Heterodimerization of the prostaglandin E2 receptor EP2 and the calcitonin receptor CTR

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.

Calcitonin receptor antibody validation and expression in the rodent brain

“…These bands were absent in lysates prepared from cells transfected with empty vector. The band patterns are broadly consistent with previous western blots using rat kidney and cell lysates (21,31,34). Interestingly, some low molecular weight bands were also present.…”

The regulation of oocyte maturation and ovulation in the closest sister group of vertebrates

“…Total RNA was extracted from isolated follicles, purified, and treated with TURBO DNase as previously described (Matsubara et al, 2017). A total of 500 ng or 150 ng of quality-verified RNA from sieved follicles or further purified follicles, respectively, was subjected to RNA-seq analysis using a HiSeq1500 instrument (Illumina, San Diego, CA) in the rapid mode, as previously described (Kawada et al, 2017; Kurihara et al, 2017).…”

International Union of Basic and Clinical Pharmacology. CIX. Differences and Similarities between Human and Rodent Prostaglandin E2Receptors (EP1–4) and Prostacyclin Receptor (IP): Specific Roles in Pathophysiologic Conditions

“…Similarly, a single mutation in PTGIR reduces cAMP production (Stitham et al, 2007) and is associated with platelet activation and cerebral infarction (Shimizu et al, 2013). The prostanoid receptors, including IP and EP receptors, are known to have the ability to form homodimers or heterodimers (Midgett et al, 2011;Matsubara et al, 2017). Relatively few studies have been devoted to this issue, making comparisons between humans and rodents concerning homodimers and heterodimers difficult (Giguère et al, 2004;McGraw et al, 2006;Osborne et al, 2009;Ibrahim et al, 2013).…”

“…Coexpression of EP1 and β -adrenergic receptors results in formation of heterodimers, which may contribute to β -agonist resistance in asthma ( McGraw et al, 2006 ). The mouse EP2 receptor has been found to form heterodimers with the calcitonin receptor, thereby reducing its ability to induce Ca 2+ flux ( Matsubara et al, 2017 ).…”