Evidence for the shuttle model for G Sα activation of adenylyl cyclase

Abstract

Knowledge of the nature of the interaction between the stimulatory G protein (G s) and the adenylyl cyclase catalytic unit (C) is essential for interpreting the effects of G s mutations and expression levels on cellular response to a wide variety of hormones, drugs, and neurotransmitters. It has been proposed that ß-adrenergic receptor activation of adenylyl cyclase occurs either by a two-step “shuttle” mechanism where the receptor activates G s independently of cyclase followed by G sα activation of cyclase independent of the receptor; or the receptor activates a “precoupled” G s—C complex in a single step. Simulations of the two models revealed that the two forms of activation are distinguishable by the effect of G s levels on epinephrine-stimulated EC 50 values for cyclase activation; specifically, the shuttle model predicts an increased potency of epinephrine stimulation as levels of G sα increase. To address this problem, S49 cyc − cells were stably transfected with the gene for G sα long regulated by the MMTV LTR promoter, which allowed for an induction of G sα long expression levels over a 40-fold range by incubation of the cells for various times with 5 μM dexamethasone. Expression of G sα was strongly correlated to the appearance of GTP shifts in the competitive binding of epinephrine with [ 125I]iodocyanopindolol to the β-adrenergic receptors and epinephrine-stimulated adenylyl cyclase activity. Most importantly, high expression of G sα resulted in lower EC 50 values for epinephrine and prostaglandin E 1 stimulation of adenylyl cyclase activity. The decrease in EC 50 did not occur as a result of a change in β 2-adrenergic receptor, G iα, Gβα, or adenylyl cyclase levels. These novel findings demonstrate that a change in the level of a protein downstream of a plasma membrane receptor can influence hormone potency. We explain these results by using kinetic arguments to suggest that some fraction of hormone-activated adenylyl cyclase occurs via a shuttle mechanism, and not a purely precoupled mechanism.