Gly 166 in the NK 1 receptor regulates tachykinin selectivity and receptor conformation

Abstract

We have studied the pharmacological properties of genetically engineered human NK 1 tachykinin receptors in which residues at the extracellular surface of the fourth transmembranal domain were substituted with the corresponding amino acids from the NK 2 receptor. We show that substitution of G166C:Y167F in the human NK 1 receptor induces high affinity binding of a group of tachykinin ligands, known as `septides' (i.e. neurokinin A, neurokinin B, [pGlu 6,Pro 9]-substance P 6-11 and substance P-methylester). In contrast, binding of substance P and non-peptide antagonists is unaffected by these mutations. This effect parallels that found on the rat receptor and is therefore species specific. Second, we demonstrate that mutation of Gly 166 to Cys alone is both necessary and sufficient to create this pan-reactive tachykinin receptor, whereas replacement of Tyr 167 by Phe has no detectable effect on the pharmacological properties of the receptor. Furthermore, analysis of the effect of N-ethylmaleimide and dithiothreitol on binding of radiolabelled substance P documents differences in the mode in which this ligand interacts with wild-type and mutant receptors and supports the existence of a mutational induced change in the conformational status of the NK 1 receptor.