Application of RGS box proteins to evaluate G-protein selectivity in receptor-promoted signaling.
Regulator of G-protein signaling (RGS) domains bind directly to GTP-bound Galpha subunits and accelerate their intrinsic GTPase activity by up to several thousandfold. The selectivity of RGS proteins for individual Galpha subunits has been illustrated. Thus, the expression of RGS proteins can be used to inhibit signaling pathways activated by specific G protein-coupled receptors (GPCRs). This article describes the use of specific RGS domain constructs to discriminate among G(i/o), Gq-and G(12/13)-mediated activation of phospholipase C (PLC) isozymes in COS-7 cells. Overexpression of the N terminus of GRK2 (amino acids 45-178) or p115 RhoGEF (amino acids 1-240) elicited selective inhibition of Galphaq- or Galpha(12/13)-mediated signaling to PLC activation, respectively. In contrast, RGS2 overexpression was found to inhibit PLC activation by both G(i/o)- and Gq-coupled GPCRs. RGS4 exhibited dramatic receptor selectivity in its inhibitory actions; of the G(i/o)- and Gq-coupled GPCRs tested (LPA1, LPA2, P2Y1, S1P3), only the Gq-coupled lysophosphatidic acid-activated LPA2 receptor was found to be inhibited by RGS4 overexpression.
Department of Pharmacology, The University of North Carolina School of Medicine, Chapel Hill 27599, USA.
Melinda D
MD
Hains
David P
DP
Siderovski
T Kendall
TK
Harden
D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D004789:Enzyme Activation; D043802:GTP-Binding Protein alpha Subunits, Gq-G11 / Q000037:antagonists & inhibitors / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism*; D020690:GTPase-Activating Proteins / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D017434:Protein Structure, Tertiary; D020710:RGS Proteins / Q000737:chemistry* / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D012680:Sensitivity and Specificity; D015398:Signal Transduction; D010738:Type C Phospholipases / Q000378:metabolism
D020690:GTPase-Activating Proteins; D007527:Isoenzymes; D020710:RGS Proteins; D043562:Receptors, G-Protein-Coupled; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins; D043802:GTP-Binding Protein alpha Subunits, Gq-G11