Abstract
Follicle-stimulating hormone (FSH) and its G protein-coupled receptor, FSHR, represents a paradigm for receptor signaling systems that activate multiple and complex pathways. Classically, FSHR activates Gαs to increase intracellular levels of cAMP, but its ability to activate other G proteins, and β-arrestin-mediated signaling is well documented in many different cell systems. The pleiotropic signal capacity of FSHR offers a mechanism for how FSH drives multiple and dynamic downstream functions in both gonadal and non-gonadal cell types, including distinct diseases, and how signal bias may be achieved at a pharmacological and cell system-specific manner. In this study, we identify an additional mechanism of FSH-mediated signaling and downstream function in the endometrial adenocarcinoma Ishikawa cell line. While FSH did not induce increases in cAMP levels, this hormone potently activated pertussis toxin sensitive Gαi/o signaling. A selective allosteric FSHR ligand, B3, also activated Gαi/o signaling in these cells, supporting a role for receptor-mediated activation despite the low levels of FSHR mRNA. The low expression levels may attribute to the lack of Gαs/cAMP signaling as increasing FSHR expression resulted in FSH-mediated activation of the Gαs pathway. Unlike prior reports for FSH-mediated Gαs/cAMP signaling, FSH-mediated Gαi/o signaling was not affected by inhibition of dynamin-dependent receptor internalization. While chronic FSH did not alter cell viability, FSH was able to increase lipid droplet size. The β-arrestins are key adaptor proteins known to regulate FSHR signaling. Indeed, a rapid, FSH-dependent increase in interactions between β-arrestin1 and Gαi1 was observed via NanoBiT complementation in Ishikawa cells. Furthermore, both inhibition of Gαi/o signaling and siRNA knockdown of β-arrestin 1/2 significantly reduced FSH-induced lipid droplet accumulation, implying a role for a Gαi/o/β-arrestin complex in FSH functions in this cell type. As FSH/FSHR has been implicated in distinct hormone-dependent cancers, including endometrial cancer, analysis of the cancer genome database from 575 human endometrial adenocarcinoma tumors revealed that a subpopulation of samples expressed FSHR. Overall, this study highlights a novel mechanism for FSHR signal pleiotropy that may be exploited for future personalized therapeutic approaches.
Highlights
The gonadotropin hormone, follicle-stimulating hormone (FSH) is secreted from the anterior pituitary and plays key role in reproduction via activation of its receptor, FSHR in the gonads
FSHR can interact as heteromers with distinct G protein-coupled receptors (GPCRs) to modulate its signaling [25,26,27] and we have recently demonstrated that this receptor requires internalization to fully activate Gas signaling from specific intracellular compartments termed very early endosomes (VEE) [28, 29]
To determine whether distinct additional Gas-coupled GPCRs could activate this pathway in this cell type, Ishikawa cells were treated with either isoproterenol (10 μM), a b-adrenergic receptor agonist, or prostaglandin E2 (PGE2, 100 nM), to activate EP2/EP4
Summary
The gonadotropin hormone, follicle-stimulating hormone (FSH) is secreted from the anterior pituitary and plays key role in reproduction via activation of its receptor, FSHR in the gonads. FSH-mediated activation of its receptor expressed in ovarian granulosa cells, induces steroidogenesis and stimulates growth of the follicle. The b-arrestins play numerous roles in regulating GPCR activity in part due to its ability to adopt multiple distinct conformations in complex with the receptor [7,8,9,10,11], its ability to associate with other signal proteins, including G proteins, have been reported [12,13,14,15,16,17]. In addition to classical roles in G protein-uncoupling and receptor internalization, b-arrestins can form signalosomes that interact with multiple partners and induce diverse downstream signaling pathways
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