Identification of the interactome of the DP1 receptor for Prostaglandin D2: Regulation of DP1 receptor signaling and trafficking by IQGAP1

Abstract

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.

Keywords: DP1; GPCR; IQGAP1; Interactome; Signaling; Trafficking.