IDENTIFICATION OF CONSERVED AROMATIC RESIDUES ESSENTIAL FOR AGONIST BINDING AND 2ND-MESSENGER PRODUCTION AT 5-HYDROXYTRYPTAMINE(2A) RECEPTORS

Several models of agonist binding to G protein-coupled 5-hydroxytrypta mine [5-HT] (serotonin) receptors have highlighted the potential impor tance of highly conserved aromatic residues for ligand binding and ago nist efficacy. In this study, we tested these models by constructing a nd characterizing a number of point mutations of conserved and noncons erved aromatic residues using the 5-HT2A receptor as a model system. M utations of three highly conserved tryptophans (W200A, W336A, and W367 A) proposed to reside near the binding pocket markedly reduced agonist affinity and efficacy at 5-HT2A receptors. Mutations of two other hig hly conserved aromatic residues postulated to be near the agonist bind ing site (F340L and Y370A) also had dramatic effects on agonist bindin g and efficacy. Point mutations of neighboring conserved phenylalanine s (F339L and F365L) had minimal effects on agonist binding, although t he F365L mutation diminished agonist efficacy. Finally, mutations of t wo nonconserved aromatic residues (F125L and F383A) not predicted to b e near the binding packet had no effects on agonist binding, potency, or efficacy. Our results are best explained by models that suggest tha t helices III, V, VI, and VII can form a unit of interacting helices i n which highly conserved aromatic residues are oriented toward the cen ter of the helical aggregate to form an aromatic pocket. In addition, our novel results identify a series of aromatic residues essential for agonist-induced second messenger production. These results demonstrat e that highly conserved aromatic residues residing in neighboring heli ces provide the optimum environment for both agonist binding and activ ation of 5-HT2A receptors.