Nonenzymatic palmitoylation at Cys 3 causes extra-activation of the α-subunit of the stimulatory GTP-binding protein Gs

Treatment of crude stimulatory GTP-binding protein of adenylyl cyclase (G(s)) from turkey erythrocyte membranes with hydroxylamine results in twofold enhancement of adenylyl cyclase activity following reconstitution with adenylyl cyclase type V expressed in Spodoptera frugiperda cells (SF9) cells. Enhancement by hydroxylamine of immunoaffinity purified G(s) was still 1.5-fold, while that of G(s) purified according to the multiple-step procedure by Northup, J. K., Sternweis, P. C., Smigel, M. D., Schleifer, L. S., Ross, E. M. & Gilman, A. G. (Proc. Natl Acad Sci. USA 78, 6516-6520, 1980) was close to unity. The alpha-subunit of the stimulatory GTP-binding protein expressed in Escherichia coli (r alpha(s)). likewise, failed to show an effect by hydroxylamine. Surprisingly, guanosine 5'-O-(3-thiotriphosphate) (GTP[S])-liganded r alpha(s), treated with palmitoyl-CoA for 14 h at 4 degrees C resulted in sixfold enhancement of reconstitutive activity. In contrast, that of the GDP-liganded r alpha(s)beta gamma heterotrimer was not improved by palmitoylation and consecutive activation with GTP[S], although incorporation of [H-3]palmitate into monomer and heterotrimer was identical. While adenylyl cyclase type-V activity reconstituted by r alpha(s) GTP[S] was not influenced by beta gamma-subunits, that activated by palmitoylated r alpha(s) . GTP[S] was considerably inhibited, suggesting a higher affinity of palmitoylated r alpha(s) for beta gamma-subunits. On treatment of either form with the proteinase Lys-C. less than 25 % of the label was found in a stable M-r 38 000 fragment with intact C terminus, but lacking the N-terminal portion. Absence of the latter did not affect activation by r alpha(s), but caused a >90% loss of extra-activation by palmitoylated r alpha(s). The results also indicate that nonenzymatic, much in the same way as physiological enzymatic, palmitoylation of alpha(s) occurs predominantly on Cps 3.