The effects of agonist stimulation and beta(2)-adrenergic receptor level on cellular distribution of Gs(alpha) protein

This study examines the effects of adrenergic ligands, cholera toxin, forsk olin, and varying levels of beta(2) adrenergic receptors (beta(2)AR) on the cellular distribution of Gs(alpha) subunits in CHO cells. Localization of Gs(alpha) was evaluated by confocal microscopy and beta(2)AR-mediated signa lling was assessed by adenylyl cyclase (AC) activity. In cells expressing 0 .2 pmol/mg protein beta(2)ARs (WT18), the localization of Gs(alpha) subunit was restricted to the plasma membrane region. Isoproterenol (ISO), cholera toxin or forskolin elicited redistribution of cellular Gs(alpha) so that G s(alpha) appeared as intense spots throughout the plasma membrane as well a s the cytoplasm. Exposure to a neutral beta(2)AR antagonist, alprenolol, pr evented the ISO-stimulated Gs(alpha) translocation from peripheral to inner cyto plasm. In cells expressing high level of beta(2)ARs (8.2 pmol/mg) (WT 4), basal and ISO-stimulated AC activities were significantly elevated when compared to the values detected in WT18 clone, suggesting a positive corre lation between receptor expression and receptor-mediated signalling. Basal Gs(alpha) distribution in this group was similar to that observed in ISO-, cholera toxin-, or forskolin-stimulated WT18 clone. ISO, cholera toxin, or forskolin did not change the distribution of Gs(alpha) significantly when t ested in WT4 clone. No difference in the cellular level of Gs(alpha) protei n between WT18 and WT4 clones was detected. Alprenolol did not affect the d istribution of Gs(alpha) in WT4 clone. ICI 118,551, a negative beta(2)AR an tagonist, altered Gs(alpha) distribution from a dispersed basal pattern to a membrane-confined pattern. The latter appearance was similar to that obse rved in unstimulated WT18 clone. Taken together, these data suggest that: ( 1) enhanced beta(2)AR-Gs alpha coupling induced by agonist stimulation or b y increased expression of beta(2)ARs remodel the cellular distribution of G s(alpha); (2) the alteration in Gs(alpha) distribution induced by beta(2)AR overexpression provides evidence for agonist-independent interaction of be ta(2)AR and Gs(alpha), that can be inhibited by a negative antagonist but n ot by a neutral antagonist; and (3) forskolin influences the activity state of Gs(alpha) that displays a Gs(alpha) distribution pattern comparable to that observed when Gs(alpha) is activated via beta(2)AR stimulation or dire ctly by cholera toxin. (C) 2000 Elsevier Science Inc. All rights reserved.