β‐Adrenergic receptor desensitization of wild‐type but not cyc lymphoma cells unmasked by submillimolar Mg 2+

Abstract

Treatment with low physiological concentrations of epinephrine (5-50 nM) rapidly desensitizes β-adrenergic stimulation of cAMP formation in S49 wild-type (WT) lymphoma cells. Previous attempts to detect this early phase of desensitization in cell-free assays of adenylate cyclase (EC 4.6.1.1) after intact cell treatment were unsuccessful. We have now found that reducing the Mg²⁺ concentrations in the adenylate cyclase assays to 2+ concentrations (5-10 mm) largely obscured the desensitization. Submillimolar Mg²⁺ conditions also revealed a two- to threefold decrease in the affinity of epinephrine binding to the β-adrenergic receptor after desensitization with 20 nm epinephrine. Detection of 4β-phorbol 12-myristate 13-acetate (PMA) desensitization of the WT β-adrencrgic receptor was also dependent on low Mg²⁺ as measured either by the decrease in epinephrine stimulation of adenylate cyclase or by the reduction in the affinity of epinephrine binding. Unexpectedly, when cyc⁻ cells were pretreated with 50 nm epinephrine, the β-adrenergic stimulation of reconstituted adenylate cyclase was not desensitized. The characteristics of the Mg²⁺ effect on epinephrine- and PMA-induced desensitizations suggest a similar mechanism of action with the most likely events being phosphorylations of the β-adrenergic receptors. Our data indicate that cAMP-dependent protein kinase (EC 2.7.1.37) may play a role in the desensitization caused by low epinephrine concentrations inasmuch as this phase of desensitization did not occur in the cyc⁻. For the PMA-induced desensitization, the phosphorylation may be mediated by protein kinase C (EC 2.7.1.37).—Clark, R. B.; Friedman, J.; Johnson, J. A.; Kunkel, M. W. β-Adrenergic receptor desensitization of wild-type but not cyc lymphoma cells unmasked by submillimolar Mg²⁺. FASEB J. 1: 289-297; 1987.