Cloning, sequence analysis, and permanent expression of a human alpha 2-adrenergic receptor in Chinese hamster ovary cells. Evidence for independent pathways of receptor coupling to adenylate cyclase attenuation and activation.
The gene encoding a human alpha 2-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 beta 2-adrenergic receptor and 57% identity with the human alpha 2-adrenergic receptor. The nucleotide sequence of a fragment containing the human alpha 2-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 alpha 2-adrenergic receptor by homology with published sequences and by pharmacological characterization of the protein expressed in cultured cells. Permanent expression of the alpha 2-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 alpha 2-receptor expressed in CHO cells displayed pharmacology characteristic of an alpha 2 A-receptor subtype with a high affinity for yohimbine (Ki = 1 nM) and a low affinity for prazosin (Ki = 10,000 nM). Agonists displayed a rank order of potency in radioligand binding assays of para-aminoclonidine greater than or equal to UK-14304 greater than (-)-epinephrine greater than (-)-norepinephrine greater than (-)-isoproterenol, consistent with the identification of this protein as an alpha 2-receptor. The role of the alpha 2-receptor in modulating intracellular cyclic AMP concentrations was investigated in three transfected 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 alpha 2-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 alpha 2-adrenergic agonists, UK-14304 and para-aminoclonidine, and blocked by the alpha 2-adrenergic antagonist yohimbine, but not by the alpha 1-adrenergic antagonist prazosin or the beta-adrenergic antagonist propranolol.(ABSTRACT TRUNCATED AT 400 WORDS)
Fraser
C M
CM
Section of Receptor Biochemistry and Molecular Biology, National Institute of Neurological Disorders and Stroke, Bethesda, Maryland 20892.
Arakawa
S
S
McCombie
W R
WR
Venter
J C
JC
D037361:Adenylate Cyclase Toxin; D000316:Adrenergic alpha-Agonists / Q000378:metabolism; D000317:Adrenergic alpha-Antagonists / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D005576:Colforsin / Q000494:pharmacology; D006224:Cricetinae; D003412:Cricetulus; D000242:Cyclic AMP / Q000378:metabolism; D004330:Drosophila / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D037342:Pertussis Toxin; D011942:Receptors, Adrenergic, alpha / Q000235:genetics*; D012689:Sequence Homology, Nucleic Acid; D010566:Virulence Factors, Bordetella
D037361:Adenylate Cyclase Toxin; D000316:Adrenergic alpha-Agonists; D000317:Adrenergic alpha-Antagonists; D011942:Receptors, Adrenergic, alpha; D010566:Virulence Factors, Bordetella; D005576:Colforsin; D000242:Cyclic AMP; D037342:Pertussis Toxin