs are inadequate. A good example is targeting of -adrenergic receptors. 2-Adrenergic receptor agonists are widely used for the treatment of bronchospasm, but these drugs can stimulate cardiac -adrenergic receptors as well. -Adrenergic receptor antagonists are commonly used for the treatment of various cardiovascular disorders. Difficulties can arise when an individual requires a -agonist for bronchospasm but has associated cardiovascular disease.101 Conversely, nonselective -blockers can be contraindicated in asthma and chronic obstructive pulmonary disease because of induced bronchoconstriction, and even 1-selective antagonists can cause problems in some patients.102 At higher doses, the selectivity of the "cardioselective" -blockers is lost and an increase in pulmonary side effects are seen. In the treatment of asthma, the use of 2-agonists leads to an increase in heart rate and is associated with an increased risk of adverse cardiovascular events.101 Thus, although -adrenergic receptors are important targets for the treatment of bronchospasm and cardiovascular disorders, treatment of one disease can precipitate dangerous side effects in the other organ system, thereby complicating therapy directed to the GPCR. RGS proteins, as potent regulators of GPCR signaling, potentially provide a new target for the regulation of GPCR signaling in such settings. A possible target could be RGS1, which shows strong mRNA expression in the lung but not the heart.103 In addition, RGS4 is found at moderate levels in the heart but low levels in the lung.103,104
In some reconstituted and in vitro systems, RGS proteins can exhibit GAP activity toward a variety of G-subunits. However, data in more physiological systems suggest that RGS proteins act with much more specificity. As the unique specificity profile of each RGS protein is determined, in particular in the in vivo setting, pharmacological manipulation of RGS proteins seem likely to become more enticing. Pharmacological regulation of RGS proteins could: (1) potentiate or attenuate the actions of an endogenous agonist; (2) complement a GPCR pharmacological agonist or antagonist, thereby reducing the dose needed; or 3) combat the drug-induced side effects produced by GPCR agonism or antagonism.
The mechanistic effects of pharmacological manipulation of RGS proteins can be grouped into two categories: (1) alteration of RGS activity; and 2) addition or removal of an RGS protein from a particular pathway, perhaps by altering subcellular localization. Drugs developed to alter RGS activity may be preferred to drugs that alter localization because drugs with the latter action have the potential to promote RGS protein interaction with other signaling pathways.
RGS proteins of the R4 subfamily are the most studied because of their "simple" structure. However, these "simple" proteins exert profound physiological effects and, perhaps as a consequence, are highly regulated via a variety of mechanisms. For example, RGS4 is regulated by phosphorylation, palmitoylation, PIP3, Ca2+/calmodulin, G-proteins, and GPCRs. Other RGS proteins with a more complex structure would presumably be subject to an even larger number of regulatory factors. The complexity of RGS protein structure or regulation may present problems in developing drugs that selectively alter a specific pathway without influencing other pathways. However, from another perspective, the numerous methods of regulation could provide additional drug targets for the manipulation of RGS activity.
One could argue that every physiological process uses a unique subset of signaling events. The challenge in developing drugs to treat disease is in finding unique targets within a specific signaling pathway that would yield efficacy without toxicity. Drugs targeted to GPCRs have proven to be extremely important for the treatment of many diseases, in part as a consequence of unique patterns of tissue expression and accessibility on the cell surface. RGS proteins appear to offer an alternative target to alter GPCR and G-proteineCmediated signaling. With a tissue distribution different from that of GPCRs, RGS proteins may prove to be useful targets. Work is already under way to develop agents that alter RGS function. Mosberg et al105 have begun developing RGS4 inhibitors that could be useful in treatment of cardiovascular disorders.79 However, RGS proteins may not prove immune to specificity problems, and accessibility of drugs to key sites on the proteins will need to be achieved. In the case of RGS4, it would be important that RGS4 activity in the brain is not altered because it may lead to schizophrenic symptoms.106
The "gaps" in current understanding of the unique mechanisms by which each RGS protein alters signaling make it challenging to predict the impact that drugs targeted to RGS proteins would have on human cardiovascular physiology. However, in our view, the significance of RGS proteins in the regulation of cell signaling warrants drug discovery efforts.
Acknowledgments
This work was supported by National Institutes of Health (NIH) grants HL007261 (to E.L.R.), and NIH AG16613 and NIH HL56256 (to M.B.), American Heart Association postdoctoral fellowship (to R.S.), and NIH grants NIH HL69758, HL66941, HL58120, and HL53773 (to P.A.I.).
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