Knowledge of the nature of the interaction between the stimulatory G protein (Gs) and the adenylyl cyclase catalytic unit (C) is essential for interpreting the effects of Gs mutations and expression levels on cellular response to a wide variety of hormones, drugs, and neurotransmitters. It has been proposed that beta-adrenergic receptor activation of adenylyl cyclase occurs either by a two-step "shuttle" mechanism where the receptor activates Gs independently of cyclase followed by Gs alpha activation of cyclase independent of the receptor; or the receptor activates a "precoupled" Gs-C complex in a single step. Simulations of the two models revealed that the two forms of activation are distinguishable by the effect of Gs levels on epinephrine-stimulated EC50 values for cyclase activation; specifically, the shuttle model predicts an increased potency of epinephrine stimulation as levels of Gs alpha increase. To address this problem, S49 cyc- cells were stably transfected with the gene for Gs alpha(long) regulated by the MMTV LTR promoter, which allowed for an induction of Gs alpha(long) expression levels over a 40-fold range by incubation of the cells for various times with 5 microM dexamethasone. Expression of Gs alpha was strongly correlated to the appearance of GTP shifts in the competitive binding of epinephrine with [125I]iodocyanopindolol to the beta-adrenergic receptors and epinephrine-stimulated adenylyl cyclase activity. Most importantly, high expression of Gs alpha resulted in lower EC50 values for epinephrine and prostaglandin E1 stimulation of adenylyl cyclase activity. The decrease in EC50 did not occur as a result of a change in beta2-adrenergic receptor, Gi alpha, G betagamma, 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.