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Cycle of VIP in the human transformed colonic epithelial cells (HT-29) in culture
$33 MOLECULAR IDENTIFICATION AND STRUCTURAL REQUIREMENT OF VIP RECEPTORS IN RAT BRAIN SYNAPTOSOMES. Alain Couvineau, Steen Gammeltoft*, Marc Laburthe Equipe de Neuroendocrinologie INSERM U178, HSpital St Antoine, Paris, France ; and * Dept of Clinical Chemistry Rigshospitalet, Copenhagen, Denmark. The molecular size and peptide specificity of VIP receptors in the central nervous system are unknown. Using purified synaptosomes from rat cerebral cortex, we show here that the specific binding of 1251-VIP to synaptosomes is competitively inhibited by several peptides with the following order of potency : VIP (IC50=0.6nM) >PH I ( IC 50=10nM) >VlP 2-28 ( IC 50=40nM) >secretin ( IC 50=223nM) >hGRF ( IC 50=300nM). Central VIP receptors thus display the same pharmacological properties as peripheral VlP receptors, characterized in intestine and liver. The cleavable cross-linker dithiobis(succinimidyl propionate) (DTSP) is used to investigate the molecular nature of VIP binding sites in synaptosomes. Solubilization of synaptosomes covalently labeled with 1251-VIP followed by SDS-PAGE analysis yields a major Mr 49,000 component. Labeling of this component is specific since it is completely abolished by incubation of synaptosomes with 1 IJM native VIP while not affected by 1 IJM glucagon or octa CCK. The covalent labeling of the Mr 49,000 1251-VIP-protein complex is inhibited by native VlP in the range of concentration between 10-10 and 10-7M (IC50=lnM). Assuming one molecule of VIP (Mr 3,300) bound per molecule of protein, a protein of Mr 46,000 is identified as the cerebral VIP receptor. The molecular size of this receptor compares well with that described in rat liver : i.e. 48,000 (Couvineau & Laburthe, Biochem. J., 225:473, 1985) but differs from the VIP receptor in rat intestine, i.e. 73,000 (Laburthe et al., Eur. J. Biochem.,139 181,1984). These structural discrepancies among tissues may be tentatively rei-~'~d to the d i f f e re n t patterns of coupling between VlP receptor and adenylate cyclase, i.e. efficient coupling in intestine and inefficient coupling in liver and brain.
CYCLE OF VIP IN THE HUMAN TRANSFORMED COLONIC EPITHELIAL CELLS (HT-29) IN CULTURE J.C. Marie, D. Hui Bon Hoa, G. Hejblum, G. Rosselin.Unit~ INSERM U.55, Centre de Recherches Saint-Antoine, 184, rue du Fg. St. Antoine, 75571 Paris Cedex 12, France. Many polypeptides ligands such as insulin and EGF have been shown to be internalized via a receptor-mediated process and subsequently degraded. Therefore, we have studied the processing of VlP in a transformed epithelial clone (Louvard) of HT-29 that possess a functional receptor specific of VIP. The cycle of VIP was studied with a pure mono ]251-VIP (MI251-VIP) obtained by HPLC. A two steps procedure was used: |) an ;h incubation at |O°C maximally bind M|251-VIP on cell surface receptors; 2) after washing an incubation at 37°C. Thereafter, three compartments that contain radioactivity (R) are separated: 1) the incubation medium (IM); 2) the surface bound of acido-soluble R (SD) and 3) internalized R (IR). The kinetic studies of the VIP processing at 37°C indicated that the surface bound VlP rapidly decreases resulting in an internalization of the ligand that is maximum within 10 minutes and a more progressive increase of R in the medium. Lowering the temperature at 20°C, results in increasing the IR and diminishing the release of the ligand into the medium. The overall kinetics indicates that the surface bound VlP is first rapidly internalized and thereafter partly released in the medium. R of each compartment was analyzed by HPLC and rebinding procedure for measuring the percent of VlP remaining intact. After 0, 20, and 120 min the percentage of intact VIP is |00, 66 and 30%, respectively. Degradation of VIP in the medium is only observed when cells and medium are present and not with medium preincubated with cells and is therefore cell-dependent. Those results indicate the existence of a cycle of VlP into the cells. Since the amount of intact andunalter~d VIPvary according to the kinetics of distribution and the temperature, the cycle of VIP into the cells and its degradation are supported by different and specific structures.
CYCLE OF VIP IN THE HUMAN TRANSFORMED COLONIC EPITHELIAL CELLS (HT-29) IN CULTURE J.C. Marie, D. Hui Bon Hoa, G. Hejblum, G. Rosselin.Unit~ INSERM U.55, Centre de Recherches Saint-Antoine, 184, rue du Fg. St. Antoine, 75571 Paris Cedex 12, France. Many polypeptides ligands such as insulin and EGF have been shown to be internalized via a receptor-mediated process and subsequently degraded. Therefore, we have studied the processing of VlP in a transformed epithelial clone (Louvard) of HT-29 that possess a functional receptor specific of VIP. The cycle of VIP was studied with a pure mono ]251-VIP (MI251-VIP) obtained by HPLC. A two steps procedure was used: |) an ;h incubation at |O°C maximally bind M|251-VIP on cell surface receptors; 2) after washing an incubation at 37°C. Thereafter, three compartments that contain radioactivity (R) are separated: 1) the incubation medium (IM); 2) the surface bound of acido-soluble R (SD) and 3) internalized R (IR). The kinetic studies of the VIP processing at 37°C indicated that the surface bound VlP rapidly decreases resulting in an internalization of the ligand that is maximum within 10 minutes and a more progressive increase of R in the medium. Lowering the temperature at 20°C, results in increasing the IR and diminishing the release of the ligand into the medium. The overall kinetics indicates that the surface bound VlP is first rapidly internalized and thereafter partly released in the medium. R of each compartment was analyzed by HPLC and rebinding procedure for measuring the percent of VlP remaining intact. After 0, 20, and 120 min the percentage of intact VIP is |00, 66 and 30%, respectively. Degradation of VIP in the medium is only observed when cells and medium are present and not with medium preincubated with cells and is therefore cell-dependent. Those results indicate the existence of a cycle of VlP into the cells. Since the amount of intact andunalter~d VIPvary according to the kinetics of distribution and the temperature, the cycle of VIP into the cells and its degradation are supported by different and specific structures.