The alpha-helical domain of Galphat determines specific interaction with regulator of G protein signaling 9

J Biol Chem. 1999 Mar 26;274(13):8770-8. doi: 10.1074/jbc.274.13.8770.

Abstract

RGS proteins (regulators of G protein signaling) are potent accelerators of the intrinsic GTPase activity of G protein alpha subunits (GAPs), thus controlling the response kinetics of a variety of cell signaling processes. Most RGS domains that have been studied have relatively little GTPase activating specificity especially for G proteins within the Gi subfamily. Retinal RGS9 is unique in its ability to act synergistically with a downstream effector cGMP phosphodiesterase to stimulate the GTPase activity of the alpha subunit of transducin, Galphat. Here we report another unique property of RGS9: high specificity for Galphat. The core (RGS) domain of RGS9 (RGS9) stimulates Galphat GTPase activity by 10-fold and Galphai1 GTPase activity by only 2-fold at a concentration of 10 microM. Using chimeric Galphat/Galphai1 subunits we demonstrated that the alpha-helical domain of Galphat imparts this specificity. The functional effects of RGS9 were well correlated with its affinity for activated Galpha subunits as measured by a change in fluorescence of a mutant Galphat (Chi6b) selectively labeled at Cys-210. Kd values for RGS9 complexes with Galphat and Galphai1 calculated from the direct binding and competition experiments were 185 nM and 2 microM, respectively. The gamma subunit of phosphodiesterase increases the GAP activity of RGS9. We demonstrate that this is because of the ability of Pgamma to increase the affinity of RGS9 for Galphat. A distinct, nonoverlapping pattern of RGS and Pgamma interaction with Galphat suggests a unique mechanism of effector-mediated GAP function of the RGS9.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • 3',5'-Cyclic-GMP Phosphodiesterases / metabolism
  • Eye Proteins / genetics
  • Eye Proteins / metabolism
  • Fluorescence
  • GTP Phosphohydrolases / metabolism
  • GTP-Binding Proteins / genetics
  • GTP-Binding Proteins / metabolism
  • GTPase-Activating Proteins
  • Kinetics
  • Models, Molecular
  • Protein Binding
  • Protein Structure, Secondary*
  • Proteins / genetics*
  • Proteins / metabolism
  • Transducin / genetics*
  • Transducin / metabolism

Substances

  • Eye Proteins
  • GTPase-Activating Proteins
  • Proteins
  • 3',5'-Cyclic-GMP Phosphodiesterases
  • GTP Phosphohydrolases
  • GTP-Binding Proteins
  • Transducin