Zh. Gao et al., Palmitoylation of the recombinant human A(1) adenosine receptor: enhanced proteolysis of palmitoylation-deficient mutant receptors, BIOCHEM J, 342, 1999, pp. 387-395
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.