Binding chemistry and molecular heterogeneity of neurotensin binding protein(s)/receptor in adult chicken tissues.
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 (D-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-C1, p-F and p-NH2 results in significant change of the binding affinity (Kd); p-OH approximately equal p-NH2 (approximately 10 pM), p-Cl (approximately 100 pM), p-F (approximately 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 approximately 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 approximately 20 pM) with varying capacities (Bmax). The order for Bmax is: liver (1.2 pMol/mg) > or = gall bladder (1.03 pMol/mg) > spleen (0.43 pMol/mg) > brain (0.3 pMol/mg) > colon > or = lung (0.15 pMol/mg). In all cases, the binding was reduced by GTPgammaS (ED50 to approximately 0.05 nM), NEM (ED50 to approximately 0.50 mM) and NaCl (ED50 to approximately 30 mM), indicating the existence of NTR identical to the mammalian type-1.
Sankar P
SP
Mitra
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001386:Azides / Q000737:chemistry; D001667:Binding, Competitive; D002462:Cell Membrane / Q000378:metabolism; D002645:Chickens; D005033:Ethylmaleimide / Q000494:pharmacology; D005260:Female; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D008099:Liver / Q000166:cytology; D008297:Male; D008970:Molecular Weight; D009496:Neurotensin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D011720:Pyrazoles / Q000494:pharmacology; D011804:Quinolines / Q000494:pharmacology; D018028:Receptors, Neurotensin / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D012965:Sodium Chloride / Q000494:pharmacology; D013237:Stereoisomerism; D014443:Tyrosine
D001386:Azides; D011720:Pyrazoles; D011804:Quinolines; D018028:Receptors, Neurotensin; C079087:SR 48692; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D009496:Neurotensin; D014443:Tyrosine; D012965:Sodium Chloride; D005033:Ethylmaleimide