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, G protein-coupled neurokinin-1 receptor (NK-1R). Previous studies from our laboratory have shown that ¹²⁵I-Bolton-Hunter reagent-labeled p-benzoylphenylalanine⁸-SP (Bpa⁸SP) covalently attaches to Met¹⁸¹, whereas ¹²⁵I-Bolton-Hunter reagent-labeled Bpa⁴SP covalently attaches to Met¹⁷⁴, both of which are located on the second extracellular loop (EC2) of the NK-1R. In this study, evidence has been obtained that at equilibrium, the photoreactive SP analogue ¹²⁵I-[D-Tyr⁰]Bpa³SP covalently labels residues in two distinct extracellular regions of the NK-1R. One site of ¹²⁵I-[D-Tyr⁰]Bpa³SP photoinsertion is located on EC2 within a segment of the receptor extending from residues 173 to 177; a second site of ¹²⁵I-[D-Tyr⁰]Bpa³SP 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.