PHOTOAFFINITY-LABELING THE SUBSTANCE-P RECEPTOR USING A DERIVATIVE OF SUBSTANCE-P CONTAINING P-BENZOYLPHENYLALANINE

A novel photoreactive substance P (SP) analogue has been synthesized by solid-phase peptide synthesis methodology to incorporate the amino acid p-benzoyl-L-phenylalanine [L-Phe(pBz)] in place of the Phe8 residue of SP. [Phe8(pBz)]SP was equipotent with SP in competing for SP binding sites on rat submaxillary gland membranes and had potent sialagogic activity in vivo. In the absence of light, the I-125-labeled Bolton-Hunter conjugate of [Phe8(pBz)]SP bound in a saturable and reversible manner to an apparently homogeneous class of binding sites (B(max) = 0.2 pmol/mg of membrane protein) with an affinity K(D) = 0.4 nM. The binding of I-125-[Phe8(pBz)]SP was inhibited competitively by various tachykinin peptides and analogues with the appropriate specificity for SP/NK-1 receptors. Upon photolysis, up to 70% of the specifically bound I-125-[Phe8(pBz)]SP underwent covalent linkage to two polypeptides of M(r) = 53 000 and 46 000, identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Quantitative analysis of the inhibitory effects of SP and related peptides on I-125-[Phe8(pBz)]SP photoincorporation indicated that the binding sites of the two photolabeled polypeptides have the same peptide specificity, namely, that typical of NK-1-type SP receptors. In addition, the labeling of the two polypeptides was equally sensitive to inhibition by guanyl-5'-yl imidodiphosphate, a nonhydrolyzable analogue of GTP. Further information on the relationship between the two labeled SP binding sites was provided by enzymatic digestion studies: the M(r) = 46 000 polypeptide contains N-linked carbohydrates and is derived most likely from the higher molecular weight species by proteolytic nicking. The highly specific and remarkably efficient photolabeling achieved with I-125-[Phe8(pBz)]SP suggests that this photoaffinity probe will be of considerable value for the characterization of the molecular structure of the SP receptor.