G Protein Selectivity Is a Determinant of RGS2 Function

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RGS proteins serve as GTPase-activating proteins and/or effector antagonists to modulate G␣ signaling events. In live cells, members of the B/R4 subfamily of RGS proteins selectively modulate G protein signaling depending on the associated receptor (GPCR). Here we examine whether GPCRs selectively recruit RGS proteins to modulate linked G protein signaling. We report the novel finding that RGS2 binds directly to the third intracellular (i3) loop of the G q/11 -coupled M1 muscarinic cholinergic receptor (M1 mAChR; M1i3). This interaction is selective because closely related RGS16 does not bind M1i3, and neither RGS2 nor RGS16 binds to the G i/o -coupled M2i3 loop. When expressed in cells, RGS2 and M1 mAChR co-localize to the plasma membrane whereas RGS16 does not. The N-terminal region of RGS2 is both necessary and sufficient for binding to M1i3, and RGS2 forms a stable heterotrimeric complex with both activated G q ␣ and M1i3. RGS2 potently inhibits M1 mAChR-mediated phosphoinositide hydrolysis in cell membranes by acting as an effector antagonist. Deletion of the N terminus abolishes this effector antagonist activity of RGS2 but not its GTPase-activating protein activity toward G 11 ␣ in membranes. These findings predict a model where the i3 loops of GPCRs selectively recruit specific RGS protein(s) via their N termini to regulate the linked G protein. Consistent with this model, we find that the i3 loops of the mAChR subtypes (M1-M5) exhibit differential profiles for binding distinct B/R4 RGS family members, indicating that this novel mechanism for GPCR modulation of RGS signaling may generally extend to other receptors and RGS proteins.Cells rely upon G protein-coupled receptors (GPCRs) 1 to convey signals from extracellular hormones and neurotransmitters to intracellular effectors and linked signaling pathways. Agonist occupancy of the GPCR activates a heterotrimeric G protein (G␣␤␥) by catalyzing the exchange of GDP for GTP on the G␣ subunit (1). This initiates dissociation of the trimer into free G␣ and G␤␥, which independently or in coordinated fashion activate downstream effectors and linked signaling pathways. Members of the regulators of G protein signaling (RGS) family of proteins are direct modulators of G protein activity. RGS proteins are best understood as GTPaseactivating proteins (GAPs), which bind to the activated form of G␣ and accelerate its GTPase activity thereby promoting the termination of G protein signaling (2-5). By virtue of their interactions with activated G␣, RGS proteins also serve as effector antagonists to block activation of downstream effector molecules (6, 7).All RGS proteins share a conserved RGS core domain of ϳ130 amino acids that contains binding sites for G␣ and is responsible for their GAP activity (5,8,9). Outside of the RGS domain, however, the more than 30 family members are widely divergent. Some RGS proteins are quite complex and contain multiple domains for binding a variety of signaling proteins. Other RGS proteins are simple, with relatively short, featureless N...

Section: Differential Binding Profiles Of Various Rgs Proteins With Tmentioning

confidence: 78%

RGS2 Binds Directly and Selectively to the M1 Muscarinic Acetylcholine Receptor Third Intracellular Loop to Modulate Gq/11α Signaling

Bernstein

Ramineni

Hague

et al. 2004

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205

“…It is known that RGS2 localizes to the mammalian cell plasma membrane more efficiently than other R4/B subfamily members and also functions as a more potent inhibitor of muscarinic receptor G␣ q signaling (15)(16)(17). However, the relative contribution of three previously reported membrane targeting mechanisms to its localization and receptor inhibition has not been determined.…”

mentioning

confidence: 79%

“…Plasma Membrane Targeting Is a More Important Functional Determinant than Intrinsic RGS-box Function-Previous data from our laboratory showed that RGS2 contained three unique amino acid residues in its RGS-box domain that mediate its selective inhibition of G␣ q -versus G␣ i -mediated signaling (15). To determine whether G␣ q selectivity could explain the enhanced relative function of RGS2, we compared the ability of the RGS-box domains of RGS2 and RGS5 to act as inhibitors of M1 muscarinic receptor signaling.…”

Section: Rgs2 Is a More Potent Inhibitor Of M1 Muscarinic Receptormedmentioning

confidence: 99%

“…First, the amino terminus of RGS2 can bind to the third intracellular loop of the M1 muscarinic receptor, whereas other R4/B subfamily members cannot (17). Second, RGS2 contains three unique residues in its RGS-box that confer G␣ q selectivity (15) compared with other R4/B subfamily members. Third, the amino-terminal amphipathic helix of RGS2 is sufficient to confer lipid binding and plasma membrane localization in mammalian cells, whereas the same domain in RGS4 localizes evenly throughout the cell (13).…”

mentioning

confidence: 99%

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Unique Hydrophobic Extension of the RGS2 Amphipathic Helix Domain Imparts Increased Plasma Membrane Binding and Function Relative to Other RGS R4/B Subfamily Members

et al. 2007

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RGS2 and RGS5 are inhibitors of G-protein signaling belonging to the R4/B subfamily of RGS proteins. We here show that RGS2 is a much more potent attenuator of M1 muscarinic receptor signaling than RGS5. We hypothesize that this difference is mediated by variation in their ability to constitutively associate with the plasma membrane (PM). Compared with full-length RGS2, the RGS-box domains of RGS2 and RGS5 both show reduced PM association and activity. Prenylation of both RGSbox domains increases activity to RGS2 levels, demonstrating that lipid bilayer targeting increases RGS domain function. Amino-terminal domain swaps confirm that key determinants of localization and function are found within this important regulatory domain. An RGS2 amphipathic helix domain mutant deficient for phospholipid binding (L45D) shows reduced PM association and activity despite normal binding to the M1 muscarinic receptor third intracellular loop and activated G␣ q . Replacement of a unique dileucine motif adjacent to the RGS2 helix with corresponding RGS5 residues disrupts both PM localization and function. These data suggest that RGS2 contains a hydrophobic extension of its helical domain that imparts high efficiency binding to the inner leaflet of the lipid bilayer. In support of this model, disruption of membrane phospholipid composition with N-ethylmaleimide reduces PM association of RGS2, without affecting localization of the M1 receptor or G␣ q . Together, these data indicate that novel features within the RGS2 amphipathic ␣ helix facilitate constitutive PM targeting and more efficient inhibition of M1 muscarinic receptor signaling than RGS5 and other members of the R4/B subfamily.Heterotrimeric G-protein-coupled receptors mediate cellular responses to a variety of extracellular ligands, including hormones, neurotransmitters, and sensory stimuli. Proper coordination of G-protein-coupled receptor signaling at the cellular and tissue level is required to ensure appropriate physiologic responses to rapidly changing environmental conditions. An important component of G-protein-coupled receptors signal coordination in human cells is the RGS (regulator of G-protein signaling) superfamily of proteins. RGS proteins inhibit G-protein signaling via their activities as GTPase-activating proteins for G-protein ␣ subunits (1-4).The human genome contains 37 RGS proteins (5) characterized by a ϳ120-amino acid domain called the "RGS-box." The RGS protein superfamily can be further subclassified based on the architectural organization and RGS-box function of its members (6). Many subfamilies are made up of larger RGS-boxcontaining proteins that include the RGS7-like, RGS12-like, RhoGEF-containing, and G-protein-coupled receptor kinases. These subfamilies are mainly composed of members with multiple modular signaling domains. By contrast, the RGSZ-like and RGS4-like (R4/B) subfamilies are made up of smaller proteins containing a RGS-box domain flanked by short aminoterminal and carboxyl-terminal extensions. These two families are distinguishe...

“…Axin contacts with the APC peptide are green (52). Residues in RGS2 that, when converted to their equivalents in RGS4, enhance GAP activity toward G␣ i (74) are indigo. Mutations in p115RhoGEF that decrease G␣ 13 GAP activity are hot pink (53).…”

Section: Fig 1 Protein Binding Surfaces Of Rh Domainsmentioning

confidence: 99%

G Protein-coupled Receptor Kinase 2/Gαq/11 Interaction

Sterne‐Marr

Tesmer

Day

et al. 2003

113

G protein-coupled receptors (GPCRs) transduce cellular signals from hormones, neurotransmitters, light, and odorants by activating heterotrimeric guanine nucleotide-binding (G) proteins. For many GPCRs, short term regulation is initiated by agonist-dependent phosphorylation by GPCR kinases (GRKs), such as GRK2, resulting in G protein/receptor uncoupling. GRK2 also regulates signaling by binding G␣ q/ll and inhibiting G␣ q stimulation of the effector phospholipase C␤. The binding site for G␣ q/ll resides within the amino-terminal domain of GRK2, which is homologous to the regulator of G protein signaling (RGS) family of proteins. To map the G␣ q/ll binding site on GRK2, we carried out site-directed mutagenesis of the RGS homology (RH) domain and identified eight residues, which when mutated, alter binding to G␣ q/ll . These mutations do not alter the ability of full-length GRK2 to phosphorylate rhodopsin, an activity that also requires the amino-terminal domain. Mutations causing G␣ q/ll binding defects impair recruitment to the plasma membrane by activated G␣ q and regulation of G␣ q -stimulated phospholipase C␤ activity when introduced into full-length GRK2. Two different protein interaction sites have previously been identified on RH domains. The G␣ binding sites on RGS4 and RGS9, called the "A" site, is localized to the loops between helices ␣3 and ␣4, ␣5 and ␣6, and ␣7 and ␣8. The adenomatous polyposis coli (APC) binding site of axin involves residues on ␣ helices 3, 4, and 5 (the "B" site) of its RH domain. We demonstrate that the G␣ q/ll binding site on the GRK2 RH domain is distinct from the "A" and "B" sites and maps primarily to the COOH terminus of its ␣5 helix. We suggest that this novel protein interaction site on an RH domain be designated the "C" site.