title: Agonist induced conformation alteration of neurotensin receptor and the mechanism behind Na+ inhibition of 125I-NT binding.
abstract: In the absence of Na+, 125I-Neurotensin (125I-NT) binding to the Neurotensin receptor (NTR) produces a stable noncovalent 125I-NT-NTR complex whose dissociation rate is extremely low even after the addition of 1 microM NT, 100 microM SR48692 (antagonist), 100 microM GPPNHP or 100 mM NaCl. Lowering the medium pH to 4.5 enhances the process (approximately 70% in 10 minutes). Labeling by photoactivatable 125I-Tyr3-Azo4-NT identifies a approximately 50 KD Mr band along with several other minor components. Interestingly, the labeling intensity is drastically reduced when binding is performed in the presence of Na+ or GPPNHP. However, a minor reduction is noticed when Na+ or GPPNHP is added to the medium after binding. The binding kinetics indicates that Na+ lowers the rate of 125I-NT association by acting as a noncompetitive inhibitor. On the contrary, Na+ favors the interaction of antagonist, SR48692 by lowering the value of Ki. GTPgamma35S binding to membranes in the presence of 30 mM NaCl suggests that Na+ inhibition of 125I-NT binding is due to the uncoupling of NTR associated G protein(s). In order to explain the entire phenomenon, a two-step, binding model has been proposed. In Step-1, interaction between NT and NTR produces a transient complex, which attains a stable state in the absence of NaCl via step-2, thereby altering the native NTR conformation. The presence of Na+ prevents step-2 by dissociating the transition complex.
authors.affiliation: Division of Urology, University of Massachusetts Medical Center, Worcester 01655, USA.
authors.forename: S P
authors.initials: SP
authors.lastname: Mitra
authors.forename: R E
authors.initials: RE
authors.lastname: Carraway
authors.forename: R
authors.initials: R
authors.lastname: Blute
authors.forename: J
authors.initials: J
authors.lastname: Luber-Narod
mesh_terms: D000818:Animals; D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism; D002645:Chickens; D008099:Liver / Q000378:metabolism*; D009496:Neurotensin / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D018028:Receptors, Neurotensin / Q000737:chemistry* / Q000378:metabolism*; D012964:Sodium / Q000378:metabolism*
chemical_list: D018028:Receptors, Neurotensin; D009496:Neurotensin; D012964:Sodium