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

In the absence of Na+, I-125-Neurotensin (I-125-NT) binding to the Neuroten sin receptor (NTR) produces a stable noncovalent I-125-NT-NTR complex whose dissociation rate is extremely low even after the addition of 1 mu M NT, 1 00 mu M SR48692 (antagonist), 100 mu M GPPNHP or 100mM NaCl. Lowering the m edium pH to 4.5 enhances the process (similar to 70% in 10 minutes). Labeli ng by photoactivatable I-125-Tyr(3)-Azo(4)-NT identifies a similar to 50 KD Mr band along with several ether minor components. Interestingly, the labe ling intensity is drastically reduced when binding is performed in the pres ence of Na or GPPNHP. However, a minor reduction is noticed when Na+ or GPP NHP is added to the medium after binding. The binding kinetics indicates th at Na+ lowers the rate of I-125-NT association by acting as a noncompetitiv e inhibitor. On the contrary, Na+ favors the interaction of antagonist, SR4 8692 by lowering the value of K-i. GTP gamma(35)S binding to membranes in t he presence of 30mM NaCl suggests that Na+ inhibition of I-125-NT binding i s 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 Ste p-1, interaction between NT and NTR produces a transient complex, which att ains 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 dissociati ng the transition complex.