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 (125)I-Bolton-Hunter reagent-labeled p-benzoylphenylalanine(8)-SP (Bpa(8)SP) covalently attaches to Met(181), whereas (125)I-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 NK-1R. In this study, evidence has been obtained that at equilibrium, the photoreactive SP analogue (125)I-[D-Tyr(0)]Bpa(3)SP covalently labels residues in two distinct extracellular regions of the NK-1R. One site of (125)I-[D-Tyr(0)]Bpa(3)SP photoinsertion is located on EC2 within a segment of the receptor extending from residues 173 to 177; a second site of (125)I-[D-Tyr(0)]Bpa(3)SP 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.
Bremer
A A
AA
Department of Pharmacology and Experimental Therapeutics, Boston University School of Medicine, Boston, Massachusetts 02118, USA.
Leeman
S E
SE
Boyd
N D
ND
D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D006868:Hydrolysis; D010449:Peptide Mapping; D010649:Phenylalanine / Q000031:analogs & derivatives* / Q000378:metabolism*; D019920:Photoaffinity Labels; D011485:Protein Binding; D051381:Rats; D018040:Receptors, Neurokinin-1 / Q000737:chemistry / Q000378:metabolism*; D013373:Substance P / Q000378:metabolism*; D014357:Trypsin / Q000378:metabolism
D019920:Photoaffinity Labels; D018040:Receptors, Neurokinin-1; D013373:Substance P; D010649:Phenylalanine; C053764:benzoylphenylalanine; D014357:Trypsin