Heterotrimeric G proteins couple cell surface receptors to intracellular effectors at the cytoplasmic face of membranes. Palmitoylation, the reversible addition of palmitic acid to cysteine residues, occurs on most alpha subunits and participates in their membrane localization. Palmitate turnover occurs upon receptor activation of the Gs protein suggesting that it regulates this signaling pathway. Since our previous studies indicated that depalmitoylation was the critical step in palmitate turnover, we have attempted to identify the G protein palmitoyl thioesterase. We are using a molecular biology approach, RT-PCR with degenerate primers or the RACE procedure, based on sequences of known palmitoyl thioesterases. We have identified two sequences. The first was highly homologous to the mammalian protein palmitoyl thioesterase with an amino acid sequence suggesting that it was a lysosomal protein and therefore unlikely to depalmitoylate alpha subunits. The other sequence is homologous to Acyl Protein Thioesterase, a soluble enzyme that can remove palmitate groups from G protein alpha subunits. We are currently analyzing its tissue distribution and enzymology. We have also evaluated the effect of nitric oxide (NO) on alpha subunit and beta adrenergic receptor palmitoylation because NO can inhibit palmitoylation of other proteins. NO decreased the incorporation of [3H]palmitate onto the beta adrenergic receptor and the G protein alpha subunit with agonist stimulation but had no effect on the palmitoylation of the basal or cholera toxin-treated alpha subunits. NO decreased the intracellular cAMP response to beta adrenergic receptor stimulation. The likely site of action was a change in the palmitate turnover of the receptor by NO. Palmitoylation may be involved in targeting G proteins to membrane microdomains enriched in cholesterol and sphingolipids. We are investigating the functional significance of these domains by depleting cellular cholesterol and performing signaling assays. The cAMP response to receptor agonists appears to be diminished with cholesterol depletion. We are locating the site on the signaling cascade effected by cholesterol depletion.
