THE PEPTIDE BINDING-SITE OF THE SUBSTANCE-P (NK-1) RECEPTOR LOCALIZEDBY A PHOTOREACTIVE ANALOG OF SUBSTANCE-P - PRESENCE OF A DISULFIDE BOND

Substance P (SP) is a neuropeptide that mediates multiple physiologica l responses including transmission of painful stimuli and inflammation via an interaction with a receptor of known primary sequence, To iden tify the regions of the SP receptor, also termed the NK-1 receptor, in volved in peptide recognition, we are using analogues of SP containing the photoreactive amino acid p-benzoyl-L-phenylalanine (Bpa), In the present study, we used radioiodinated Bpa(8)-SP to covalently label wi th high efficiency the rat SP receptor expressed in a transfected mamm alian cell line, To identify the amino acid residue that serves as the site of covalent attachment, a membrane preparation of labeled recept or was subjected to partial enzymatic cleavage by trypsin, A major dig estion product of 22 kDa was identified. Upon reduction with 2-mercapt oethanol the mass of this product decreased to 14 kDa. The 22-kDa tryp tic fragment was purified in excellent yield by preparative SDS/PAGE u nder nonreducing conditions. Subcleavage with Staphylococcus aureus VS protease and endoproteinase ArgC yielded fragments of 8.2 and 9.0 kDa , respectively, Upon reductive cleavage, the V8 protease fragment decr eased to 3.0 kDa while the endoproteinase ArgC fragment decreased to 3 .2 kDa, Taking into consideration enzyme specificity, molecular size, determination of the presence or absence of N-glycosylation sites, and recognition by antibodies to specific sequences of the SP receptor, t he V8 protease fragment is Thr-173 to Glu-183, while the endoproteinas e ArgC fragment is Val-178 to Arg-190, These two fragments share the c ommon sequence Val-Val-Cys-Met-Ile-Glu (residues 178-183). The site of covalent attachment of radioiodinated Bpa(8)-SP is thus restricted to a residue within this overlap sequence, The data presented here also establish that the cysteine residue in this sequence Cys-180, which is positioned in the middle of the second extracellular loop, participat es in a disulfide bond that links the first and second extracellular l oops of the receptor.