Experiments were conducted to examine the molecular basis for the high affinity and potency of a new class of 5-HT 2A receptor agonists, N-benzyl phenethylamines. Competition binding assays at several serotonin receptors confirmed that an N-arylmethyl substitution was necessary for affinity increases up to 300-fold over simple N-alkyl homologs, as well as enhanced selectivity for 5-HT 2A versus 5-HT 2C and 5-HT 1A receptors. PI hydrolysis functional assays confirmed that these Nbenzyl phenethylamines are potent and highly efficacious agonists at the rat 5-HT 2A receptor. Virtual docking of these compounds into a human 5-HT 2A receptor homology model indicated that the N-benzyl moiety might be interacting with Phe339 (6.51) , whereas the phenethylamine portion was likely to be interacting with Phe340 (6.52) . Experiments in h5-HT 2A receptors with Phe339 (6.51) L and Phe340 (6.52) L mutations seem to support this hypothesis. Dramatic detrimental effects on affinity, potency, and intrinsic activity were observed with the Phe339 (6.51) L mutation for all N-benzyl analogs, whereas most N-unsubstituted phenethylamines and traditional agonists were only weakly affected, if at all. Consistent with other published studies, the Phe340 (6.52) L mutation detrimentally affected affinity, potency, and intrinsic activity of nearly all compounds tested, although a strong change in intrinsic activity was not seen with most N-aryl analogs. These data further validate the topology of our h5-HT 2A receptor homology model. It is noteworthy that this study is the first to identify a hitherto unrecognized role for residue 6.51 in agonist activation of a serotonin G protein-coupled receptor (GPCR), whereas most previous reports have suggested a varied and sometimes contradictory role in homologous GPCRs.Agonist activity at the serotonin 2A (5-HT 2A ) receptor is essential for the psychopharmacology of serotonergic psychedelics such as LSD, DOI, psilocin, and 5-MeO-DMT, compounds with unique and dramatic effects on certain aspects of consciousness (Nichols 2004). Moreover, we have recently identified a functionally selective 5-HT 2A receptor agonist that selectively activates phosphoinositide turnover over production of eicosanoids (McLean et al., 2006a). A key aspect to understanding the effects on consciousness of psychedelics is the study of the receptor-ligand interaction at the molecular level and how it modulates second messenger generation subsequent to receptor activation.We have been particularly interested in experimental val-

The trace amine-associated receptor 1 (TAAR 1 ) is an aminergic G protein-coupled receptor (GPCR) potently activated by 3-iodothyronamine (1), an endogenous derivative of thyroid hormone. Structure activity relationship studies on 1 and related agonists showed that the rat and mouse species of TAAR 1 accommodated structural modifications and functional groups on the ethylamine portion and the biaryl ether moiety of the molecule. However, the two receptors clearly exhibited distinct, speciesspecific ligand preferences despite being remarkably similar with 93% sequence similarity. In this study, we generated single and double mutants of rat and mouse TAAR 1 to probe the molecular recognition of agonists and the underlying basis for the ligand selectivity of rat and mouse TAAR 1 . Key, non-conserved specificity determinant residues in transmembranes helices 4 and 7 within the ligand binding site appear to be the primary source of a number of the observed ligand preferences. Residue 7.39 in transmembrane 7 dictated the preference for a β-phenyl ring while residue 4.56 in transmembrane 4 was partially responsible for the lower potency of 1 and tyramine for the mouse receptor. Additionally, 1 and tyramine were found to have the same binding mode in rat TAAR 1 despite structure activity relationship data suggesting the possibility of each molecule having different binding orientations. These findings provide valuable insights into the critical binding site residues involved in the ligand-receptor interaction that can influence compound selectivity and functional activity of aminergic GPCRs.