Center for Substance Abuse Research and Department of Pharmacology, Temple University Lewis Katz School of Medicine, Philadelphia, PA, USA.
Department of Molecular and Cellular Biochemistry, University of Kentucky College of Medicine, Lexington, KY, USA.
Handb Exp Pharmacol. 2022;271:83-96. doi: 10.1007/164_2020_398.
We reported previously that GEC1 (glandular epithelial cell 1), a member of microtubule-associated proteins (MAPs), interacted directly with the C-tail of KOR (KCT) and tubulin and enhanced cell surface expression of KOR in CHO cells by facilitating its trafficking along the export pathway. Two GEC1 analogs (GABARAP and GATE16) were also shown to increase KOR expression. In addition, to understand the underlying mechanism, we demonstrated that N-ethylmaleimide-sensitive factor (NSF), an essential component for membrane fusion, co-immunoprecipitated with GEC1 from brain extracts. In this study, using pull-down techniques, we have found that (1) GEC1 interacts with NSF directly and prefers the ADP-bound NSF to the ATP-bound NSF; (2) D1 and/or D2 domain(s) of NSF interact with GEC1, but the N domain of NSF does not; (3) NSF does not interact with KCT directly, but forms a protein complex with KCT via GEC1; (4) NSF and/or α-SNAP do not affect KCT-GEC1 interaction. Thus, GEC1 (vs the α-SNAP/SNAREs complex) binds to NSF in distinctive ways in terms of the ADP- or ATP-bound form and domains of NSF involved. In conclusion, GEC1 may, via its direct interactions with KOR, NSF, and tubulin, enhance trafficking and fusion of KOR-containing vesicles selectively along the export pathway, which leads to increase in surface expression of KOR. GABARAP and GATE16 may enhance KOR expression in a similar way.
我们之前曾报道过，GEC1（腺上皮细胞 1）是微管相关蛋白（MAPs）的成员，它与 KOR（KCT）的 C 尾和微管直接相互作用，并通过促进其沿输出途径运输，增强 CHO 细胞表面 KOR 的表达。两种 GEC1 类似物（GABARAP 和 GATE16）也被证明可以增加 KOR 的表达。此外，为了了解潜在的机制，我们证明了 N-乙基马来酰亚胺敏感因子（NSF），一种对于膜融合至关重要的成分，从脑提取物中与 GEC1 共同免疫沉淀。在这项研究中，我们使用下拉技术发现：（1）GEC1 与 NSF 直接相互作用，并且更喜欢 ADP 结合的 NSF 而不是 ATP 结合的 NSF；（2）NSF 的 D1 和/或 D2 结构域与 GEC1 相互作用，但 NSF 的 N 结构域不与 GEC1 相互作用；（3）NSF 不与 KCT 直接相互作用，但通过 GEC1 与 KCT 形成蛋白复合物；（4）NSF 和/或 α-SNAP 不影响 KCT-GEC1 相互作用。因此，GEC1（相对于 α-SNAP/SNAREs 复合物）在结合 NSF 时在 ADP 或 ATP 结合形式以及涉及的 NSF 结构域方面以独特的方式结合。总之，GEC1 可能通过其与 KOR、NSF 和微管的直接相互作用，选择性地增强含有 KOR 的囊泡的运输和融合，从而导致 KOR 表面表达增加。GABARAP 和 GATE16 可能以类似的方式增强 KOR 的表达。
