Covalent cross-linking of vasoactive intestinal polypeptide to its receptors on intact human lymphoblasts.
125I-labeled vasoactive intestinal polypeptide (125I-VIP) was covalently cross-linked with its binding sites on intact cultured human lymphoblasts by each of three bifunctional reagents: disuccinimidyl suberate (DSS), ethylene glycol bis(succinimidyl succinate) (EGS), and N-succinimidyl 6-(4'-azido-2'-nitrophenylamino) hexanoate (SANAH). A fourth cross-linking agent with a shorter chain length, N-hydroxysuccinimidyl 4-azidobenzoate (HSAB), was much less effective in cross-linking 125I-VIP to the site. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography demonstrated a band of Mr approximately equal to 50,000 +/- 3,000, regardless of which cross-linker was used. The labeling of this band was specific in that it was prevented by 10(-6) M unlabeled VIP and was partially blocked by the homologous hormones secretin and glucagon. The relative potencies of these peptides in blocking the cross-linking of 125I-VIP to the Mr approximately equal to 50,000 band of the lymphoblasts (VIP greater than secretin greater than or equal to glucagon) were similar to those previously found for competitive inhibition of 125I-VIP binding to its putative high-affinity receptor on these cells. The covalent cross-linking required a bifunctional reagent; it was dependent on both the number of Molt cells and the concentration of 125I-VIP. The apparent molecular weight of the cross-linked species was unchanged by treatment with dithiothreitol. These observations suggest that the Mr = 50,000 species represents 125I-VIP cross-linked to a specific plasma membrane receptor and that the receptor does not contain interchain disulfide bonds.
Wood
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O'Dorisio
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MS
D000345:Affinity Labels / Q000378:metabolism; D001386:Azides / Q000494:pharmacology; D002460:Cell Line; D003432:Cross-Linking Reagents / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008970:Molecular Weight; D011956:Receptors, Cell Surface / Q000378:metabolism*; D018005:Receptors, Vasoactive Intestinal Peptide; D013388:Succinimides / Q000494:pharmacology; D013601:T-Lymphocytes / Q000378:metabolism*; D014660:Vasoactive Intestinal Peptide / Q000378:metabolism*
D000345:Affinity Labels; D001386:Azides; D003432:Cross-Linking Reagents; D011956:Receptors, Cell Surface; D018005:Receptors, Vasoactive Intestinal Peptide; D013388:Succinimides; D014660:Vasoactive Intestinal Peptide; C029534:hydroxysuccinimidyl-4-azidobenzoate; C037733:N-succinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate; C028722:ethylene glycolylbis(succinimidyl succinate); C019358:disuccinimidyl suberate