Evidence for spatial proximity of two distinct receptor regions in the substance P (SP)• neurokinin-1 receptor (NK-1R) complex obtained by photolabeling the NK-1R with p-benzoylphenylalanine3-SP

Substance P (SP) belongs to the tachykinin family of bioactive peptides and exerts its many biological effects through functional interaction with its cell-surface, Gr protein-coupled neurokinin-l receptor (NK-1R). Previous studies from our laboratory have shown that I-125-Bolton-Hunter reagent-labeled p-benzoylphenylalanine(8)-SP (Bpa(8)SP) covalently attaches to Met(181), whereas I-125-Bolton-Hunter reagent-labeled Bpa(4)SP covalently attaches to Met(174), both of which are located on the second extracellular loop (EC2) of the NR-1R, In this study, evidence has been obtained that at equilibrium, the photoreactive SP analogue I-125-[D-Tyr(0)]Bpa(3)Sp covalently labels residues in two distinct extracellular regions of the NK-1R, One site of I-125-[D-Tyr(0)]Epa3Sp photoinsertion is located on EC2 within a segment of the receptor extending from residues 173 to 177; a second site of I-125-[D-Tyr(0)]BpaSP photoinsertion is located on the extracellular N terminus within a segment of the receptor extending from residues 11 to 21, a sequence that contains both potential sites for N-linked glycosylation, Since competition binding data presented in this study do not suggest the existence of multiple peptide NK-1R complexes, it is reasonable to assume that the receptor sequences within EC2 and N terminus identified by peptide mapping are in close proximity in the equilibrium complex.