A CONSTITUTIVELY ACTIVE MUTANT OF THE ALPHA(1B)-ADRENERGIC RECEPTOR CAN CAUSE GREATER AGONIST-DEPENDENT DOWN-REGULATION OF THE G-PROTEINS G(Q)ALPHA AND G(11)ALPHA THAN THE WILD-TYPE RECEPTOR

Rat 1 fibroblasts transfected to express either the wild-type hamster alpha(1B)-adrenergic receptor or a constitutively active mutant (CAM) form of this receptor resulting from the alteration of amino acid resi dues 288-294 to encode the equivalent region of the human beta(2)-adre nergic receptor were examined. The basal level of inositol phosphate g eneration in cells expressing the CAM alpha(1B)-adrenergic receptor wa s greater than for the wild-type receptor. The addition of maximally e ffective concentrations of phenylephrine or noradrenaline resulted in substantially greater levels of inositol phosphate generation by the C AM alpha(1B)-adrenergic receptor, although this receptor was expressed at lower steady-state levels than the wild-type receptor. The potency of both phenylephrine and noradrenaline to stimulate inositol phospha te production was approx. 200-fold greater at the CAM alpha(1B)-adrene rgic receptor than at the wild-type receptor. In contrast, endothelin 1, acting at the endogenously expressed endothelin ET(A) receptor, dis played similar potency and maximal effects in the two cell lines. The sustained presence of phenylephrine resulted in down-regulation of the alpha subunits of the phosphoinositidase C-linked, pertussis toxin-in sensitive, G-proteins G(q) and G(11) in cells expressing either the wi ld-type or the CAM alpha(1B)-adrenergic receptor. The degree of down-r egulation achieved was substantially greater in cells expressing the C AM alpha(1B)-adrenergic receptor at all concentrations of the agonist. However, in this assay phenylephrine displayed only a slightly greate r potency at the CAM alpha(1B)-adrenergic receptor than at the wild-ty pe re ceptor. There were no detectable differences in the basal rate o f G(q) alpha/G(11)alpha degradation between cells expressing the wild- type or (-) the CAM alpha(1B)-adrenergic receptor. In both cell lines the addition of phenylephrine substantially increased the rate of degr adation of these G proteins, with a greater effect at the CAM alpha(1B )-adrenergic receptor. The enhanced capacity of agonist both to stimul ate second-messenger production at the CAM alpha(1B)-adrenergic recept or and to regulate cellular levels of its associated G-proteins by sti mulating their rate of degradation is indicative of an enhanced stoich iometry of coupling of this form of the receptor to G(q) and G(11).