Agonist induced conformation alteration of neurotensin receptor and the mechanism behind Na~+ inhibition of ~(125)I-NT binding

摘要

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 #mu#M NT, 100 #mu#M SR48692 (antagonist), 100 #mu#M GPPNHP or 100 mM NaCl. Lowering the medium pH to 4.5 enhances the process (approx 70% in 10 minutes). Labeling by photoactivatable ~(125)I-Tyr~3-Azo~4-NT identifies a approx 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 ~(125)I-NT association by acting as a noncompetitive inhibitor. On the contrary, Na~+ favors the interaction of antagonist, SR48692 by lowering the value of K_i. GTP_#gamma#~(35)S binding to membranes in the presence of 30 mM NaCl suggests that Na~+ inhibition of ~(125)I-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 table 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.