Expression and analysis of G(sα) mutants with decreased ability to activate adenylylcyclase

H. Itoh, A. G. Gilman

Research output: Contribution to journalArticle

58 Citations (Scopus)

Abstract

We have constructed mutants of the α subunit of G(s) in an attempt to identify sites in the protein that are important for its interaction with adenylylcylase. Some residues specific for those G proteins that activate adenylylcyclase were replaced with residues characteristic of G(iα). Mutant proteins were expressed in Escherichia coli, and two of these were purified to homogeneity and characterized in detail. Mutation of trp263, leu268, or arg269 caused a significant loss of the capacity of G(sα) to stimulate adenylylcyclase, and the triple mutant had less than 1% of the ability of wild type G(sα) to activate the enzyme. Guanine nucleotide binding and gtp hydrolysis by the mutant proteins were unaltered, as was guanosine 5'-3-O-(thio)triphosphate-induced enhancement of intrinsic tryptophan fluorescence. Mutant proteins also appeared to have a reduced affinity for the G protein βγ subunit complex. Secondary structure analysis and comparison with the structure of p21(ras) suggests that the region of G(sα) that we have identified is part of a loop that may be involved in interaction of the protein with adenylylcyclase. Although these residues are essential for activation of adenylylcyclase, they are not sufficient to do this when placed in the context of another G protein α subunit.

Original languageEnglish (US)
Pages (from-to)16226-16231
Number of pages6
JournalJournal of Biological Chemistry
Volume266
Issue number24
StatePublished - 1991

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Mutant Proteins
GTP-Binding Proteins
Protein Subunits
Proto-Oncogene Proteins p21(ras)
Guanosine 5'-O-(3-Thiotriphosphate)
Guanine Nucleotides
Tryptophan
Escherichia coli
Hydrolysis
Proteins
Fluorescence
Chemical activation
Mutation
Enzymes

ASJC Scopus subject areas

  • Biochemistry

Cite this

Expression and analysis of G(sα) mutants with decreased ability to activate adenylylcyclase. / Itoh, H.; Gilman, A. G.

In: Journal of Biological Chemistry, Vol. 266, No. 24, 1991, p. 16226-16231.

Research output: Contribution to journalArticle

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