Palmitoylation is required for signaling functions and membrane attachment of Gq alpha and Gs alpha.
We have identified the palmitoylated cysteine residues of alpha q and alpha s, alpha subunits of two heterotrimeric G proteins. Mutational substitutions of serines for cysteines 9 and 10 in alpha q and cysteine 3 in alpha s profoundly alter behavior of the subunits expressed in HEK293 cells. Neither mutant alpha subunit incorporates palmitate; both mutant proteins are found in the soluble rather than the particulate fraction; mutant alpha q or alpha s cannot couple a co-expressed receptor to stimulation of phospholipase C or adenylylcyclase, respectively; cysteine substitution prevents a mutationally activated alpha q (R183C) from stimulating phospholipase C directly, and reduces but does not abolish the ability of a similarly activated alpha s (R201C) to stimulate cAMP synthesis. Substitution of a myristoylation sequence for the palmitoylation sites leads to labeling of alpha q and alpha s by myristate, rather than by palmitate. Myristoylation restores the abilities of both nonpalmitoylated alpha q and alpha s to attach to membranes and, in the case of alpha q, restores its ability to stimulate phospholipase C, whether triggered by the R183C mutation or by receptor activation. These findings identify palmitoylation as a critical determinant of membrane attachment for alpha q and alpha s and show that this modification is required for normal signaling by these proteins.
Wedegaertner
P B
PB
Department of Pharmacology, University of California, San Francisco 94143.
Chu
D H
DH
Wilson
P T
PT
Levis
M J
MJ
Bourne
H R
HR
D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D004252:DNA Mutational Analysis; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D019814:Myristic Acid; D009227:Myristic Acids / Q000378:metabolism; D019308:Palmitic Acid; D010169:Palmitic Acids / Q000378:metabolism*; D015398:Signal Transduction
D009227:Myristic Acids; D010169:Palmitic Acids; D019814:Myristic Acid; D019308:Palmitic Acid; D019204:GTP-Binding Proteins