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

ABSTRACT

The study focuses on the importance of Tyr11 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 125I-NT with several chicken tissues, it is identified that one of the crucial factors behind all high affinity (Kd ~10 pM) interactions is due to phenolic-OH (Φ-OH) at the para (p) position of Tyr11 within RRPYIL-CO2H (NT8-13) sequence. Replacing the p-OH only in Tyr11 by substituting with p-Cl, p-F and p-NH2 results in significant change of the binding affinity (Kd); p-OH ≈ p-NH2 (~10 pM), p-Cl (~100 pM), p-F (~120 pM). Interestingly, p-NH2 equals to p-OH displaying the highest affinity. Experiments conducted by binding several of the 125I-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 Tryp11 in place of Tyr11 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 125I-Y3-azido-NT analogs shows several specifically labeled bands with considerable range of molecular weight (Mr ~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 (Kd ~20 pM) with varying capacities (Bmax). The order for Bmax 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 GTPgS (ED50 ~ 0.05 nM), NEM (ED50 ~ 0.50 mM) and NaCl (ED50 ~30 mM), indicating the existence of NTR identical to the mammalian type-1.