Regulation of hamster α1B-adrenoceptors expressed in Chinese hamster ovary cells

Abstract

Chinese hamster ovary (CHO) cells were stably transfected to express the hamster α 1B-adrenoeceptor, and the function and agonist-induced regulation of the binding properties of these receptors were characterized. The cells expressed approximately 230 000 receptors per cell, with a K D for [ 3H]prazosin of 140 pM. In assays of competition by epinephrine for [ 3H]prazosin binding to receptors on intact cells, 88% of the receptors were in a low affinity form. The protein kinase C activator phorbol 12-myristate, 13-acetate (PMA) did not further increase the fraction in the low affinity form, but the protein kinase C inhibitor staurosporine reduced the low affinity fraction to 51%. In sucrose density gradient centrifugation assays of receptor internalization, the percentage of receptors in the light vesicle fraction was 25% for control cells, 53% for epinephrine-pretreated cells, 44% for PMA-pretreated cells, and 53% for cells pretreated with epinephrine plus PMA. Staurosporine completely blocked PMA-induced internalization, but only partially inhibited epinephrine-induced internalization. These results suggest a relationship between low affinity binding and internalization for α 1B-adrenoceptors and the involvement of protein kinase C in both processes. Longer-term (24 h) exposure of cells to epinephrine induced an unexpected up-regulation of receptor density of approximately 2-fold that was accompanied by an increase in maximal agonist-stimulated phosphoinositide turnover. These studies document several regulatory differences between α 1B-adrenoceptors expressed in transfected CHO cells and those natively expressed in DDT 1 MF-2 hamster smooth muscle cells, and they provide additional information on the molecular mechanisms involved in agonist-induced regulation of α 1B-adrenoceptors.