MUTAGENESIS AND MODELING OF THE NEUROTENSIN RECEPTOR NTR1 - IDENTIFICATION OF RESIDUES THAT ARE CRITICAL FOR BINDING SR-48692, A NONPEPTIDENEUROTENSIN ANTAGONIST

The two neurotensin receptor subtypes known to date, NTR1 and NTR2, be long to the family of G-protein-coupled receptors with seven putative transmembrane domains (TM). SR 48692, a nonpeptide neurotensin antagon ist, is selective for the NTR1. In the present study we attempted, thr ough mutagenesis and computer-assisted modeling, to identify residues in the rat NTR1 that are involved in antagonist binding and to provide a tentative molecular model of the SR 48692 binding site. The seven p utative TMs of the NTR1 were defined by sequence comparison and alignm ent of bovine rhodopsin and G-protein-coupled receptors. Thirty-five a mino acid residues within or flanking the TMs were mutated to alanine. Additional mutations were performed for basic residues. The wild type and mutant receptors were expressed in COS M6 cells and tested for th eir ability to bind I-125-NT and [H-3]SR 48692. A tridimensional model of the SR 48692 binding site was constructed using frog rhodopsin as a template. SR 48692 was docked into the receptor, taking into account the mutagenesis data for orienting the antagonist. The model shows th at the antagonist binding pocket lies near the extracellular side of t he transmembrane helices within the first two helical turns. The data identify one residue in TM 4, three in TM 6, and four in TM 7 that are involved in SR 48692 binding, Two of these residues, Arg(327) in TM 6 and Tyr(351) in TM 7, play a key role in antagonist/receptor interact ions. The former appears to form an ionic link with the carboxyIic gro up of SR 48692, as further supported by structure-activity studies usi ng SR 48692 analogs. The data also show that the agonist and antagonis t binding sites in the rNTR1 are different and help formulate hypothes es as to the structural basis for the selectivity of SR 48692 toward t he NTR1 and NTR2.