Heterotrimeric G proteins couple cell surface receptors with intracellular effectors at the cytoplasmic face of membranes. The heterotrimer consists of a subunits which bind the guanine nucleotide and bg subunits which are tightly bound together. The membrane localization of G proteins is critical for their ability to relay signal between membrane-bound receptors and effectors. The a subunits undergo the post-translational modifications of myristoylation and palmitoylation. Myristoylation, the addition of a 14 carbon fatty acid to the amino-terminal glycine, is critical for the membrane localization of a subset of a subunits primarily because it increases the affinity of the a subunit for the bg complex. Palmitoylation is the reversible addition of the 16 carbon palmitic acid to cysteine residues through a thioester bond. Using a HPLC method, we found that approximately 75% of the subunits are modified with palmitate. Mutation of the cysteine at residue 3 in ai1 prevents palmitoylation. Palmitoylation like myristoylation is critical for the membrane attachment of ai1 but is not necessary for its formation of a heterotrimer with bg. The ai1 subunit undergoes palmitoylation when membrane localized and does not require myristoylation though myristoylation can slow the turnover of palmitoylation on ai1. Mutation of cysteine 11 in a12 prevents its palmitoylation but does not change its membrane localization. The loss of palmitoylation in a constitutively active mutant of a12 prevents the oncogenic transformation seen in NIH 3T3 cells after stable expression of the proteins. However, palmitoylation is not specifically required because myristoylation can substitute for palmitoylation and restore the transformed phenotype. Tunicamycin and nitric oxide (NO) were evaluated as possible modulators of palmitoylation. Tunicamycin can inhibit the incorporation of [3H]palmitate into as and ai and may act to decrease depalmitoylation. Its effect on cAMP responses is too nonspecific to be useful as a tool to study G protein palmitoylation. Treatment with a NO donor slightly decreased palmitoylation of as and ai and caused a marked rightward shift in the isoproterenol dose response curve. This latter effect is unlikely to be due to changes in palmitoylation.
