Background

Mechanisms governing localization, trafficking and signaling of G protein-coupled receptors (GPCRs) are critical in cell function. Protein-protein interactions are determinant in these processes. However, there are very little interacting proteins known to date for the DP1 receptor for prostaglandin D₂.

Methods

We performed LC-MS/MS analyses of the DP1 receptor interactome in HEK293 cells. To functionally validate our LC-MS/MS data, we studied the implications of the interaction with the IQGAP1 scaffold protein in the trafficking and signaling of DP1.

Results

In addition to expected interacting proteins such as heterotrimeric G protein subunits, we identified proteins involved in signaling, trafficking, and folding localized in various cell compartments. Endogenous DP1-IQGAP1 co-immunoprecipitation was observed in colon cancer HT-29 cells. The interaction was augmented by DP1 agonist activation in HEK293 cells and GST-pulldown assays showed that IQGAP1 binds to intracellular loops 2 and 3 of DP1. Co-localization of the two proteins was observed by confocal microscopy at the cell periphery and in intracellular vesicles in the basal state. PGD₂ treatment resulted in the redistribution of the DP1-IQGAP1 co-localization in the perinuclear vicinity. DP1 receptor internalization was promoted by overexpression of IQGAP1, while it was diminished by IQGAP1 knockdown with DsiRNAs. DP1-mediated ERK1/2 activation was augmented and sustained overtime by overexpression of IQGAP1 when compared to DP1 expressed alone. IQGAP1 knockdown decreased ERK1/2 activation by DP1 stimulation. Interestingly, ERK1/2 signaling by DP1 was increased when IQGAP2 was silenced, while it was impaired by IQGAP3 knockdown.

Conclusions

Our findings define the putative DP1 interactome, a patho-physiologically important receptor, and validated the interaction with IQGAP1 in DP1 function. Our data also reveal that IQGAP proteins may differentially regulate GPCR signaling.

General significance

The identified putative DP1-interacting proteins open multiple lines of research in DP1 and GPCR biology in various cell compartments.

中文翻译：

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前列腺素 D2 DP1 受体相互作用组的鉴定：IQGAP1 对 DP1 受体信号传导和贩运​​的调节

背景

控制 G 蛋白偶联受体 (GPCR) 的定位、运输和信号传导的机制对细胞功能至关重要。蛋白质-蛋白质相互作用是这些过程中的决定因素。然而，迄今为止已知的与前列腺素 D ₂的 DP1 受体相互作用的蛋白质非常少。

方法

我们对 HEK293 细胞中的 DP1 受体相互作用组进行了 LC-MS/MS 分析。为了在功能上验证我们的 LC-MS/MS 数据，我们研究了与 IQGAP1 支架蛋白相互作用对 DP1 的运输和信号传导的影响。

结果

除了预期的相互作用蛋白质（如异源三聚体 G 蛋白亚基）外，我们还鉴定了参与信号、运输和折叠的蛋白质，这些蛋白质位于各种细胞区室中。在结肠癌 HT-29 细胞中观察到内源性 DP1-IQGAP1 共免疫沉淀。HEK293 细胞中 DP1 激动剂激活增强了这种相互作用，GST 下拉测定表明 IQGAP1 与 DP1 的细胞内环 2 和 3 结合。通过共聚焦显微镜在细胞外周和基础状态的细胞内囊泡中观察到两种蛋白质的共定位。PGD ₂处理导致 DP1-IQGAP1 共定位在核周附近的重新分布。DP1 受体内化由 IQGAP1 的过表达促进，而 IQGAP1 与 DsiRNA 的敲低会减少它。与单独表达的 DP1 相比，通过 IQGAP1 的过表达，DP1 介导的 ERK1/2 激活得到增强并持续加班。IQGAP1 敲低通过 DP1 刺激降低了 ERK1/2 的激活。有趣的是，当 IQGAP2 沉默时，DP1 的 ERK1/2 信号会增加，而 IQGAP3 敲低会削弱它。

结论

我们的研究结果定义了假定的 DP1 相互作用组，这是一种重要的病理生理受体，并验证了与 IQGAP1 在 DP1 功能中的相互作用。我们的数据还显示 IQGAP 蛋白可能差异调节 GPCR 信号。

一般意义

确定的假定 DP1 相互作用蛋白开启了 DP1 和 GPCR 生物学在不同细胞区室中的多条研究线。