Chapter 3 Regulators of G Protein Signaling Proteins as Central Components of G Protein‐Coupled Receptor Signaling Complexes

Regulators of G Protein Signaling Proteins as Central Components of G Protein‐Coupled Receptor Signaling Complexes Kelly L. McCoy and John R. Hepler Department of Pharmacology, G205 Rollins Research Center, Emory University School of Medicine, Atlanta, Georgia 30322

I. Introduction .................................................................................. II. Overview of RGS Proteins................................................................. A. RGS Protein Structure Determines Function .................................... III. RGS Protein Interactions with GPCRs ................................................. A. GPCRs Interact Directly with RGS Proteins ..................................... B. Indirect GPCR/RGS Protein Interactions......................................... C. Implied RGS Protein and GPCR Interactions ................................... D. RGS Proteins also Interact with Non‐GPCR Receptors and Ion Channels ......................................................... E. Factors that Dictate RGS Protein Localization at the Plasma Membrane.............................................................. IV. GPCRs Serve as Platforms for Molecular Signaling ................................. V. Summary and Perspectives ................................................................ References ....................................................................................

50 50 51 52 54 57 59 61 62 63 66 67

The regulators of G protein signaling (RGS) proteins bind directly to G protein alpha (Ga) subunits to regulate the signaling functions of Ga and their linked G protein‐coupled receptors (GPCRs). Recent studies indicate that RGS proteins also interact with GPCRs, not just G proteins, to form preferred functional pairs. Interactions between GPCRs and RGS proteins may be direct or indirect (via a linker protein) and are dictated by the receptors, rather than the linked G proteins. Emerging models suggest that GPCRs serve as platforms for assembling an overlapping and distinct constellation of signaling proteins that perform receptor‐specific signaling tasks. Compelling evidence now indicates that RGS proteins are central components of these GPCR signaling complexes. This review will outline recent discoveries of GPCR/RGS pairs as well as new data in support of the idea that GPCRs serve as platforms for the formation of multiprotein signaling complexes.

Progress in Molecular Biology and Translational Science, Vol. 86 DOI: 10.1016/S1877-1173(09)86003-1

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I. Introduction Neurotransmitters and hormones exert their activity by relaying messages across the plasma membrane and inside cells via specific G protein‐coupled receptors (GPCRs). In turn, GPCRs activate heterotrimeric G proteins and linked intracellular signaling pathways.1 Early models of G protein signaling proposed that GPCRs preferentially bound and activated one specific G protein. However, our understanding of these pathways has evolved in recent years to include a new appreciation for an unexpected complexity of GPCR/G protein signal transduction. Emerging evidence suggests that following agonist stimulation, some receptors can activate multiple G proteins and regulatory proteins to trigger various signaling pathways.2 In some cases, signaling occurs in the absence of agonist due to constitutive receptor activity.3,4 Extensive cross talk between G protein‐linked and other signaling pathways also is well documented, further complicating GPCR signal transduction.5–8 To preserve specificity and fidelity, these complex receptor‐initiated signals must be tightly regulated at multiple levels. A large number of regulatory proteins have been identified in recent years that modulate GPCR and G protein signaling. Prominent among these are the regulators of G protein signaling (RGS) proteins, a diverse family of multifunctional proteins that regulate GPCR signal transduction at the level of the receptor, the G protein and the effector. This review will focus on our current understanding of RGS protein interactions with receptors and their regulation of receptor signaling.

II. Overview of RGS Proteins A primary function of RGS proteins is to regulate the lifetime of G protein signaling events. Agonist activation of a GPCR triggers the exchange of GDP for GTP on a bound Ga, thereby stimulating the protein to initiate a downstream signaling cascade. The duration of the signaling event is determined by the lifetime of GTP bound to the Ga subunit that, in turn, is dictated by the intrinsic GTPase activity of the Ga. In some cases, Ga GTPase activity may be accelerated when a Ga interacts with its effector protein.9,10 However, in most cases, RGS proteins serve in this capacity as GTPase activating proteins, or GAPs, for active Ga subunits to limit their signaling. In a cellular context, RGS proteins serve to fine‐tune GPCR and G protein signal transduction. RGS proteins are both modulators and integrators of receptor and G protein signaling.11 The RGS family has more than 30 members, all of which share a conserved 120 amino acid RGS domain that defines the family and confers the capacity to bind one or more active Ga–GTP subunits of

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heterotrimeric G proteins.11–13 Early recognition of Ga/RGS interactions provided an appreciation for the important role of RGS proteins in cellular signaling.13 As a consequence of an RGS binding to a Ga subunit, signal duration was limited, which shed light on how RGS proteins are mechanistically involved in GPCR and G protein signaling. Recent studies suggest models whereby GPCRs act as docking platforms for G proteins and functionally related binding partners, including RGS proteins.5,6,14–16 Together, the proteins that make up these complexes share a common goal of targeted signal transduction. Below, we will summarize evidence that details the important role of RGS proteins in these GPCR/G protein complexes.

A. RGS Protein Structure Determines Function Apart from their conserved RGS domain, RGS proteins have diverse tertiary structures and functions that vary widely. The 37 identified proteins that contain RGS domains or RGS‐like domains have been divided into subfamilies according to the shared sequence identities within these domains. Two different nomenclatures have emerged for classifying RGS proteins: a nondescript alphabetical designation (subfamily A–H, etc.) and alternatively, abbreviations signifying a representative family member (e.g., the RZ subfamily, represented by RGSZ and the R4 family represented by RGS4).12,13 Members of the A/RZ and B/R4 subfamilies are the smallest RGS proteins and consist of RGS domains flanked by small but variable N‐ and C‐terminal regions. Because these proteins consist of little more than an RGS domain, their primary function is to bind active Ga–GTP and serve as GAPs, though evidence for other diverse signaling functions of these small RGS proteins has emerged.17 By contrast, members of the C/R7, D/R12, E/RA, F/GEF, G/GRK, and H/SNX subfamilies are large, multidomain proteins that range in size from 60 to 160 kDa and have assorted functions that are not limited to modulating GPCR and G protein signal transduction.18,19 The GAP activity of RGS proteins is contained within the RGS domain. Like some other GAPs, RGS proteins are not responsible for the actual hydrolysis of the GTP molecule but induce a change in the active Ga–GTP complex which creates a much more favorable conformation for the complex to act as its own efficient hydrolase.13 RGS domains also have the capacity to serve as binding sites for Ga and as effector antagonists.9,10 In the case of the RGS domains of the G/GRK subfamily,20 this is their primary role as these proteins block Gq/11a signaling without any apparent GAP activity for Ga. The N‐ and C‐terminal regions flanking the RGS domain also are important, as they provide RGS proteins with the capacity to form protein–protein and membrane interactions. It is through these domains and their interactions that RGS

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proteins vary widely from one another and gain a large degree of their specificity of function. As we will discuss further, these regions serve as binding sites for specific receptors and effectors. The larger, more complex RGS proteins also contain additional domains that bind other signaling proteins as well. For example RGS12 and RGS14, members of the D/R12 subfamily, have multiple domains that provide a capacity to interact with additional binding partners including inactive Gai‐ GDP, Ras/Rap GTPases, Raf kinases and,11,19,21–23 in the case of RGS12, ion channels.24,25 Members of the C/R7, subfamily contain G protein gamma‐like (GGL) domains that bind Gb5 and, the SCG10 neuronal growth associated protein and the DMAP1 transcriptional repressor.26–30 Novel binding partners also have been reported for other RGS family members (reviewed in Refs. 11, 18, 19). In most of these cases, the functional consequences of these protein interactions have yet to be fully elucidated. However, studies suggest that some RGS proteins serve as multifunctional scaffolds either on their own or as part of a higher order complex with receptors and linked G proteins. In doing so, RGS proteins bridge GPCR/G proteins to other signaling pathways and events. We will discuss current evidence demonstrating that RGS proteins form functional pairs with GPCRs to modulate and integrate receptor and G protein signaling.

III. RGS Protein Interactions with GPCRs Compelling evidence from many independent studies now indicates that RGS proteins selectively interact with GPCRs to form functional pairs (Table I). These studies have demonstrated that RGS protein interactions with receptors may be G protein‐dependent, G protein‐independent, or both—though which of these mechanisms applies in individual cases remains to be clearly established. Considerable information is now available regarding how RGS proteins interact with G proteins.13 Early studies using purified proteins in reconstituted systems provided initial evidence that the RGS domain of specific RGS proteins can selectively bind and regulate preferred Ga subunits. For example, members of the F/GEF subfamily exhibit high‐binding selectivity for G12a and G13a, and members of the C/R7 and D/R12 subfamilies selectively bind to members of the Gi/oa family. By contrast, certain members of the B/R4 subfamily (RGS1‐5, 8, 13, 16, and 18) have been shown to nonselectively bind to Ga subunits of the Gq/11a and Gi/oa subfamilies. Among these RGS proteins, RGS2 exhibits a strong apparent selectivity for Gq/11a,31 though this specificity may be receptor and/or cell type‐dependent.32,33 The preference of RGS2 for binding to Gq/11a over other Ga subunits is determined by only a few defined amino acids in the RGS/Ga interface. Likewise, specificity of F/GEF RGS proteins for G12/13 also is

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TABLE I INTERACTIONS BETWEEN GPCRS AND RGS PROTEINS Direct RGS protein/GPCR interactions GPCR

RGS

Receptor binding region

References

a1A‐Adrenergic

RGS2

i3 loop

41

‐Opioid

RGS4

C terminus

46

m‐Opioid

RGS4

C terminus

43, 46

CCK2

RGS2

C terminus

47

CXCR2

RGS12

C terminus

38

M1 mAChR

RGS2, RGS8

i3 loop

40,43

MCH1

RGS8

i3 loop

42

ORL1

RGS19 (GAIP)

unknown

48

RGS protein/GPCR interactions mediated by intermediate proteins GPCR

RGS

Intermediate protein

References

a1B‐Adrenergic

RGS2, RGS4

Spinophilin

53, 58

m‐Opioid

RGS9‐2

Spinophilin, beta‐arrestin‐2

56

D2 dopamine

RGS19 (GAIP)

GIPC

49

M1 mAChR

RGS8

Spinophilin

57

Implied RGS protein/GPCR interactions CPCR

RGS

Reference

‐Opioid

RGS9

55

m‐Opioid

RGS1, RGS2, RGS4, RGS9, RGS10, RGS14, RGSZ1, RGSZ2

55, 59–63

b2‐Adrenergic

RGS2

66

5‐HT1A

RGS4, RGS10, RGSZ1

65

5‐HT2A

RGS2, RGS7

65

AT1A angiotensin II

RGS2, RGS5

64, 66

D2 dopamine

RGS9‐2

65

D3 dopamine

RGS19 (GAIP)

49

Endothelin‐1 (ET‐1)

RGS3, RGS4

64

GnRHR

RGS2, RGS3, RGS4

70

LPA

PDZrhoGEF

68

M2 mAChR

RGS4

66

M3 mAChR

RGS2, RGS3, RGS4

70

S1P1

RGS2, RGS3

64 (Continued)

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TABLE I (Continued) CPCR

RGS

Reference

S1P2

RGS1–3

64

S1P3

RGS1, RGS3, RGS4

64

Substance P

RGS8

44

Thrombin

LARG

68

defined by specific amino acids. Taken together, these findings and others (reviewed in Ref. 17) clearly indicate that some level of signaling specificity is built into the RGS/Ga interaction. Although some RGS proteins selectively interact with only certain Ga subunits, many others do not, and this apparent ‘‘promiscuity’’ raised the question of exactly how RGS/G protein selectivity is determined in a cellular environment. In the absence of cellular and molecular mechanisms with the capacity to dictate RGS/Ga selectivity, chaotic signaling would ensue. The first clue that such mechanisms exist came from studies on RGS regulation of receptor signaling in pancreatic acinar cells.34 Introduction of RGS1, RGS4, and RGS16 into these cells inhibited calcium signaling by Gq/11‐linked muscarinic acetylcholine receptors (mAChRs) with different potencies. However, these same RGS proteins inhibited cholecystokinin (CCK) receptor calcium signaling (also mediated by Gq/11) with a much lower (30–100‐fold) potency or not at all.34 In stark contrast, RGS2 did not exhibit the same selectivity for inhibition of CCK‐calcium signaling but, instead, it blocked signaling by both muscarinic and CCK receptors in this system.34 In summary, while each of these RGS proteins had been shown to bind and inhibit Gq/11 signaling in isolation, their striking selectivity for inhibition of Gq/11 signaling depended upon their linked receptor when they were in a cellular context. In other words, RGS regulation of G protein signaling appeared to be dictated by the receptor, not the G protein. These studies provided the first indication that RGS proteins and receptors form preferred functional pairs to differentially regulate cellular signaling. In doing so, such GPCR/RGS pairs (shown in Table I) impart specificity and order to what otherwise could be chaotic signaling in cells. Based on these findings, a number of studies have focused on understanding cellular and molecular mechanisms underlying RGS interactions with receptors.

A. GPCRs Interact Directly with RGS Proteins GPCRs contain seven‐transmembrane‐spanning domains, an extracellular N‐terminus, three extracellular loops, three intracellular loops, and an intracellular C‐terminus. Many signaling and regulatory proteins, most notably G

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proteins, G protein receptor kinases (GRKs) and arrestins, have been shown to have precise binding sites on particular receptors’ intracellular loops and C‐tails (reviewed in Refs. 35, 36). Recent data indicates that in other cases; interactions between GPCRs and proteins may be indirect and occur through intermediate scaffolding proteins (reviewed in Ref. 6). RGS proteins have been shown to modulate receptors in both manners—directly and indirectly. Considerable evidence now suggests that, at least for some GPCR/RGS functional pairs, specific regions on the GPCR and RGS protein are responsible for dictating the direct binding that occurs between these two proteins, though no consensus domains shared among receptors have been defined so far. Early evidence demonstrated that the N‐terminal portion of some RGS proteins might be responsible for selective receptor binding.37 When RGS4 was truncated, with only its RGS domain intact, its inhibition of receptor and Gq/11‐stimulated calcium signaling was 10,000‐fold less potent compared to that of full‐length RGS4 in pancreatic acinar cells.37 These studies demonstrated a requirement for the N‐terminus of RGS4 in determining RGS4 regulation of GPCR signaling. Independent of this work, the N‐terminus of RGS12 has been shown to directly interact with the C‐tail of the interleukin‐ 8 receptor, CXCR2.38 RGS12 and the CXCR2 GPCR have complimentary PDZ domain and binding motifs, respectively, which facilitate the direct interaction that occurs at the C‐terminus of the receptor. The physiological significance of this pairing remains to be demonstrated in cells since only the isolated receptor C‐tail was shown to interact with the PDZ domain of RGS12. Besides RGS12, the only other RGS protein that contains a PDZ domain is a splice variant of RGS3, which has been shown to interact directly with the PDZ‐ binding motif for the ephrin‐B receptor, a non‐GPCR (discussed below and Table II).39 While PDZ domains are not a general mechanism for RGS/GPCR coupling, these findings did suggest that RGS proteins can directly interact with GPCRs.

TABLE II INTERACTIONS BETWEEN NON‐GPCR RECEPTORS AND RGS PROTEINS Receptors

RGS proteins

References

Ephrin‐B

RGS3

39, 71

IGF‐1

LARG

72

TrKA

RGS19 (GAIP)

73

PDGFb

RGS12

74

BKCa

CRBN

75

Cav2.2

RGS12

24, 25

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Other direct RGS/GPCR interactions occur independent of a PDZ domain. The first study that documented such an interaction showed that RGS2, but not RGS16, binds directly to the third intracellular (i3) loop of the Gq/11‐coupled m1 mAChR.40 RGS2 formed a stable complex with the i3 loop of m1 mAChR and Gqa indicating that RGS2 can serve as a bridge to bind both receptor and G protein, simultaneously. The N‐terminus of the RGS protein was reported to be responsible for binding to the receptor while the RGS domain of the protein bound to active, but not inactive Gqa. Furthermore, phosphatidyl inositol 4,5‐ bisphosphate (PIP2) hydrolysis triggered by stimulation of the m1 mAChR was significantly decreased in the presence of purified RGS2, and this inhibition was dependent on the N‐terminus of RGS2. By contrast, RGS2 did not bind to the i3 loops of either the Gi/o‐linked m2 or m4 mAChRs. These findings supported the notion that this RGS2/m1 mAChR interaction is direct, selective, and receptor‐ dependent, and that the N‐terminus of RGS2 and the i3 loop of the receptor define the complex interface.40 In follow‐up studies,41 RGS2 also exhibited selectivity for certain adrenergic receptors (ARs). Specifically, RGS2 was shown to bind directly to the i3 loop of the a1A‐AR but not the a1B‐AR. This interaction was demonstrated using purified protein pull downs of the i3 loop of the receptor and the full‐length RGS protein and was supported in cells by receptor‐mediated recruitment of GFP‐tagged RGS2 from the cytosol/nucleus to the plasma membrane by the a1A‐AR but not the a1B‐AR. Three discrete amino acids within the i3 loop of a1A‐AR were identified that were shown to be necessary for RGS recruitment to the receptor. When substituted with the corresponding amino acids from the i3 loop of the a1B‐ARs, a1A‐AR no longer bound RGS2, and RGS2 no longer modulated mutant receptor signaling in cells.41 Consistent with these reports are others showing that RGS proteins bind to receptor i3 loops. For example, RGS8 binds to the melanin concentrating hormone receptor 1 (MCH1R)42 and to the m1 mAChR.43 In the latter case, earlier studies had shown that RGS8 selectively modulated m1 mAChR signaling.44 In follow‐up work, to examine mechanism, the authors found that RGS8 bound directly to the i3 loop of the m1 mAChR, that this interaction was mediated by a specific sequence (MPRR) in the N‐terminus of RGS8, and that this binding was responsible for RGS8 modulation of receptor signaling (Table I).43,45 This same group also examined RGS8 interactions with the MCH1R.42 Both RGS8 and the MCH1R are highly expressed in the brain, thus indicating that they may physiologically interact in a normal cellular environment. RGS8 was shown to directly associate with the i3 loop of the MCH1R in vitro, similar to what has been observed for RGS8 and RGS2 modulation of m1 mAChR and of RGS2 modulation of a1A‐AR, as discussed above. Co-localization of these proteins at the plasma membrane in HEK‐293T and the attenuation of receptor‐mediated calcium mobilization in the presence of RGS8 also were reported.42 Together, these studies demonstrated direct

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interactions between GPCRs and RGS proteins, and defined important roles for the N‐terminus of the RGS protein and for the i3 loop of the receptor as contact sites and determinants for selective GPCR/RGS protein interactions. Recent studies have shown that RGS2 also interacts directly with certain GPCRs at regions other than the receptor i3 loop, most notably the C‐tail. RGS4 was reported to bind directly to the C‐termini of both the m‐ and ‐opioid receptors in a complex with Gia (Table I),46 the first demonstration of an RGS protein directly interacting with the C‐tails of receptors. In this study, RGS4 was reported to block m‐opioid receptor (MOR)‐mediated inhibition of forskolin‐stimulated adenylyl cyclase. However, these effects of RGS4 were not observed upon activation of the ‐opioid receptor, suggesting that they are receptor‐dependent.46 Independent of these findings, RGS2 recently was shown to bind to the C‐terminus of the cholecystokinin receptor‐2 (CCK2R).47 When activated with agonist, the CCK2R binds to discrete residues that lie within the N‐terminus of RGS2. The residues on the CCK2R responsible for this interaction are located on its C‐tail. An increased affinity for the binding of RGS2 was observed when two specific CCK2R amino acid residues, S434 and T439, were phosphorylated. The functional role of RGS2 in CCK2R signaling was demonstrated by its involvement in reducing CCK2R‐ mediated inositol phosphate production. In contrast, CCK2R‐mediated signaling was reported to be insensitive to RGS8, also a member of the B/R4 family.47 Still other studies have further confirmed an important role for the N‐terminus of RGS protein in GPCR/RGS complex formation, without defining the involved receptor region. The opioid‐receptor‐like (ORL1) receptor was shown to preferentially bind RGS19 (GAIP), while the m‐, ‐, and k‐opioid receptors exhibit a greatly decreased affinity for this RGS protein.48 Results from this study showed that an N‐terminally truncated form of RGS19 (GAIP) did not bind to the receptor, offering yet another example where the N‐terminal region of the protein is necessary for this interaction.48 RGS4 also was shown to exhibit a range of affinities for these opioid receptors, binding with highest affinity for the MOR and with the least affinity for the ORL1 receptor.48 In summary, this collective body of work with various GPCRs (m1 mAChR, a1‐AR, CCK2, MCHR‐1, MOR, ‐OR, ORL‐1) and various RGS proteins (RGS2, RGS8, RGS4, RGS19) provide compelling evidence that certain RGS proteins directly and selectively interact with certain receptors to form preferred functional pairs.

B. Indirect GPCR/RGS Protein Interactions In some cases, RGS proteins also can functionally interact with specific GPCR’s indirectly with the assistance of an intermediate scaffolding protein. The first such report showed that, following agonist activation, the D2

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dopamine receptor (D2R) recruits RGS19 (GAIP) to the plasma membrane.49 The authors demonstrated that this recruitment required a scaffold protein, GIPC (GAIP‐interacting protein, C‐terminus). GIPC also was shown to be necessary for RGS19 to modulate D2R‐mediated inhibition of forskolin‐ stimulated cAMP accumulation. Although no other examples of GIPC bridging GPCRs to RGS proteins have been reported to date, these results provide the first evidence that some RGS proteins require scaffolding proteins in order for them to bind certain receptors and to function effectively.49 Recently, considerable attention has focused on the role of different scaffolding proteins in mediating GPCR/RGS interactions. Spinophilin, a large (
90 kDa) multifunctional scaffolding protein, has been shown to facilitate indirect RGS protein interactions with GPCRs. Previous work had established that this protein binds the i3 loops of a number of receptors including the D2R and the a2‐ARs.50–52 A subsequent study reported that spinophilin is involved in GPCR/ RGS functional coupling.53 In this study, spinophilin was shown to directly interact with the N‐terminus of RGS2 and to bind RGS1, RGS4, RGS16, and RGS19/GAIP as well. This interaction also was shown to have functional consequences since RGS2 modulation of AR signaling was enhanced in the presence of spinophilin. When coexpressed, RGS2 and spinophilin block the receptor‐ activated calcium‐activated chloride current in Xenopus laevis oocytes. These data indicate that RGS2, spinophilin, and a1B‐ARs form stable ternary complexes that allow them to signal optimally in cells.53 Separate studies suggest that it’s through spinophilin that certain opioid receptors functionally interact with RGS proteins. Initial reports showed that the striatum‐specific splice variant of RGS9, RGS9‐2, blocks signaling by the MOR (as does the retina‐specific RGS9‐1).54,55 The functional effects of RGS9‐2 on the MOR are to delay the receptor’s agonist‐induced internalization. Importantly, this study also showed that the MOR/RGS9‐2 complex can be coimmunoprecipitated out of PC12 cell lysates suggesting that these proteins form a stable complex. This interaction recently was shown to be part of a larger, result multiprotein complex that includes the MOR, spinophilin, the G protein receptor kinase‐2 (GRK2), RGS9‐2, and the Gai subunit.56 Different GPCR/RGS interactions are modulated by spinophilin as well. As discussed above, RGS8 binds the i3 loop of the m1 mAChR.43 A more recent follow‐up report indicates an unexpectedly complicated interaction between RGS/spinophilin and the receptor. Spinophilin binds to RGS8 at the same N‐terminal residues of RGS8 (MPRR) that binds the m1 mAChR i3 loop. Interestingly, in the presence of spinophilin, RGS8 binding to the m1 mAChR is decreased but, its inhibition of receptor signaling is enhanced.57 Indirect evidence also supports a role for spinophilin in mediating RGS protein regulation of AR signaling. In this case modulation of NMDA receptors in neuronal cortex derived from mice lacking the spinophilin gene and protein

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was altered.58 In prefrontal cortical neurons, activating the a1‐AR (linked to Gq/11) and the a2‐AR (linked to Gi/o) results in a net decrease in the NMDA receptor excitatory postsynaptic current amplitude and whole‐cell NMDA receptor current amplitude. The effects of a1‐AR on NMDA receptors were shown to be dependent on inositol phosphate and calcium, whereas the effects of a2‐AR on NMDA receptors relied on protein kinase A and downstream ERK signaling. RGS2 and RGS4 each were tested for their capacity to negatively regulate the effect of these receptors on NMDA receptor signaling. Both RGS2 and RGS4 inhibited a1‐AR‐regulated NMDA receptor currents, but only RGS4 had the capacity to block a2‐AR regulation of NMDA receptor currents. Of note, in brain slices from spinophilin knockout mice, a1‐AR regulation of the NMDA receptors was not observed but the effect of RGS4 on a2‐AR signaling was unaffected. These data suggest that the effects of the two ARs on NMDA receptors are differentially regulated by RGS proteins58 and that spinophilin mediates RGS2 actions. In summary, considerable evidence now indicates that certain RGS proteins can form stable functional complexes with preferred GPCRs. These interactions can occur either through direct contact between N‐terminus of the RGS protein and the receptor i3 loop and/or C‐tail, or indirectly, though the assistance of an intermediate scaffolding protein such as GIPC or spinophilin.

C. Implied RGS Protein and GPCR Interactions A number of findings demonstrate the existence of stable functional complexes between specific RGS proteins and GPCRs (outlined above and Table I). Several other studies also show functional pairings suggestive of similar direct or indirect interactions but without evidence of a defined underlying mechanism. Studies with opioid receptors showed that m‐ and ‐opioid receptors, which link to the Gi/o family of G proteins, are differentially regulated by RGS proteins.55 RGS proteins from different subfamilies (RGSZ of the A/RZ family, RGS1, 2, and 4 of the B/R4 family, RGS9 of the C/R7 family, and RGS10 of the D/R12 family) all were tested for their capacity to block signaling by either the m‐ or ‐ opioid receptor. Every RGS protein investigated inhibited signaling by the MOR but only RGS9 blocked signaling by the ‐opioid receptor. Whether these interactions between the RGS proteins and opioid receptors were direct or indirect was not investigated or reported.55 A different study examining the effects of knockdown of mRNA (and presumably protein) for RGSZ1, suggested that this RGS protein assisted in the development of MOR tolerance to morphine, suggesting that RGSZ1 selectively regulates signaling by the MOR.59 The RGSZ2 protein has also been reported to exist in a complex with these receptors and may assist in their desensitization.60–62 This is in contrast to RGS14 which, in a separate study, was reported to block MOR internalization.63

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RGS8 also interacts with some receptors in a manner that is not yet fully understood. One study showed that RGS8 blocks the calcium‐activated chloride current activated by the M1 mAChR and the Substance P receptor (both of which couple to Gq/11a) in Xenopus oocytes.44 However, RGS8 does not block signaling by the m3 mAChR in this system44 demonstrating RGS protein selectivity between the closely related Gq/11‐linked M1 mAChR and M3 mAChR receptors. While subsequent studies (outlined above) showed that RGS8 directly binds spinophilin and the i3 loop of M1 mAChR, RGS8 interactions with Substance P receptors were not reported. Therefore, the underlying mechanisms of RGS regulation of this receptor’s signaling remain undefined. Other reports demonstrate that certain members of the smaller B/R4 family (RGS2, RGS3, RGS4, RGS5) selectively regulate receptor signaling by an unknown mechanism. One study showed that RGS3 has a selective role in regulating S1P1 receptor signaling in the cardiovascular system.64 In this report, RGS1, RGS2, and RGS3 all were implicated in regulation of S1P2 signaling, but differences were noted in RGS protein regulation of S1P3, endothelin‐1 (ET‐1), and angiotensin II type 1 (AT1) receptor signaling. RGS1, RGS3, and RGS4 inhibited the S1P3 receptor while only RGS2 was shown to regulate the AT1 receptor. RGS3 and (to a lesser extent) RGS4 were reported to be involved in ET‐1 receptor signaling.64 This one study established that several GPCRs are differentially regulated by RGS proteins but whether these interactions are direct or indirect has not been elucidated. Still other findings demonstrate selective coupling between B/R4 family members and other GPCRs (Table I). For example, RGS4, RGS10, and RGSZ1 modulate signaling by the 5‐HT1A receptor and two different RGS proteins, RGS2 and RGS7, selectively affect 5‐HT2A receptor signaling.65 The specificity of these interactions was confirmed when the RGS proteins tested did not alter signaling by D2 dopamine receptors.65 A separate report demonstrated selective functional coupling between RGS2 and b2‐adrenergic and AT1A receptors, and between RGS4 and m2 mAChR receptors. In this case, RGS proteins were recruited to the plasma membrane by particular receptors visualized by confocal microscopy and biochemical techniques.66 Independent of these studies, knockdown of RGS3 and RGS5 mRNA showed that these RGS protein selectively regulated the signaling functions of the M3 mAChR and the AT1 receptor, respectively.67 Although the majority of studies outlined above focused on interactions between GPCRs and small simple RGS proteins (A/RZ and B/R4 family members), implied interactions between receptors and the larger, more complex RGS proteins also have been reported (Table I). One study68 investigated whether the G12/13‐linked thrombin and lysophosphatidic acid (LPA) receptors are regulated by various rhoGEFs that contain RGS domains including

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RGS‐LARG (leukemia‐associated rhoGEF), p115rhoGEF, and PDZrhoGEF. The authors found that PDZrhoGEF selectively regulates LPA signaling whereas LARG selectively regulates thrombin signaling, thereby establishing that LARG and PDZrhoGEF differentially mediate downstream G12/13‐activated rho signaling by two GPCRs.68 Taken together, these various reports establish that signaling by various GPCRs is differentially regulated by RGS proteins, suggesting a functional pairing, but shed no light on whether this regulation is mediated by direct or indirect RGSprotein/GPCR interactions. While considerable evidence has emerged to indicate that RGS proteins and certain GPCRs can form stable complexes and preferred functional pairs (outlined above), there also is some evidence to the contrary. One study demonstrated that RGS2 and RGS4 fail to exhibit selectivity for inhibition of signaling by various Gq/11‐linked muscarinic (m1, m3, and m5) receptors when controlled for protein expression.69 A separate study also suggests that members of the B/R4 subfamily of RGS proteins do not selectively inhibit signaling by different Gq/11‐linked GPCRs.70 This study showed that RGS2, RGS3, and RGS4 do not discriminate between binding to m3 receptor and the gonadotropin‐releasing hormone receptor (GnRHR).70 These examples of failures of RGS proteins to discriminate among receptors may be a result of the specific RGS protein/GPCR pairs examined or of the specific cellular systems used in these studies. It also is reasonable to propose that, in some cases, RGS proteins do not selectively interact with certain GPCRs, but inhibit receptor signaling by recognizing the linked G protein (shared in many cases among receptors), or the receptor/G protein complex, rather than the receptor alone.

D. RGS Proteins also Interact with Non‐GPCR Receptors and Ion Channels Some RGS proteins also have been shown to selectively interact with cell surface receptors that are not GPCRs (Table II), suggesting that RGS proteins have unexpected and multifunctional roles in larger signaling complexes. RGS3, one of only two RGS protein family members that contains a PDZ domain (the other being RGS12, as discussed above), has been reported to interact with the ephrin‐B receptor, a tyrosine kinase receptor important for neuronal development.39 The PDZ domain at the N‐terminus of RGS3 was shown to interact with the PDZ‐binding motif on the C‐tail of the ephrin‐B receptor (Table II). RGS3 also was shown to mediate signaling by the ephrin‐B receptor. When expressed at appropriate levels, ephrin‐B promotes cell deadhesion in Xenopus embryos.71 However, when ephrin‐B is expressed at suboptimal levels, the effect is lost but can be rescued by the addition of RGS3 into the cells suggesting that RGS3 helps promote—and is perhaps required for—this receptor’s signaling.39

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Whether these effects of RGS3 on the ephrin‐B receptor involved a G protein and/or a GPCR was not apparent. However, these data indicate that RGS3 may possess novel functions unrelated to GPCR signaling. Other examples of non‐GPCR receptor/RGS protein couplings also have been reported. Early studies showed that the insulin‐like growth factor receptor‐1 (IGF‐1), a tyrosine kinase growth factor receptor, is modulated by LARG, a member of the F/RL subfamily of RGS proteins.72 Also, similar to D2R interactions with RGS19/GAIP discussed above,49 the TrkA nerve growth factor receptor also interacts with RGS19 (GAIP) through GIPC, as part of a larger signaling complex that links the TrkA receptor to Gi/oa signaling.73 In airway smooth muscle cells, PDGFb receptors and RGS12 have been shown to colocalize in cytoplasmic vesicles.74 Functionally, these proteins also were shown to interact since RGS12 overexpression decreased PDGF‐stimulated p42/44 MAPK activation.74 Together, these studies demonstrate that RGS proteins can participate as part of a large multiprotein signaling complex that integrates G protein signaling with non‐GPCR signaling. RGS proteins have been shown to interact directly with certain ion channels as well. The newly described protein Cereblon (CRBN), an RGS domain‐containing protein, has been shown to interact with the C‐terminus of a large‐conductance calcium‐activated potassium channel, BKCa.75 These proteins were shown to interact by coimmunoprecipitation experiments in brain lysates and to colocalize in rat hippocampal neurons. CRBN prevented receptor assembly and surface expression and consequently reduced the ion current.75 In separate studies, RGS12 was reported to form a stable complex with N‐type calcium channels and to modulate voltage‐independent inhibition of currents by these channels.24,25 Others studies showed that smaller RGS proteins of the B/R4 family enhance the activation/deactivation kinetics of G protein‐gated inward rectifier potassium (GIRK) channels, perhaps by separate mechanisms which may involve a large signaling complex.76,77

E. Factors that Dictate RGS Protein Localization at the Plasma Membrane The findings reviewed here support a model whereby RGS proteins and their cell surface receptor partners each contain specific targeting domains that allow them to form preferred stable pairings. Of course, these specific interactions also are dictated by localization of RGS proteins and their binding partners within shared host cells and proximity to each other within those cells. Except for a few cases, RGS proteins are not integral membrane proteins and must rely on cellular mechanisms to translocate and to attach at the plasma membrane. Not surprisingly, G proteins likely contribute to this process. However, much of the evidence outlined above suggests that receptors are able to recruit RGS

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protein partners independent of their linked G protein. But, the linked G protein is the functional target of the RGS protein; as such, the optimal substrate recognized by the RGS protein in cells may be the GPCR/G protein complex. The specificity of a G protein for a particular RGS protein also may assist in recruiting the RGS protein to the plasma membrane where it is in close proximity with its preferred GPCR.78–81 Furthermore, the N‐ and/or C‐termini of some RGS proteins also contain biochemical factors and/or modifications that serve to target and attach RGS proteins to the cell cortex and to microdomains within the plasma membrane.22,82–85 For example, RGS4 contains a charged amphipathic helix and is palmitoylated to dictate plasma membrane attachment and localization, independent of either the linked G protein or receptor.86,87 Likewise, RGS2 contains charged N‐terminal residues that are essential for membrane targeting.81 Among larger RGS proteins, RGS9 contains a DEP (for Disheveled, EGL‐10, Pleckstrin) homology domain responsible for targeting this and other proteins to specific locations on subcellular membranes.83 RGS12 and RGS14 each contain GoLoco/GPR motifs that specifically bind inactive Gia subunits to dictate membrane localization.22,84 Thus, specific RGS protein recognition of partner receptors at the plasma membrane is determined by multiple factors on the RGS protein, the receptor, and the linked G protein.

IV. GPCRs Serve as Platforms for Molecular Signaling As outlined above, RGS proteins and GPCRs form preferred functional pairs, either through direct or indirect interactions. This information changes the way that we think about GPCR signaling and impacts working models of how these receptors and their linked G proteins and downstream signaling pathways are regulated. Recently solved crystal structures of GPCRs indicate that these seven‐transmembrane‐spanning proteins have the surface area and capacity to form multiprotein complexes. In this way, GPCRs serve as a nucleation center for various proteins to come together and perform a shared, though receptor‐specific signaling task.36,88 RGS proteins are newly appreciated contributors to these complexes that interact with receptor i3 loops and/or C‐tails, in a manner similar to GRKs and arrestins. In emerging models of GPCR signaling, regulatory proteins are in close proximity with receptors, perhaps even preassociated with inactive receptor. Established models of G protein signaling89–92 propose that, upon agonist stimulation, receptors and G proteins undergo a conformational change that dissociate G protein subunits to reveal binding sites on the receptor and G proteins to which regulatory and signaling proteins could attach. However, recent evidence suggests that, at least in some cases, GPCRs and G protein subunits remain complexed following agonist stimulation, and merely

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rearrange in situ to present newly revealed binding interfaces for effectors and transduce signals to nearby signaling partners.89–92 In this model, signaling and regulatory proteins must be prepositioned within close proximity of the GPCR prior to receptor stimulation and move to the GPCR/G protein complex for signal transduction to occur. Several studies focused on rhodopsin and AR agonism and consequential structural changes suggest that ligand‐induced movements of the third, fifth, and/or sixth transmembrane domains (TMIII, TMV, TMVI) may be responsible for such conformational rearrangements.93–98 Due to our growing awareness of the scaffolding roles of GPCRs, the involved protein complexes have become the focus of intensive investigation in recent years. As outlined, RGS proteins are selectively recruited to GPCRs to fine‐tune G protein signaling. Many proteins also are recruited to interact with GPCRs at the plasma membrane as well. Besides heterotrimeric G proteins, proteins involved in desensitization of receptor signaling—arrestins and GRKs in particular—also interact with receptors as part of the multiprotein signaling complex. While these proteins are best known for their involvement in the termination of signaling, compelling evidence now shows that arrestins and GRKs also bind other signaling molecules when linked to receptors.99–105 For example, considerable evidence shows that arrestins can recruit various components of the MAP Kinase signaling pathways to initiate ERK signaling outside of the nucleus.106 More recently, GPCRs were linked to PIP2 production via an arrestin‐mediated interaction with 4‐phosphate 5‐kinase (PIP5K) which converts PIP a PIP2.107 Other scaffolding proteins that interact with GPCRs include spinophilin and GIPC, which link various other signaling molecules (e.g., certain RGS proteins) to GPCRs and G proteins as well. Thus, GPCRs serve as platforms for multiple scaffolding proteins that engage a variety of signaling proteins and pathways that, in combination, initiate a unique profile of shared, overlapping, and distinct signaling outputs specific to that receptor. This working model of inactive GPCRs as plasma membrane signaling centers is supported by reported structures of multiprotein complexes of known GPCR regulatory proteins. The structure of RGS4 complexed with Gia1108 revealed that the RGS domain is composed of two subdomains that include nine a‐helices. Via one of these domains (helices 4–7), RGS4 was shown to interact with the switch domain of Gia1. A more recent study109 suggests that the binding groove for Gia1 actually is in a more open conformation when it is not bound to RGS4. This finding indicates that the G protein may undergo a conformational change upon RGS binding—from being more to less accessible. The crystal structures of RGS domains from several other RGS proteins (RGS9, axin, GRK2, p115RhoGEF, RGS16, and PDZRhoGEF) have been solved16,72,108–113 and will help to shed new light on the mechanisms of RGS/G protein and receptor interactions.

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The structures of other protein components of the G protein ternary complexes have been solved recently14,16 and provide evidence in support of multiprotein interactions. These reports describe the structures of Gqa‐ containing complexes that included effector molecules. The first study14 reported Gqa in a complex with p63RhoGEF and RhoA, and showed that the Gqa effector binding site and its C‐terminal region interacts with p63RhoGEF on its Dbl and pleckstrin homology domains. RhoGEF, in turn, binds to RhoA on a distinct interface. This finding provides the first structural evidence of an indirect linkage existing between GPCRs and RhoA.14 This same group also subsequently reported the structure of Gqa and p63RhoGEF in complex with GRK2,16 and confirmed its functionality with flow‐cytometry protein interaction and GAP assays. In this article, the investigators showed that two effector proteins, p63RhoGEF and GRK2, interact with Gqa at precise and distinct binding sites.16 Importantly, these sites are different from the RGS binding site on this G protein—RGS2 and RGS4 were crystallized separately, as part of the larger complex.16 The results of this study indicate that RGS proteins and effector proteins can bind to G proteins simultaneously and are positioned to interact with the receptor. Independently, the crystal structure of the GRK2/Gqa/Gbg complex also was reported.114 Interestingly, GRK2, an RGS‐like protein that is recruited to the plasma membrane by Gbg, also is known to act as an effector antagonist and inhibit the function of Gqa.20 Based on the data from these studies, it was proposed by the authors that the arrangement and orientation of this multiprotein complex leaves open a binding pocket for an RGS protein to bind to a receptor and Ga subunit simultaneously15 as is observed in biochemical studies of RGS2 interactions receptor i3 loops and Gqa.40 Further indirect evidence that supports this model comes from computational studies that examine reduced‐order modeling of GPCR/G protein/RGS protein interactions and the kinetics of GPCR signaling. Using such methods, the predicted structures of ternary complexes involving RGS4 binding to both the m1 mAChR and Gqa are consistent with the idea that GPCRs and GAPs (like RGS proteins) must interact in order to accurately describe the observed kinetics of signaling events in cells.115 These data also are consistent with biochemical studies showing that RGS2 can simultaneously bind to active Gqa and to the i3 loop of the Gq‐linked m1 mAChR.40 Perhaps the most compelling evidence that GPCRs and RGS proteins form preferred functional pairs comes from experiments performed in Arabidopsis,116,117 a simple plant. In this organism, the DNA coding sequence of an RGS domain is encoded in frame within the coding sequence of novel GPCR‐like plant receptor. Amazingly, the RGS domain resides within the C‐tail of the receptors116,117 and would be prepositioned to modulate G protein signaling upon receptor activation, demonstrating quite convincingly that GPCRs and RGS proteins are

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functionally coupled. It is reasonable then to postulate that higher organisms evolved to express each protein on separate and multiples genes to allow for greater fidelity in regulation of complex signaling, as is outlined above. Together, these studies support a model whereby GPCRs serve as platforms to nucleate signaling complexes, and RGS proteins are newly appreciated and important components of these complexes. Evidence indicates that GPCRs form preferred functional pairs with RGS proteins that, in turn, determine RGS specificity for Ga. Such a model is consistent with previous findings showing that RGS proteins interact nonselectively with Ga as purified proteins in isolation, but exhibit strict selectivity for GPCR/Ga pairs in cells, irrespective of the linked G protein.34,66,67,118 In this model, RGS/Ga coupling is dictated by the receptor that recruits a preferred RGS protein to regulate the linked Ga. The RGS protein does not need to be selective for the Ga since the GAP activity is only restricted to the nearby G protein coupled to the receptor. Beyond that, GPCR nucleation of functionally related and cooperating proteins (e.g., G protein, RGS, spinophilin, GRK, effector, arrestin) that are not otherwise in close proximity with one another is an essential function of these receptor platforms. Clearly, such an arrangement would allow a GPCR to transmit a unique message reflecting the particular constellation of signaling proteins it recruits (and the proteins they recruit). It is likely that RGS proteins and other regulatory proteins that GPCRs interact with are receptor‐ and cell type‐specific, depending on the profile of proteins expressed in those cells.

V. Summary and Perspectives This review has summarized the many reports of RGS protein involvement in signaling by GPCRs and other cell surface receptors. From these reports, investigators are gaining an appreciation for the essential role that RGS proteins play in receptor signaling beyond their recognized role as simple inhibitors of G protein signaling. New models that portray GPCRs as multifunctional platforms that mediate diverse and overlapping signaling pathways are supported by a large and growing body of research. RGS proteins are now accepted as significant components of this GPCR signaling complex. The full range of roles of RGS proteins in these GPCR signaling complexes are not yet fully elucidated, but remain an important topic for discovery going forward. The functional coupling and pairing of RGS proteins and GPCRs may have broad implications for future therapeutic interventions. GPCRs regulate nearly all aspects of cell and organ physiology, and exhibit discrete tissue distribution patterns making them ideal as front‐line therapeutic targets. Like GPCRs, RGS proteins also exhibit discrete cellular and tissue distribution patterns and have been shown to play important roles in receptor functions critical for

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the cardiovascular,64,119–124 immune,123–133 and central/peripheral nervous systems.59,134–139 In the CNS, RGS proteins play key roles in relating to receptors involved with drug abuse, addiction, and drug tolerance.134,135,138,139 Small molecule inhibitors of RGS protein/GPCR interactions and RGS regulation of GPCRs could help to reduce ‘‘dirty’’ drug cross reactivity and extend the specificity of existing drugs that act on GPCRs, or perhaps offer new therapies to boost GPCR function where it is diminished.135 Thus the identification of new RGS protein/GPCR pairs and an understanding of the underlying mechanisms of how they interact are important goals for future research.

Acknowledgments K.L.M. was supported by an American Heart Association predoctoral fellowship (AHA0715465B). J.R.H. was supported by grants from the National Institutes of Health (R01NS037112 and R01NS049195).

References 1. Hepler JR, Gilman AG. G proteins. Trends Biochem Sci 1992;17:383–7. 2. Hermans E. Biochemical and pharmacological control of the multiplicity of coupling at G‐protein‐coupled receptors. Pharmacol Ther 2003;99:25–44. 3. Milligan G. Constitutive activity and inverse agonists of G protein‐coupled receptors: a current perspective. Mol Pharmacol 2003;64:1271–6. 4. Smit MJ, Vischer HF, Bakker RA, Jongejan A, Timmerman H, Pardo L, et al. Pharmacogenomic and structural analysis of constitutive G protein‐coupled receptor activity. Annu Rev Pharmacol Toxicol 2007;47:53–87. 5. Brady AE, Limbird LE. G protein‐coupled receptor interacting proteins: emerging roles in localization and signal transduction. Cell Signal 2002;14:297–309. 6. Hall RA, Lefkowitz RJ. Regulation of G protein‐coupled receptor signaling by scaffold proteins. Circ Res 2002;91:672–80. 7. Hubbard KB, Hepler JR. Cell signalling diversity of the Gqalpha family of heterotrimeric G proteins. Cell Signal 2006;18:135–50. 8. Riobo NA, Manning DR. Receptors coupled to heterotrimeric G proteins of the G12 family. Trends Pharmacol Sci 2005;26:146–54. 9. Hepler JR, Berman DM, Gilman AG, Kozasa T. RGS4 and GAIP are GTPase‐activating proteins for Gq alpha and block activation of phospholipase C beta by gamma‐thio‐GTP‐Gq alpha. Proc Natl Acad Sci USA 1997;94:428–32. 10. Yan Y, Chi PP, Bourne HR. RGS4 inhibits Gq‐mediated activation of mitogen‐activated protein kinase and phosphoinositide synthesis. J Biol Chem 1997;272:11924–7. 11. Hollinger S, Hepler JR. Cellular regulation of RGS proteins: modulators and integrators of G protein signaling. Pharmacol Rev 2002;54:527–59. 12. De VL, Zheng B, Fischer T, Elenko E, Farquhar MG. The regulator of G protein signaling family. Annu Rev Pharmacol Toxicol 2000;40:235–71.

68

McCOY AND HEPLER

13. Ross EM, Wilkie TM. GTPase‐activating proteins for heterotrimeric G proteins: regulators of G protein signaling (RGS) and RGS‐like proteins. Annu Rev Biochem 2000;69:795–827. 14. Lutz S, Shankaranarayanan A, Coco C, Ridilla M, Nance MR, Vettel C, et al. Structure of Galphaq‐p63rhoGEF‐rhoA complex reveals a pathway for the activation of rhoA by GPCRs. Science 2007;318:1923–7. 15. Neitzel KL, Hepler JR. Cellular mechanisms that determine selective RGS protein regulation of G protein‐coupled receptor signaling. Semin Cell Dev Biol 2006;17:383–9. 16. Shankaranarayanan A, Thal DM, Tesmer VM, Roman DL, Neubig RR, Kozasa T, et al. Assembly of high order Galphaq‐effector complexes with RGS proteins. J Biol Chem 2008;283:34923–34. 17. Tinker A. The selective interactions and functions of regulators of G‐protein signalling. Semin Cell Dev Biol 2006;17:377–82. 18. Willars GB. Mammalian RGS proteins: multifunctional regulators of cellular signalling. Semin Cell Dev Biol 2006;17:363–76. 19. Siderovski DP, Willard FS. The GAPs, GEFs, and GDIs of heterotrimeric G‐protein alpha subunits. Int J Biol Sci 2005;1:51–66. 20. Carman CV, Parent JL, Day PW, Pronin AN, Sternweis PM, Wedegaertner PB, et al. Selective regulation of Galpha(q/11) by an RGS domain in the G protein‐coupled receptor kinase, GRK2. J Biol Chem 1999;274:34483–92. 21. Mittal V, Linder ME. Biochemical characterization of RGS14: RGS14 activity towards G‐protein alpha subunits is independent of its binding to Rap2A. Biochem J 2006;394:309–15. 22. Shu FJ, Ramineni S, Amyot W, Hepler JR. Selective interactions between Gi alpha1 and Gi alpha3 and the GoLoco/GPR domain of RGS14 influence its dynamic subcellular localization. Cell Signal 2007;19:163–76. 23. Traver S, Splingard A, Gaudriault G, De GJ. The RGS (regulator of G‐protein signalling) and GoLoco domains of RGS14 co‐operate to regulate Gi‐mediated signalling. Biochem J 2004;379:627–32. 24. Richman RW, Diverse‐Pierluissi MA. Mapping of RGS12‐Cav2.2 channel interaction. Methods Enzymol 2004;390:224–39. 25. Schiff ML, Siderovski DP, Jordan JD, Brothers G, Snow B, De V.L, et al. Tyrosine‐kinase‐ dependent recruitment of RGS12 to the N‐type calcium channel. Nature 2000;408:723–7. 26. Liu Z, Fisher RA. RGS6 interacts with DMAP1 and DNMT1 and inhibits DMAP1 transcriptional repressor activity. J Biol Chem 2004;279:14120–8. 27. Liu Z, Chatterjee TK, Fisher RA. RGS6 interacts with SCG10 and promotes neuronal differentiation. Role of the G gamma subunit‐like (GGL) domain of RGS6. J Biol Chem 2002;277:37832–9. 28. Zhang JH, Simonds WF. Copurification of brain G‐protein beta5 with RGS6 and RGS7. J Neurosci 2000;20:RC59. 29. Cabrera JL, de FF, Satpaev DK, Slepak VZ. Identification of the Gbeta5‐RGS7 protein complex in the retina. Biochem Biophys Res Commun 1998;249:898–902. 30. Snow BE, Krumins AM, Brothers GM, Lee SF, Wall MA, Chung S, et al. A G protein gamma subunit‐like domain shared between RGS11 and other RGS proteins specifies binding to Gbeta5 subunits. Proc Natl Acad Sci USA 1998;95:13307–12. 31. Heximer SP, Watson N, Linder ME, Blumer KJ, Hepler JR. RGS2/G0S8 is a selective inhibitor of Gqalpha function. Proc Natl Acad Sci USA 1997;94:14389–93. 32. Heximer SP, Srinivasa SP, Bernstein LS, Bernard JL, Linder ME, Hepler JR, et al. G protein selectivity is a determinant of RGS2 function. J Biol Chem 1999;274:34253–9. 33. Ingi T, Krumins AM, Chidiac P, Brothers GM, Chung S, Snow BE, et al. Dynamic regulation of RGS2 suggests a novel mechanism in G‐protein signaling and neuronal plasticity. J Neurosci 1998;18:7178–88.

RGS PROTEINS AS COMPONENTS OF GPCR SIGNALING COMPLEXES

69

34. Xu X, Zeng W, Popov S, Berman DM, Davignon I, Yu K, et al. RGS proteins determine signaling specificity of Gq‐coupled receptors. J Biol Chem 1999;274:3549–56. 35. Ferguson SS. Phosphorylation‐independent attenuation of GPCR signalling. Trends Pharmacol Sci 2007;28:173–9. 36. Moore CA, Milano SK, Benovic JL. Regulation of receptor trafficking by GRKs and arrestins. Annu Rev Physiol 2007;69:451–82. 37. Zeng W, Xu X, Popov S, Mukhopadhyay S, Chidiac P, Swistok J, et al. The N‐terminal domain of RGS4 confers receptor‐selective inhibition of G protein signaling. J Biol Chem 1998;273:34687–90. 38. Snow BE, Hall RA, Krumins AM, Brothers GM, Bouchard D, Brothers CA, et al. GTPase activating specificity of RGS12 and binding specificity of an alternatively spliced PDZ (PSD‐95/Dlg/ZO‐1) domain. J Biol Chem 1998;273:17749–55. 39. Lu Q, Sun EE, Klein RS, Flanagan JG. Ephrin‐B reverse signaling is mediated by a novel PDZ‐RGS protein and selectively inhibits G protein‐coupled chemoattraction. Cell 2001;105: 69–79. 40. Bernstein LS, Ramineni S, Hague C, Cladman W, Chidiac P, Levey AI, et al. RGS2 binds directly and selectively to the M1 muscarinic acetylcholine receptor third intracellular loop to modulate Gq/11alpha signaling. J Biol Chem 2004;279:21248–56. 41. Hague C, Bernstein LS, Ramineni S, Chen Z, Minneman KP, Hepler JR. Selective inhibition of alpha1a‐adrenergic receptor signaling by RGS2 association with the receptor third intracellular loop. J Biol Chem 2005;280:27289–95. 42. Miyamoto‐Matsubara M, Saitoh O, Maruyama K, Aizaki Y, Saito Y. Regulation of melanin‐ concentrating hormone receptor 1 signaling by RGS8 with the receptor third intracellular loop. Cell Signal 2008;20:2084–94. 43. Itoh M, Nagatomo K, Kubo Y, Saitoh O. Alternative splicing of RGS8 gene changes the binding property to the M1 muscarinic receptor to confer receptor type‐specific Gq regulation. J Neurochem 2006;99:1505–16. 44. Saitoh O, Murata Y, Odagiri M, Itoh M, Itoh H, Misaka T, et al. Alternative splicing of RGS8 gene determines inhibitory function of receptor type‐specific Gq signaling. Proc Natl Acad Sci USA 2002;99:10138–43. 45. Fujii S, Yamazoe G, Itoh M, Kubo Y, Saitoh O. Spinophilin inhibits the binding of RGS8 to m1 mAChR but enhances the regulatory function of RGS8. Biochem Biophys Res Commun 2008;377:200–4. 46. Georgoussi Z, Leontiadis L, Mazarakou G, Merkouris M, Hyde K, Hamm H. Selective interactions between G protein subunits and RGS4 with the C‐terminal domains of the mu‐ and delta‐opioid receptors regulate opioid receptor signaling. Cell Signal 2006;18:771–82. 47. Langer I, Tikhonova IG, Boulegue C, Esteve JP, Vatinel S, Ferrand A, et al. Evidence for a direct and functional interaction between the regulators of G protein signaling‐2 and phosphorylated C terminus of cholecystokinin‐2 receptor. Mol Pharmacol 2009;75:502–13. 48. Xie GX, Yanagisawa Y, Ito E, Maruyama K, Han X, Kim KJ, et al. N‐terminally truncated variant of the mouse GAIP/RGS19 lacks selectivity of full‐length GAIP/RGS19 protein in regulating ORL1 receptor signaling. J Mol Biol 2005;353:1081–92. 49. Jeanneteau F, Guillin O, Diaz J, Griffon N, Sokoloff P. GIPC recruits GAIP (RGS19) to attenuate dopamine D2 receptor signaling. Mol Biol Cell 2004;15:4926–37. 50. Brady AE, Wang Q, Colbran RJ, Allen PB, Greengard P, Limbird LE. Spinophilin stabilizes cell surface expression of alpha 2B‐adrenergic receptors. J Biol Chem 2003;278:32405–12. 51. Smith FD, Oxford GS, Milgram SL. Association of the D2 dopamine receptor third cytoplasmic loop with spinophilin, a protein phosphatase‐1‐interacting protein. J Biol Chem 1999;274:19894–900.

70

McCOY AND HEPLER

52. Richman JG, Brady AE, Wang Q, Hensel JL, Colbran RJ, Limbird LE. Agonist‐regulated interaction between alpha2‐adrenergic receptors and spinophilin. J Biol Chem 2001;276: 15003–8. 53. Wang X, Zeng W, Soyombo AA, Tang W, Ross EM, Barnes AP, et al. Spinophilin regulates Ca2þ signalling by binding the N‐terminal domain of RGS2 and the third intracellular loop of G‐protein‐coupled receptors. Nat Cell Biol 2005;7:405–11. 54. Psifogeorgou K, Papakosta P, Russo SJ, Neve RL, Kardassis D, Gold SJ, et al. RGS9‐2 is a negative modulator of mu‐opioid receptor function. J Neurochem 2007;103:617–25. 55. Xie Z, Li Z, Guo L, Ye C, Li J, Yu X, et al. Regulator of G protein signaling proteins differentially modulate signaling of mu and delta opioid receptors. Eur J Pharmacol 2007;565:45–53. 56. Charlton JJ, Allen PB, Psifogeorgou K, Chakravarty S, Gomes I, Neve RL, et al. Multiple actions of spinophilin regulate mu opioid receptor function. Neuron 2008;58:238–47. 57. Fujii S, Yamazoe G, Itoh M, Kubo Y, Saitoh O. Spinophilin inhibits the binding of RGS8 to m1 mAChR but enhances the regulatory function of RGS8. Biochem Biophys Res Commun 2008;377:200–4. 58. Liu W, Yuen EY, Allen PB, Feng J, Greengard P, Yan Z. Adrenergic modulation of NMDA receptors in prefrontal cortex is differentially regulated by RGS proteins and spinophilin. Proc Natl Acad Sci USA 2006;103:18338–43. 59. Garzon J, Rodriguez‐Munoz M, Lopez‐Fando A, Garcia‐Espana A, Sanchez‐Blazquez P. RGSZ1 and GAIP regulate mu‐ but not delta‐opioid receptors in mouse CNS: role in tachyphylaxis and acute tolerance. Neuropsychopharmacology 2004;29:1091–104. 60. Garzon J, Rodriguez‐Munoz M, Lopez‐Fando A, Sanchez‐Blazquez P. The RGSZ2 protein exists in a complex with mu‐opioid receptors and regulates the desensitizing capacity of Gz proteins. Neuropsychopharmacology 2005;30:1632–48. 61. Rodriguez‐Munoz M, Torre‐Madrid E, Sanchez‐Blazquez P, Garzon J. Morphine induces endocytosis of neuronal mu‐opioid receptors through the sustained transfer of Galpha subunits to RGSZ2 proteins. Mol Pain 2007;3:19. 62. Rodriguez‐Munoz M, Bermudez D, Sanchez‐Blazquez P, Garzon J. Sumoylated RGS‐Rz proteins act as scaffolds for Mu‐opioid receptors and G‐protein complexes in mouse brain. Neuropsychopharmacology 2007;32:842–50. 63. Rodriguez‐Munoz M, de lT‐M, Gaitan G, Sanchez‐Blazquez P, Garzon J. RGS14 prevents morphine from internalizing Mu‐opioid receptors in periaqueductal gray neurons. Cell Signal 2007;19:2558–71. 64. Cho H, Harrison K, Schwartz O, Kehrl JH. The aorta and heart differentially express RGS (regulators of G‐protein signalling) proteins that selectively regulate sphingosine 1‐phosphate, angiotensin II and endothelin‐1 signalling. Biochem J 2003;371:973–80. 65. Ghavami A, Hunt RA, Olsen MA, Zhang J, Smith DL, Kalgaonkar S, et al. Differential effects of regulator of G protein signaling (RGS) proteins on serotonin 5‐HT1A, 5‐HT2A, and dopamine D2 receptor‐mediated signaling and adenylyl cyclase activity. Cell Signal 2004;16:711–21. 66. Roy AA, Lemberg KE, Chidiac P. Recruitment of RGS2 and RGS4 to the plasma membrane by G proteins and receptors reflects functional interactions. Mol Pharmacol 2003;64:587–93. 67. Wang Q, Liu M, Mullah B, Siderovski DP, Neubig RR. Receptor‐selective effects of endogenous RGS3 and RGS5 to regulate mitogen‐activated protein kinase activation in rat vascular smooth muscle cells. J Biol Chem 2002;277:24949–58. 68. Wang Q, Liu M, Kozasa T, Rothstein JD, Sternweis PC, Neubig RR. Thrombin and lysophosphatidic acid receptors utilize distinct rhoGEFs in prostate cancer cells. J Biol Chem 2004;279:28831–4.

RGS PROTEINS AS COMPONENTS OF GPCR SIGNALING COMPLEXES

71

69. Bodenstein J, Sunahara RK, Neubig RR. N‐terminal residues control proteasomal degradation of RGS2, RGS4, and RGS5 in human embryonic kidney 293 cells. Mol Pharmacol 2007;71:1040–50. 70. Karakoula A, Tovey SC, Brighton PJ, Willars GB. Lack of receptor‐selective effects of either RGS2, RGS3 or RGS4 on muscarinic M3‐ and gonadotropin‐releasing hormone receptor‐ mediated signalling through G alpha q/11. Eur J Pharmacol 2008;587:16–24. 71. Jones TL, Chong LD, Kim J, Xu RH, Kung HF, Daar IO. Loss of cell adhesion in Xenopus laevis embryos mediated by the cytoplasmic domain of xlerk, an erythropoietin‐producing hepatocellular ligand. Proc Natl Acad Sci USA 1998;20:576–81. 72. Taya S, Inagaki N, Sengiku H, Makino H, Iwamatsu A, Urakawa I, et al. Direct interaction of insulin‐like growth factor‐1 receptor with leukemia‐associated rhoGEF. J Cell Biol 2001;155: 809–20. 73. Lou X, Yano H, Lee F, Chao MV, Farquhar MG. GIPC and GAIP form a complex with trka: a putative link between G protein and receptor tyrosine kinase pathways. Mol Biol Cell 2001;12:615–27. 74. Sambi BS, Hains MD, Waters CM, Connell MC, Willard FS, Kimple AJ, et al. The effect of RGS12 on PDGFbeta receptor signalling to p42/p44 mitogen activated protein kinase in mammalian cells. Cell Signal 2006;18:971–81. 75. Jo S, Lee KH, Song S, Jung YK, Park CS. Identification and functional characterization of cereblon as a binding protein for large‐conductance calcium‐activated potassium channel in rat brain. J Neurochem 2005;94:1212–24. 76. Doupnik CA, Davidson N, Lester HA, Kofuji P. RGS proteins reconstitute the rapid gating kinetics of gbetagamma‐activated inwardly rectifying Kþ channels. Proc Natl Acad Sci USA 1997;94:10461–6. 77. Sadja R, Alagem N, Reuveny E. Gating of GIRK channels: details of an intricate, membrane‐ delimited signaling complex. Neuron 2003;39:9–12. 78. Druey KM, Sullivan BM, Brown D, Fischer ER, Watson N, Blumer KJ, et al. Expression of GTPase‐deficient Gialpha2 results in translocation of cytoplasmic RGS4 to the plasma membrane. J Biol Chem 1998;273:18405–10. 79. Dulin NO, Sorokin A, Reed E, Elliott S, Kehrl JH, Dunn MJ. RGS3 inhibits G protein‐ mediated signaling via translocation to the membrane and binding to Galpha11. Mol Cell Biol 1999;19:714–23. 80. Masuho I, Itoh M, Itoh H, Saitoh O. The mechanism of membrane‐translocation of regulator of G‐protein signaling (RGS) 8 induced by Galpha expression. J Neurochem 2004;88:161–8. 81. Heximer SP, Blumer KJ. RGS proteins: Swiss army knives in seven‐transmembrane domain receptor signaling networks. Sci STKE 2007;2007:e2. 82. Martemyanov KA, Arshavsky VY. Noncatalytic domains of RGS9‐1.Gbeta 5L play a decisive role in establishing its substrate specificity. J Biol Chem 2002;277:32843–8. 83. Martemyanov KA, Lishko PV, Calero N, Keresztes G, Sokolov M, Strissel KJ, et al. The DEP domain determines subcellular targeting of the gtpase activating protein RGS9 in vivo. J Neurosci 2003;23:10175–81. 84. Mittal V, Linder ME. The RGS14 goloco domain discriminates among Galphai isoforms. J Biol Chem 2004;279:46772–8. 85. Skiba NP, Martemyanov KA, Elfenbein A, Hopp JA, Bohm A, Simonds WF, et al. RGS9‐G beta 5 substrate selectivity in photoreceptors. Opposing effects of constituent domains yield high affinity of RGS interaction with the G protein‐effector complex. J Biol Chem 2001;276: 37365–72. 86. Bernstein LS, Linder ME, Hepler JR. Analysis of RGS protein palmitoylation. Methods Mol Biol 2004;237:195–204.

72

McCOY AND HEPLER

87. Gu S, He J, Ho WT, Ramineni S, Thal DM, Natesh R, et al. Unique hydrophobic extension of the RGS2 amphipathic helix domain imparts increased plasma membrane binding and function relative to other RGS R4/B subfamily members. J Biol Chem 2007;282:33064–75. 88. Ferguson SS. Evolving concepts in G protein‐coupled receptor endocytosis: the role in receptor desensitization and signaling. Pharmacol Rev 2001;53:1–24. 89. Bunemann M, Frank M, Lohse MJ. Gi protein activation in intact cells involves subunit rearrangement rather than dissociation. Proc Natl Acad Sci USA 2003;100:16077–82. 90. Hein P, Bunemann M. Coupling mode of receptors and G proteins. Naunyn Schmiedebergs Arch Pharmacol 2009;379:435–43. 91. Hoffmann C, Zurn A, Bunemann M, Lohse MJ. Conformational changes in G‐protein‐ coupled receptors‐the quest for functionally selective conformations is open. Br J Pharmacol 2008;153(Suppl 1): S358–66. 92. Zurn A, Zabel U, Vilardaga JP, Schindelin H, Lohse MJ, Hoffmann C. Fluorescence resonance energy transfer analysis of {alpha}2a‐adrenergic receptor activation reveals distinct agonist‐ specific conformational changes. Mol Pharmacol 2009;75:534–41. 93. Gether U. Uncovering molecular mechanisms involved in activation of G protein‐coupled receptors. Endocr Rev 2000;21:90–113. 94. Okada T, Ernst OP, Palczewski K, Hofmann KP. Activation of rhodopsin: new insights from structural and biochemical studies. Trends Biochem Sci 2001;26:318–24. 95. Hubbell WL, Altenbach C, Hubbell CM, Khorana HG. Rhodopsin structure, dynamics, and activation: a perspective from crystallography, site‐directed spin labeling, sulfhydryl reactivity, and disulfide cross‐linking. Adv Protein Chem 2003;63:243–90. 96. Rosenkilde MM, David R, Oerlecke I, Benned‐Jensen T, Geumann U, Beck‐Sickinger AG, et al. Conformational constraining of inactive and active states of a seven transmembrane receptor by metal ion site engineering in the extracellular end of transmembrane segment V. Mol Pharmacol 2006;70:1892–901. 97. Marjamaki A, Frang H, Pihlavisto M, Hoffren AM, Salminen T, Johnson MS, et al. Chloroethylclonidine and 2‐aminoethyl methanethiosulfonate recognize two different conformations of the human alpha(2A)‐adrenergic receptor. J Biol Chem 1999;274:21867–72. 98. Nyronen T, Pihlavisto M, Peltonen JM, Hoffren AM, Varis M, Salminen T, et al. Molecular mechanism for agonist‐promoted alpha(2A)‐adrenoceptor activation by norepinephrine and epinephrine. Mol Pharmacol 2001;59:1343–54. 99. Pfleger KD, Dalrymple MB, Dromey JR, Eidne KA. Monitoring interactions between G‐protein‐coupled receptors and beta‐arrestins. Biochem Soc Trans 2007;35:764–6. 100. DeFea KA, Zalevsky J, Thoma MS, Dery O, Mullins RD, Bunnett NW. Beta‐arrestin‐ dependent endocytosis of proteinase‐activated receptor 2 is required for intracellular targeting of activated ERK1/2. J Cell Biol 2000;20(148), 1267–81. 101. Macey TA, Lowe JD, Chavkin C. Mu opioid receptor activation of ERK1/2 is GRK3 and arrestin dependent in striatal neurons. J Biol Chem 2006;281:34515–24. 102. Scott MG, Pierotti V, Storez H, Lindberg E, Thuret A, Muntaner O, et al. Cooperative regulation of extracellular signal‐regulated kinase activation and cell shape change by filamin A and beta‐arrestins. Mol Cell Biol 2006;26:3432–45. 103. Shenoy SK, Drake MT, Nelson CD, Houtz DA, Xiao K, Madabushi S, et al. Beta‐arrestin‐ dependent, G protein‐independent ERK1/2 activation by the beta2 adrenergic receptor. J Biol Chem 2006;281:1261–73. 104. Tohgo A, Choy EW, Gesty‐Palmer D, Pierce KL, Laporte S, Oakley RH, et al. The stability of the G protein‐coupled receptor‐beta‐arrestin interaction determines the mechanism and functional consequence of ERK activation. J Biol Chem 2003;278:6258–67.

RGS PROTEINS AS COMPONENTS OF GPCR SIGNALING COMPLEXES

73

105. Zheng H, Loh HH, Law PY. Beta‐arrestin‐dependent mu‐opioid receptor‐activated extracellular signal‐regulated kinases (erks) translocate to nucleus in contrast to G protein‐dependent ERK activation. Mol Pharmacol 2008;73:178–90. 106. DeWire SM, Ahn S, Lefkowitz RJ, Shenoy SK. Beta‐arrestins and cell signaling. Annu Rev Physiol 2007;69:483–510. 107. Nelson CD, Kovacs JJ, Nobles KN, Whalen EJ, Lefkowitz RJ. Beta‐arrestin scaffolding of phosphatidylinositol 4‐phosphate 5‐kinase Ialpha promotes agonist‐stimulated sequestration of the beta2‐adrenergic receptor. J Biol Chem 2008;283:21093–101. 108. Tesmer JJ, Berman DM, Gilman AG, Sprang SR. Structure of RGS4 bound to alf4–activated G(i alpha1): stabilization of the transition state for GTP hydrolysis. Cell 1997;89:251–61. 109. Moy FJ, Chanda PK, Cockett MI, Edris W, Jones PG, Mason K, et al. NMR structure of free RGS4 reveals an induced conformational change upon binding Galpha. Biochemistry 2000;20(39), 7063–73. 110. Chen Z, Wells CD, Sternweis PC, Sprang SR. Structure of the rgRGS domain of p115rhoGEF. Nat Struct Biol 2001;8:805–9. 111. Lodowski DT, Pitcher JA, Capel WD, Lefkowitz RJ, Tesmer JJ. Keeping G proteins at bay: a complex between G protein‐coupled receptor kinase 2 and Gbetagamma. Science 2003;300: 1256–62. 112. Slep KC, Kercher MA, He W, Cowan CW, Wensel TG, Sigler PB. Structural determinants for regulation of phosphodiesterase by a G protein at 2.0 A. Nature 2001;409:1071–77. 113. Slep KC, Kercher MA, Wieland T, Chen CK, Simon MI, Sigler PB. Molecular architecture of Galphao and the structural basis for RGS16‐mediated deactivation. Proc Natl Acad Sci USA 2008;105:6243–8. 114. Tesmer VM, Kawano T, Shankaranarayanan A, Kozasa T, Tesmer JJ. Snapshot of activated G proteins at the membrane: the Galphaq‐GRK2‐Gbetagamma complex. Science 2005;310: 1686–90. 115. Maurya MR, Bornheimer SJ, Venkatasubramanian V, Subramaniam S. Reduced‐order modelling of biochemical networks: application to the GTPase‐cycle signalling module. Syst Biol (Stevenage) 2005;152:229–42. 116. Chen JG, Willard FS, Huang J, Liang J, Chasse SA, Jones AM, et al. A seven‐transmembrane RGS protein that modulates plant cell proliferation. Science 2003;19(301): 1728–31. 117. Willard FS, Siderovski DP. Purification and in vitro functional analysis of the Arabidopsis thaliana regulator of G‐protein signaling‐1. Methods Enzymol. 2004;389:320–38. 118. Hepler JR. RGS protein and G protein interactions: a little help from their friends. Mol Pharmacol 2003;64:547–9. 119. Cifelli C, Rose RA, Zhang H, Voigtlaender‐Bolz J, Bolz SS, Backx PH, et al. RGS4 regulates parasympathetic signaling and heart rate control in the sinoatrial node. Circ Res 2008;103: 527–35. 120. Harris DM, Cohn HI, Pesant S, Eckhart AD. GPCR signalling in hypertension: role of grks. Clin Sci (Lond) 2008;115:79–89. 121. Hendriks‐Balk MC, Peters SL, Michel MC, Alewijnse AE. Regulation of G protein‐coupled receptor signalling: focus on the cardiovascular system and regulator of G protein signalling proteins. Eur J Pharmacol 2008;585:278–91. 122. Tang KM, Wang GR, Lu P, Karas RH, Aronovitz M, Heximer SP, et al. Regulator of G‐protein signaling‐2 mediates vascular smooth muscle relaxation and blood pressure. Nat Med 2003;9: 1506–12. 123. Cho H, Harrison K, Kehrl JH. Regulators of G protein signaling: potential drug targets for controlling cardiovascular and immune function. Curr Drug Targets Immune Endocr Metabol Disord 2004;4:107–18.

74

McCOY AND HEPLER

124. Huang X, Fu Y, Charbeneau RA, Saunders TL, Taylor DK, Hankenson KD, et al. Pleiotropic phenotype of a genomic knock‐in of an RGS‐insensitive G184S Gnai2 allele. Mol Cell Biol 2006;26:6870–9. 125. Agenes F, Bosco N, Mascarell L, Fritah S, Ceredig R. Differential expression of regulator of G‐protein signalling transcripts and in vivo migration of CD4þ naive and regulatory T cells. Immunology 2005;115:179–88. 126. Bansal G, Xie Z, Rao S, Nocka KH, Druey KM. Suppression of immunoglobulin E‐mediated allergic responses by regulator of G protein signaling 13. Nat Immunol 2008;9:73–80. 127. Hamzah J, Jugold M, Kiessling F, Rigby P, Manzur M, Marti HH, et al. Vascular normalization in Rgs5‐deficient tumours promotes immune destruction. Nature 2008;453:410–4. 128. Moratz C, Harrison K, Kehrl JH. Regulation of chemokine‐induced lymphocyte migration by RGS proteins. Methods Enzymol 2004;389:15–32. 129. Moratz C, Hayman JR, Gu H, Kehrl JH. Abnormal B‐cell responses to chemokines, disturbed plasma cell localization, and distorted immune tissue architecture in Rgs1/ mice. Mol Cell Biol 2004;24:5767–75. 130. Moratz C, Harrison K, Kehrl JH. Role of RGS proteins in regulating the migration of B lymphocytes. Arch Immunol Ther Exp (Warsz) 2004;52:27–35. 131. Druey KM. Regulators of G protein signalling: potential targets for treatment of allergic inflammatory diseases such as asthma. Expert Opin Ther Targets 2003;7:475–84. 132. Lombardi MS, Kavelaars A, Heijnen CJ. Role and modulation of G protein‐coupled receptor signaling in inflammatory processes. Crit Rev Immunol 2002;22:141–63. 133. Kehrl JH. Heterotrimeric G protein signaling: roles in immune function and fine‐tuning by RGS proteins. Immunity 1998;8:1–10. 134. Hooks SB, Martemyanov K, Zachariou V. A role of RGS proteins in drug addiction. Biochem Pharmacol 2008;75:76–84. 135. Neubig RR. Regulators of G protein signaling (RGS proteins): novel central nervous system drug targets. J Pept Res 2002;60:312–6. 136. Burchett SA. Psychostimulants, madness, memory. . . and RGS proteins? Neuromol Med 2005;7:101–27. 137. Hepler JR. R7BP: a surprising new link between G proteins, RGS proteins, and nuclear signaling in the brain. Sci STKE 2005;2005:e38. 138. Garzon J, Rodriguez‐Munoz M, Torre‐Madrid E, Sanchez‐Blazquez P. Effector antagonism by the regulators of G protein signalling (RGS) proteins causes desensitization of mu‐opioid receptors in the CNS. Psychopharmacology (Berl) 2005;180:1–11. 139. Xie GX, Palmer PP. RGS proteins: new players in the field of opioid signaling and tolerance mechanisms. Anesth Analg 2005;100:1034–42.