Molecular identification of receptors for vasoactive intestinal peptide in rat intestinal epithelium by covalent cross‐linking

Abstract

The cleavable cross-linking reagent dithiobis (succinimidyl propionate, DTSP) linked 125I-labeled vasoactive intestinal peptide (125I-VIP) covalently to its receptors in rat intestinal epithelial membranes. DTSP treatment of 125I-VIP-labeled membranes inhibited the dissociation of VIP-receptor complexes in a way which was dependent on both time and concentration (ED50 = 200 .mu.M). Polyacrylamide gel electrophoresis of membrane proteins revealed 3 125I-VIP-protein complexes of MW 76,000, 36,000 and 17,000. The labeling of those compounds was not observed when: treatment of membranes by DTSP was omitted; the reagent quench, ammonium acetate, was added together with DTSP; DTSP-treated membranes were incubated with 2-mercaptoethanol which reduces the disulfide bond present within DTSP. Labeling of MW 76,000 and MW 36,000 complexes was specific in that it could be abolished by native VIP, while the labeling of the MW 17,000 complex was not. Densitometric scanning of autoradiographs indicated that: labeling of the MW 76,000 complex was abolished by low VIP concentrations (0.03-10 nM), by VIP agonists with the relative potency VIP > a peptide having N-terminal histidine and C-terminal isoleucine amide > secretin, and by GTP (10-5-1 mM) but was unaffected by various other peptide hormones; labeling of the MW 36,000 complex was inhibited by high VIP concentrations (1-300 nM), by VIP agonists at high concentrations but was not affected by GTP and various peptide hormones. Assuming 1 molecule of 125I-VIP was bound per molecule of protein, 2 proteins with MW 73,000 and 33,000 were identified as VIP binding sites. The MW 73,000 protein displays many characteristics (affinity, specificity, discriminating power toward agonists, sensitivity to GTP regulation) of the high-affinity VIP receptors mediating adenylate cyclase activation. The MW 33,000 proteins displays the characteristics (affinity, specificity) of a low-affinity VIP binding site. The molecular characteristics of the VIP receptor are shown and the molecular heterogeneity of VIP binding sites is discussed.