HIGH-LEVEL OF EXPRESSION OF FUNCTIONAL HUMAN PLATELET ALPHA-2-ADRENERGIC RECEPTORS IN A STABLE MOUSE C127 CELL-LINE

We have generated, by transfection and proper selection, a stable mouse C127 cell line which expresses the human α2-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) · 106 receptors per cell. Antagonist competition experiments displayed the order of potency: yohimbine > rauwolscine > phentolamine ≫ prazosin. Agonist competitions demonstrated the order of potency: p-aminoclonidine > (-)epinephrine ≫ (+)epinephrine ≫ (-)isoproterenol. This pharmacological profile is characteristic of the human platelet α2-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 μM epinephrine decreased forskolin-stimulated cellular cyclic AMP accumulation by 72%. The response was biphasic, and the attenuation effect disappeared at 100 μM epinephrine. A transfected clone which did not demonstrate detectable α2-adrenergic receptor mRNA displayed low levels of α2-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 α2-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 α2-adrenergic receptor and should aid in separating the components of various signal transduction mechanisms putatively attributed to this receptor. © 1990.