MUTATIONS OF THE ALPHA-2A-ADRENERGIC RECEPTOR THAT ELIMINATE DETECTABLE PALMITOYLATION DO NOT PERTURB RECEPTOR-G-PROTEIN COUPLING

The alpha2A-adrenergic receptor (alpha(2A)AR) is coupled to a variety of effectors via pertussis toxin-sensitive GTP-binding proteins. Like most members of the G-protein-coupled receptor superfamily, the primary structure of the alpha(2A)AR possesses a putative consensus sequence for palmitoylation in the COOH terminus at Cys-442. This study demonstrates that the alpha(2A)AR incorporates [H-3] palmitic acid in metabolic labeling studies and that mutation of Cys-442 to Ala or Ser eliminates detectable H-3-palmitoylation. However, mutation of Cys-442 does not alter adrenergic ligand specificity or allosteric modulation by amphipathic agents, such as amiloride analogs. Since reports in the literature suggest that a homologous mutation in the beta2-adrenergic receptor attenuates coupling to G(s) (O'Dowd, B. F., Hnatowich, M., Caron, M. G., Lefkowitz, R. J., and Bouvier, M. (1989) J. Biol. Chem. 264, 7564-7569) whereas chemical removal of palmitate from bovine rhodopsin enhances coupling to G(t) (Morrison, D. F., O'Brien, P. J., and Pepperberg, D. R. (1991) J. Biol. Chem. 266, 20118-20123), we examined if mutation of Cys-442 and parallel loss of detectable palmitoylation alter alpha(2A)AR coupling to G-proteins. Several independent cell lines of Madin-Darby canine kidney II cells expressing wild-type (Cys-442) or mutant (Ala-442 and Ser-442) alpha(2A)ARs were established. Metabolic labeling of Madin-Darby canine kidney cells expressing wild-type (Cys442) or mutant (Ala-442) alpha(2A)ARs with [H-3]palmitic acid indicated that only wild-type Cys-442-containing receptors incorporated [H-3]palmitate, monitored following isolation of the alpha(2A)AR detergent extracts using yohimbine-agarose chromatography. Receptor-G-protein coupling was assessed by evaluating sensitivity of receptor-agonist interactions to guanine nucleotides in competition for [H-3]yohimbine antagonist binding, guanyl-5'-yl imidotrisphosphate sensitivity of pertussis toxin-sensitive p-[I-125]iodoclonidine agonist binding, and agonist-stimulated guanosine 5'-O-(thiotriphosphate) binding. Using all three approaches, no detectable change in alpha(2A)AR-G-protein coupling was apparent, in contrast to apparent opposite effects on the beta2-adrenergic receptor-G(s) and rhodopsin-G(t) coupling reported previously by others. One interpretation is that this conserved cysteine may play differing roles at different receptor-G-protein interfaces. Alternatively, this shared structural motif may play a role in not yet investigated pathways, such as receptor expression, turnover, and localization.