Open AccessMolecular characteristics and evidence for internalization of vasoactive‐intestinal‐peptide (VIP) receptors in the tumoral rat‐pancreatic acinar cell line AR 4‐2 J
Author(s) -
SVOBODA Michal,
NEEF Philippe,
TASTENOY Michèle,
CHRISTOPHE Jean
Publication year - 1988
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1988.tb14334.x
Subject(s) - vasoactive intestinal peptide , secretin , internalization , receptor , chemistry , peptide , cycloheximide , phenylarsine oxide , acinar cell , cell culture , medicine , endocrinology , biochemistry , biology , neuropeptide , pancreas , protein biosynthesis , genetics
1 Vasoactive intestinal peptide (VIP) receptors were investigated in the tumoral acinar cell line AR 4‐2 J derived from rat pancreas [ 125 I]Iodo‐VIP binding to cell membranes showed the following IC 50 values for unlabeled peptides: VIP, 0.3 nM; peptide His‐IleNH 2 , 2 nM; helodermin, 30 nM; secretin, 100 nM. After incubation with 20 nM dexamethasone, the binding capacity increased twofold but affinities were unchanged. External [ 125 I]iodo‐VIP binding to intact cells reached steady state after 5 min at 37°C, while the sequestration‐internalization of the [ 125 I]iodo‐VIP ‐receptor complex (tested by cold acid washing) increased progressively, reaching 75% of total binding after 1 h. This phenomenon was blocked at 4°C. Further data with dexamethasone, tunicamycin, cycloheximide, low temperature, and/or phenylarsine oxide, suggested a half‐life of 2 days for VIP receptors and the necessity of N ‐glycosylation for proper translocation. 2 For chemical [ 125 I]iodo‐VIP cross‐linking bis[2‐(succinimidooxycarbonyloxy)ethyl]sulfone gave the best yield when compared with five other bifunctional reagents. In membranes, the main specifically cross‐linked peptide had M T 66000 under nonreducing conditions, and migrated with lower velocity (–5%) under reducing conditions. Cross‐linking was suppressed by VIP, peptide His‐IleNH 2 and helodermin (competitively) and also by GTP. In intact cells, the M r of [ 125 I]iodo‐VIP‐cross‐linked peptides depended on the mode of cell solubilization. After direct solubilization, the major cross‐linked radioactivity migrated as a smear of M r 130000–180000 but an M T ‐66000 peptide was also detectable. In contrast, the solubilization of cross‐linked cells detached by mild trypsinisation gave mainly the M T ‐66000 labeled peptide. This suggests that most VIP receptors in intact, attached cells were in a high‐ M T complex and that mild cell treatment was sufficient to disrupt this complex.