HT 29, a model cell line: stimulation by the vasoactive intestinal peptide (VIP); VIP receptor structure and metabolism.
HT 29, a cell line derived from a human colonic adenocarcinoma, is highly responsive to the vasoactive intestinal peptide (VIP) as shown by a more than 100-fold intracellular cAMP increase (Ka = 0.3 nM), the stimulations of protein kinase A (Ka = 0.1 nM) and the low-Km cAMP phosphodiesterase (Ka = 40 nM). Remarkably, adenylate cyclase, cAMP-dependent kinase and cAMP-specific phosphodiesterase are activated in a sequential manner. Binding studies with [125I]-labeled VIP indicate a high affinity site with a Kd value (0.5 nM) close to the activation constant value (Ka) of the three enzymes. The molecular structure of the VIP receptor was studied by immunological and chemical approaches. A monoclonal antibody (mAb 109-10-16) which partially decreased the binding of VIP to its receptor allowed the characterization of Mr = 53,000 and Mr = 48-49,000 polypeptides. More precise identification of protein components of the VIP receptor resulted from covalent cross-linking on intact HT 29 cells by four bifunctional reagents: dithiobis-(succinimidyl propionate) and its non-cleavable analog disuccinimidyl suberate, the photoactivable azido phenyl glyoxal and dimethylpimelimidate. Analysis by SDS-polyacrylamide gel electrophoresis demonstrated a major band of Mr = 67,000 regardless of which cross-linker was used. The same band and an Mr = 49,000 species were found in experiments using a crude membrane fraction of HT 29 cells. Assuming one molecule of VIP (Mr = 3326) linked per polypeptide, these observations suggest that an Mr = 64,000 species belongs to the VIP specific plasma membrane receptor. This protein contains an Mr = 20,000 N-linked sialic acid rich oligosaccharidic moiety.(ABSTRACT TRUNCATED AT 250 WORDS)
Marchis-Mouren
G
G
Institut de Chimie Biologique, CNRS UA202, Université d'Aix-Marseille, France.
Martin
J M
JM
Luis
J
J
el Battari
A
A
Muller
J M
JM
Marvaldi
J
J
Pichon
J
J
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000378:metabolism; D000230:Adenocarcinoma / Q000378:metabolism*; D000262:Adenylyl Cyclases / Q000378:metabolism; D003110:Colonic Neoplasms / Q000378:metabolism*; D000242:Cyclic AMP / Q000096:biosynthesis / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D006801:Humans; D007118:Immunoassay; D008563:Membrane Lipids / Q000502:physiology; D011494:Protein Kinases / Q000378:metabolism; D011964:Receptors, Gastrointestinal Hormone / Q000378:metabolism*; D018005:Receptors, Vasoactive Intestinal Peptide; D014407:Tumor Cells, Cultured / Q000378:metabolism*; D014660:Vasoactive Intestinal Peptide / Q000378:metabolism / Q000494:pharmacology*
D008563:Membrane Lipids; D011964:Receptors, Gastrointestinal Hormone; D018005:Receptors, Vasoactive Intestinal Peptide; D014660:Vasoactive Intestinal Peptide; D000242:Cyclic AMP; D011494:Protein Kinases; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D000262:Adenylyl Cyclases