SYNTHETIC PEPTIDES AS PROBES FOR G-PROTEIN FUNCTION - CARBOXYL-TERMINAL G-ALPHA(S) PEPTIDES MIMIC G(S) AND EVOKE HIGH-AFFINITY AGONIST BINDING TO BETA-ADRENERGIC RECEPTORS

The molecular interfaces between G(s) and the beta-adrenergic receptor were investigated using synthetic peptides corresponding to various r egions of its alpha subunit, alpha(s). These experiments were carried out on saponin-permeable C6 glioma cells in which the beta-adrenergic receptor appears tightly coupled to G(s). Synthetic site-specific pept ides from alpha(s) (corresponding to amino acids 15-29, 354-372, and 3 84-394) and alpha(i) (8-22, 315-524, and 345-455) were tested for thei r ability to interfere with coupling between the beta-adrenergic recep tor and G(s). The two carboxyl-terminal peptides from alpha(s) blocked beta-adrenergic stimulation of adenylyl cyclase in permeable cells. H owever, only alpha(s)-354-372 had this effect in C6 membranes. It is s uggested that the partial uncoupling of G(s), which occurs subsequent to cell disruption, may be related to a change in the interaction of t he alpha(s) carboxyl terminus with the beta-adrenoreceptor, Two carbox yl-terminal peptides, 354-372 and 384-394, could also mimic the effect of G(s) to increase agonist affinity for the beta-adrenergic receptor . In combination, alpha(s)-354-372 and alpha(s)-384-394 increased the ability of isoproterenol to compete with I-125-pindolol binding in a p artially additive manner. Synthetic peptides from alpha(i) and amino-t erminal peptides from alpha(s) had no effect on beta-agonist binding, suggesting a high specificity of peptide effects. Two findings suggest that these peptides bind directly to the beta-adrenergic receptor and stabilize its high agonist affinity conformation. First, GTP and hydr olysis-resistant GTP analogs did not alter the high affinity binding i n the presence of high concentrations of the peptides. Second, in S49 lymphoma cyc(-) cells, which lack G(s), these peptides evoked the high affinity agonist binding state of the beta-receptor. Neither peptide had an effect on antagonist binding affinity, as measured by propranol ol displacement of I-125-pindolol. These data suggest that at least tw o regions on the alpha subunit of G(s) participate in high affinity G( s) binding to the beta-adrenergic receptor. The fact that these small peptides could mimic the holo-G(s) effect on the receptor is rather su rprising, and the specificity of the effect suggests that the primary and secondary structure of small regions of alpha(s) contain much of t he information for specific interaction with beta-adrenergic receptors .