A cysteine-11 to serine mutant of Gα12 impairs activation through the thrombin receptor

We have recently reported that G alpha(12) is acylated with palmitic acid [Veit et al,, FEES Lett. 339 (1994) 160-164], Here we identify cysteine 11 as the sole palmitoylation site and assess the function of G alpha(12) palmitoylation after expression of wild type and acylation-deficient mutant in insect cells. Our experimental approach yielded the following results. (1) Palmitoylation of G alpha(12) has no influence on the subunit interactions, (2) Palmitoylation promotes membrane binding of G alpha(12) when this protein is expressed alone. Membrane attachment of the heterotrimer occurs independent of the presence of fatty acids in G alpha(12). (3) Assays for agonist-stimulated binding of [S-35]GTP gamma S after expression of the human thrombin receptor (PAR1) along with G alpha(12) and the beta gamma subunits revealed a 70% inhibition with the palmitoyl-deficient mutant. (C) 1998 Federation of European Biochemical Societies.