Chimeric rat/human neurotensin receptors localize a region of the receptor sensitive to binding of a novel, species-specific, picomolar affinity peptide

Recently, we reported the development of a species-specific neurotensin analog that displays selective binding affinity at the rat and human neurotensin (NT) receptor, L-[3,2'-Nal¹¹]NT(8-13) (where Nal is naphthylalanine) (NT19). We have developed another neurotensin analog, L- [3,1'-Nal¹¹]NT(8-13), (NT34), that exhibits a 126-fold difference in binding affinities between the rat and human receptors. This compound differs from our previous reported species-specific ligand in the steric positioning of the naphthyl ring on the L-alanine side chain. For NT34, the observed K(d) values at the rat and human neurotensin receptors were 0.046 and 5.8 nM, respectively. In stimulating phosphatidylinositol turnover, the observed EC₅₀ values were 2.8 nM and 130 nM in rat and human, respectively. We constructed a series of chimeric rat/human neurotensin receptor genes and expressed them by transient transfection into human embryonic kidney (HEK- 293) cells. Radioligand binding assays were then performed using neurotensin and NT34. Our results led us to propose a region of the neurotensin receptor that may be involved in determining species specificity, i.e. the transmembrane VI, the third extracellular loop, and transmembrane VII regions of the neurotensin receptor.