A mutation changes ligand selectivity and transmembrane signaling preference of the neurokinin-1 receptor

studied the biochemical properties of a genetically engineered neurokinin-1 receptor (NK(1)R) in which two residues lying on the extracellular edge of the fourth transmembrane domain were replaced by equivalently located elements of the neurokinin-2 receptor (G166C, Y167F NK(1)R mutant), The mutation produced two effects, The first is enhancement of the apparent binding affinity for heterologous tachykinins (substance K and neurokinin B) and for N- or C-terminal modified analogues of substance P, but not for substance P itself, its full-length analogues, and several peptide and nonpeptide antagonists, Only two antagonists, as exceptions, were found 60 exhibit a diminished affinity for the mutant receptor. The second effect is a shift in NK(1)R preference for distinct G protein-mediated signaling pathways, NK(1)R mediated phosphoinositide hydrolysis was enhanced both in transiently and permanently transfected cells, while stimulation of cAMP accumulation did not change in transient expression experiments and was reduced in permanently expressing cells. The effect of the mutation on ligand affinity was not related to any obvious structural commonality, nor to the selectivity for different neurokinin receptors or the agonistic/antagonistic nature of the ligand, However, all ligands responding to the mutation appear to share the ability to induce phosphoinositide signaling more efficiently than cAMP responses when binding to NK(1)R. We suggest that the mutation shifts the internal equilibria of different functional forms of NK(1)R. A theoretical analysis according to a multistate allosteric model suggests that the link between binding and biological changes can result from altered stability constants of substates in the conformational space of the receptor.