SYNERGISM OF CONSTITUTIVE ACTIVITY IN ALPHA(1)-ADRENERGIC RECEPTOR ACTIVATION

Recently a number of mutations have been found in vitro which maintain cll-adrenergic receptors (ARs) in a partially activated form, We have previously identified two amino acid residue positions in the alb-adr energic receptor (AR), Cys(128) and Ala(204), one in each of the third and fifth transmembrane segments, that constitutively activate the re ceptor when substituted for a phenylalanine or valine, respectively [P erez et al. (1996) Mel. Pharmacol. 49, 112-122; Hwa et al. (1996) J. B iol, Chem. 271, 7956-7964]. Another mutation analyzed previously, Ala( 293)Glu, located in the third intracellular loop, also constitutively activates the receptor [Kjelsborg et al. (1992) J, Biol. Chem. 267, 14 30-1433]. All three mutations displayed similar manifestations of cons titutive activity such as higher binding affinity and potency for agon ists as well as higher basal signal transduction as predicted by the r evised ternary complex model of receptor activation. We hypothesized t hat the individual mutations because of their critical location alter the conformation of the transmembrane helices such that mimicry occurs that partially conforms to the activated state, R. To explore whethe r these potential conformations are independent, we combined these thr ee mutations in all possible permutations. The combined triple mutatio n displays 700-fold higher binding affinity for (-)-epinephrine and 20 -fold higher basal IP3 release than the wild-type receptor, We also ob served that each mutation contributed independently and synergisticall y to both receptor agonist binding and activation with the combined mu tations' basal activity exceeding that of the fully-stimulated wild-ty pe receptor. There was also a direct correlation between epinephrine's binding affinity and the degree of constitutive activity. Because the mutations affect different transmembrane domains, these results are c onsistent with a mechanism that helical movement acts in a concerted f ashion in agonist-induced activation, a synergism predicted if multipl e helix movement is involved in receptor activation.