Abstract
The alpha 2A-adrenergic receptor (alpha 2AAR) 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 2AAR possesses a putative consensus sequence for palmitoylation in the COOH terminus at Cys-442. This study demonstrates that the alpha 2AAR incorporates [3H] palmitic acid in metabolic labeling studies and that mutation of Cys-442 to Ala or Ser eliminates detectable 3H-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 beta 2-adrenergic receptor attenuates coupling to Gs (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 Gt (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 2AAR 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 2AARs were established. Metabolic labeling of Madin-Darby canine kidney cells expressing wild-type (Cys-442) or mutant (Ala-442) alpha 2AARs with [3H]palmitic acid indicated that only wild-type Cys-442-containing receptors incorporated [3H]palmitate, monitored following isolation of the alpha 2AAR 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 [3H]yohimbine antagonist binding, guanyl-5'-yl imidotrisphosphate sensitivity of pertussis toxin-sensitive p-[125I]iodoclonidine agonist binding, and agonist-stimulated guanosine 5'-O-(thiotriphosphate) binding. Using all three approaches, no detectable change in alpha 2AAR-G-protein coupling was apparent, in contrast to apparent opposite effects on the beta 2-adrenergic receptor-Gs and rhodopsin-Gt 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.
Highlights
The azA-adrenergic receptor(cYZAARi)s coupled to a TheporcinebrainazA-adrenergic receptor (a2~AR)lis a variety of effectors via pertussis toxin-sensitive GTP-member of alarge superfamily of G-protein-coupled membinding proteins
Palmitate-We examined two independent clonal cell lines of LLC-PK1 cells that habdeen stablytransfected with the pCMV4vector containingthe aZAARgene to determine the ability of the aZAARto incorporate ['Hlpalmitate
T o assessfurthertheability of wild-type andmutant ~ZAARSto couple to G-proteins, we evaluated the guanine nucleotide sensitivity of agonist binding to the aPAARusing p-[12sI]iodoclonidine.The rationale for these studies is that asGpp(NH)p reverses thereceptor-G-protein complex in into the CY~AANRo. radiolabeled band was detectable in yohimbine- membranes, withaparallel loss inagonist affinity of the agarose eluates from an equivalent number of 100-mm dishes of [”] palmitate-labeled MDCK I1 parental cells as the Ala-442-74 clone
Summary
Palmitate-We examined two independent clonal cell lines of LLC-PK1 cells (designated "0" and "E" clones) that habdeen stablytransfected with the pCMV4vector containingthe aZAARgene to determine the ability of the aZAARto incorporate ['Hlpalmitate. Analysis of 5 pmol of aZAAReluted from yohimbine-agarose eluates from [3H]palmitate-labeled LLC-. PK1 0 clone cells via SDS-PAGE, fluorography, and autoradiography revealed aband migrating with an apparenmt olecular massof 67 kDa that had incorporatedradioactivity (Fig. 1).Several lines of evidence suggest that this 67-kDa bandis the aZAARF. Irst, itwas uniquely adsorbedto and elutedfrom the yohimbine-agaroseresin.Second, the[3H]palmitate-labeled band co-migrated with the protein labeled by [IZ5I]rau-. Palmitate-labeled band that was eluted from the yohimbineagarose matrix was not detected in preparationsderived from. - ent smallemr olecular mass of 17 kDa, whicahlso incorporated [3H]palmitate, was present in yohimbine-agarose eluates. It is probable that this band represents a proteolytic fragment of the azAAR since it was not present in untransfected cells,
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