A single amino acid of the human and rat neurotensin receptors (subtype 1)determining the pharmacological profile of a species-selective neurotensinagonist

The neurotensin (NT) receptor, subtype 1 (NTR1), is a 7-transmembrane-spann ing receptor, forming 3 extracellular and 3 intracellular loops. Previously , we showed that the third outer loop (E3) is the binding site for NT and i ts analogs, several of which bind with higher affinity to rat NTR1 (rNTR1) than to human NTR1 (hNTR1). In particular, NT34 {[3,1'-naphthyl-1-Ala(11)]N T(8-13)} has greater than 60-fold higher affinity for rNTR1 (46 and 60 pM f or transiently- and stably-transfected cells, respectively) than for hNTR1 (2.8 and 5.8 nM, for transiently- and stably-transfected cells, respectivel y) isolated from transfected cell membranes. Previously, our molecular mode ling studies of rNTR1 and hNTR1 showed that the binding pocket in the human receptor for NT34 is smaller in volume from the bulky residue Tyr(339) in the pocket center, as compared with the corresponding residue Phe(344) in t he rat binding pocket. Therefore, with site-directed mutagenesis, we derive d mutant forms of rNTR1 (F344Y) and hNTR1 (Y339F). Examination of the mutan t receptors from membranal preparations of transfected cells in radioligand binding assays and with intact cells in functional assays (phosphatidyl-4, 5-bisphosphate turnover) showed that the human-like rat receptor and the ra t-like human receptor bound NT34 with a predicted reverse of binding compar ed with its binding to the wild-type receptors. These results strongly affi rm our molecular modeling studies and demonstrate the importance of the stu dy of even minor structural variations in proteins to determine the basis o f significantly different drug responses, an area of focus for pharmacologi cal research in the 21st century. (C) 2000 Elsevier Science Inc.