Acylation of G alpha(13) is important for its interaction with thrombin receptor, transforming activity and actin stress fiber formation

Palmitoylation of alpha-subunits in heterotrimeric G proteins has become a research object of growing attention. Following our recent report on the ac ylation of the mono-palmitoylated G alpha(12) [Ponimaskin et al,, FEES Lett , 429 (1998) 370-374], we report here on the identification of three palmit oylation sites in the second member of the G(12) family, G alpha(13), and o n the biological significance of fatty acids on the particular sites. Using mutants of alpha(13) in which the potentially palmitoylated cysteine resid ues (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 fat ty acids are functionally defective. The following biological functions of Gals were found to be inhibited: coupling to the PAR1 thrombin receptor, ce ll transformation and actin stress fiber formation, Results from establishe d assays for the above functions with a series of mutants, including deriva tives of the constitutively active mutant G alpha(13)Q226L, revealed a grad ed inhibitory response on the above mentioned parameters, As a rule, it app ears that palmitoylation of the N-proximal sites (e.g. Cys-14 and Cys-18) c ontributes more effectively to biological function than of the acylation si te located more internally (Cys-37), However, the mutant with Cys-37 replac ed by serine is more severely inhibited in stress fiber formation (80%) tha n in cell transformation (50%), pointing to the possibility of a differenti al involvement of the three palmitoylation sites in G alpha(13). (C) 2000 F ederation of European Biochemical Societies, Published by Elsevier Science B.V. All rights reserved.