Abstract

G protein-coupled receptors (GPCRs) have been found to form heterodimers and modulate or fine-tune the functions of GPCRs. However, the involvement of GPCR heterodimerization and its functional consequences in gonadal tissues, including granulosa cells, have been poorly investigated, mainly due to the lack of efficient method for identification of novel GPCR heterodimers. In this paper, we identified a novel GPCR heterodimer between prostaglandin E2 (PGE2) receptor 2 (EP2) and calcitonin (CT) receptor (CTR). High-resolution liquid chromatography (LC)-tandem mass spectrometry (MS/MS) of protease-digested EP2-coimmunoprecipitates detected protein fragments of CTR in an ovarian granulosa cell line, OV3121. Western blotting of EP2- and CTR-coimmunoprecipitates detected a specific band for EP2-CTR heterodimer. Specific heterodimerization between EP2 and CTR was also observed by fluorescence resonance energy transfer analysis in HEK293MSR cells expressing cyan- and yellow-fluorescent protein-fused EP2 and CTR, respectively. Collectively, these results provided evidence for heterodimerization between EP2 and CTR. Moreover, Ca2+ mobilization by CT was approximately 40% less potent in HEK293MSR cells expressing an EP2-CTR heterodimer, whereas cAMP production by EP2 or CT was not significantly altered compared with cells expressing EP2- or CTR alone. These functional analyses verified that CTR-mediated Ca2+ mobilization is specifically decreased via heterodimerization with EP2. Altogether, the present study suggests that a novel GPCR heterodimer, EP2-CTR, is involved in some functional regulation, and paves the way for investigation of novel biological roles of CTR and EP2 in various tissues.

Highlights

  • Most receptors of neurotransmitters, neuropeptides, and hormones are G protein-coupled receptors (GPCRs), and their pharmacological properties are targets of drug development [1]

  • No GPCR heterodimer has yet been identified using this method [23, 24], these findings suggest that Co-IP-based liquid chromatography (LC)-MS/MS may be used to screen for novel GPCR heterodimers

  • This study presents the identification of a novel GPCR heterodimer between the E2 (PGE2) receptor 2 (EP2) and the calcitonin (CT) receptor (CTR) in cultured cell lines using high-resolution LC-MS/MS of EP2-coimmunoprecipitates, and fluorescent resonance energy transfer (FRET)

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Summary

Introduction

Neuropeptides, and hormones are G protein-coupled receptors (GPCRs), and their pharmacological properties are targets of drug development [1]. This study presents the identification of a novel GPCR heterodimer between the EP2 and the calcitonin (CT) receptor (CTR) in cultured cell lines using high-resolution LC-MS/MS of EP2-coimmunoprecipitates, and fluorescent resonance energy transfer (FRET). We showed that Ci-GnRHR1 (R1) and Ci-GnRHR4 (R4) formed heterodimers following their transfection into HEK293MSR cells [11, 12].

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