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#M NT, 100#M SR48692 (antagonist), 100#M GPPNHP or 100mM NaCl. Lowering the medi...

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Bibliographic Details
Published in:Journal of receptors and signal transduction 1999-11, Vol.19 (6), p.995-1021
Main Authors: Mitra, Sankar P., Carraway, Robert E., Blute, Robert, Luber-Narod, Judith
Format: Article
Language:English
Subjects:
Animals
Binding Sites
Cell Membrane - metabolism
Chickens
Liver - metabolism
Neurotensin - chemistry
Neurotensin - metabolism
Protein Binding
Protein Conformation
Receptors, Neurotensin - chemistry
Receptors, Neurotensin - metabolism
Sodium - metabolism
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Summary: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#M NT, 100#M SR48692 (antagonist), 100#M GPPNHP or 100mM NaCl. Lowering the medium pH to 4.5 enhances the process (∼70% in 10 minutes). Labeling by photoactivatable 125I-Tyr3-Azo4-NT identifies a ∼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 K+. GTPγ35S binding to membranes in the presence of 30mM NaCI 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 NaCI via step-2, thereby altering the native NTR conformation. The presence of Na+ prevents step-2 by dissociating the transition complex.
ISSN:1079-9893
1532-4281
DOI:10.3109/10799899909038436