Smyth E M, Austin S C, Reilly M P, FitzGerald G A
Center for Experimental Therapeutics, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
J Biol Chem. 2000 Oct 13;275(41):32037-45. doi: 10.1074/jbc.M003873200.
Prostacyclin (PGI(2)), the major product of cyclooxygenase in macrovascular endothelium, mediates its biological effects through its cell surface G protein-coupled receptor, the IP. PKC-mediated phosphorylation of human (h) IP is a critical determinant of agonist-induced desensitization (Smyth, E. M., Hong Li, W., and FitzGerald, G. A. (1998) J. Biol. Chem. 273, 23258-23266). The regulatory events that follow desensitization are unclear. We have examined agonist-induced sequestration of hIP. Human IP, tagged at the N terminus with hemagglutinin (HA) and fused at the C terminus to the green fluorescent protein (GFP), was coupled to increased cAMP (EC(50) = 0.39 +/- 0.09 nm) and inositol phosphate (EC(50) = 86. 6 +/- 18.3 nm) generation when overexpressed in HEK 293 cells. Iloprost-induced sequestration of HAhIP-GFP, followed in real time by confocal microscopy, was partially colocalized to clathrin-coated vesicles. Iloprost induced a time- and concentration-dependent loss of cell surface HA, indicating receptor internalization, which was prevented by inhibitors of clathrin-mediated trafficking and partially reduced by cotransfection of cells with a dynamin dominant negative mutant. Sequestration (EC(50) = 27.6 +/- 5.7 nm) was evident at those concentrations of iloprost that induce PKC-dependent desensitization. Neither the PKC inhibitor GF109203X nor mutation of Ser-328, the site for PKC phosphorylation, altered receptor sequestration indicating that, unlike desensitization, internalization is PKC-independent. Deletion of the C terminus prevented iloprost-induced internalization, demonstrating the critical nature of this region for sequestration. Internalization was unaltered by cotransfection of cells with G protein-coupled receptor kinases (GRK)-2, -3, -5, -6, arrestin-2, or an arrestin-2 dominant negative mutant, indicating that GRKs and arrestins do not play a role in hIP trafficking. The hIP is sequestered in response to agonist activation via a PKC-independent pathway that is distinct from desensitization. Trafficking is dependent on determinants located in the C terminus, is GRK/arrestin-independent, and proceeds in part via a dynamin-dependent clathrin-coated vesicular endocytotic pathway although other dynamin-independent pathways may also be involved.
前列环素（PGI₂）是大血管内皮细胞中环氧化酶的主要产物，它通过其细胞表面G蛋白偶联受体IP介导其生物学效应。蛋白激酶C（PKC）介导的人（h）IP磷酸化是激动剂诱导脱敏的关键决定因素（史密斯，E.M.，李鸿伟，W.，和菲茨杰拉德，G.A.（1998年）《生物化学杂志》273，23258 - 23266）。脱敏后随之发生的调节事件尚不清楚。我们研究了激动剂诱导的hIP隔离。在HEK 293细胞中过表达时，在N端用血凝素（HA）标记并在C端与绿色荧光蛋白（GFP）融合的人IP与cAMP生成增加（EC₅₀ = 0.39 ± 0.09纳米）和肌醇磷酸生成增加（EC₅₀ = 86.6 ± 18.3纳米）相关。用共聚焦显微镜实时跟踪，伊洛前列素诱导的HAhIP - GFP隔离部分定位于网格蛋白包被小泡。伊洛前列素诱导细胞表面HA随时间和浓度依赖性丢失，表明受体内化，这被网格蛋白介导的转运抑制剂所阻止，并且通过共转染细胞与发动蛋白显性负突变体而部分减少。在那些诱导PKC依赖性脱敏的伊洛前列素浓度下，隔离（EC₅₀ = 27.6 ± 5.7纳米）很明显。PKC抑制剂GF109203X和PKC磷酸化位点Ser - 328的突变均未改变受体隔离，表明与脱敏不同，内化不依赖PKC。C端缺失阻止了伊洛前列素诱导的内化，证明了该区域对于隔离的关键性质。通过共转染细胞与G蛋白偶联受体激酶（GRK）-2、-3、-5、-6、抑制蛋白-2或抑制蛋白-2显性负突变体，内化未改变，表明GRK和抑制蛋白在hIP转运中不起作用。hIP通过一条与脱敏不同的不依赖PKC的途径响应激动剂激活而被隔离。转运依赖于位于C端的决定因素，不依赖GRK/抑制蛋白，并且部分通过依赖发动蛋白的网格蛋白包被小泡内吞途径进行，尽管也可能涉及其他不依赖发动蛋白的途径
