Molecular characteristics and evidence for internalization of vasoactive-intestinal-peptide (VIP) receptors in the tumoral rat-pancreatic acinar cell line AR 4-2 J.
1. Vasoactive intestinal peptide (VIP) receptors were investigated in the tumoral acinar cell line AR 4-2 J derived from rat pancreas [125I]Iodo-VIP binding to cell membranes showed the following IC50 values for unlabeled peptides: VIP, 0.3 nM; peptide His-IleNH2, 2 nM; helodermin, 30 nM; secretin, 100 nM. After incubation with 20 nM dexamethasone, the binding capacity increased twofold but affinities were unchanged. External [125I]iodo-VIP binding to intact cells reached steady state after 5 min at 37 degrees C, while the sequestration-internalization of the [125I]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 degrees 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 [125I]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 Mr 66,000 under nonreducing conditions, and migrated with lower velocity (-5%) under reducing conditions. Cross-linking was suppressed by VIP, peptide His-IleNH2 and helodermin (competitively) and also by GTP. In intact cells, the Mr of [125I]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 Mr 130,000-180,000 but an Mr-66,000 peptide was also detectable. In contrast, the solubilization of cross-linked cells detached by mild trypsinisation gave mainly the Mr-66,000 labeled peptide. This suggests that most VIP receptors in intact, attached cells were in a high-Mr complex and that mild cell treatment was sufficient to disrupt this complex.
Svoboda
M
M
Department of Biochemistry and Nutrition, Medical School, Université Libre de Bruxelles, Belgium.
De Neef
P
P
Tastenoy
M
M
Christophe
J
J
D000818:Animals; D001345:Autoradiography; D001665:Binding Sites / Q000187:drug effects; D002460:Cell Line; D002462:Cell Membrane / Q000032:analysis; D003513:Cycloheximide / Q000494:pharmacology; D003907:Dexamethasone / Q000494:pharmacology; D004586:Electrophoresis; D010190:Pancreatic Neoplasms / Q000032:analysis*; D051381:Rats; D011964:Receptors, Gastrointestinal Hormone / Q000032:analysis*; D018005:Receptors, Vasoactive Intestinal Peptide; D013696:Temperature; D014415:Tunicamycin / Q000494:pharmacology; D014660:Vasoactive Intestinal Peptide / Q000494:pharmacology
D011964:Receptors, Gastrointestinal Hormone; D018005:Receptors, Vasoactive Intestinal Peptide; D014415:Tunicamycin; D014660:Vasoactive Intestinal Peptide; D003907:Dexamethasone; D003513:Cycloheximide