Mutations in the GTP-binding site of G(sα) alter stimulation of adenylyl cyclase

S. B. Masters, R. T. Miller, M. H. Chi, F. H. Chang, B. Beiderman, N. G. Lopez, H. R. Bourne

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149 Scopus citations

Abstract

Mutational replacements of specific residues in the GTP-binding pocket of the 21-kDa ras proteins (p21(ras)) reduce their GTPase activity. To test the possibility that the cognate regions of G protein α chains participate in GTP binding and hydrolysis, we compared signaling functions of normal and mutated α chains (termed α(s)) of G(s), the stimulatory regulator of adenylyl cyclase. α(s) chains were expressed in an α(s)-deficient S49 mouse lymphoma cell line, cyc- α(s) in which leucine replaces glutamine 227 (corresponding to glutamine 61 of p21(ras)) constitutively activates adenylyl cyclase and reduces the k(cat) for GTP hydrolysis more than 100-fold. There is a smaller reduction in GTPase activity in another mutant in which valine replaces glycine 49 (corresponding to glycine 12 of p21(ras)). This mutant α(s) is a poor activator of adenylyl cyclase. Moreover, the glycine 49 protein, unlike normal α(s), is not protected against tryptic cleavage by hydrolysis resistant GTP analogs; this finding suggests impairment of the mutant protein's ability to attain the active (GTP-bound) conformation. We conclude that α(s) residues near glutamine 227 and glycine 49 participate in binding and hydrolysis of GTP, although the GTP binding regions of α(s) and p21(ras) are not identical.

Original languageEnglish (US)
Pages (from-to)15467-15474
Number of pages8
JournalJournal of Biological Chemistry
Volume264
Issue number26
StatePublished - Jan 1 1989

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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    Masters, S. B., Miller, R. T., Chi, M. H., Chang, F. H., Beiderman, B., Lopez, N. G., & Bourne, H. R. (1989). Mutations in the GTP-binding site of G(sα) alter stimulation of adenylyl cyclase. Journal of Biological Chemistry, 264(26), 15467-15474.