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Several residues of the human neurokinin-2 receptor have been identified to be critical for the binding of peptide agonists and non-peptide antagonists. Amino acid substitutions in the first and second extracellular segments and the second transmembrane segment led to substantial reduction in peptide affinity without affecting the affinity of antagonist SR48968. These effects are identical to those observed for homologous residues in the neurokinin-1 receptor, suggesting that these three regions are involved in high-affinity peptide binding to both receptor 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 location of these receptor residues suggest that the distance between bound 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 thus receptor subtype selectivity. While many single-residue substitutions did not affect the binding affinity of the antagonist SR48968, two double mutants in the sixth and seventh transmembrane segments were found to reduce its affinity substantially. Therefore, receptor residues participate cooperatively in the binding of SR48968. These results demonstrate the usefulness of combining single-residue substitutions in studying and confirming the role of receptor residues in ligand binding. Finally, the overlapping nature of agonist and antagonist binding sites is consistent with the observation that substitutions of some residues modify the binding affinities of both peptide agonists and non-peptide antagonists.

Peptide= correspondinR, to the amino tLcid ~equenee or the hamster/~=.~drenergi¢ receptor If/eAR.) were synthesized and their ability to activate purified O.proteins determined. Two peptides. ¢omprismll the N, and C-terminal I$ m'nino arid= of the putative third intraeellular loop region or the p~AR were found to aetivale the G.protein O, but not to activate a preparation of G,/G,. Other p~ptides ¢orre~pondin= to the intern=l portion= of this loop and the C.terminal lab region fitiled to activate either G.protein. The presence of pho=pholipid vesicles was required for thil activation. The ob~:rvation that peptides with ~quences eorrespondinii to the ends of the third intraeelhflar loop of the,BAR can spccifcally activate O, co)~firms the results of prev=ous muta$enesis studies on tho receptor and demonstrates that the ~econdary strugttlre ¢onferred by the amino acid =equ=nces in these regions is sufficient for the activation of Q,proteins.

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