title: High level of expression of functional human platelet alpha 2-adrenergic receptors in a stable mouse C127 cell line.
abstract: We have generated, by transfection and proper selection, a stable mouse C127 cell line which expresses the human alpha 2-adrenergic receptor gene. The size of the mRNA produced by the cloned gene is 1.8 kb. Electrophoretic analysis and autoradiography of cell membrane proteins photoaffinity labeled with p-[3H]azidoclonidine gave a broad protein band of molecular mass of approx. 64 kDa. Saturation binding with [3H]rauwolscine as ligand gave an equilibrium dissociation constant of 1.29 +/- 0.46 nM (mean +/- S.D.) and binding capacity range of 18-35 pmol/mg membrane protein, with (3-6) x 10(6) receptors per cell. Antagonist competition experiments displayed the order of potency: yohimbine greater than rauwolscine greater than phentolamine much greater than prazosin. Agonist competitions demonstrated the order of potency: p-aminoclonidine greater than (-)epinephrine much greater than (+)epinephrine much greater than (-)isoproterenol. This pharmacological profile is characteristic of the human platelet alpha 2-adrenergic receptor. The expressed receptor is able to couple to the Gi protein. Thus, when epinephrine competition for specific binding of [3H]rauwolscine was performed in the presence of 1 mM MgCl2, 1 mM Gpp[NH]p increased the Ki for epinephrine from 164 to 315 nM. Following preincubation of cultures with 1 mM isobutylmethylxanthine, 1 microM epinephrine decreased forskolin-stimulated cellular cyclic AMP accumulation by 72%. The response was biphasic, and the attenuation effect disappeared at 100 microM epinephrine. A transfected clone which did not demonstrate detectable alpha 2-adrenergic receptor mRNA displayed low levels of alpha 2-adrenergic receptor, (less than 50 fmol/mg membrane protein), similar to those found in the parent C127 cell line. In this clone, epinephrine did not attenuate but, rather, enhanced forskolin-stimulated cyclic AMP accumulation. This new C127 cell line expressing high levels of alpha 2-adrenergic receptor provides an abundant source of a single human adrenergic receptor subtype in membrane-bound conformation which is able to couple to the Gi protein and inhibit forskolin-stimulated adenylate cyclase activity. This cell line will facilitate studies of the structure: function relationship of the alpha 2-adrenergic receptor and should aid in separating the components of various signal transduction mechanisms putatively attributed to this receptor.
authors.affiliation: Section of Hypertension and Atherosclerosis, Boston University School of Medicine, MA 02118.
authors.forename: M R
authors.initials: MR
authors.lastname: Bresnahan
authors.forename: C S
authors.initials: CS
authors.lastname: Flordellis
authors.forename: D K
authors.initials: DK
authors.lastname: Vassilatis
authors.forename: S C
authors.initials: SC
authors.lastname: Makrides
authors.forename: V I
authors.initials: VI
authors.lastname: Zannis
authors.forename: H
authors.initials: H
authors.lastname: Gavras
mesh_terms: D000316:Adrenergic alpha-Agonists / Q000494:pharmacology; D000317:Adrenergic alpha-Antagonists / Q000494:pharmacology; D000818:Animals; D001667:Binding, Competitive; D001792:Blood Platelets / Q000187:drug effects / Q000378:metabolism*; D002460:Cell Line; D019204:GTP-Binding Proteins / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D008565:Membrane Proteins / Q000096:biosynthesis / Q000235:genetics; D051379:Mice; D012333:RNA, Messenger / Q000032:analysis; D011942:Receptors, Adrenergic, alpha / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
chemical_list: D000316:Adrenergic alpha-Agonists; D000317:Adrenergic alpha-Antagonists; D008565:Membrane Proteins; D012333:RNA, Messenger; D011942:Receptors, Adrenergic, alpha; D019204:GTP-Binding Proteins