TY - JOUR
T1 - A single amino acid of the human and rat neurotensin receptors (subtype 1) determining the pharmacological profile of a species-selective neurotensin agonist
AU - Cusack, Bernadette
AU - Jansen, Karen
AU - McCormick, Daniel J.
AU - Chou, Thomas
AU - Pang, Yuan Ping
AU - Richelson, Elliott
N1 - Funding Information:
This work was funded by Grant MH 27692 from the National Institute of Mental Health and by the Mayo Foundation for Medical Education and Research.
PY - 2000/9/15
Y1 - 2000/9/15
N2 - The neurotensin (NT) receptor, subtype 1 (NTR1), is a 7-transmembrane-spanning receptor, forming 3 extracellular and 3 intracellular loops. Previously, we showed that the third outer loop (E3) is the binding site for NT and its analogs, several of which bind with higher affinity to rat NTR1 (rNTR1) than to human NTR1 (hNTR1). In particular, NT34 {[3,1'-naphthyl-1-Ala11]NT(8-13)} has greater than 60-fold higher affinity for RNTR1 (46 and 60 Pm for transiently- and stably-transfected cells, respectively) than for HNTR1 (2.8 and 5.8 Nm for transiently- and stably-transfected cells, respectively) isolated from transfected cell membranes. Previously, our molecular modeling studies of RNTR1 and HNTR1 showed that the binding pocket in the human receptor for NT34 is smaller in volume from the bulky residue Tyr339 in the pocket center, as compared with the corresponding residue Phe344 in the rat binding pocket. Therefore, with site-directed mutagenesis, we derived mutant forms of RNTR1(F344Y) and HNTR1(Y339F). Examination of the mutant 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 rat-like human receptor bound NT34 with a predicted reverse of binding compared with its binding to the wild-type receptors. These results strongly affirm our molecular modeling studies and demonstrate the importance of the study of even minor structural variations in proteins to determine the basis of significantly different drug responses, an area of focus for pharmacological research in the 21st century. (C) 2000 Elsevier Science Inc.
AB - The neurotensin (NT) receptor, subtype 1 (NTR1), is a 7-transmembrane-spanning receptor, forming 3 extracellular and 3 intracellular loops. Previously, we showed that the third outer loop (E3) is the binding site for NT and its analogs, several of which bind with higher affinity to rat NTR1 (rNTR1) than to human NTR1 (hNTR1). In particular, NT34 {[3,1'-naphthyl-1-Ala11]NT(8-13)} has greater than 60-fold higher affinity for RNTR1 (46 and 60 Pm for transiently- and stably-transfected cells, respectively) than for HNTR1 (2.8 and 5.8 Nm for transiently- and stably-transfected cells, respectively) isolated from transfected cell membranes. Previously, our molecular modeling studies of RNTR1 and HNTR1 showed that the binding pocket in the human receptor for NT34 is smaller in volume from the bulky residue Tyr339 in the pocket center, as compared with the corresponding residue Phe344 in the rat binding pocket. Therefore, with site-directed mutagenesis, we derived mutant forms of RNTR1(F344Y) and HNTR1(Y339F). Examination of the mutant 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 rat-like human receptor bound NT34 with a predicted reverse of binding compared with its binding to the wild-type receptors. These results strongly affirm our molecular modeling studies and demonstrate the importance of the study of even minor structural variations in proteins to determine the basis of significantly different drug responses, an area of focus for pharmacological research in the 21st century. (C) 2000 Elsevier Science Inc.
KW - Binding site
KW - Neurotensin receptor
KW - Peptide analogs
KW - Site-directed mutagenesis
KW - Structure-activity studies
KW - Subtype 1
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U2 - 10.1016/S0006-2952(00)00409-3
DO - 10.1016/S0006-2952(00)00409-3
M3 - Article
C2 - 10930533
AN - SCOPUS:0034666715
SN - 0006-2952
VL - 60
SP - 793
EP - 801
JO - Biochemical Pharmacology
JF - Biochemical Pharmacology
IS - 6
ER -