Abstract
Cells of clones of rat 1 fibroblasts transfected to express the molecularly defined alpha 1A/D, alpha 1B, or alpha 1C adrenoreceptors and prelabeled with myo-[3H]inositol were each shown to generate high levels of inositol phosphates when exposed to the alpha 1 adrenoreceptor agonist phenylephrine. Maintained exposure of each of these cells to phenylephrine resulted in a large down-regulation of the receptors and also a marked down-regulation of cellular levels of both of the phosphoinositidase C-linked G-proteins Gq alpha and G11 alpha. To examine the mechanism of phenylephrine-induced down-regulation of Gq alpha and G11 alpha, pulse-chase 35S-amino acid labeling experiments were performed with each of the alpha 1A/D, alpha 1B, and alpha 1C adrenoreceptor-expressing cell lines. The rate of degradation of G11 alpha/Gq alpha, which was adequately modeled by a monoexponential with half-life between 33 and 40 h in each of the cell lines in the absence of agonist, was accelerated substantially (some 4-fold) in the presence of phenylephrine. By contrast, the rate of degradation of the G-protein Gi2 alpha, which would not be anticipated to be activated by members of the alpha 1 adrenoreceptor family, was unaltered by the presence of phenylephrine. Levels of mRNA encoding Gq alpha and G11 alpha were not substantially altered by exposure of the cells to phenylephrine in any of the cell lines studied.
Highlights
From the Molecular Pharmacology Group, Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, Scotland, United Kingdom
Three distinct al adrenoreceptor cDNA species have currently been isolated [2,3,4,5], aIAID, alB' and alC, but there has been considerable debate as to how closely these reflect the pharmacologically defined subtypes, even if all of these cDNA species show the same signal transduction mechanisms following their heterologous expression in cell lines
It has been well established that sustained exposure of many G-protein-coupled receptors to agonist can result in a reduction in cellular levels of the receptor, it has only been in the recent past that agonist-mediated reduction in cellular levels of G-proteins has been observed
Summary
Vol 270, No 29, Issue of July 21, pp. I7196-l7203, 1995 Printed in U.S.A. Three distinct al adrenoreceptor cDNA species have currently been isolated [2,3,4,5], aIAID, alB' and alC, but there has been considerable debate as to how closely these reflect the pharmacologically defined subtypes (see Ref. 1 for review), even if all of these cDNA species show the same signal transduction mechanisms following their heterologous expression in cell lines. We note that in cells expressing each ofthe three receptor species, sustained exposure to phenylephrine results in a large, selective down-regulation ofGlla and Gqa as well as in down-regulation of the receptors These are the G-proteins that have been demonstrated to couple a l adrenoreceptors to phosphoinositidase C activity and the hydrolysis of inositolcontaining phospholipids [7]. These data indicate that G-proteins activated by a receptor are degraded considerably more rapidly than those in the inactive state and provide a mechanistic explanation for how receptor agonists can control the cellular content of G-proteins, which interact with that receptor
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