During short term agonist exposure, the alpha 2A-adrenergic receptor (alpha 2AAR) undergoes rapid functional desensitization caused by phosphorylation of the receptor by the beta-adrenergic receptor kinase (beta ARK). This signal quenching is similar in nature to that found with a number of G-protein coupled receptors in which agonist-promoted desensitization is due to beta ARK phosphorylation; like these other receptors, the precise molecular determinants of the receptor required for beta ARK phosphorylation are not known. To delineate such a motif in the human alpha 2AAR (alpha 2C10), we constructed six mutated receptors consisting of deletions or substitutions of Ser-296-299 in the EESSSS sequence of the third intracellular loop of the receptor. These were expressed in Chinese hamster ovary and COS-7 cells, and agonist-promoted desensitization and receptor phosphorylation were assessed. Deletion of the EESSSS sequence and substitution of alanine for all four serines resulted in a total loss of phosphorylation and desensitization. Mutant receptors that retained two of the original serines (AASS and SSAA) underwent agonist-promoted phosphorylation of 55 +/- 7% and 57 +/- 8% of the phosphorylation found for wild type alpha 2C10. Additional substitution mutants (SSSA and SAAA) underwent 77 +/- 1% and 27 +/- 4% of wild type phosphorylation, respectively. Thus, substitution of alanine for each additional serine decreased overall phosphorylation as compared with wild type alpha 2C10 by approximately 25%, which is consistent with all 4 serines being phosphorylated. Mutated receptors that only partially phosphorylated (as compared with wild type) failed to undergo agonist-promoted desensitization. Thus, beta ARK-mediated phosphorylation of alpha 2C10 occurs at Ser-296-299 in the third intracellular loop, and this represents the critical step in rapid agonist-promoted desensitization. A number of other G-protein coupled receptors that undergo desensitization have a sequence motif similar to that which we have found for beta ARK-mediated phosphorylation of alpha 2C10, suggesting that these receptors may also be substrates for beta ARK.

We have investigated potential G i and G s coupling domains within the intracellular regions of the ␣ 2A AR subtype using a series of nine chimeric mutations. The second intracellular loop (ICL2, amino acids 133-149) and the amino-and carboxyl-terminal regions of the third intracellular loop (ICL3, amino acids 218 -235 and 355-371, respectively) of the cloned human ␣ 2A AR were substituted with the analogous sequence from either the G s -coupled ␤ 2 AR or the G i -coupled serotonin type 1A receptor (5-HT 1A R). Mutant and wild type ␣ 2A AR were stably expressed in Chinese hamster ovary cells and functional coupling of each receptor to G i and G s was assessed in membrane adenylyl cyclase assays. Substitution of 5-HT 1A R sequence into ICL2 ablated coupling to G s but not to G i , whereas substitution of ␤ 2 AR sequence significantly depressed coupling to G i but not to G s . Thus, the ICL2 of the ␣ 2A AR contains elements essential for both signaling pathways. Substitution of either the amino-or carboxyl-terminal segments of ICL3 with 5-HT 1A R sequence ablated agonist stimulation of adenylyl cyclase activity (without affecting inhibition), suggesting that both domains are necessary for ␣ 2A AR coupling to G s . In contrast, individual substitution of ␤ 2 AR sequence into ICL3 amino or carboxyl termini had no appreciable effect on G i coupling. Concomitant substitution of ␤ 2 AR sequence into both regions substantially impaired G i coupling, implying that each is capable of independently supporting functional coupling. Substitution of 5-HT 1A R at either locus had no effect on G i coupling. Thus, for G s coupling, these two domains within ICL3 are both required for functional coupling. However, for G i coupling, the ␣ 2A AR appears to have two distinct regions within ICL3 that are capable of supporting G i coupling independently. There has been no previous elucidation of a receptor having redundant, fully competent domains for coupling to a single class of Gprotein. Such duplicity of functional domains within ␣ 2 AR may suggest strong evolutionary pressure to maintain G i coupling. Adrenergic receptors (AR)1 are members of a superfamily of integral membrane proteins that signal to the interior of the cell through heterotrimeric guanine nucleotide binding proteins or G-proteins. The AR are divided into three classes, ␣ 1 , ␣ 2 , and ␤, which are differentiated by their relative selectivity for certain ligands, signal transduction pathways, and molecular structure. Each class of AR has been further divided into several pharmacological subtypes, each of which represents distinct receptors, as demonstrated by the cloning and recombinant expression of their respective genes from a variety of species, including human. There are three cloned human ␣ 2 AR, ␣ 2 C10, ␣ 2 C4, and ␣ 2 C2, that correspond to the pharmacologically defined subtypes ␣ 2A , ␣ 2C , and ␣ 2B , respectively (1-3). The ␣ 2 AR display a wide distribution in both peripheral (4, 5) and central (5-7) tissues and mediate a variety of physiological respon...

␣ 2 -Adrenergic receptors (␣ 2 AR) functionally couple not only to G i but also to G s . We investigated the aminoterminal portion of the third intracellular loop of the human ␣ 2A AR (␣ 2 C10) for potential G s coupling domains using site-directed mutagenesis and recombinant expression in several different cell types. A deletion mutant and four chimeric receptors consisting of the ␣ 2A AR with the analogous sequence from the 5-HT 1A receptor (a G i -coupled receptor) and the ␤ 2 AR (a G s -coupled receptor) were expressed in Chinese hamster ovary cells, Chinese hamster fibroblasts, or COS-7 cells and examined for their ability to mediate stimulation or inhibition of membrane adenylyl cyclase activity or whole cell cAMP accumulation.In stably expressing Chinese hamster ovary cells, deletion of amino acids 221-231, which are in close proximity to the fifth transmembrane domain, eliminated ␣ 2 C10-mediated stimulation of adenylyl cyclase activity, while ␣ 2 C10-mediated inhibition was only moderately affected. This suggested that this region is important for G s coupling, prompting construction of the chimeric receptor mutants. Substitution of amino acids 218 -235 with 5-HT 1A receptor sequence entirely ablated agonistpromoted G s coupling, as compared with a 338 ؎ 29% stimulation of adenylyl cyclase activity observed with the wild-type ␣ 2 C10. In contrast, G i coupling for this mutant remained fully intact (57 ؎ 2% versus 52 ؎ 1% inhibition for wild-type ␣ 2 C10). Similar substitution with ␤ 2 AR sequence had no effect on G i coupling but did reduce G s coupling. Two additional mutated ␣ 2 C10 containing smaller substitutions of the amino-terminal region with 5-HT 1A receptor sequence at residues 218 -228 or 229 -235 were then studied. While G i coupling remained intact with both mutants, G s coupling was ablated in the former but not the latter mutant receptor. Similar results were obtained using transfected Chinese hamster fibroblasts (which exclusively display ␣ 2 AR-G i coupling) and COS-7 cells (which exclusively display ␣ 2 AR-G s coupling). Thus, a critical determinant for G s coupling is contained within 11 amino acids (218 -228) of the amino-terminal region of the third intracellular loop localized directly adjacent to the fifth transmembrane domain.Taken together, these studies demonstrate the presence of a discrete structural determinant for agonistpromoted ␣ 2 AR-G s coupling, which is distinct and separable from the structural requirements for ␣ 2 AR-G i coupling.Activation of cellular signaling pathways by many hormones and neurotransmitters occurs via interaction with members of a superfamily of integral cellular membrane receptors that physically bind and activate heterotrimeric guanine nucleotide binding proteins (G-proteins).1 G-protein coupled receptors have an extracellular amino terminus and intracellular carboxyl terminus and are thought to span the cellular membrane seven times producing three extracellular and three intracellular loops. Chimeric receptor (1-6), site-directed mutagenesis (...

Most guanine nucleotide binding protein (G protein)-coupled receptors have a conserved cysteine in the C-terminal cytoplasmic tail near the seventh transmembrane spnning region. This cysteine is known to be palmitoylated in rhodopsin, the P2-adrenergic receptor (AJAR) and the a2A-adrenergic receptor (a2AAR). For the J2AR, this cysteine has been shown to be important for stimulatory G protein (Gs) coupling and agonist-promoted desensitization. For the a 2AAR (human a2C10) (32AR) (5), and the a2AAR (6). The sites for palmitoylation of these three receptors are at conserved cysteine residues in the proximal portion of the cytoplasmic tail. For these receptors, it has been proposed that palmitoylation of this cysteine promotes association of this portion of the cytosolic tail with the cellular membrane, forming a fourth intracellular loop region (2,5,6).While studies have suggested the structural importance of this palmitoylated cysteine in the function ofthe PIAR (5) and perhaps rhodopsin (3, 7), no functional significance of palmitoylation of the a2AAR has been assigned (6). Interestingly, the human a2cAR (8) lacks this conserved cysteine found in the human a2AAR (9), having instead a phenylalanine in this position. We have recently delineated a number of different properties between these two subtypes, particularly relating to G-protein coupling (10) and agonist regulation (11,12 11178The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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