RGS-insensitive G-protein mutations to study the role of endogenous RGS proteins.
Regulator of G-protein signaling (RGS) proteins are very active GTPase-accelerating proteins (GAPs) in vitro and are expected to reduce signaling by G-protein coupled receptors in vivo. A novel method is presented to assess the in vivo role of RGS proteins in the function of a G protein in which Galpha subunits do not bind to RGS proteins or respond with enhanced GTPase activity. A point mutation in the switch I region of Galpha subunits (G184S Galpha(o) and G183S Galpha(i1)) blocks the interaction with RGS proteins but leaves intact the ability of Galpha to couple to betagamma subunits, receptors, and downstream effectors. Expression of the RGS-insensitive mutant G184S Galpha(o) in C6 glioma cells with the micro-opioid receptor dramatically enhances adenylylcyclase inhibition and activation of extracellular regulated kinase. Introducing the same G184S Galpha(o) protein into embryonic stem (ES) cells by gene targeting allows us to assess the functional importance of the endogenous RGS proteins using in vitro differentiation models and in intact mice. Using ES cell-derived cardiocytes, spontaneous and isoproterenol-stimulated beating rates were not different between wild-type and G184S Galpha(o) mutant cells; however, the bradycardiac response to adenosine A1 receptor agonists was enhanced significantly (seven-fold decrease EC50) in Galpha(o)RGSi mutant cells compared to wild-type Galpha(o), indicating a significant role of endogenous RGS proteins in cardiac automaticity regulation. The approach of using RGS-insensitive Galpha subunit knockins will reveal the role of RGS protein-mediated GAP activity in signaling by a given G(i/o) protein. This will reveal the full extent of RGS regulation and will not be confounded by redundancy in the function of multiple RGS proteins.
Fu
Ying
Y
Department of Pharmacology, University of Michigan, Ann Arbor 48105, USA.
Zhong
Huailing
H
Nanamori
Masakatsu
M
Mortensen
Richard M
RM
Huang
Xinyan
X
Lan
Kengli
K
Neubig
Richard R
RR
D000818:Animals; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000235:genetics / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000235:genetics*; D020690:GTPase-Activating Proteins / Q000378:metabolism; D006801:Humans; D017354:Point Mutation; D020710:RGS Proteins / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction
D020690:GTPase-Activating Proteins; D020710:RGS Proteins; D019204:GTP-Binding Proteins; D019206:GTP-Binding Protein alpha Subunits, Gi-Go