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

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)

URL: http://www.ncbi.nlm.nih.gov/pubmed/2568356

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.

Item Type: Paper
Uncontrolled Keywords: adenylate cyclase alpha 2 adrenergic receptor cyclic amp animal cell cell culture cho cell gene expression genetic engineering human human cell molecular cloning nonhuman priority journal Adrenergic alpha-Agonists Adrenergic alpha-Antagonists Amino Acid Sequence Animal Base Sequence Cloning, Molecular Cricetulus Drosophila Forskolin Hamsters Molecular Sequence Data Receptors, Adrenergic, alpha Sequence Homology, Nucleic Acid Virulence Factors, Bordetella
Subjects: bioinformatics > genomics and proteomics > analysis and processing
bioinformatics > genomics and proteomics > genetics & nucleic acid processing
bioinformatics > genomics and proteomics
Investigative techniques and equipment
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification
Investigative techniques and equipment > assays
Investigative techniques and equipment > cloning
Investigative techniques and equipment > assays > cloning

bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > genes, structure and function > gene expression
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein expression
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein receptor
CSHL Authors:
Communities: CSHL labs > McCombie lab
Depositing User: Matt Covey
Date Deposited: 25 Apr 2013 18:00
Last Modified: 25 Apr 2013 18:00
Related URLs:
URI: http://repository.cshl.edu/id/eprint/28201

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