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 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Department of Physiology, University of Massachusetts Medical Center, Worcester 01655.
N D
ND
Boyd
C F
CF
White
R
R
Cerpa
E T
ET
Kaiser
S E
SE
Leeman
D000345:Affinity Labels; D000818:Animals; D001667:Binding, Competitive; D002462:Cell Membrane / Q000378:metabolism; D006165:Guanylyl Imidodiphosphate / Q000494:pharmacology; D066298:In Vitro Techniques; D010649:Phenylalanine / Q000031:analogs & derivatives / Q000737:chemistry; D010777:Photochemistry; D051381:Rats; D011919:Rats, Inbred Strains; D018040:Receptors, Neurokinin-1; D017981:Receptors, Neurotransmitter / Q000378:metabolism*; D013363:Submandibular Gland; D013373:Substance P / Q000031:analogs & derivatives / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*
D000345:Affinity Labels; D018040:Receptors, Neurokinin-1; D017981:Receptors, Neurotransmitter; D013373:Substance P; D006165:Guanylyl Imidodiphosphate; D010649:Phenylalanine; C053764:benzoylphenylalanine