Palmitoylation of the recombinant human A(1) adenosine receptor: enhanced proteolysis of palmitoylation-deficient mutant receptors

Palmitoylation of the recombinant human A, adenosine receptor (A,AR) expres sed in HEK-293 cells is demonstrated by showing that hexahistidine (His(6)) /Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (FLAG) (H/F) A(1)ARs, purified to homogene ity from cells metabolically labelled with [H-3]palmitate, incorporate trit ium into a 38-42kDa receptor glycoprotein. The amount of palmitoylation is not affected by incubation of cells with the A,AR-selective agonist N-6-cyc lopentyladenosine (CPA). A,AR palmitoylation is abolished by treatment with neutral hydroxylamine or by mutation of Cys-309 to Ala (C-309 --> A). Base d on Western blotting and pulse-chase experiments with [S-35]methionine, at least 90 % of wild-type receptors are palmitoylated and turn over with a t (1/2) of 6.4 h. Of the C-309 --> A mutated receptors, 40 % appear to turn o ver like wild-type receptors, with a t(1/2) of 7.1 h, and 60 % appear to be rapidly cleaved to form a 25 kDa receptor fragment that turns over with a t(1/2) of 0.8 h. In HEK-293 cell lines expressing similar numbers of wild-t ype or C-309, A mutant A(1)Rs, there is little difference in the kinetics o f CPA-induced receptor internalization (1 h), down-regulation (24 h), inhib ition of forskolin-stimulated cAMP accumulation, or activation of co-transf ected G-protein-activated inward rectifier K+/cardiac inward rectifying K(GIRK1/CIR K+) channels. Also unaffected by palmitoylation is guanosine 5'- [gamma-thio]triphosphate ([S]GTP)-sensitive binding to membranes by the ago nist I-125-labelled aminobenzyladenosine. The results suggest that palmitoy lation has little effect on receptor-effector coupling, agonist-induced int ernalization or down-regulation. We speculate that palmitoylation may diver t newly synthesized A(1)ARs from a pathway leading to rapid degradation.