Proposed ligand binding site of the transmembrane receptor for neurotensin(8-13)

We report here the first proposed ligand binding site of the transmembrane receptor for neurotensin(8-13) in human and rat, the corresponding bound conformation of the peptide ligand, and site-directed mutagenesis studies that support the binding site model. These three-dimensional structures were generated by using a heuristic approach in conjunction with experimental data. The proposed neurotensin(8-13) binding site is primarily composed of eight residues (i.e., Phe³²⁶, Ile³²⁹, Trp³³⁴, Phe³³⁷, Tyr³³⁹, Phe³⁴¹, Ty³⁴², and Tyr³⁴⁴ in the human receptor; Phe³³¹, Ile³³⁴, Trp³³⁹, Phe³⁴², Phe³⁴⁴, Phe³⁴⁶, Tyr³⁴⁷, and Tyr³⁴⁹ in the rat receptor) located in the third extracellular loop. The seven aromatic residues form an aromatic pocket on the extracellular surface of the neurotensin receptor to accommodate its ligands apparently by cation- pi, pi-pi, and hydrogen bonding interactions. The neurotensin(8-13) ligand adopts a compact conformation at the proposed binding site. In the bound conformation of neurotensin(8-13), the backbone of Arg⁹-Pro¹⁰-Tyr¹¹- Ile¹² forms the proline type I turn, and the hydroxy group of Tyr¹¹ interacts with the two guanidinium groups of Arg⁸ and Arg⁹. These guanidinium groups are curled toward the hydroxy group so that they interact electrostatically with the hydroxy group, and that the guanidinium group of Arg⁹ forms an intra-hydrogen bond with the hydroxy group. The proposed three-dimensional structure may not only provide a basis for rationalizing mutations of the neurotensin receptor gene but also offer insights into understanding the binding of many neurotensin analogs, biological functions of the neurotensin receptors, and structural elements for species specificity of the neurotensin receptors, and may expedite developing nonpeptidic neurotensin mimetics for the potential treatment of the neuropsychiatric diseases.