Covalent cross‐linking of vasoactive intestinal peptide (VIP) to its receptor in intact colonic adenocarcinoma cells in culture (HT 29)

Muller, Jean-Marc; Luis, José; Fantini, Jacques; Abadie, B.; Giannellini, Fernand; Marvaldi, Jacques; Pichon, Jacques

doi:10.1111/j.1432-1033.1985.tb09117.x

Cited by 57 publications

(24 citation statements)

References 30 publications

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“…These conditions were selected and used to repeat the experiments described above. Under these new conditions, a rapid loss ( t l j 2 "N 2 min) of VIP binding was also observed (Fig.2; open [23]. This value is in good agreement with the present data.…”

Section: %)supporting

confidence: 91%

“…The disappearance of cell-surfacebound 1251-VIP after incubation of the cells at 37"C, was further demonstrated by cross-linking experiments. The amount of the M , 64000 polypeptide, previously described as the high-affinity VIP binding site [23], was reduced by 68% in HT 29 cells exposed for 10 min at 37°C. The values obtained by the two experimental approaches were thus in very good agreement.…”

Section: Discussionmentioning

confidence: 82%

“…'2sI-VIP was initially bound at 4°C to the cell surface, then cells were incubated at 37°C or 4°C for 10 rnin and '2sI-VIP was crosslinked to its receptor with DTSP under the conditions already described [23]. Data from Fig.1 shows clearly that the labelling of the major polypeptide of M , 64000 was greatly reduced (68% of labelling reduction) in cells incubated at 37 'C ( Fig.…”

Section: %)mentioning

confidence: 76%

“…Conditions for cross-linking experiments have been described elsewhere [23]. Briefly, monolayers of HT 29 cells (3 x lo6 cells) were incubated for 3 h at 4°C with 0.5 nM lZ5I-VIP.…”

Section: Cross-linking Of Bound "I-vip To Ht 29 Cellsmentioning

confidence: 99%

“…These data demonstrated that lZ5I-VIP bound to its receptors was rapidly (less than 10 min) recovered in an intracellular compartment and then released in the incubation medium in a degraded form [21, 221. When HT 29 cells were pretreated with VIP at 37°C the VIP receptors were no longer detectable on the cell surface [23]. This suggests that VIP is internalized together with its receptor or in other words by receptor-mediated endocytosis.…”

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confidence: 99%

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Cycle of the vasoactive intestinal peptide and its binding site in a human adenocarcinoma cell line (HT 29)

et al. 1986

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The disappearance of vasoactive-intestinal-peptide (VIP) binding sites at the cell surface of a cultured target cell, originating from a human colonic adenocarcinoma (HT 29 cell line), was studied, after preexposition of the cell to the peptide, as a function of time, VIP concentration and temperature. Maximum effect (60 -8O0/o loss of binding capacity) was obtained after a 5-10 min exposure of the cells at 37°C with a VIP concentration of 100 nM. The tljz of maximum disappearance was less than 2 min and the concentration of native VIP giving half-maximum decrease in lZ5I-VIP binding was 6 nM. The affinity of remaining binding sites for VIP was not affected compared to that of control cells (Kd = 0.3 nM). Disappearance of VIP binding sites was spccific sincc, with the same conditions of preincubation, the specific binding of '251-labeled epidermal growth factor to HT 29 cells was not modified. The phenomenon was reversible and 90% of binding capacity could be restored in less than 60 min by incubating cells in VIP-free medium. Correlatively we showed, by two independent experimental procedures, that lZ5I-VIP, initially bound to HT 29 cells, was maximally internalized after 10 min of incubation at 37"C. All the data strongly suggest that: (1) internalization of VIP is receptor-mediated; (2) upon exposure to native VIP, VIP receptors are down-regulated or at least sequestered within HT 29 cells.The regulation of the concentration of cell-surface receptors is a general way for the cell to modulate its responsiveness to hormones, growth factors or neurotransmitters. Loss of cell-surface receptor binding is generally obtained by exposure of target cells to the effector itself. This phenomenon occurs with a number of polypeptidic hormones including insulin [l], epidermal growth factor (EGF) [2], platelet-derived growth factor [3,4], human growth hormone [5], thyroliberin [6] and thyrotropin [7, 81. The reduced capacity of cells to bind the ligand was not attributable, in most cases, to a decreased affinity of the receptor for its ligand but to a reduction in the number of cell surface receptors [l, 5, 7, 9, 101.In several cases, the internalization of the ligand-receptors complexes, followed by the degradation of the receptor and of the ligand have been demonstrated [3,. These events have been proposed to represent the down-regulation of cellular receptors. The consequence of receptor down-regulation is generally a refractoriness of cells to agents acting at the cell-surface receptors. For example, the down-regulation of EGF receptors leads to a desensitization of the cells to the growth factor and to a reduced mitogenic response [14]. Likewise, in pathological states, such as some cases of obesity, large concentrations of insulin in the blood stream involve a decreased sensitivity of target tissues to the hormone [15, 161.The down-regulation of /?-adrenergic receptors has been extensively studied and it appears from two recent reports [I 7, 181 that one may distinguish between sequestration and downregulation of j-re...

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Section: %)supporting

confidence: 91%

Section: Discussionmentioning

confidence: 82%

Section: %)mentioning

confidence: 76%

“…Conditions for cross-linking experiments have been described elsewhere [23]. Briefly, monolayers of HT 29 cells (3 x lo6 cells) were incubated for 3 h at 4°C with 0.5 nM lZ5I-VIP.…”

Section: Cross-linking Of Bound "I-vip To Ht 29 Cellsmentioning

confidence: 99%

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confidence: 99%

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