Open AccessMolecular identification of receptors for vasoactive intestinal peptide in rat intestinal epithelium by covalent cross‐linking
Author(s) -
LABURTHE Marc,
BREANT Bernadette,
ROUYERFESSARD Christiane
Publication year - 1984
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1984.tb07992.x
Subject(s) - vasoactive intestinal peptide , chemistry , peptide , receptor , membrane , biochemistry , secretin , neuropeptide , pancreas
The cleavable cross‐linking reagent dithiobis (succinimidyl propionate) or DTSP was shown to link 125 I‐labeled vasoactive intestinal peptide ( 125 I‐VIP) covalently to its receptors in rat intestinal epithelial membranes. DTSP treatment of 125 I‐VIP‐labeled membranes inhibited the dissociation of VIP‐receptor complexes in a way which was dependent on both time and concentration (ED 50 = 200 μM). Polyacrylamide gel electrophoresis of membrane proteins revealed three 125 I‐VIP‐protein complexes of M r 76000, 36000 and 17000. The labeling of those compounds was not observed when: (a) treatment of membranes by DTSP was omitted; (b) the reagent quench, ammonium acetate, was added together with DTSP; (c) DTSP‐treated membranes were incubated with 2‐mercaptoethanol which reduces the disulfide bond present within DTSP. Labeling of M r ‐76000 and M r ‐36000 complexes was specific in that it could be abolished by native VIP, while the labeling of the M r ‐17000 was not. Densitometric scanning of autoradiographs indicated that: (a) labeling of the M r ‐76000 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; (b)labeling of the M r ‐36000 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 one molecule of 125 I‐VIP was bound per molecule of protein, two proteins with M r ‐73000 and 33000 were identified as VIP binding sites. The M r ‐73000 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 M r ‐33000 protein displays the characteristics (affinity, specificity) of a low‐affinity VIP binding site. This study thus shows the molecular characteristics of the VIP receptor and further argues for the molecular heterogeneity of VIP binding sites.