Nonenzymatic palmitoylation at Cys 3 causes extra-activation of the alpha-subunit of the stimulatory GTP-binding protein Gs.
Treatment of crude stimulatory GTP-binding protein of adenylyl cyclase (Gs) 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 Gs was still 1.5-fold, while that of Gs 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 [3H]palmitate into momomer and heterotrimer was identical. While adenylyl cyclase type-V activity reconstituted by r(alpha)s x GTP[S] was not influenced by betagamma-subunits, that activated by palmitoylated r(alpha)s x GTP[S] was considerably inhibited, suggesting a higher affinity of palmitoylated r(alpha)s for betagamma-subunits. On treatment of either form with the proteinase Lys-C, less than 25% of the label was found in a stable M(r) 38000 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 Cys 3.
Mollner
S
S
Department of Physiological Chemistry II, Heinrich-Heine University, Düsseldorf, Germany.
Ferreira
P
P
Beck
K
K
Pfeuffer
T
T
D000215:Acylation; D000818:Animals; D002460:Cell Line; D003545:Cysteine / Q000378:metabolism*; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D019308:Palmitic Acid / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D014316:Tritium; D014422:Turkeys
D011994:Recombinant Proteins; D014316:Tritium; D019308:Palmitic Acid; D019205:GTP-Binding Protein alpha Subunits, Gs; D003545:Cysteine