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 language||English (US)|
|Number of pages||6|
|Journal||Journal of Biological Chemistry|
|State||Published - Oct 29 1991|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology