Palmitoylation of the recombinant human A1 adenosine receptor (A1AR) expressed in HEK-293 cells is demonstrated by showing that hexahistidine (His6)/Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (FLAG) (H/F) A1ARs, purified to homogeneity from cells metabolically labelled with [3H]palmitate, incorporate tritium into a 38-42 kDa receptor glycoprotein. The amount of palmitoylation is not affected by incubation of cells with the A1AR-selective agonist N6-cyclopentyladenosine (CPA). A1AR palmitoylation is abolished by treatment with neutral hydroxylamine or by mutation of Cys-309 to Ala (C309 → A). Based on Western blotting and pulse-chase experiments with [35S]methionine, at least 90% of wild-type receptors are palmitoylated and turn over with a t½ of 6.4 h. Of the C309 → A mutated receptors, 40% appear to turn over like wild-type receptors, with a t½ of 7.1 h, and 60% appear to be rapidly cleaved to form a 25 kDa receptor fragment that turns over with a t½ of 0.8 h. In HEK-293 cell lines expressing similar numbers of wild-type or C309 → A mutant A1Rs, there is little difference in the kinetics of CPA-induced receptor internalization (1 h), down-regulation (24 h), inhibition of forskolin-stimulated cAMP accumulation, or activation of co-transfected G-protein-activated inward rectifier K+/cardiac inward rectifying K+ (GIRK1/CIR K+) channels. Also unaffected by palmitoylation is guanosine 5′-[γ-thio]-triphosphate ([S]GTP)-sensitive binding to membranes by the agonist 125I-labelled aminobenzyladenosine. The results suggest that palmitoylation has little effect on receptor-effector coupling, agonist-induced internalization or down-regulation. We speculate that palmitoylation may divert newly synthesized A1ARs from a pathway leading to rapid degradation.

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