The rat liver vasoactive intestinal peptide binding site. Molecular characterization by covalent cross-linking and evidence for differences from the intestinal receptor

Abstract

To identify the molecular components of the vasoactive intestinal peptide (VIP) binding sites in the liver, 125I-labelled VIP was covalently linked to liver membranes by using the cleavable cross-linker dithiobis(succinimidylpropionate). Purified rat liver plasma membranes were incubated with 125I-VIP, washed and treated with 1 mM-cross-linker. Polyacrylamide-gel electrophoresis of membrane proteins followed by autoradiography revealed a major 125I-VIP-protein complex of Mr 51 000. A minor Mr 89 000 complex was also observed. An identical pattern of protein labelling was obtained using crude membranes from rat liver. Labelling of the Mr 51 000 and 89 000 species was specific in that it could be abolished by native VIP, but was unaffected by 1 microM-glucagon and cholecystokinin octapeptide. Densitometric scanning of autoradiographs indicated that the labelling of the two species was abolished by similar low VIP concentrations (0.1-100 nM). It was also reduced by two VIP agonists, peptide histidine isoleucine amide and secretin, with a potency that is 1/7 and 1/200 that of native VIP, respectively. The guanine nucleotide GTP in the concentration range between 10(-7) and 10(-3) M reduces the labelling of the major Mr 51 000 protein and that of the minor Mr 89 000 protein, but with a slightly higher potency. Assuming one molecule of 125I-VIP was bound per molecule of protein, a major Mr 48 000 protein and a minor Mr 86 000 protein were identified as components of the high-affinity VIP binding sites in liver. This contrasts markedly with the pattern of labelling of rat intestinal epithelial membranes, where a Mr 73 000 protein was identified as a high-affinity VIP receptor and a Mr 33 000 protein as a low-affinity VIP binding site [Laburthe, Bréant & Rouyer-Fessard (1984) Eur. J. Biochem. 139, 181-187], suggesting structural differences between VIP binding sites in rat liver and intestinal epithelium.