Fraser, C. M., Arakawa, S., McCombie, W. R., Venter, J. C. (1989) Cloning, sequence analysis, and permanent expression of a human α2-adrenergic receptor in Chinese hamster ovary cells. Evidence for independent pathways of receptor coupling to adenylate cyclase attenuation and activation. Journal of Biological Chemistry, 264 (20). pp. 11754-11761. ISSN 00219258 (ISSN)
Abstract
The gene encoding a human α2-adrenergic receptor was isolated from a human genomic DNA library using a 367-base pair fragment of Drosophila genomic DNA that exhibited 54% identity with the human β2-adrenergic receptor and 57% identity with the human α2-adrenergic receptor. The nucleotide sequence of a fragment containing the human α2-receptor gene and 2.076 kilobases of untranslated 5' sequence was determined, and potential upstream regulatory regions were identified. This gene encodes a protein of 450 amino acids and was identified as an α2-adrenergic receptor by homology with published sequences and by pharmacological characterization of the protein expressed in cultured cells. Permanent expression of the α2-receptor was achieved by transfecting Chinese hamster ovary (CHO) cells which lack adrenergic receptors with a 1.5-kilobase NcoI-HindIII fragment of the genomic clone containing the coding region of the gene. The α2-receptor expressed in CHO cells displayed pharmacology characteristic of an α2A-receptor subtype with a high affinity for yohimbine (K(i) = 1 nM) and a low affinity for prazosin (K(i) = 10,000 nM). Agonists displayed a rank order of potency in radioligand binding assays of para-aminoclonidine ≥ UK-14304 > (-)-epinephrine > (-)-norepinephrine > (-)-isoproterenol, consistent with the identification of this protein as an α2-receptor. The role of the α2-receptor in modulating intracellular cyclic AMP concentrations was investigated in three transferred cell lines expressing 50, 200, and 1200 fmol of receptor/mg membrane protein. At low concentrations (1-100 nM), (-)-epinephrine attenuated forskolin-stimulated cyclic AMP accumulation by up to 60% in a receptor density-dependent manner. At epinephrine concentrations above 100 nM, cyclic AMP levels were increased up to 140% of the forskolin-stimulated level. Pertussis toxin pretreatment of cells eliminated α2-receptor-mediated attenuation of forskolin-stimulated cyclic AMP levels and enhanced the receptor density-dependent potentiation of forskolin-stimulated cyclic AMP concentrations from 3 to 8-fold. Potentiation of forskolin-stimulated cyclic AMP levels was also elicited by the α2-adrenergic agonists, UK-14304 and para-aminoclonidine, and blocked by the α2-adrenergic antagonist yohimbine, but not by the α1-adrenergic antagonist prazosin or the β-adrenergic antagonist propranolol. α2-receptor-mediated potentiation of forskolin-stimulated adenylate cyclase activity is apparently not due to activation of phospholipase C, as epinephrine had no effect on phosphoinositide hydrolysis in transfected cells, or to Na+/H+ exchange, as the potentiation was unaffected by ethylisopropylamiloride at concentrations up to 100 μM. The nonselective phospholipase A2 inhibitor quinacrine antagonized the α2-receptor-mediated stimulation of cyclic AMP production in pertussis toxin-treated cells in a dose-dependent manner. In cells treated with quinacrine in the absence of pertussis toxin, epinephrine produced up to a 90% inhibition of forskolin-stimulated cyclic AMP concentrations in a dose-dependent manner with no increases in cyclic AMP production. These data suggest that the human α2-adrenergic receptor in CHO cells may simultaneously couple to more than one effector including a pertussis toxin-sensitive attenuation of adenylate cyclase and a pertussis toxin-insensitive pathway that results in potentiation of intracellular cyclic AMP levels.
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