A single mutation Asp229 --> Ser confers upon Gs alpha the ability to interact with regulators of G protein signaling.
RGS proteins (regulators of G protein signaling) are GTPase activating proteins (GAPs) for Gi and Gq families of heterotrimeric G proteins but have not been found to interact with Gs alpha. The Gs alpha residue Asp229 has been suggested to be responsible for the inability of RGS proteins to interact with Gs alpha [Natochin, M., and Artemyev, N. O. (1998) J. Biol. Chem. 273, 4300-4303]. To test this hypothesis, we have investigated the possibility of generating an interaction between Gs alpha and RGS proteins by substituting Gs alpha Asp229 with Ser and replacing the potential Gs alpha Asp229 contact residues in RGS16, Glu129 and Asn131, by Ala and Ser, respectively. RGS16 and its mutants failed to interact with Gs alpha. A single mutation of Gs alpha, Asp229Ser, rendered the Gs alpha subunit with the ability to interact with RGS16 and RGS4. Like RGS protein binding to Gi and Gq alpha-subunits, RGS16 preferentially recognized the AlF4--bound conformation of Gs alpha Asp229Ser. In a single-turnover assay, RGS16 maximally stimulated GTPase activity of Gs alpha Asp229Ser by approximately 5-fold with an EC50 value of 7.5 microM. Our findings demonstrate that Asp229 of Gs alpha represents a major barrier for Gs alpha interaction with known RGS proteins.
Natochin
M
M
Department of Physiology and Biophysics, University of Iowa College of Medicine, Iowa City 52242, USA.
Artemyev
N O
NO
D000077410:Aluminum Chloride; D017607:Aluminum Compounds / Q000494:pharmacology; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D001224:Aspartic Acid / Q000235:genetics*; D002417:Cattle; D002712:Chlorides / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects / Q000235:genetics; D020558:GTP Phosphohydrolases / Q000378:metabolism; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000502:physiology*; D020690:GTPase-Activating Proteins; D015870:Gene Expression; D016297:Mutagenesis, Site-Directed; D011485:Protein Binding; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D020710:RGS Proteins; D012694:Serine / Q000235:genetics*; D015398:Signal Transduction / Q000235:genetics*; D012969:Sodium Fluoride / Q000494:pharmacology; D015293:Transducin / Q000378:metabolism
D017607:Aluminum Compounds; D002712:Chlorides; D020690:GTPase-Activating Proteins; D011506:Proteins; D020710:RGS Proteins; C107413:RGS16 protein; C098799:RGS4 protein; D001224:Aspartic Acid; D000077410:Aluminum Chloride; D012694:Serine; D012969:Sodium Fluoride; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins; D019205:GTP-Binding Protein alpha Subunits, Gs; D015293:Transducin