Acylation of Gα13is important for its interaction with thrombin receptor, transforming activity and actin stress fiber formation

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Abstract
Palmitoylation of α-subunits in heterotrimeric G proteins has become a research object of growing attention. Following our recent report on the acylation of the mono-palmitoylated Gα12 [Ponimaskin et al., FEBS Lett. 429 (1998) 370–374], we report here on the identification of three palmitoylation sites in the second member of the G12 family, Gα13, and on the biological significance of fatty acids on the particular sites. Using mutants of α13 in which the potentially palmitoylated cysteine residues (Cys) were replaced by serine residues, we find that Cys-14, Cys-18 and Cys-37 all serve as palmitoylation sites, and that the mutants lacking fatty acids are functionally defective. The following biological functions of Gα13 were found to be inhibited: coupling to the PAR1 thrombin receptor, cell transformation and actin stress fiber formation. Results from established assays for the above functions with a series of mutants, including derivatives of the constitutively active mutant Gα13Q226L, revealed a graded inhibitory response on the above mentioned parameters. As a rule, it appears that palmitoylation of the N-proximal sites (e.g. Cys-14 and Cys-18) contributes more effectively to biological function than of the acylation site located more internally (Cys-37). However, the mutant with Cys-37 replaced by serine is more severely inhibited in stress fiber formation (80%) than in cell transformation (50%), pointing to the possibility of a differential involvement of the three palmitoylation sites in Gα13.