THE PALMITOYLATED CYSTEINE OF THE CYTOPLASMIC TAIL OF ALPHA(2A)-ADRENERGIC RECEPTORS CONFERS SUBTYPE-SPECIFIC AGONIST-PROMOTED DOWN-REGULATION

Most guanine nucleotide binding protein (G protein)-coupled receptors have a conserved cysteine in the C-terminal cytoplasmic tail near the seventh transmembrane spanning region. This cysteine is known to be pa lmitoylated in rhodopsin, the beta(2)-adrenergic receptor (beta(2)AR) and the alpha(2A)-adrenergic receptor (alpha(2A)AR). For the beta(2)AR , this cysteine has been shown to be important for stimulatory G prote in (G(s)) coupling and agonist-promoted desensitization. For the alpha (2A)AR (human alpha(2)C10) palmitoylation occurs at Cys-442, but it is not known what function such fatty acid acylation subserves. The clos ely related alpha(2C)AR subtype denoted alpha(2)C4 lacks a cysteine in this region and has different G-protein-coupling characteristics and agonist regulatory properties as compared to alpha(2)C10. To assess th e role of the palmitoylcysteine in alpha(2)AR function, we constructed a mutated alpha(2)C10 having a phenylalanine (the analogous amino aci d in the alpha(2)C4 in this position) substituted for Cys-442, denoted alpha(2)C10(Phe-442), and expressed this along with wild-type alpha(2 )C10 and alpha(2)C4 in CHO cells. Functional coupling to inhibitory G protein (G(i)) and to G(s) was identical between wild-type alpha(2)C10 and alpha(2)C10(Phe-442). Agonist-promoted desensitization of both th e G(i) and G(s)-mediated pathways was also found to be unaffected by t his mutation. Cellular trafficking induced by agonist exposure was eva luated by delineation of intracellular (sequestered) versus cell surfa ce receptors and by determination of net receptor loss. Mutation of Cy s-442 did not alter the extent or rate of agonist-promoted sequestrati on induced by agonists or the recovery from sequestration. However, th e downregulation of receptor number after prolonged agonist exposure w as completely abolished by this mutation and converted alpha(2)C10 to an alpha(2)C4 phenotype in regard to this adaptive response. Another m utated alpha(2)C10, in which Cys-442 was replaced by alanine, also fai led to downregulate. Thus, the function of this cytoplasmic palmitoylc ysteine is distinctly different between the alpha(2)AR and other G-pro tein-coupled receptors such as the beta(2)AR and rhodopsin, and this s uggests that this molecular attribute may subserve diverse roles among members of this family of receptors. For the alpha(2)ARs, this may re present an evolved feature that provides for differing needs for regul ation of the alpha(2)C10 and alpha(2)C4 subtypes by agonist.