title: Assembly and function of the regulator of G protein signaling 14 (RGS14)·H-Ras signaling complex in live cells are regulated by Gαi1 and Gαi-linked G protein-coupled receptors.
abstract: Regulator of G protein signaling 14 (RGS14) is a multifunctional scaffolding protein that integrates heterotrimeric G protein and H-Ras signaling pathways. RGS14 possesses an RGS domain that binds active Gα(i/o)-GTP subunits to promote GTP hydrolysis and a G protein regulatory (GPR) motif that selectively binds inactive Gα(i1/3)-GDP subunits to form a stable heterodimer at cellular membranes. RGS14 also contains two tandem Ras/Rap binding domains (RBDs) that bind H-Ras. Here we show that RGS14 preferentially binds activated H-Ras-GTP in live cells to enhance H-Ras cellular actions and that this interaction is regulated by inactive Gα(i1)-GDP and G protein-coupled receptors (GPCRs). Using bioluminescence resonance energy transfer (BRET) in live cells, we show that RGS14-Luciferase and active H-Ras(G/V)-Venus exhibit a robust BRET signal at the plasma membrane that is markedly enhanced in the presence of inactive Gα(i1)-GDP but not active Gα(i1)-GTP. Active H-Ras(G/V) interacts with a native RGS14·Gα(i1) complex in brain lysates, and co-expression of RGS14 and Gα(i1) in PC12 cells greatly enhances H-Ras(G/V) stimulatory effects on neurite outgrowth. Stimulation of the Gα(i)-linked α(2A)-adrenergic receptor induces a conformational change in the Gα(i1)·RGS14·H-Ras(G/V) complex that may allow subsequent regulation of the complex by other binding partners. Together, these findings indicate that inactive Gα(i1)-GDP enhances the affinity of RGS14 for H-Ras-GTP in live cells, resulting in a ternary signaling complex that is further regulated by GPCRs.
authors.affiliation: Department of Pharmacology, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
authors.forename: Christopher P
authors.initials: CP
authors.lastname: Vellano
authors.forename: Nicole E
authors.initials: NE
authors.lastname: Brown
authors.forename: Joe B
authors.initials: JB
authors.lastname: Blumer
authors.forename: John R
authors.initials: JR
authors.lastname: Hepler
mesh_terms: D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002470:Cell Survival; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D008954:Models, Biological; D016501:Neurites / Q000378:metabolism; D016716:PC12 Cells; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism*; D020710:RGS Proteins / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D018341:Receptors, Adrenergic, alpha-2 / Q000378:metabolism*; D018343:Receptors, Adrenergic, beta-2 / Q000378:metabolism*; D015398:Signal Transduction
chemical_list: C579276:Gnai1 protein, mouse; C522851:Gnai1 protein, rat; D020710:RGS Proteins; D018341:Receptors, Adrenergic, alpha-2; D018343:Receptors, Adrenergic, beta-2; C491399:Rgs14 protein, mouse; C106374:Rgs14 protein, rat; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; C495971:Hras protein, mouse; D016283:Proto-Oncogene Proteins p21(ras)