The palmitoylated cysteine of the cytoplasmic tail of alpha 2A-adrenergic receptors confers subtype-specific agonist-promoted downregulation.
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 palmitoylated in rhodopsin, the beta 2-adrenergic receptor (beta 2AR) and the alpha 2A-adrenergic receptor (alpha 2AAR). For the beta 2AR, this cysteine has been shown to be important for stimulatory G protein (Gs) coupling and agonist-promoted desensitization. For the alpha 2AAR (human alpha 2 C10) palmitoylation occurs at Cys-442, but it is not known what function such fatty acid acylation subserves. The closely related alpha 2CAR subtype denoted alpha 2C4 lacks a cysteine in this region and has different G-protein-coupling characteristics and agonist regulatory properties as compared to alpha 2C10. To assess the role of the palmitoylcysteine in alpha 2AR function, we constructed a mutated alpha 2C10 having a phenylalanine (the analogous amino acid in the alpha 2C4 in this position) substituted for Cys-442, denoted alpha 2C10(Phe-442), and expressed this along with wild-type alpha 2C10 and alpha 2C4 in CHO cells. Functional coupling to inhibitory G protein (Gi) and to Gs was identical between wild-type alpha 2C10 and alpha 2C10(Phe-442). Agonist-promoted desensitization of both the Gi and Gs-mediated pathways was also found to be unaffected by this mutation. Cellular trafficking induced by agonist exposure was evaluated by delineation of intracellular (sequestered) versus cell surface receptors and by determination of net receptor loss. Mutation of Cys-442 did not alter the extent or rate of agonist-promoted sequestration induced by agonists or the recovery from sequestration. However, the downregulation of receptor number after prolonged agonist exposure was completely abolished by this mutation and converted alpha 2C10 to an alpha 2C4 phenotype in regard to this adaptive response. Another mutated alpha 2C10, in which Cys-442 was replaced by alanine, also failed to downregulate. Thus, the function of this cytoplasmic palmitoylcysteine is distinctly different between the alpha 2AR and other G-protein-coupled receptors such as the beta 2AR and rhodopsin, and this suggests that this molecular attribute may subserve diverse roles among members of this family of receptors. For the alpha 2ARs, this may represent an evolved feature that provides for differing needs for regulation of the alpha 2C10 and alpha 2C4 subtypes by agonist.
Department of Medicine (Pulmonary), University of Cincinnati College of Medicine, OH 45267-0564.
M G
MG
Eason
M T
MT
Jacinto
C T
CT
Theiss
S B
SB
Liggett
D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003545:Cysteine / Q000031:analogs & derivatives*; D015536:Down-Regulation; D004837:Epinephrine / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000502:physiology; D006801:Humans; D066298:In Vitro Techniques; D016297:Mutagenesis, Site-Directed; D010168:Palmitates; D018341:Receptors, Adrenergic, alpha-2 / Q000737:chemistry / Q000502:physiology*; D015398:Signal Transduction; D013329:Structure-Activity Relationship
D010168:Palmitates; D018341:Receptors, Adrenergic, alpha-2; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases; D003545:Cysteine; D004837:Epinephrine