IDENTIFICATION OF RESIDUES INVOLVED IN LIGAND-BINDING TO THE NEUROKININ-2 RECEPTOR

Several residues of the human neurokinin-2 receptor have been identifi ed to be critical for the binding of peptide agonists and non-peptide antagonists. Amino acid substitutions in the first and second extracel lular segments and the second transmembrane segment led to substantial reduction in peptide affinity without affecting the affinity of antag onist SR48968. These effects are identical to those observed for homol ogous residues in the neurokinin-l receptor, suggesting that these thr ee regions are involved in high-affinity peptide binding to both recep tor subtypes. On the other hand, some conserved residues in the fourth to seventh transmembrane segments are required for peptide binding to only one receptor subtype but not both. The conserved nature and loca tion of these receptor residues suggest that the distance between boun d peptide and helices 4-7 varies depending on the receptor subtype. It is likely that the conformational compatibility between a ligand and a given receptor determines the magnitude of binding affinity, and thu s receptor subtype selectivity. While many single-residue substitution s did not affect the binding affinity of the antagonist SR48968, two d ouble mutants in the sixth and seventh transmembrane segments were fou nd to reduce its affinity substantially. Therefore, receptor residues participate cooperatively in the binding of SR48968. These results dem onstrate the usefulness of combining single-residue substitutions in s tudying and confirming the role of receptor residues in ligand binding . Finally, the overlapping nature of agonist and antagonist binding si tes is consistent with the observation that substitutions of some resi dues modify the binding affinities of both peptide agonists and non-pe ptide antagonists.