Nielsen F C, Gammeltoft S, Westermark B, Fahrenkrug J
Department of Clinical Chemistry, Bispebjerg Hospital, Copenhagen NV, Denmark.
Peptides. 1990 Nov-Dec;11(6):1225-31. doi: 10.1016/0196-9781(90)90156-y.
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 mumol/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 phenanthroline and pepstatin-A. Surface binding and internalization of 125I-VIP was increased 3 times by phenanthroline, and pepstatin-A caused a 5 times increase in surface binding. Chloroquine reduced surface-bound 125I-VIP, but caused retention of internalized 125I-VIP.
血管活性肠肽（VIP）以高亲和力（解离常数Kd为0.13 nmol/l）与人胶质瘤细胞系U - 343 MG Cl 2:6上的受体结合。这些受体与相关肽类蛙皮素、垂体腺苷酸环化酶激活肽（PHM）和促胰液素的结合亲和力分别低10倍、400倍和5000倍。脱酰胺VIP（VIP - COOH）和[去组氨酸1]VIP的结合亲和力分别低10倍和100倍。片段VIP(7 - 28)可取代25%与受体结合的125I - VIP，而VIP(16 - 28)和VIP(1 - 22 - NH2)无活性。10 μmol/l的拮抗剂[N - 乙酰 - 酪氨酸1，D - 苯丙氨酸2]促肾上腺皮质激素释放因子(1 - 29) - NH2、[对氯 - D - 苯丙氨酸6，亮氨酸17]VIP和VIP(10 - 28)可完全抑制125I - VIP的结合；相反，拮抗剂L - 8 - K无活性。亲和标记显示VIP分别与分子量为75 kDa、66 kDa和50 kDa的蛋白质结合。结合后，该肽迅速内化，在稳态时，细胞相关的125I - VIP中只有20%与细胞表面的受体结合。内化的125I - VIP完全降解为125I - 酪氨酸并从细胞中释放出来。氯喹、菲咯啉和胃蛋白酶抑制剂A可显著减少内化的125I - VIP的降解。菲咯啉使125I - VIP的表面结合和内化增加3倍，胃蛋白酶抑制剂A使表面结合增加5倍。氯喹减少了表面结合的125I - VIP，但导致内化的125I - VIP滞留。
