Palmitoylation of the recombinant human A1 adenosine receptor: enhanced proteolysis of palmitoylation-deficient mutant receptors.
Palmitoylation of the recombinant human A(1) adenosine receptor (A(1)AR) expressed 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 homogeneity from cells metabolically labelled with [(3)H]palmitate, incorporate tritium into a 38-42 kDa receptor glycoprotein. The amount of palmitoylation is not affected by incubation of cells with the A(1)AR-selective agonist N(6)-cyclopentyladenosine (CPA). A(1)AR palmitoylation is abolished by treatment with neutral hydroxylamine or by mutation of Cys-309 to Ala (C(309)-->A). Based on Western blotting and pulse-chase experiments with [(35)S]methionine, at least 90% of wild-type receptors are palmitoylated and turn over with a t1/2 of 6.4 h. Of the C(309)-->A mutated receptors, 40% appear to turn over like wild-type receptors, with a t1/2 of 7.1 h, and 60% appear to be rapidly cleaved to form a 25 kDa receptor fragment that turns over with a t1/2 of 0.8 h. In HEK-293 cell lines expressing similar numbers of wild-type or C(309)-->A mutant A(1)Rs, 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'-[gamma-thio]-triphosphate ([S]GTP)-sensitive binding to membranes by the agonist (125)I-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 A(1)ARs from a pathway leading to rapid degradation.
Gao
Z
Z
Department of Molecular Physiology and Biological Physics, University of Virginia Health Sciences Center, Charlottesville, VA 22908, USA.
Ni
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Szabo
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Linden
J
J
D000241:Adenosine / Q000031:analogs & derivatives / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D017931:DNA Primers / Q000235:genetics; D015536:Down-Regulation / Q000187:drug effects; D019204:GTP-Binding Proteins / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D019308:Palmitic Acid / Q000378:metabolism; D017354:Point Mutation; D018047:Receptors, Purinergic P1 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D017931:DNA Primers; D018047:Receptors, Purinergic P1; D011994:Recombinant Proteins; D019308:Palmitic Acid; C048599:N(6)-cyclopentyladenosine; D019204:GTP-Binding Proteins; D000241:Adenosine