Mutation of cysteine 214 in Gi1 alpha subunit abolishes its endogenous GTPase activity.
The functional consequences of the mutation of a conserved Cys-214 in Galpha(i1) have been investigated. We reported herein that substitutions of Cys-214 of Galpha(i1) to either alanine or tryptophan abolished the intrinsic GTPase activity. Free phosphate release from [32P]GTP-bound Galpha(i1) C214A or [32P]GTP-bound Galpha(i1) C214W was at least 30-fold lower than that of the wild-type Galpha(i1) in single-turnover GTPase assays. Consistently, tryptic proteolysis of C214A and C214W proteins showed that they were partially protected by GTP, further confirming that the GTPase activity in both mutant proteins was impaired. Expression of C214A or C214W mutants in Chinese hamster ovary K1 cells caused significant inhibition of forskolin-stimulated adenylate cyclase activity. However, the mutations did not significantly affect the GTP[S] (guanosine 5'-[gamma-[35S]thio]triphosphate)-binding activity. Both C214A and C214W mutants serve as good substrates for pertussis toxin-catalysed ADP ribosylation, indicating that they interact well with betagamma subunits. Moreover, RGS4 protein, a GTPase-activating protein for Galpha(i1), cannot interact with Cys-214 mutants even in the presence of AlF4-, which induces the transition state of Galpha. In summary, our findings suggest that C214A or C214W are GTPase-deficient mutants and can functionally serve as constitutively active forms of Galpha(i1) in cells.
Wang
Yuren
Y
Neuroscience Discovery Research, Wyeth Research, Princeton, NJ 08543-8000, USA. wangy4@wyeth.com
Tawa
Gregory
G
Smith
Deborah
D
Krishnamurthy
Girija
G
Young
Kathleen H
KH
D000244:Adenosine Diphosphate / Q000378:metabolism; D000262:Adenylyl Cyclases / Q000378:metabolism; D000409:Alanine / Q000235:genetics / Q000378:metabolism; D019943:Amino Acid Substitution / Q000235:genetics; D000818:Animals; D016466:CHO Cells; D005576:Colforsin / Q000494:pharmacology; D006224:Cricetinae; D000242:Cyclic AMP / Q000378:metabolism; D003545:Cysteine / Q000235:genetics* / Q000378:metabolism*; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D009154:Mutation / Q000235:genetics*; D037342:Pertussis Toxin / Q000494:pharmacology; D011485:Protein Binding; D021122:Protein Subunits / Q000378:metabolism; D020710:RGS Proteins / Q000378:metabolism; D013816:Thermodynamics; D014357:Trypsin / Q000378:metabolism; D014364:Tryptophan / Q000235:genetics / Q000378:metabolism
D021122:Protein Subunits; D020710:RGS Proteins; C098799:RGS4 protein; D005576:Colforsin; D000244:Adenosine Diphosphate; D006160:Guanosine Triphosphate; D014364:Tryptophan; D000242:Cyclic AMP; D037342:Pertussis Toxin; D014357:Trypsin; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D000262:Adenylyl Cyclases; D003545:Cysteine; D000409:Alanine