© 2018. Published by The Company of Biologists Ltd | Journal of Cell Science (2018) 131, jcs212522. doi:10.1242/jcs.212522 RESEARCH ARTICLE The VLDL receptor regulates membrane progesterone receptor trafficking and non-genomic signaling Nancy Nader, Maya Dib, Raphael Courjaret, Rawad Hodeify, Raya Machaca, Johannes Graumann and Khaled Machaca* ABSTRACT the plasma membrane and interact with the classical P4 receptor, is Progesterone mediates its physiological functions through activation of nonetheless effective at mediating non-genomic P4 signaling both transcription-coupled nuclear receptors and seven-pass- (Bandyopadhyay et al., 1998; Dressing et al., 2011; Peluso et al., transmembrane progesterone receptors (mPRs), which transduce 2002). These results argued for the presence of membrane P4 the rapid non-genomic actions of progesterone by coupling to various receptors that are distinct from the nuclear P4 receptors. In 2003, the signaling modules. However, the immediate mechanisms of action Thomas laboratory identified a family of membrane progesterone downstream of mPRs remain in question. Herein, we use an untargeted receptors (mPRs) from fish ovaries (Zhu et al., 2003a,b) that belong quantitative proteomics approach to identify mPR interactors to better to the progestin and adiponectin (AdipoQ) receptor family (also define progesterone non-genomic signaling. Surprisingly, we identify named PAQ receptors). However, the signal transduction cascade the very-low-density lipoprotein receptor (VLDLR) as an mPRβ downstream of mPRs that mediates the non-genomic actions of P4 (PAQR8) partner that is required for mPRβ plasma membrane remains unclear. localization. Knocking down VLDLR abolishes non-genomic The non-genomic action of mPR and the ensuing signaling progesterone signaling, which is rescued by overexpressing VLDLR. cascade have been studied extensively over the years in frog and fish Mechanistically, we show that VLDLR is required for mPR trafficking oocytes, where progesterone releases oocyte meiotic arrest. from the endoplasmic reticulum to the Golgi. Taken together, our data Vertebrate oocytes must undergo a maturation period before they define a novel function for the VLDLR as a trafficking chaperone become fertilization competent and are able to support embryonic required for the mPR subcellular localization and, as such, non- development. Oocyte maturation encompasses progression through genomic progesterone-dependent signaling. meiosis and arrest at metaphase II until fertilization (Machaca, 2007). It is defined by drastic cellular remodeling characterized by This article has an associated First Person interview with the first author the dissolution of the nuclear envelope (referred to as germinal of the paper. vesicle breakdown; GVBD), extrusion of the first polar body, chromosome condensation and arrest in metaphase of meiosis II KEY WORDS: Membrane progesterone receptor, VLDLR, Meiotic (Bement and Capco, 1990; Nader et al., 2013; Sadler and Maller, arrest, Oocyte, Trafficking, Oocyte maturation, Endoplasmic 1985; Smith, 1989; Voronina and Wessel, 2003). Prior to oocyte reticulum, Golgi, Progesterone maturation and for prolonged periods of time, fully grown vertebrate oocytes are arrested at prophase of meiosis I, as they grow and stock INTRODUCTION molecular components essential for future development (Smith, Progesterone (P4) is a steroid hormone that regulates various 1989; Voronina and Wessel, 2003). P4 releases Xenopus oocyte reproductive processes in females, including ovulation, meiotic arrest by ultimately activating maturation-promoting factor implantation and sexual differentiation. Signaling downstream of (MPF; the cyclin-B–cdc2 complex), the key driver of meiosis P4 has been studied primarily through the activation of nuclear (Nader et al., 2013; Nebreda and Ferby, 2000). In amphibian receptors that act as transcription factors to stimulate P4-dependent oocytes, treatment with the membrane-impermeant P4 conjugated gene expression (Ellmann et al., 2009). However, P4 is also known to BSA (P4–BSA), efficiently releases meiotic arrest. These and to transduce rapid non-genomic signals that link to cAMP, Ca2+ and other results indicate that P4 acts through a membrane receptor the mitogen-activated protein kinase (MAPK) cascade, among other rather than the classical nuclear receptor (Bandyopadhyay et al., signaling modules (Valadez-Cosmes et al., 2016). Initially, 1998; Blondeau and Baulieu, 1984; Josefsberg Ben-Yehoshua activation of these signaling cascades was attributed to the et al., 2007), although there is evidence for a potential role for the classical ability of the progesterone receptor to interact with nuclear receptor as well (Bayaa et al., 2000; Tian et al., 2000). There cytoplasmic factors (Dressing et al., 2011). However, non- is also evidence that other steroids release frog oocyte meiotic arrest, genomic actions of progestins have been demonstrated in several with testosterone being the physiological inducer of oocyte tissues that do not express classical progesterone receptors, and P4 maturation in vivo (Lutz et al., 2001). In 2007, Ben-Yehoshua coupled to bovine serum albumin (BSA), which is unable to cross et al. reported the identification of a Xenopus laevis mPR ortholog, mPRβ (also known as PAQR8, herein referred to as mPR for simplicity) as the receptor functionally responsible for the Department of Physiology and Biophysics, Weill Cornell Medicine Qatar, Education City – Qatar Foundation, P.O. Box 24144, Doha, Qatar. resumption of meiosis in response to P4 (Josefsberg Ben- Yehoshua et al., 2007). It is well documented that high levels of *Author for correspondence ([email protected]) cAMP and protein kinase A (PKA) are important in maintaining K.M., 0000-0001-6215-2411 meiotic arrest in vertebrates (Bravo et al., 1978; Cho et al., 1974; Conti et al., 2002; Gallo et al., 1995; Lutz et al., 2000; Maller and Received 25 October 2017; Accepted 17 April 2018 Krebs, 1977; Meijer and Zarutskie, 1987; Sadler et al., 2008; Sheng Journal of Cell Science 1 RESEARCH ARTICLE Journal of Cell Science (2018) 131, jcs212522. doi:10.1242/jcs.212522 et al., 2001; Stern and Wassarman, 1974). In Xenopus oocytes, the We then checked the trafficking of the two constructs GFP–mPR high levels of cAMP–PKA are maintained, at least in part, through and mPR–GFP by performing confocal imaging of live oocytes. To the action of the constitutively active Gαs-coupled G protein- mark the plasma membrane, we also expressed the resident plasma coupled receptor GPR185 (Ríos-Cardona et al., 2008), the homolog membrane (PM) protein TMEM–mCherry, which encodes a Ca2+- of mammalian GPR3, which has been found to block oocyte activated Cl− channel (Yu et al., 2010). A z-stack of confocal images maturation in mouse oocytes (Freudzon et al., 2005; Mehlmann was acquired across the oocyte (Fig. S1), with the orthogonal et al., 2002, 2004; Norris et al., 2007). mPR activation does not section through this stack representing the subcellular distribution of seem to signal through Gαi to inhibit adenylate cyclase since the proteins as previously described (Yu et al., 2010). We have treatment with pertussis toxin was ineffective at blocking P4- previously shown that TMEM–mCherry targets to the PM and that induced oocyte maturation (Mulner et al., 1985; Olate et al., 1984; its membrane residence is not affected throughout oocyte Sadler et al., 1984). Similarly, maturation in mouse oocytes also maturation (Nader et al., 2014; Yu et al., 2010). Interestingly, the seems to be independent from the action of the Gi subunit GFP–mPR construct, which is unable to release meiotic arrest, was (Mehlmann et al., 2006). Moreover, although high levels of mostly intracellular and unable to localize to the PM (Fig. 1E; Fig. cAMP are essential in maintaining oocyte meiotic arrest, oocyte S1B), whereas a significant proportion of the functional mPR–GFP maturation progresses without any changes in cAMP or PKA levels localized to the PM (microvilli) in addition to localizing (Nader et al., 2016). These findings argue for a positive signal intracellularly (Fig. 1E; Fig. S1A). downstream of mPR that overrides the cAMP–PKA inhibitory GFP–mPR- and mPR–GFP-expressing oocytes were stained with signal (Nader et al., 2016). This positive signal is likely to be wheat germ agglutinin (WGA) to label the plasma membrane, initiated through the P4–mPR axis to release the oocyte from allowing for the quantification of the distribution of mPR at the PM meiotic arrest. (Courjaret et al., 2016; El-Jouni et al., 2008). mPR–GFP was found Here, we employ an untargeted proteomics approach to identify to be significantly enriched at the PM (38.91±1.89%, mean±s.e.m.; the mPR interactome to better define signaling downstream of n=10), whereas GFP–mPR was mainly restricted to the cytosol, mPR. We identify the very-low-density lipoprotein receptor with only 16.18±0.98% (n=12) at the PM (Fig. 1F). The (VLDLR) as an mPR-interacting protein, and show that VLDLR intracellular component of mPR–GFP could represent the is essential for mPR plasma membrane localization, and, as such, proportion of the receptor that is undergoing biogenesis, and/or its signaling function. VLDLR acts as a molecular chaperone that saturation of the trafficking machinery when mPR is overexpressed. is required for the trafficking of mPR from the endoplasmic Furthermore, our conservative quantification approach using the reticulum (ER) to the Golgi. In the absence of VLDLR, mPR peak of WGA staining