Biochemical purification and functional analysis of complexes between the G-protein subunit Gbeta5 and RGS proteins

Methods Enzymol. 2004;390:149-62. doi: 10.1016/S0076-6879(04)90010-9.

Abstract

Regulator of G-protein signaling (RGS) proteins of the R7 subfamily (RGS6, 7, 9, and 11) contain a unique Ggamma-like (GGL) domain that enables their association with the G-protein beta subunit Gbeta5. The existence of these complexes was demonstrated by their purification from native tissues as well as by reconstitution in vitro. According to pulse-chase analysis, Gbeta5 and RGS7 monomers undergo rapid proteolytic degradation in cells, whereas the dimer is stable. Studies of the functional role of Gbeta5-RGS dimers using GTPase activity, ion channel, and calcium mobilization assays showed that, similarly to other RGS proteins, they can negatively regulate G-protein-mediated signal transduction. Protein-protein interactions involving the Gbeta5-RGS7 complex can be studied in cells using fluorescence resonance energy transfer utilizing Gbeta5, RGS, and Galpha subunits fused to the cyan and yellow versions of green fluorescent protein.

MeSH terms

  • Animals
  • Brain / metabolism
  • Cell Line
  • Dimerization
  • Fluorescence Resonance Energy Transfer
  • GTP-Binding Protein alpha Subunits / genetics
  • GTP-Binding Protein alpha Subunits / metabolism
  • GTP-Binding Protein beta Subunits / chemistry
  • GTP-Binding Protein beta Subunits / genetics
  • GTP-Binding Protein beta Subunits / isolation & purification*
  • GTP-Binding Protein beta Subunits / metabolism*
  • RGS Proteins / chemistry
  • RGS Proteins / genetics
  • RGS Proteins / isolation & purification*
  • RGS Proteins / metabolism*
  • Rats
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Retina / chemistry
  • Second Messenger Systems / physiology

Substances

  • GTP-Binding Protein alpha Subunits
  • GTP-Binding Protein beta Subunits
  • Gnb5 protein, rat
  • RGS Proteins
  • Recombinant Fusion Proteins