Organellar Gβγ signaling—GPCR signaling beyond the cell surface

Chapter 13

Organellar Gbg signalingdGPCR signaling beyond the cell surface Ryan D. Martina, Ce´lia A. Bouazzaa and Terence E. He´bert Department of Pharmacology and Therapeutics, McGill University, Montréal, Québec, Canada

13.1 Introduction The classical view of cellular signaling through G protein-coupled receptors (GPCRs) is that they signal primarily from the cell surface. Recently, a large number of GPCRs have been demonstrated to be targeted to endomembrane locations, as have their associated signaling cascades. Many studies have now demonstrated that not all of these receptors are transported to the cell surface and may in fact be targeted to internal cellular structures such as the nucleus, mitochondria, endoplasmic reticulum (ER), and Golgi apparatus where they regulate a number of cellular events in a distinct manner from those they regulate at the cell surface (Irannejad and von Zastrow, 2014; Vaniotis et al., 2011; Tadevosyan et al., 2012; Branco and Allen, 2015; Ma et al., 2015; Audet et al., 2018). Downstream partners, such as heterotrimeric G proteins, are also localized to distinct intracellular sites. They may be associated with these GPCRs in a manner similar to how they are organized at the cell surface or they may be released from GPCRs at different sites and be translocated to other distal subcellular sites. Here, we will review recent advances in our understanding of selected novel, noncanonical roles of Gbg subunits, with a focus on events occurring in the Golgi apparatus, mitochondria, and the nucleus. Although much is known regarding roles that Gbg subunits generally play in signal transduction (reviewed in (Dupré et al., 2009; Khan et al., 2013; Lehmann et al., 2008)), the impact of mammalian Gbg subunit diversity remains understudied. Gb1-4 subunits share 78%e88% identity over their approximately 340 amino acid sequences (reviewed in (Khan et al., 2013; Schwindinger and Robishaw, 2001)) while Gb5 is structurally distinct from the other Gb subunits, sharing only 50% sequence identity. Gg subunits are even more structurally diverse than the Gb subunits, sharing between 27% and 76% sequence identity (Khan et al., 2013; Schwindinger and Robishaw, 2001). A number of studies have reported specific roles for individual Gb and Gg subunits, but we still have an incomplete view of their individual functions. Roles for individual Gb and Gg subunits have been suggested using antisense and RNA interference (RNAi) approaches and the roles they play in receptor signaling pathways as well as in embryonic development have been characterized in animal knockout models (Ching-Kang et al., 2003; Hosohata et al., 2000; Kalkbrenner et al., 1995; Kleuss et al., 1993; Krispel et al., 2003; Lobanova et al., 2008; Okae, 2010; Schwindinger et al., 2003, 2004, 2009, 2011; Varga et al., 2005; Wang et al., 2011; Xie et al., 2012; Zhang et al., 2011a; Khan et al., 2015). Most of these studies have focused on canonical GPCR effectors, mainly localized to the plasma membrane such as ion channels and enzymes like adenylyl cyclase and phospholipase Cb. Here, we will focus on a growing number of noncanonical GPCR effectors identified as targets of Gbg signaling, with a focus on pathways identified in the Golgi apparatus, mitochondria, and nucleus (reviewed in (Khan et al., 2013), Fig. 13.1). In the text below, when we refer to a specific Gb or Gg subunit (with the exception of Gb5), we assume they are partnered in the context of a Gbg dimer. We know of no instances where they have been shown to function alone.

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These authors contributed equally to this manuscript.

GPCRs. https://doi.org/10.1016/B978-0-12-816228-6.00013-1 Copyright © 2020 Elsevier Inc. All rights reserved.

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FIGURE 13.1 A sampling of noncanonical organellar functions of Gbg. In addition to canonical functions of Gbg in signal transduction following the activation of receptors at the plasma membrane, Gbg regulate many noncanonical effectors at distinct subcellular locations as depicted in this figure and denoted by letters referred to in the text. These effectors highlight various functions of Gbg in cellular signaling with a focus on protein trafficking through the Golgi, mitochondrial dynamics, direct or indirect transcriptional regulation, and mRNA processing.

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13.2 Gbg signaling in the ER and Golgi apparatus A number of studies have shown that many of the proteins involved in GPCR signaling interact initially in the ER, including receptor equivalents in GPCR oligomers (Salahpour et al., 2004), receptor and Gbg subunits (Dupré et al., 2006), and effectors such as inwardly rectifying potassium (Kir3) channels and adenylyl cyclase with nascent Gbg (Rebois et al., 2006; Dupré et al., 2007). The interactions between adenylyl cyclase or the b2-adrenergic receptor (b2AR) and Gbg subunits, or between receptor equivalents in the b2AR homodimer were insensitive to dominant negative Rab1 or Sar1 GTPases (Dupré et al., 2006, 2007) which regulate receptor trafficking ((Dupré and Hébert, 2006), reviewed in (Dong et al., 2006)). However, these latter studies highlight the fact that the Ga subunit is assembled with nascent receptor/Gbg/ effector complexes either at ER exit sites or in the Golgi apparatus as these interactions were blocked by dominant negative versions of Sar1 and Rab1 GTPases critical for anterograde protein trafficking (Dupré et al., 2006, 2007). Is there a specific role for individual Gbg subunits in organizing the assembly or trafficking of such complexes? One tantalizing observation for a precocious role for Gbg in assembling receptor dimers is that when Gbg function is inhibited in membranes by using a membrane-localized GPCR kinase (GRK2) carboxyl-terminus (GRK2-CT) construct, the formation of b2AR homodimers could be reduced (Dupré et al., 2009). Do Gbg subunits play an analogous role in the reassembly of signaling complexes when receptors are recycled? These pathways may be more difficult to dissect out than the relatively straightforward de novo synthesis pathway. Modulation of subtype-specific Gbg-dependent events with regard to assembly or subsequent signaling using RNAi or CRISPR/Cas9 approaches will perhaps identify roles for specific Gbg subunits. As many Gg subunits are prenylated, it was believed that Gbg subunits were localized strictly to the plasma membrane. Recent studies of Gbg subcellular localization indicates that they are also present on various endomembrane compartments and organelles such as the Golgi apparatus, ER, mitochondria, and nucleus ((Saini et al., 2009; Saini et al., 2010; Jiang et al., 2010; Robitaille et al., 2010; Campden et al., 2015a; Garcia-Regalado et al., 2008), reviewed in (Dupré et al., 2009; Hewavitharana and Wedegaertner, 2012; Khan et al., 2016)). Gbg translocation to the Golgi was previously thought to be limited to certain combinations of Gbg containing particular Gg subunits. However, it was noted that all 12 Gg subunits are capable of supporting Gbg translocation, albeit with varying kinetics under basal conditions and following GPCR stimulation ((Ajith Karunarathne et al., 2012), reviewed in (Hewavitharana and Wedegaertner, 2012)). This suggests that Gbg translocation is a general phenomenon following receptor activation and may provide explanations for the many noncanonical roles Gbg dimers play in cellular signaling. Examples of such noncanonical roles include, but are not limited to, the regulation of microtubule dynamics, control of intracellular anterograde and retrograde trafficking from the Golgi apparatus and signaling complex assembly in the ER. Gbg dimers have been demonstrated to have specific functional roles in the Golgi apparatus, such as regulation of protein trafficking in the trans-Golgi network (TGN) and maintaining Golgi structure. Initial studies identified a novel role of Gbg dimers in Golgi vesiculation, independent of the Ga subunit (Jamora et al., 1997). Addition of free Gbg dimers to permeabilized cells was sufficient to vesiculate Golgi stacks, which was blocked when Gbg dimers were sequestered with Ga-GDP. Gbg regulates Golgi vesiculation via interaction with phospholipase Cb (PLCb3), which stimulates production of diacylglycerol (DAG) leading to activation of protein kinase Ch (PKCh) and the recruitment of protein kinase D (PKD) (Fig. 13.1A). PKCh phosphorylates the activation loop of PKD, which goes on to regulate vesicle fission (Diaz Anel, 2007; Diaz Anel and Malhotra, 2005; Liljedahl et al., 2001). Interestingly, specific combinations of Gbg subunits were identified to regulate this pathway. Of the Gb subunits, only overexpression of Gb1g2 and Gb3g2 led to increased PKD activation and Golgi vesiculation (Diaz Anel and Malhotra, 2005). On the other hand, overexpression of Gg2, g3, g4, g5, g7, and g10 were able to stimulate PKD activity (Lau et al., 2013). Furthermore, inhibition of Gbg specifically in the Golgi through localized inhibition with overexpressed GRK2-CT demonstrated selective regulation of the delivery of basolaterally targeted, but not apically targeted, cargo (Klayman and Wedegaertner, 2017). Gbg-dependent regulation of Golgi vesiculation is critical for recycling of the protease-activated receptor 2 (PAR2) to the plasma membrane following activation and agonist-dependent autocleavage and endocytosis. Gbg translocated to the Golgi and promoted mobilization of new PAR2 from the TGN to the plasma membrane (Jensen et al., 2016). This mechanism allows for sustained signaling downstream of PAR2 through trafficking of replacement pools of receptor to the plasma membrane. As many GPCRs are able to activate PKD, it remains to be determined if this is a ubiquitous mechanism regulating receptor trafficking or particular to protease-activated receptors. Localization of Gbg to the Golgi apparatus in response to receptor activation is dependent on Raf kinase in trapping to the Golgi (RKTG; also known as the Progestin and AdipoO Receptor 3 or PAQR3) (Jiang et al., 2010). RKTG is a Golgiresident membrane protein that interacts with Gbg through its amino-terminus, tethering the dimer to the Golgi (Fig. 13.1B). Overexpression of RKTG abrogates canonical signaling of plasma membrane Gbg, as evident by the decreased protein kinase B (Akt) phosphorylation and attenuated recruitment of GRK2 following activation of the b2AR.

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While RKTG inhibits Gbg signaling at the plasma membrane, it has been shown to stimulate Gbg signaling in the Golgi (Hewavitharana and Wedegaertner, 2015). Overexpression of RKTG led to Golgi vesiculation in HeLa cells, while a Gbg-binding deficient RKTG was unable to elicit fragmentation and prevented Golgi to plasma membrane transport of the vesicular stomatitis virus protein G (VSV-G). Further dependence on Gbg/PKD pathway was demonstrated through small molecule inhibition of both PKD and Gbg and GRK2-CT sequestration of Gbg. These studies demonstrate the regulatory dichotomy for RKTG for Gbg signaling, dependent on the subcellular localization of the effector pathway being observed. Further, Gbg has also been implicated in regulation of phospholipase Cε (PLCε) in perinuclear regions of the Golgi apparatus. PLCε is a recently discovered PLC isoform downstream of GPCR and receptor tyrosine kinase (RTK) activation of both Ras small GTPase family members (Ras, Rap, and Rho) and Gbg signaling (Lorenz et al., 2009; Vidal et al., 2012). The regulation of PLCε by Gbg was first elucidated in the context of cardiac hypertrophy. Knockdown of PLCε blunted cardiomyocyte hypertrophy in response to the GPCR agonists endothelin-1 (ET-1), norepinephrine, and isoproterenol (Zippin et al., 2003). Furthermore, PLCε knockout in mice prevented development of cardiac hypertrophy following transverse aortic constriction (Acin-Perez et al., 2011). An interaction with muscle-specific A kinase anchoring protein (mAKAP) was shown to anchor PLCε to the nuclear envelope. Disruption of this scaffolding interaction prevented nuclear PKD activation and agonist-induced hypertrophy. This indicated that localized production of second messengers by PLCε was critical for cardiomyocyte hypertrophy (Zippin et al., 2003). Interestingly, it was demonstrated that phosphatidylinositol 4-phosphate (PI4P) was enriched at the Golgi and was the substrate for this pool of PLCε, rather than the classical PIP2, leading to production of DAG in close proximity to the nuclear envelope for regulation of nuclear PKD (Acin-Perez et al., 2011). Although PLCε activity was shown to be critical for development of cardiac hypertrophy downstream of GPCRs, it was not known how the signal was transmitted from the plasma membrane to the Golgi. Gbg was discovered as the signal transducer between GPCR activation and PLCε residing at the perinuclear space (Acin-Perez et al., 2009). Inhibition of Gbg through expression of a Golgi targeted GRK2-CT, and not a plasma membrane GRK2-CT, prevented ET-1 mediated depletion of PI4P in the Golgi (Acin-Perez et al., 2009). Inducing translocation of Gbg to the Golgi, through rapamycin-induced Gg translocation, stimulated PI4P hydrolysis and nuclear PKD activation independent of GPCR activation (Acin-Perez et al., 2009). Furthermore, although it was previously known that Gbg was able to activate PLCε, the mechanism was unknown (Ryu et al., 2005). Recently, it was discovered that Gbg directly activates PLCε through an interaction with both the CDC25 and RA2 domains (Benard et al., 2012) (Fig. 13.1C). These studies demonstrate the importance of free Gbg translocation to the Golgi in regulating pathological signaling pathways leading to heart failure. With respect to how Gbg might be involved in the translocation of other signaling proteins and complexes, translocation of extracellular signal-regulated kinase 1/2 (ERK1/2) to the nucleus serves as an excellent example. A noncanonical autophosphorylation event on ERK1/2 at Thr188 resulting in phosphorylation of nuclear targets involved in cardiac hypertrophy (Andreeva et al., 2008) was found to be induced by Gbg signaling. Hypertrophic stimuli induced interaction of Gbg with Raf1 and ERK1/2 and subsequent nuclear localization of ERK1/2 (Andreeva et al., 2008; Beninca et al., 2014) (Fig. 13.1D). What happened to Gbg itself in this process (i.e., whether it shuttles to the nucleus alongside the Thr188-phosphorylated ERK1/2) remains unknown.

13.3 Gbg signaling dynamics in mitochondria In addition to the increasing body of research supporting the presence of GPCRs and G proteins in the mitochondria, several groups have also shown evidence of potential downstream signaling effectors in this organelle, including adenylyl cyclase (Lyssand and Bajjalieh, 2007), phosphodiesterase (Dagda et al., 2005; Zhang et al., 2010), and protein kinase A (Li et al., 2014). Type-1 cannabinoid receptors (CB-1) have been found at the surface of neuronal mitochondria in mice (Fig. 13.1E). Their direct activation regulates complex I activity and mitochondrial respiration through cAMP accumulation and protein kinase A activity. Stimulation of CB-1 receptors leads to changes in mitochondrial energetics, which may play a role in endocannabinoid-dependent synaptic plasticity in the central nervous system (Ozdemir et al., 2017). There is an increasing body of evidence supporting the presence and diverse functions of G proteins in the mitochondria, including Ga12, Gaq, Ga11, Gai, Gao1, Gai2-3, Gb1, Gb4, and Gg2 (Abadir et al., 2011; Zaballos et al., 2008; Yost et al., 2015). For instance, silencing of Ga12 in human umbilical vein endothelial cells and in COS-7 cells using siRNA resulted in changes in mitochondria morphology, motility, and membrane permeability (Abadir et al., 2011; Zaballos et al., 2008). In one study, knockout of Gaq or Ga11 led to defects in mitochondrial morphology, regulation of fusion and/or fission events, as well as a decrease in membrane potential, overall respiratory capacity, ATP production, and

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growth dependent on oxidative phosphorylation. Interestingly, mitochondrial targeting of Gaq/11 requires an interaction with Gbg (Zaballos et al., 2008). While the presence of Ga subunits has been detected in the mitochondrial lumen, where they may exert some of their signaling functions, Gbg subunits cannot cross through translocase of the outer membrane (TOM) complexes, likely due to its WD propeller domain (Zaballos et al., 2008; Mizuno et al., 2013). Translocation of Gaq and Ga12 could be explained by their association with Hsp90 leading to directed unfolding by the chaperone, allowing cargo passage through TOM complexes. While Gbg is restricted to the outer membrane, it still plays a part in regulation of mitochondrial dynamics through interactions with mitofusins (Mfn), dynamin 1-like (Drp1), and optic atrophy 1 (OPA1) proteins (Zaballos et al., 2008) (Fig. 13.1F). More specifically, Gb2 directly interacts with Mfn-1 to impact mitochondrial morphology and fusion rates. In one study, Mfn-1 was required for Gb2 localization to mitochondria (Fig. 13.1G). Furthermore, Gb2 sequestered Mfn-1, limiting its mobility, resulting in a perturbed mitochondrial morphology and decreased fusion rate. Gb1 knockdown also resulted in some aggregation of mitochondria in 10% of the HeLa cell population, suggesting a potential conserved function between both isoforms (Tyutyunnykova et al., 2017). These findings suggest that, although still poorly understood, downstream signaling by Gbg dimers might be important in events ultimately regulating mitochondrial respiration and energy production. For example, a specific Gbg dimer complex, Gb2g10, has been found to function in mitochondria. Indeed, Gb2g10 is involved in tumor necrosis factor-a (TNF)-mediated necroptosis, although the mechanism by which TNF activates the dimer remains poorly understood (Fig. 13.1H). This interaction was detected in heterotypic membrane fractions containing mitochondria fraction. Furthermore, cells treated with gallein, a small molecule Gbg inhibitor, were less sensitive to TNF-induced necroptosis supporting a function for Gb2g10 in this pathway (Smrcka et al., 2012). The authors suggested that Gb2g10 may modulate Src kinase activity, as TNF-induced activation of Src was diminished upon Gg10 knockdown in L929 cells. Based on these findings, they showed that Src may serve as the downstream effector of Gb2g10 in TNF-induced necroptosis, and they identified a regulatory signaling role for Gb2g10-Src in translocation of RIP1/RIP3/MLKL-containing necrosomes on heterotypic membrane fractions. The authors suggested that these may represent signaling junctions between the endoplasmic reticulum and mitochondria, as well as other platforms where necrosomes facilitate cell death (Smrcka et al., 2012). Further work is needed to establish the roles of the Gb2g10-Src pathway on necrosome trafficking and how such vesicles function on heterotypic membranes. In another study, transgenic mice overexpressing Gb3 were shown to have an altered gene expression profile of white and brown adipose tissue and were characterized by an obese phenotype and type II diabetes. Expression levels of Ucp1, which functions to dissipate energy in the form of heat in mitochondria, are decreased in adipocytes upon overexpression of Gb3. This leads to remodeling of the white adipose tissue, and a loss of brown properties in brown adipose tissue, resulting in increased adiposity in mice fed regular chow (Zhang et al., 2011b). Further studies are needed to understand the mechanisms by which Gb3 alters energy expenditure, tissue metabolism, and signaling. In addition to Gbg subunit involvement in mitochondrial energetics, Ga subunits may also impact respiration in mitochondria. Indeed, knockout of Gaq and Ga11 resulted in lower potentials at the inner mitochondrial membrane which could be rescued by reintroducing Gaq in these cells. The loss of Gaq and Ga11 also resulted in 15% less cellular ATP and in a lower oxygen consumption rate, both at baseline and under maximal respiration capacity as induced by mitochondrial depolarization. The molecular mechanism underlying these findings involved reduced amounts of dimer complex V in the respiratory chain. Lastly, the doubling time for Gaq/11 knockout cells was increased when grown in galactose-enriched media, which renders cells highly dependent on ATP, supporting involvement of Gaq/11 in oxidative phosphorylation (Zaballos et al., 2008). Supporting these findings of direct involvement of G proteins with the oxidative phosphorylation pathway, our lab conducted a proteomic screen using tandem affinity purification (TAP) in HEK 293 cells identifying 18 potential interaction partners of Gbg in the oxidative phosphorylation pathway in complexes I, II, IV, and the ATP synthase (Campden et al., manuscript in preparation) (Fig. 13.1I). These interactions were identified both under basal conditions, and upon stimulation of the endogenous M3 muscarinic acetylcholine receptor (M3-R). These findings suggest G proteins may be involved in signaling that is both dependent and independent of upstream GPCR activation in mitochondria. While all these studies suggest a potential direct involvement of G proteins in the mitochondria, a novel reninangiotensin system involving the GPCR angiotensin II receptor type II (AT2R) at the inner mitochondrial membrane was described (Fig. 13.1E and (Zhang et al., 2013)). Stimulation of mitochondrial AT2R led to a dose-dependent increase in nitric oxide production and decrease in mitochondrial respiration (Zhang et al., 2013). Further characterization of this signaling pathway is necessary to establish potential involvement of G proteins and to support their functions in mitochondrial energetics, but the presence of functional GPCR systems strongly supports energy metabolism as a novel noncanonical role of G proteins in the cell.

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13.4 Gbg-mediated regulation of transcriptional activity Signaling pathways downstream of GPCR activation have previously been shown to converge on regulation of gene expression. Gbg regulates transcription through activation of signaling cascades indirectly effecting transcriptional activity (Fig. 13.1J) or through direct interactions with transcription factors (Fig. 13.1K), and we will briefly discuss both. First, Gbg was implicated in thyroid differentiation following activation of the thyroid stimulating hormone (TSH) receptor (TSHR) and regulation of the Akt/PI3K pathway (Malik et al., 2015). TSH caused receptor-mediated activation of Gas and increased cAMP levels, leading to recruitment of Pax8, a member of the paired-box family of transcription factors, to the promoter of sodium iodide transporters (NIS) resulting in increased expression. Meanwhile, Gbg was found to have an opposing effect on NIS expression. TSHR activation led to a Gbg-dependent increase in activity of the Akt/PI3K pathway and exclusion of Pax8 from the nucleus, thereby decreasing NIS expression (Malik et al., 2015). Gbg regulation of intracellular calcium mobilization is also involved in the regulation of various transcription factors. For example, specific Gbg subunits were implicated with nuclear factor of activated T-cells (NFAT) family members to module interleukin 2 (IL-2) levels in CD4þ T helper cells (Wing et al., 2001). Knockdown of Gb1, but not Gb2, and smallmolecule inhibition with gallein led to enhanced IL-2 expression in response to T cell receptor (TCR) activation. Ablating Gb1g signaling potentiated release of intracellular calcium in response to TCR activation, leading to translocation of NFAT1 and NFAT2 into the nucleus where they could interact with their target gene IL-2 (Wing et al., 2001). Another example involves Gbg subunit-mediated regulation of 1,4,5-triphosphate receptor (IP3R-1) expression downstream of dopamine D2 receptor (D2R) activation (Madukwe et al., 2018). Here, D2R activation with quinpirole led to increased nuclear levels of cFos and phosphorylated Jun and subsequent translocation of NFATc4 to the nucleus. These three components were then recruited to the IP3R-1 promoter region. Inhibition of Gbg or PLC and chelation of intracellular calcium prevented D2R-mediated upregulation of IP3R-1 (Madukwe et al., 2018). Although Gb5 is the most structurally distinctive subunit in the Gb family, it is also capable of nuclear translocation (Fig. 13.1L). Gb5 preferentially forms obligate dimers with the Gg-like (GGL) domain-containing R7-regulator of G protein signaling (R7-RGS) family of proteins (Witherow and Slepak, 2003). Cellular distribution and nuclear targeting of Gb5-R7-RGS is believed to involve the R7-binding protein (R7BP) (Hepler, 2005; Drenan et al., 2005; Song et al., 2006). Palmitoylation of R7BP anchors it to the plasma membrane; however, a recent study demonstrates that mutant R7BP lacking the N-terminal DEP (dishevelled, EGL-10, pleckstrin) homology domain displays marked decreases in nuclear localization (Panicker et al., 2010). Gb5 nuclear localization was assessed in neurons and brains from R7BP knockout mice and it was found that Gb5-R7-RGS displayed a 50%e70% reduction on nuclear localization suggesting a central role for R7BP in targeting Gb5-R7-RGS to the nuclei.

13.5 Gbg-dependent regulation of transcription through interactions with transcription factors As discussed above, studies have implicated Gbg-dependent regulation of mitogen-activated protein kinases (MAPK) downstream of receptor activation in transcriptional regulation. Gbg was also implicated in regulating expression of the transcription factor Zif268 in differentiated dopaminergic-like SH-SY5Y cells and in rat midbrain slices in response to D2R activation (Ahmadiantehrani and Ron, 2013). Treatment with the D2R agonist quinpirole led to Gbg-dependent increases in ERK1/2 activation and upregulation of Zif268, which was then able to interact with the promoter for glial cell lineederived neurotrophic factor (GDNF) and increase GDNF expression (Ahmadiantehrani and Ron, 2013). Similarly, Gbg was implicated in phosphorylation-dependent activation of the transcription factor cAMP responsive element binding protein (CREB). In striatal neurons, corticotropin releasing factor (CFR) binding to CRF receptor 1 lead to Gbg and MAPK-dependent phosphorylation of CREB, independent of canonical CREB activation downstream of Gas, adenylyl cyclases, and PKA (Stern et al., 2011). There have also been many reports of Gbg directly interacting with and regulating transcription factor activity (Fig. 13.1K). A first report noted the interaction between Gg5 and the adipocyte enhancer-binding protein (AEBP1) using a yeast two hybrid assay (Fisher and Aronson, 1992). Subsequently, it was determined that Gbg5 attenuated transcriptional repression mediated by AEBP1 during adipogenesis (Park et al., 1999). Similarly, Gb1g2 also interacts with the C-terminal domain of histone deacetylase (HDAC) 4 and 5, leading to inhibition of transcriptional repression (Spiegelberg and Hamm, 2005). Under basal conditions, the myocyte enhancer factor 2C (MEF2C) interacts with HDAC5 creating a repressive transcriptional environment. Upon activation of the a2A-adrenergic receptor with epinephrine, Gb1g2 disrupts the MEF2C/ HDAC5 interaction, relieving the inhibitory effect of HDAC5 as evidenced by increased MEF2C reporter gene expression. This effect was ablated by coexpression of the GRK2-CT, indicating Gbg dependence rather than Ga signaling

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(Spiegelberg and Hamm, 2005). Gbg also attenuates glucocorticoid receptor (GR)-mediated activation of glucocorticoid responsive genes (GREs) (Kino et al., 2005). Following treatment with the GR agonist dexamethasone, Gb2 and GR were translocated to the nucleus and associated with GREs together. Gb2 was found to suppress the activation function-2 (AF-2) transcriptional activity of GR. It was previously demonstrated that Gbg interacts with and represses activator protein-1 (AP-1) gene reporter activity following activation of PKC in HEK 293 cells and following gonadotropin-releasing hormone (GNRH1) treatment of LbT2 gonadotrope cells (Robitaille et al., 2010). While the Gbg/HDAC interaction enabled MEF2C-mediated transcription (Spiegelberg and Hamm, 2005), Gbg-recruited HDACs inhibited AP-1 transcriptional activity. Interestingly, Gbg interactions with other transcription factors result in increased transcription. For example, it was demonstrated that activation of the angiotensin II type 1 receptor (AT1R) led to translocation of Gb2 to the nucleus. Following translocation, Gb2 interacted with core histones and transcriptional regulators as determined by coimmunoprecipitation. Using a heterologous expression system in HEK 293 cells, Gb2 is required for the synergistic action of MEF2A and TATA-binding protein (TBP) and the transcription activating factor (TAF) complex for reporter gene induction. A more global role was identified for Gb2 downstream of the AT1R, as Gb2 knockdown resulted in decreased expression of w400 genes with a specific set regulated by MEF2A, NFAT, STAT1, and STAT3. This corresponded with direct interactions observed between Gb2 and these transcriptions factors that were dependent on its WD repeat structure. This adds to the complexity of Gbg-mediated regulation of NFAT. As we previously discussed, NFAT translocation was dependent on Gbg signaling (Wing et al., 2001; Madukwe et al., 2018), it is also evident that there is a regulatory role mediated through a direct interaction. Other studies expand our understanding of how Gbg interacts with and regulates the signal transducer and activator of transcription (STAT) family of transcription factors. Members of the STAT protein family, specifically STAT1 and STAT3, have been shown to be activated by Gaq/11, Ga16, and Ga14 (Ferrand et al., 2005; Lo and Wong, 2004; Lo et al., 2003). More recent studies have also implicated Gbg dimers in STAT activation. Of the 48 different combinations of Gb and Gg overexpressed in HEK 293 cells, 13 different combinations led to various degrees of STAT3 activation (Yuen et al., 2010). A subsequent study furthered our understanding of Gbg-dependent activation of the STAT family member, STAT5B (Georganta et al., 2010). In heterologous HEK293 cells stably expressing the d-opioid receptor, STAT5B constitutively interacted with the carboxyl-terminus of the receptor. Upon receptor activation, Gbg served as a scaffold, directly interacting with STAT5B and recruiting c-Src kinase. Gbg-dependent recruitment of c-Src phosphorylates STAT5b, leading to subsequent transcriptional activation. The discussed examples illustrate the complexity of Gbg action in the nucleus, where it functions as both a negative and positive regulator of transcription either through eliciting signaling cascades or direct interactions with transcriptional regulators. In order to further understand the complex roles served by Gbg in the nucleus, tandem affinity purification (TAP) coupled with mass spectrometry and cell fractionation was performed to identify the interactome of Gb1 specifically in the nucleus and cytoplasm (Campden et al., 2015b). Interactions were determined under basal conditions and following activation of the HEK293 endogenous M3-R with carbachol. Interestingly, several members of the heterologous nuclear ribonucleoprotein family (hnRNP) (Fig. 13.1M) and proteins involved in nuclear import and export such as importin 7 and exportin 1 (Fig. 13.1N) were identified, adding to the growing list of noncanonical Gbg roles (Campden et al., 2015b). More recently, Gb1 occupancy was found along more than 700 promoters (Fig. 13.1O) using ChIP-on-chip in HEK 293 cells, such as the promoter of the gene for the Gb4 isoform (GNB4) (Khan et al., 2015). As Gbg had been identified to interact with many different transcription factors and associate with numerous promoter regions, it was hypothesized that Gbg interacted with a ubiquitous component of the transcriptional machinery. From this hypothesis, a novel interaction between Gbg and RNA polymerase II (RNAPII) was identified (Khan et al., 2018). This interaction was detected in both HEK293 and neonatal rat cardiac fibroblasts in response to endogenous M3-R or AT1R activation, in each cell, respectively, suggesting it may be a general feature of transcriptional regulation downstream of GPCRs. Using neonatal rat cardiac fibroblasts and the AT1R to model the fibrotic response mediated by angiotensin II (Ang II), Gb1 was identified as the predominant subunit interacting with RNAPII. Depletion of Gb1 using siRNA leads to a dysregulated profibrotic transcriptional response under basal conditions and even higher expression following Ang II stimulation. Using chromatin immunoprecipitation followed by qPCR, Gb1 was demonstrated to be preferentially recruited toward the 30 end of fibrotic genes. This was corroborated by the increased interaction with the actively elongating form of RNAPII by coimmunoprecipitation and sensitivity to inhibitors of transcriptional elongation. Previous studies predominantly focused on regulation of specific transcription factors, but the interaction with RNAPII may suggest a genome-wide role of Gbg on the active transcriptional machinery. While mammals have 5 Gb and 12 Gg subunits, which can assemble into 60 possible combinations, Arabidopsis thaliana only harbors a single Gb (AGB1) and 2 Gg (AGG1 and AGG2) orthologs in their genome (Khan et al., 2013;

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Wang et al., 2008). The unique Gbg analog AGB1, has been found to localize to various organelles, one of which is in the nucleus, where it can interact with various transcription factors and effectors (Fig. 13.1P), hinting at a more broadly conserved regulatory function in transcriptional regulation (Wang et al., 2008; Ishida et al., 2014; Anderson and Botella, 2007; Xu et al., 2017; Zhang et al., 2017). One study recently uncovered a direct interaction between AGB1 and the transcription factor B-box domain protein 21 (BBX21), which is responsible for a variety of cellular and developmental processes in plants. This results in the inhibition of the transcriptional activation function of BBX21 (Xu et al., 2017). Using a yeast one-hybrid assay, they determined that while AGB1 did not prevent BBX21 binding to DNA, it bound to the carboxyl-terminal domain of BBX21 responsible for transcriptional activity, effectively inhibiting the protein. Their findings suggest that AGB1 acts as an activation switch for hypocotyl growth in plant embryos and is part of a negative feedback loop with BBX21 where it positively promotes hypocotyl elongation by negatively regulating BBX21 (Xu et al., 2017). Another group identified the transcription factor brassinosteroid (BR) signaling positive regulator family protein (BES1) as another interactor of AGB1. AGB1 was found to positively mediate BR signaling in a BES1-dependent manner (Zhang et al., 2017). Indeed, they demonstrated that AGB1 synergistically regulates expression of BES1 target genes, including BR biosynthesis genes and genes required for promoting cell elongation. The authors suggested that AGB1 alters the phosphorylation status of BES1 toward a dephosphorylated phenotype that accumulates in the nucleus, where it also stimulates the transcriptional activity (Zhang et al., 2017). Lastly, another study showed that AGB1 interacted with the ABA-response mitogen-activated protein kinase 6 (AtMPK6), a transcription factor in the nucleus (Xu et al., 2015). They showed that AGB1 might negatively regulate the ABA signaling pathway involved in drought tolerance. AGB1 mutants led to increased expression of three ABA responsive genes: AtMPK6, AtVIP1, and the AtMYB44 transcription factor (Xu et al., 2015). These findings provide evidence for evolutionarily Gbg-conserved functions in transcriptional regulation not only across species, but even across kingdoms.

13.6 Concluding remarks In the last 20 years, we have progressed from a simplistic view of Gbg subunits as regulators of Ga signaling to a much richer tapestry of Gbg-dependent signaling events throughout the cell. It is clear that broader proteomic and genomic approaches will continue to reveal new roles for Gbg signaling in different parts of the cell, in different cells and tissues, and in a subunit- and species-specific manner.

Acknowledgments This work was supported by grants from the Canadian Institutes of Health Research (CIHR, MOP-130309, PJT-159687) and from the Natural Sciences and Engineering Research Council of Canada (NSERC). R.D.M. was supported by a scholarship from the Canadian Institutes of Health Research (CIHR) and a Faculty of Medicine Studentship from McGill University. C.A.B. was supported by a Faculty of Medicine Studentship from McGill University. Both R.D.M. and C.A.B. were supported by Graduate Excellence Fellowships from the Department of Pharmacology and Therapeutics, McGill University.

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