Binding chemistry and molecular heterogeneity of neurotensin binding protein(s)/receptor in adult chicken tissues

The study focuses on the importance of Tyr(11) amino acid (AA) and subsequent stereochemistry involved in the binding process of neurotensin (NT) with its receptor (NTR)/binding protein(s) as well as the size heterogeneity. Using the binding of I-125-NT with several chicken tissues, it is identified that one of the crucial factors behind all high affinity (K-d similar to 10 pM) interactions is due to phenolic-OH (Phi-OH) at the para (p) position of Tyr(11) within RRPYIL-CO2H (NT8-13) sequence. Replacing the p-OH only in Tyr(11) by substituting with p-Cl, p-F and p-NH2 results in significant change of the binding affinity (K-d); p-OH p-NH2 (similar to 10 pM), p-Cl (similar to 100 pM), p-F (similar to 120 pM). Interestingly, p-NH2 equals to p-OH displaying the highest affinity Experiments conducted by binding several of the I-125-azido NT analogs having azido group attached at different positions within the NT molecule have further confirmed the necessity of RRPYIL sequence for high affinity ligand-receptor interaction. The role of Tryp(11) in place of Tyr(11) in addition to the results above establishes a significant possibility of H bonding occurring between p-OH of NT and NTR inside the docking space. Photo labeling of the liver tissue by substituted I-125-Y-3-azido-NT analogs shows several specifically labeled bands with considerable range of molecular weight (M-r similar to 90-30 kDa) variations. These results indicate the existence of molecular heterogeneity concerning the sizes of NTR or else any NT binding proteins in the avian tissues. Further, the study has revealed that besides liver, several other chicken tissues also express similar specific high affinity binding (K-d similar to 20 pM) with varying capacities (B-max). The order for B-max is: liver (1.2 pMol/mg) >= gall bladder (1.03 pMol/mg) > spleen (0.43 pMol/mg) > brain (0.3 pMol/mg) > colon >= lung (0.15 pMol/mg). In all cases, the binding was reduced by GTP gamma S (ED50 similar to 0.05 nM), NEM (ED50 similar to 0.50 mM) and NaCl (ED50 similar to 30 mM), indicating the existence of NTR identical to the mammalian type-1.