High affinity receptors for vasoactive intestinal peptide on a human glioma cell line |
| |
Authors: | Finn C. Nielsen Steen Gammeltoft Bengt Westermark Jan Fahrenkrug |
| |
Affiliation: | a Department of Clinical Chemistry, Bispebjerg Hospital, DK-2400, Copenhagen NV, Denmark b Department of Pathology, University Hospital, S-75185, Uppsala, Sweden |
| |
Abstract: | Vasoactive intestinal peptide (VIP) bound with high affinity (Kd 0.13 nmol/l) to receptors on the human glioma cell line U-343 MG Cl 2:6. The receptors bound the related peptides helodermin. PHM and secretin with 10, 400 and 5000 times lower affinity, respectively. Deamidated VIP (VIP-COOH) and [des-His1]VIP bound with 10 and 100 times lower affinity. The fragment VIP(7–28) displaced 25% of the receptor-bound 125I-VIP whereas VIP(16–28) and VIP(1–22-NH2) were inactive. The binding of 125I-VIP could be completely inhibited by 10 μmol/l of the antagonists [N-Ac-Tyr1,D-Phe2]GRF(1–29)-NH2, [pCl-D-Phe6,Leu17]VIP and VIP(10–28); in contrast, the antagonist L-8-K was inactive. Affinity labeling showed that VIP bound to proteins with Mr's of 75 kDa, 66 kDa and 50 kDa, respectively. Following binding, the peptide was rapidly internalized, and at steady-state only 20% of cell-associated 125I-VIP was bound to receptors on the cell surface. The internalized 125I-VIP was completely degraded to 125I-tyrosine which was released from the cells. Degradation of internalized 125I-VIP was significantly reduced by chloroquine phenantroline and pepstatin-A. Surface binding and internalization of 125I-VIP was increased 3 times by phenantroline, and pepstatin-A caused a 5 times increase in surface binding. Chloroquine reduced surface-bound 125I-VIP, but caused retention of internalized 125I-VIP. |
| |
Keywords: | Vasoactive intestinal peptide receptors Human glioma cells Binding specificity Internalization Degradation |
本文献已被 ScienceDirect 等数据库收录! |
|