The human vasoactive intestinal peptide receptor: molecular identification by covalent cross-linking in colonic epithelium.
To characterize the molecular components of the vasoactive intestinal peptide (VIP) receptor in human intestine, [125I]VIP was covalently bound to human colonic epithelial membranes using dithio-bis(succinimidyl propionate). Sodium dodecyl sulfate-polyacrylamide gel autoradiographic studies of affinity labeled membranes revealed three major bands corresponding to [125I]VIP-protein complexes of 66,000, 33,000, and 16,000 mol wt. Labeling of the 66,000 and 33,000 mol wt complexes was specific, since it was abolished by VIP, while labeling of the 16,000 mol wt complex was not. Densitometric scanning of autoradiographs indicated that labeling of the 66,000 mol wt complex was inhibited by low VIP concentrations in the 10(-10)-10(-8) M range, but was unaffected by glucagon or octa-cholecystokinin. It was also reduced by VIP-(2-28), with a potency 1/100th that of VIP, and by GTP in the concentration range of 10(-7)-10(-3) M. The 33,000 mol wt complex behaved similarly to the 66,000 mol wt complex with respect to specificity and GTP sensitivity, but differed in one major feature, its affinity for VIP. Its labeling was inhibited by native VIP concentrations in the 10(-9)-10(-7) M range. Assuming one molecule of [125I]VIP bound per molecule of protein, two proteins of 63,000 and 30,000 mol wt were identified as VIP-binding sites. The 63,000 mol wt protein had the properties expected for the VIP receptor coupled to adenylate cyclase in human colon, while the 30,000 mol wt protein was a low affinity binding site. Treatment of human colonic membranes with the sulfhydryl reducing agent dithiothreitol before [125I]VIP binding strongly reduced the labeling of the two proteins. This finding does not support the hypothesis that the low affinity 30,000 mol wt binding site may be a monomer of the high affinity binding site.
Couvineau
A
A
Laburthe
M
M
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D003106:Colon / Q000187:drug effects / Q000378:metabolism*; D003432:Cross-Linking Reagents; D004229:Dithiothreitol / Q000494:pharmacology; D004848:Epithelium / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000494:pharmacology; D006801:Humans; D008970:Molecular Weight; D051381:Rats; D011956:Receptors, Cell Surface / Q000378:metabolism*; D018005:Receptors, Vasoactive Intestinal Peptide; D013388:Succinimides / Q000494:pharmacology*; D014660:Vasoactive Intestinal Peptide / Q000378:metabolism*
D003432:Cross-Linking Reagents; D011956:Receptors, Cell Surface; D018005:Receptors, Vasoactive Intestinal Peptide; D013388:Succinimides; D014660:Vasoactive Intestinal Peptide; D006160:Guanosine Triphosphate; C011240:dithiobis(succinimidylpropionate); D004229:Dithiothreitol