EVIDENCE FOR THE SHUTTLE MODEL FOR G(S)ALPHA ACTIVATION OF ADENYLYL-CYCLASE

Knowledge of the nature of the interaction between the stimulatory G p rotein (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 neurotr ansmitters. It has been proposed that beta-adrenergic receptor activat ion of adenylyl cyclase occurs either by a two step ''shuttle'' mechan ism where the receptor activates G(s) independently of cyclase followe d by G(s) alpha activation of cyclase independent of the receptor; or the receptor activates a ''precoupled'' G(s)-C complex in a single ste p. Simulations of the two models revealed that the two forms of activa tion are distinguishable by the effect of G(s) levels on epinephrine-s timurated EC50 values for cyclase activation; specifically, the shuttl e model predicts an increased potency of epinephrine stimulation as le vers of G(s) alpha increase. To address this problem, S49 cyc(-) cells were stably transfected with the gene for G(s) alpha(long) regulated by the MMTV LTR promoter, which allowed for an induction of G(s) alpha (long) expression levels over a 40-fold range by incubation of the cel ls for various times with 5 mu M dexamethasone. Expression of G(s) alp ha was strongly correlated to the appearance of GTP shifts in the comp etitive binding of epinephrine with [I-125]iodocyanopindolol to the be ta-adrenergic receptors and epinephrine-stimulated adenylyl cyclase ac tivity. Most importantly, high expression of G(s) alpha resulted in lo wer EC50 values for epinephrine and prostaglandin E-1 stimulation of a denylyl cyclase activity. The decrease in Ec(50) did not occur as a re sult of a change in beta(2)-adrenergic receptor, G(i) alpha, G beta ga mma, or adenylyl cyclase levels. These novel findings demonstrate that a change in the level of a protein downstream of a plasma membrane re ceptor can influence hormone potency. We explain these results by usin g kinetic arguments to suggest that some fraction of hormone-activated adenylyl cyclase occurs via a shuttle mechanism, and not a purely pre coupled mechanism. (C) 1997 Elsevier Science Inc.