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

J Biol Chem. 2013 Feb 1;288(5):3620-31. doi: 10.1074/jbc.M112.440057. Epub 2012 Dec 17.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Membrane / metabolism
  • Cell Survival
  • GTP-Binding Protein alpha Subunits, Gi-Go / metabolism*
  • HEK293 Cells
  • Humans
  • Mice
  • Models, Biological
  • Neurites / metabolism
  • PC12 Cells
  • Protein Binding
  • Protein Structure, Tertiary
  • Protein Transport
  • Proto-Oncogene Proteins p21(ras) / metabolism*
  • RGS Proteins / chemistry
  • RGS Proteins / metabolism*
  • Rats
  • Receptors, Adrenergic, alpha-2 / metabolism*
  • Receptors, Adrenergic, beta-2 / metabolism*
  • Signal Transduction

Substances

  • Gnai1 protein, mouse
  • Gnai1 protein, rat
  • RGS Proteins
  • Receptors, Adrenergic, alpha-2
  • Receptors, Adrenergic, beta-2
  • Rgs14 protein, mouse
  • Rgs14 protein, rat
  • GTP-Binding Protein alpha Subunits, Gi-Go
  • Hras protein, mouse
  • Proto-Oncogene Proteins p21(ras)