Mutations of G(sα) designed to alter the reactivity of the protein with bacterial toxins. Substitutions at Arg187 results in loss of GTPase activity

M. Freissmuth, A. G. Gilman

Research output: Contribution to journalArticle

132 Citations (Scopus)

Abstract

We have introduced two types of mutations into cDNAs that encode the α subunit of G(s), the guanine nucleotide-binding regulatory protein that stimulates adenylyl cyclase. The arginine residue (Arg187) that is the presumed site of ADP-ribosylation of G(sα) by cholera toxin has been changed to Ala, Glu, or Lys. The rate constant for hydrolysis of GTP by all of these mutants is reduced approximately 100-fold compared with the wild-type protein. As predicted from this change, these proteins activate adenylyl cyclase constitutively in the presence of GTP. Despite these substitutions, cholera toxin still catalyzes the incorporation of 0.2-0.3 mol of ADP-ribose/mol of mutant α subunit. The sequence near the carboxyl terminus of G(sα) was altered to resemble those in G(iα) polypeptides, which are substrates for pertussis toxin. Despite this change, the mutant protein is a poor substrate for pertussis toxin. Although this protein has unaltered rates of GDP dissociation and GTP hydrolysis, its ability to activate adenylyl cyclase in the presence of GTP is enhanced by 3-fold when compared with the wild-type protein but only when these assays are performed after reconstitution of G(sα) into cyc- (G(sα)-deficient) S49 cell membranes.

Original languageEnglish (US)
Pages (from-to)21907-21914
Number of pages8
JournalJournal of Biological Chemistry
Volume264
Issue number36
StatePublished - 1989

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Bacterial Toxins
GTP Phosphohydrolases
Guanosine Triphosphate
Substitution reactions
Adenylyl Cyclases
Mutation
Cholera Toxin
Pertussis Toxin
Proteins
Hydrolysis
Adenosine Diphosphate Ribose
Mutant Proteins
GTP-Binding Proteins
Guanine Nucleotides
Adenosine Diphosphate
Arginine
Substrates
Cell membranes
Carrier Proteins
Complementary DNA

ASJC Scopus subject areas

  • Biochemistry

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Mutations of G(sα) designed to alter the reactivity of the protein with bacterial toxins. Substitutions at Arg187 results in loss of GTPase activity. / Freissmuth, M.; Gilman, A. G.

In: Journal of Biological Chemistry, Vol. 264, No. 36, 1989, p. 21907-21914.

Research output: Contribution to journalArticle

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