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 residues 288-294 to encode the equivalent region of the human beta(2)-adrenergic receptor were examined. The basal level of inositol phosphate generation in cells expressing the CAM alpha(1B)-adrenergic receptor was greater than for the wild-type receptor. The addition of maximally effective concentrations of phenylephrine or noradrenaline resulted in substantially greater levels of inositol phosphate generation by the CAM 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 phosphate production was approx. 200-fold greater at the CAM alpha(1B)-adrenergic receptor than at the wild-type receptor. In contrast, endothelin 1, acting at the endogenously expressed endothelin ET(A) receptor, displayed 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-insensitive, G-proteins G(q) and G(11) in cells expressing either the wild-type or the CAM alpha(1B)-adrenergic receptor. The degree of down-regulation achieved was substantially greater in cells expressing the CAM alpha(1B)-adrenergic receptor at all concentrations of the agonist. However, in this assay phenylephrine displayed only a slightly greater potency at the CAM alpha(1B)-adrenergic receptor than at the wild-type re ceptor. There were no detectable differences in the basal rate of 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 degradation of these G proteins, with a greater effect at the CAM alpha(1B)-adrenergic receptor. The enhanced capacity of agonist both to stimulate second-messenger production at the CAM alpha(1B)-adrenergic receptor and to regulate cellular levels of its associated G-proteins by stimulating their rate of degradation is indicative of an enhanced stoichiometry of coupling of this form of the receptor to G(q) and G(11).