Coupling of the α2-adrenergic receptor to the inhibitory G-protein G(i) and adenylate cyclase in HT29 cells

Abstract

Previous studies have established that the human colon carcinoma cell line HT29 expresses an α2-adrenergic receptor of the α2A subtype, which is negatively coupled to adenylate cyclase. The purpose of the present study was to examine the mechanisms of α2-adrenergic signal transduction in these cells. [32P]ADP-ribosylation with pertussis toxin and immunoblots using antibodies specific for the G(i) α-subunits indicated that two distinct G(i)-proteins (G(i)2 and G(i)3) were present in HT29-cell membranes. Treatment of intact cells with pertussis toxin resulted in a time-dependent decrease in the amount of [32P]ADP-ribosylatable G(i)2 and G(i)3, which coincided with a diminution in the number of α2-adrenergic receptors in high-affinity state for agonists and with a progressive loss of ability of UK14304 to inhibit forskolin-stimulated accumulation of cyclic AMP. When membranes were [32P]ADP-ribosylated with cholera toxin in the absence of exogenous added guanine nucleotides, radioactivity was incorporated into a 45 kDa polypeptide representing G8 as well as into 40-41 kDa polypeptides corresponding to G(i)3 and G(i)2. The amount of radioactivity incorporated into the two G(i)s under basal conditions was decreased by addition of the α2-antagonist RX821002. It was not significantly affected by addition of clonidine (partial α2-agonist), but was doubled by the addition of UK14304 (full α2-agonist). This effect was blocked by RX821002. Study of adenylate cyclase activity indicated that preincubation of HT29 membranes with the antibody AS/7 (anti α(i)l/α(i)2), but not with the antibody EC/2 (anti α(i) 3), attenuated the inhibitory effect of UK14304 on forskolin-stimulated adenylate cyclase. These data demonstrate that the α2A-adrenergic receptor is coupled to both G(i)2 and G(i)3, and identify G(i)2 as the major mediator of inhibition of adenylate cyclase in HT29 cells.