Recent studies with gene knock-out mice indicate a prominent role for the functionally linked signaling proteins RGS2, Gq-alpha (Gqa), alpha-1A-adrenergic (a1A-AR) and m1 muscarininc cholinergic receptors (M1 AChR) in regulation of vascular hypertension, cardiac hypertrophy, sympathetic control of cardiac function, and vascular tone. However, molecular models for how these proteins interact are not well understood. RGS proteins bind directly to activated Ga subunits to modulate and integrate their functions. Very little is known about how RGS selectivity for target Ga is determined in cells. Recent studies suggest that RGS and GPCR are functionally linked in cells but direct interactions have not been shown. Based on these observations, we tested whether RGS proteins and GPCR interact directly. We found that RGS2 (but not RGS1 or RGS16) binds directly and selectively to the third intracellular loop (i3) of the Gq/11-linked M1AChR and a1A-AR, but not i3 of a1B-AR or a1D-AR or Gi/o-linked M2- or M4AChR. Our studies show that RGS2, M1-13, and Gqa form a stable heterotrimer complex, and that the N-terminus of RGS2 is responsible for RGS binding to the i3 of both receptors. My working hypothesis is that RGS proteins form stable, functional complexes with preferred GPCR to selectively modulate the signaling functions of those receptors and linked G proteins. Using molecular, cellular and biochemical approaches, the Specific Aims will be to:
Aim 1 : Identify amino acids on the M1AChR, a1A-AR and RGS2 responsible for direct binding.
Aim 2 : Determine roles for RGS2 on receptor/ligand binding and functional Gq/11 a coupling.
Aim 3 : Determine the effects of RGS2 on GRK2 binding/phosphorylation and arrestin binding to receptors on their desensitization, internalization, and intracellular trafficking.
Aim 4 : Determine the effects of suppressing native RGS2 mRNA/protein on native M1AChR and a1A-AR signaling functions in cells/VSM tissues that natively expresses a1A-AR or M1AChR and RGS2. These studies will define novel cellular mechanisms for regulating neurotransmitter and hormone signaling, and identify potential new molecular targets for therapeutic intervention in cardiovascular diseases.
|Ghil, Sungho; McCoy, Kelly L; Hepler, John R (2014) Regulator of G protein signaling 2 (RGS2) and RGS4 form distinct G protein-dependent complexes with protease activated-receptor 1 (PAR1) in live cells. PLoS One 9:e95355|
|Zhao, Peishen; Nunn, Caroline; Ramineni, Suneela et al. (2013) The Ras-binding domain region of RGS14 regulates its functional interactions with heterotrimeric G proteins. J Cell Biochem 114:1414-23|
|Vellano, Christopher P; Brown, Nicole E; Blumer, Joe B et al. (2013) Assembly and function of the regulator of G protein signaling 14 (RGS14)·H-Ras signaling complex in live cells are regulated by G?i1 and G?i-linked G protein-coupled receptors. J Biol Chem 288:3620-31|
|McCoy, Kelly L; Gyoneva, Stefka; Vellano, Christopher P et al. (2012) Protease-activated receptor 1 (PAR1) coupling to G(q/11) but not to G(i/o) or G(12/13) is mediated by discrete amino acids within the receptor second intracellular loop. Cell Signal 24:1351-60|
|Vellano, Christopher P; Shu, Feng-Jue; Ramineni, Suneela et al. (2011) Activation of the regulator of G protein signaling 14-G?i1-GDP signaling complex is regulated by resistance to inhibitors of cholinesterase-8A. Biochemistry 50:752-62|
|Vellano, Christopher P; Maher, Ellen M; Hepler, John R et al. (2011) G protein-coupled receptors and resistance to inhibitors of cholinesterase-8A (Ric-8A) both regulate the regulator of g protein signaling 14 RGS14·G?i1 complex in live cells. J Biol Chem 286:38659-69|
|McCoy, Kelly L; Traynelis, Stephen F; Hepler, John R (2010) PAR1 and PAR2 couple to overlapping and distinct sets of G proteins and linked signaling pathways to differentially regulate cell physiology. Mol Pharmacol 77:1005-15|
|Shu, Feng-jue; Ramineni, Suneela; Hepler, John R (2010) RGS14 is a multifunctional scaffold that integrates G protein and Ras/Raf MAPkinase signalling pathways. Cell Signal 22:366-76|
|Jernigan, Kristin K; Cselenyi, Christopher S; Thorne, Curtis A et al. (2010) Gbetagamma activates GSK3 to promote LRP6-mediated beta-catenin transcriptional activity. Sci Signal 3:ra37|
|McCoy, Kelly L; Hepler, John R (2009) Regulators of G protein signaling proteins as central components of G protein-coupled receptor signaling complexes. Prog Mol Biol Transl Sci 86:49-74|
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