Photoaffinity labeling the substance P receptor using a derivative of substance P containing p-benzoylphenylalanine

• Published in: Biochemistry (Easton) 1991-01, Vol.30 (2), p.336-342
• Main Authors: Boyd, N. D, Cerpa, R, Kaiser, E. T, Leeman, S. E, White, C. F
• Format: Article
• Language: English
• Subjects: Affinity Labels; Animals; Binding, Competitive; Biological and medical sciences; Cell Membrane - metabolism; Cell receptors; Cell structures and functions; Fundamental and applied biological sciences. Psychology; Guanylyl Imidodiphosphate - pharmacology; In Vitro Techniques; Miscellaneous; Molecular and cellular biology; p-benzoylphenylalanine; Phenylalanine - analogs & derivatives; Phenylalanine - chemistry; Photochemistry; Rats; Rats, Inbred Strains; Receptors, Neurokinin-1; Receptors, Neurotransmitter - metabolism; Submandibular Gland; Substance P - analogs & derivatives; Substance P - antagonists & inhibitors; Substance P - chemistry; Substance P - metabolism
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00216a005

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 125I-labeled Bolton-Hunter conjugate of [Phe8(pBz)]SP bound in a saturable and reversible manner to an apparently homogeneous class of binding sites (Bmax = 0.2 pmol/mg of membrane protein) with an affinity KD = 0.4 nM. The binding of 125I-[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 125I-[Phe8(pBz)]SP underwent covalent linkage to two polypeptides of Mr = 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 125I-[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 Mr = 46,000 polypeptide contains N-linked carbohydrates and is derived most likely from the higher molecular weight species by proteolytic nicking.