A Brief Review of GPCR Signaling with a Focus on the Drosophila GPCR Family Caitlin D

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COMMENTARY Outside-in signaling – a brief review of GPCR signaling with a focus on the Drosophila GPCR family Caitlin D. Hanlon and Deborah J. Andrew*

ABSTRACT 800 GPCRs are encoded in the human genome and well over 700 in G-protein-coupled receptors (GPCRs) are the largest family of the zebrafish (Fredriksson and Schioth, 2005). Caenorhabditis receptors in many organisms, including worms, mice and humans. elegans is predicted to encode over 1000 GPCRs, a particularly GPCRs are seven-transmembrane pass proteins that are activated impressive number, as this figure accounts for over 5% of the entire by binding a stimulus (or ligand) in the extracellular space and then worm genome. Mice also encode a large number of GPCRs (over transduce that information to the inside of the cell through 1300). Drosophila encodes over 200 GPCRs, whereas just over 50 are conformational changes. The conformational changes activate found in Dictyostelium (Prabhu and Eichinger, 2006). In contrast, – heterotrimeric G-proteins, which execute the downstream signaling yeast encode a surprisingly small number of GPCRs three in pathways through the recruitment and activation of cellular enzymes. Saccharomyces cerevisiae and nine in S. pombe. One kingdom The highly specific ligand–GPCR interaction prompts an efficient where GPCRs might be absent is the plants, which do not require cellular response, which is vital for the health of the cell and organism. GPCRs to function as guanine-nucleotide-exchange factors In this Commentary, we review general features of GPCR signaling (GEFs) due to the very high abundance of GTP (Urano et al., α and then focus on the Drosophila GPCRs, which are not as well- 2012). Instead, the G subunit of the heterotrimeric G protein complex characterized as their worm and mammalian counterparts. We is typically already bound to GTP, with regulation of activity at the discuss findings that the Drosophila odorant and gustatory level of GTP hydrolysis to halt signaling (Urano and Jones, 2014). Of receptors are not bona fide GPCRs as is the case for their the 56 putative plant GPCRs identified based on structural homology, – – mammalian counterparts. We also present here a phylogenetic only a single candidate GCR1 has features consistent with analysis of the bona fide Drosophila GPCRs that suggest potential it functioning as a bona fide GPCR with GEF activity (Taddese et al., roles for several family members. Finally, we discuss recently 2014). Whether or not GCR1 acts as a GEF remains to be determined. discovered roles of GPCRs in Drosophila embryogenesis, a field we GPCRs are involved in nearly every aspect of animal life, from expect will uncover many previously unappreciated functions for early development and heart function to neuronal activity GPCRs. (Wettschureck and Offermanns, 2005). Mutations in GPCRs are linked to a number of human diseases, such as Usher syndrome, KEY WORDS: AGS, β-arrestin, GASP, G-proteins, GPCR, GPSM, which results in variable onset deaf-blindness (Schöneberg et al., GRK, RGS, Signaling 2004). Cell migration is another process that requires GPCRs, in both beneficial and detrimental ways. The single-celled amoeba Introduction Dictyostelium uses four GPCRs, cAR1–cAR4, to detect cAMP, How cells sense and respond to outside stimuli has been a key which triggers migration and coalescence into a multicellular question in biology for well over a century. The hypothesized organism (Prabhu and Eichinger, 2006). In both zebrafish and mice, existence of receptors that spanned the cell membrane and were able germ cell migration is regulated by the chemokine receptor CXCR4 to both sense and transduce signals seems logical today, but when and its ligand stromal derived factor 1 (SDF1) (Doitsidou et al., this idea was first proposed, it was summarily rejected (Lefkowitz, 2002; Molyneaux, 2003). Neutrophils also migrate through 2013). Decades of biochemical studies have since proven that such activation of a GPCR (Becker et al., 1987). GPCR-regulated cell receptors exist, and that most are G-protein-coupled receptors migration can also be detrimental for the organism, primarily during (GPCRs). The main feature of GPCRs is their seven transmembrane- cancer (Lappano and Maggiolini, 2011). Overly activated GPCRs spanning segments, which position the N-terminus of the protein on are able to trans-activate epidermal growth factor receptor (EGFR) the outside of the cell and the C-terminus inside. GPCRs bind an and other receptors, which can cause unregulated growth and cell astoundingly diverse set of ligands – proteins, small molecules, migration (Bhola and Grandis, 2008). hormones, drugs, photons – usually by capturing the ligand with In this Commentary, we first cover the basics of GPCR signaling, their N-terminus and/or with a pocket formed by the extracellular from how the receptor is arranged in the membrane to heterotrimeric G and transmembrane domains (Fig. 1). The GPCR cycle, described in protein activation and receptor recycling. We focus then on the more detail below, is an elegant cellular solution for sensing a previously described Drosophila GPCR family, which includes three specific exogenous signal, transducing it to a signaling cascade, and major groups, the two chemosensory groups – odorant and gustatory – then terminating the signal. and the classical receptor group. Phylogenetic trees representing all GPCRs are widely represented in most life forms, from bacteria to three major groups of Drosophila GPCRs are presented. We cover fungi and animals, including all of the major model organisms. Over recent findings that insect odorant and (likely) gustatory receptors, unlike their vertebrate counterparts, are not bona fide GPCRs, leaving the classical Drosophila GPCR family with only 116 members. Department of Cell Biology, The Johns Hopkins University School of Medicine, Finally, we discuss the known roles of GPCRs in Drosophila 725 N. Wolfe St., Baltimore, MD 21205-2196, USA. development, an open and exciting field given the number of

*Author for correspondence ([email protected]) uncharacterized GPCRs encoded in the fly genome. Journal of Cell Science

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᭹ L Ligand-induced conformational change in GPRC facilitates interaction with heterotrimeric G-proteins

E E

GPCR γ γ α β α β

GDP GDP GTP

᭹ Activated GPCR acts ᭹ GPCR internalization followed by its as a GEF for Gα degradation or recycling back to the PM ᭹ Attenuation of effector signaling through feedback loops

E* E

γ α β α* β* γ* βArr GDP GTP

᭹ GTP-binding-induced separation of heterotrimeric G-proteins ᭹ Binding of β-arrestin to ᭹ Interaction of G-protein subunits phosphorylated GPCR with effectors ᭹ Re-association of heterotrimeric G-proteins

E* E

γ * γ* α α β β * P ᭹ Activation of second messengers GRK P P by effectors GDP GTP ᭹ GTP hydrolysis GDP ᭹ GPCR phosphorylation by GRK

Fig. 1. The GPCR cycle. The GPCR cycle starts on the top left of the figure. In its basal state, a GPCR is free of ligand (L). Gα binds to GDP and associates with Gβγ. The heterotrimeric protein complex might associate with the receptor at this point, or remain free in the membrane as pictured. Upon ligand binding the GPCR becomes activated and undergoes a conformational change. The activated GPCR acts as a GEF for Gα. The resulting GTP-bound Gα separates from βγ, and the heterotrimeric proteins are active (*). Activated Gα can then interact with an effector (E), such as PLC or adenylate cyclase, which results in effector activation (*) and initiation of a second-messenger cascade. The GTP in Gα is then hydrolyzed to GDP through the activity of Gα and RGS proteins (not shown), leading to Gα inactivation and reassociation of the heterotrimeric protein complex. Independently, GRKs bind to and phosphorylate the GPCR. This stimulates its binding by β-arrestin (βArr), which promotes internalization of the receptor. The GPCR can then be recycled back to the cell surface without ligand, restarting the cycle.

GPCR structure–function relationship stabilization of the transmembrane domains of the GPCR, Because the GPCR superfamily is so diverse, there is little sequence collectively termed the GPCR barrel owing to their composite conservation among families. Nonetheless, the superfamily does share shape in the membrane (Venkatakrishnan et al., 2013). Ligand several architectural features. The N-terminus and extracellular loops contact triggers movement of TM3 and causes conformational (ECLs) are responsible for ligand binding. This can involve direct changes of the intracellular loops (ICLs). In the rhodopsin family, binding of the ECL to the ligand [as is the case for metabotropic ICL2 contains an E/DRY motif near the boundary between ICL2 glutamate (mGlu) receptors] or funneling of a hydrophobic ligand and TM3 (Rovati et al., 2007). Mutations within this motif can result into a binding pocket formed by the transmembrane domains in constitutive GPCR signaling or impaired G-protein binding. (Venkatakrishnan et al., 2013). ECLs often contain disulfide bridges Interestingly, other than the E/DRY motif, very little is known about to stabilize the loops and prevent promiscuous GPCR signaling. Indeed, how GPCRs associate with specific G-proteins. Chimeras resulting ligand binding does not induce a simple on-off state for GPCRs. GPCRs from swapping of ICL3 domains between GPCRs result in a switch are dynamic proteins that fluctuate between many states (Fig. 1). Ligand in their G-protein selectivity (Kobilka et al., 1988), suggesting that binding stabilizes the GPCR into an ‘on’ position, which is further this domain imparts specificity for the downstream signaling stabilized by binding of a G-protein (Kobilka, www.nobelprize.org/ pathway of the receptor. nobel_prizes/chemistry/laureates/2012/kobilka-lecture.html). The localization of a GPCR within a cell membrane can affect its Bioinformatic analyses show that specific residues, such as ability to signal. Lateral movement of GPCRs within the plasma asparagine, tryptophan and proline, cause clustering and membrane is often restricted by the preferential localization of Journal of Cell Science

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GPCRs to a specific lipid microenvironment (Allen et al., 2007). 2015). Some AGS proteins act as guanine-nucleotide-dissociation Although GPCRs are usually shown as monomers, they can form inhibitors (GDIs) (e.g. the GPR proteins, as well as proteins that oligomers (i.e. homodimers and heterodimers) within lipid rafts and contain a GoLoCo motif), causing Gα to remain in a GDP-bound are stabilized by interactions that are mediated through their state. These GDI-AGS proteins are termed G-protein signaling transmembrane domains (Ferré et al., 2014). Planar lipid rafts and modulators (GPSMs) and affect the amount of free Gβγ that can caveolae both influence GPCR signaling by either excluding or signal because they also prevent re-association of the heterotrimeric recruiting G-proteins and their effectors. Because native GPCRs are protein complexes. Finally, the RZ family of RGS proteins has not highly expressed in cells (McCusker et al., 2007), lipid rafts recently been shown to have the dual activities of promoting Gα concentrate GPCRs and their associated proteins to promote GTP hydrolysis and inhibiting the exchange of GDP for GTP (Lin receptor dimerization and signaling. Caveolar-localized GPCRs et al., 2014). are also subject to more rapid endocytosis, which represents another Compared to Gα (for which there are 23 genes), there are fewer way to modulate GPCR signaling (Chini and Parenti, 2004). Gβ and Gγ genes in mammals, five and 12 genes, respectively. This would suggest that there are as many as 60 βγ complexes, each with Signal propagation and the GPCR cycle different preferences for specific Gα subunits (Dingus et al., 2005). The presence of a ligand–receptor binding event must then be Gβγ can affect a wide range of ion channels and other signaling propagated and responded to by the cell itself. The effectors of effectors, including pathways that are also targeted by Gα, such as GPCR activation are the heterotrimeric G-proteins Gα,Gβ and Gγ. PLC (Lau et al., 2013). Because the Gβγ dimer has long been Organisms encode several types of each G-protein, and different considered to be a less important pathway component, the exact combinations of these proteins into heterotrimers preferentially roles different combinations of Gβγ dimers have on GPCR signaling activate different signaling pathways. Gα proteins are GTPases, remain to be elucidated. which catalyzes the hydrolysis of GTP to GDP. Gα proteins are Several mechanisms exist to attenuate GPCR signaling. The same typically anchored in the membrane by N-terminal palmitoylation conformational change in the GPCR that results in the release of the and can also be myristoylated (Vögler et al., 2008). Gγ proteins are heterotrimeric complex allows accessibility for phosphorylation by isoprenylated at their C-terminal CAAX motif (Higgins and Caseys, G-protein-coupled receptor kinases (GRKs) (Palczewskiss et al., 1994). Gβ proteins do not have any membrane-anchoring post- 1991). Gβγ is able to recruit a GRK to the GPCR, thus establishing a translational modifications. Instead, they are tightly linked to Gγ negative-feedback loop (Luttrell et al., 1999). GRKs usually through hydrophobic interactions (Sondek et al., 1996). A phosphorylate GPCRs at serine or threonine residues in the ICL3 heterotrimeric complex can dock to an inactivated receptor or drift or the C-terminal tail of the activated receptor (Pitcher et al., 1998). in the membrane, but once it encounters a ligand-bound GPCR, This phosphorylation of the GPCR results in the binding of downstream signaling is initiated (Fig. 1). Activated GPCRs act β-arrestin (Drake et al., 2006), which then recruits clathrin and its as GEFs and exchange GDP for GTP in the Gα subunit, adaptor protein AP-2, to internalize the GPCR. Several different which activates the protein. Upon GTP binding, Gα changes its fates await the GPCR following internalization and GPCRs are conformation, allowing it to separate from the Gβγ dimer. The divided into two classes depending upon how strongly they subunits are then free to interact with downstream targets. When Gα maintain β-arrestin binding. Class A GPCRs lose β-arrestin hydrolyzes GTP into GDP, it becomes inactivated, allowing Gα to following internalization (Oakley et al., 2000) and can be reassociate with Gβγ. This process represents a full GPCR G-protein dephosphorylated and recycled back to the cell surface. An cycle. emerging concept in the field is the ability of GPCRs to maintain Gα proteins are divided into four subclasses with each targeting a signaling once they are endocytosed (Mullershausen et al., 2009). It specific type of signaling cascade (Wettschureck and Offermanns, is thought that by continuing to signal at endosomes instead of the 2005). Gα(s), Gα(i), and Gα(o) all regulate adenylate cyclases. Gα(s) plasma membrane, the physical distance between the GPCR and the stimulates adenylate cyclase activity, whereas Gα(i) and Gα(o) nucleus is reduced, resulting in more efficient signaling to are inhibitory. The third subclass, Gα(q/11) targets phospholipase C transcriptional pathways (Tsvetanova and von Zastrow, 2014). (PLC), which cleaves phosphatidylinositol 4,5-bisphosphate Returning a GPCR back to the plasma membrane completes the [PtdIns(4,5)P2] into inositol trisphosphate (IP3) and membrane- GPCR cycle (Fig. 1). Class B GPCRs maintain β-arrestin binding. bound diacylglycerol (DAG). Finally, Gα(12/13) activates Rho Here, GRK-mediated phosphorylation and binding to β-arrestin can GEFs, which in turn activate Rho. Previous models have suggested stimulate the ubiquitylation of GPCRs. Ubiquitylated receptors are that an individual GPCR interacts with only one specific type of Gα, then targeted to the lysozome for degradation. More recently, the but it is now established that GPCRs are able to activate several Gα so-called GPCR-associated sorting protein (GASP) family has been types, albeit with a marked preference for one (Cerione et al., 1985). identified, which assists in the decision as to whether GPCRs are Gα proteins are weak GTPases, which slows the signaling degraded or recycled (Bornert et al., 2013; Simonin et al., 2004). cascade because new signaling information cannot be integrated The amino acid sequence at the C-terminus of the GPCR is thought (Kleuss et al., 1994). To accelerate GTP hydrolysis, Gα proteins are to direct the type of adaptor protein that binds the receptor, thus targeted by the regulator of G-protein signaling (RGS) molecules influencing the fate of the receptor once it is internalized (Marchese (De Vries et al., 2000). RGS proteins are somewhat promiscuous for et al., 2008). Gα proteins, and several RGS proteins have been shown to bind to specific Gβ proteins and prevent re-formation of the heterotrimeric GPCR families complex (Witherow and Slepak, 2003). Conversely, activators of The rhodopsin-like family is the largest family of GPCRs in most G-protein signaling (AGS) can act as GEFs for Gα to prolong organisms. Members of this family are well-known for the diversity signaling (Vögler et al., 2008). Recent work has shown that Gα can of their ligands, which range from hormones and peptides to be activated by non-receptor GEFs, which themselves are activated odorants and photons of light. Rhodopsin was first described as a through associations with non-GPCR signaling pathways such as light-sensitive compound in animals (Kühne, 1877), and was cloned the receptor tyrosine kinase (RTK) pathway (Garcia-Marcos et al., over a century later (Nathans and Hogness, 1983). This finding Journal of Cell Science

3535 COMMENTARY Journal of Cell Science (2015) 128, 3533-3542 doi:10.1242/jcs.175158 incited the explosion of subsequent GPCR research. Because the systems, such as Xenopus oocytes, and HEK293 or CHO cells. In rhodopsin-like family represents over 80% of human GPCRs this way, specific GPCRs can then be exposed to individual ligands (Fredriksson and Schioth, 2005), it has been intensely studied for to test for functionality. Heterologous expression systems are potential therapeutic benefits. The huge number of mammalian preferentially used to identify ligands for GPCRs (a process called odorant or olfactory receptors (∼700 in humans and ∼1200 in ‘de-orphanizing’ or ‘deorphaning’) because it reduces the rodents) all fall into the rhodopsin-like family based on sequence possibility of endogenous proteins triggering the receptor, as most homology. Olfactory GPCRs activate a Gα(olf) subunit that GPCRs are quite specific for ligand recognition (Caers et al., 2014). activates an adenylyl cyclase, leading to increases in cAMP Many downstream effectors have not been singularly identified as (DeMaria and Ngai, 2010). Increased cAMP, in turn, activates coupling to a specific receptor and, instead, output is typically cyclic-nucleotide-gated ion channels that bring in Na+ and Ca2+, measured through changes in Ca2+ levels. Activation of Gα(q) thereby depolarizing the cell. Ca2+-gated Cl− channels are then releases Ca2+ through the downstream effector PLC, and various activated, leading to further depolarization. Although insect odorant Ca2+ reporters can be used to track this change. Although changes in receptors were initially thought to function in the same way, it cAMP and Rho GEF activity are not as easily tracked, ligand- appears that the seven-transmembrane-pass insect odorant receptors induced changes in cAMP levels have been used to link the are not bona fide GPCRs and instead function directly as ligand- neuropeptide controlling Drosophila circadium rhythms, known as gated ion channels (see below) (Ha and Smith, 2008; Leal, 2013; pigment-dispersal factor (PDF), to an orphan secretin-related Wicher et al., 2008). GPCR, known as PDF-R or Han (Hyun et al., 2005; Mertens Another important GPCR familiar is the secretin-like family. Its et al., 2005). To circumvent challenges in linking orphan receptors main feature is its large N-terminal extracellular domain (ECD) to their cognate ligands, GPCRs have typically been expressed (Watkins et al., 2012), which is crucial for the recognition and along with the promiscuous human Gα16, which readily couples binding of ligands, typically peptides or hormones. Historically, this with many GPCRs to cause changes in intracellular Ca2+. Although family was named for the intestinal hormone secretin, which, in the this system can identify ligand–receptor interactions, it does not early 20th century, was the first hormone discovered (Bayliss and identify the endogenous Gα that interacts with a GPCR. Moreover, Starling, 1902). Its receptor was first described nearly 80 years later as stated above, GPCRs are known to interact with more than one (Jensen and Gardner, 1981; Chey and Chang, 2003). Gα, causing multiple output changes. Studies that have identified mGlu receptors bind a diverse set of ligands, such as pheromones, specific Gα coupling to a receptor have often been performed using amino acids and Ca2+ (Chun et al., 2012). Members of this family physical and genetic interaction data. contain a large ECD that forms a so-called Venus flytrap (VFT) module (Bessis et al., 2002). Upon ligand binding to one lobe of the Drosophila GPCRs VFT, the other lobe closes, introducing a conformational change Drosophila encodes over 200 proteins originally considered to be that is transduced to the rest of the protein through a cysteine-rich GPCRs based on domain topology and other key structural features region. mGlu receptors function as dimers that are either covalently (Brody and Cravchik, 2000). Not surprisingly, modulators of GPCR linked by disulfide bonds or by shared ion binding. Compared to the signaling, such as GRKs, GDIs, GPCR kinases and arrestins, are other GPCR families, the mGlu family was discovered relatively also encoded in the Drosophila genome. One aspect of Drosophila late. Although glutamate was a known neurotransmitter, it was GPCR signaling that makes it particularly appealing for study is the assumed to function solely through channels or ionotropic reduced number of G-proteins – nine genes encode predicted Gα receptors, which themselves function as channels (Curtis and proteins (six have been characterized and three are known only by Watkins, 1965). However, metabotropic glutamate receptors their CG numbers), three genes encode Gβ proteins and two genes function as conventional GPCRs by binding a ligand and encode Gγ proteins (Table 1) (Katanayeva et al., 2010; modulating that signal through the membrane to downstream Anantharaman et al., 2011). Of the three Gα subunits with only G-proteins. CG designations, one appears to be a pseudogene (CG40005) and The final family of GPCRs are the atypical GPCRs, which the other two are not expressed in embryos (CG30054 and includes receptors such as Frizzled or adhesion GPCRs. Members CG17760). Indeed, only two of the Gβ proteins (Gβ5 and Gβ13f) of these families were initially thought to primarily not signal and a single G γ-subunit (Gγ1) are expressed during the first half of through heterotrimeric G-proteins (Tang et al., 2012). Frizzled Drosophila embryogenesis. With only 13 G-protein subunits, family members contain a cysteine-rich domain in their N-terminus Drosophila offers a relatively simple system for studying GPCR that binds lipoglycoproteins of the Wingless (Wnt) family (Yang- signaling. However, compared to worms and mammals, the fly Snyder et al., 1996). Upon ligand-binding, Frizzled family members GPCRs are not as well characterized. Nearly half of the Drosophila usually signal through the phosphoprotein Dishevelled (Schulte and GPCRs are orphans, suggesting that the field is relatively wide open Bryja, 2007). More recently, however, both Frizzled family for new discoveries. Here, we present new insights into this field that members and Smoothened have been shown to also function as might aid in future characterization of Drosophila GPCRs and we canonical GPCRs, with Frizzled proteins acting as GEFs for Gα(o/i) discuss the roles of the receptors in embryonic development, which proteins (Koval and Katanaev, 2011; Nichols et al., 2013), and is also a rather unstudied side of GPCR signaling. Smoothened acting as a GEF for Gα(i) (Shen et al., 2013). GPCRs of the related adhesion group often contain cadherin or integrin Insect odorant receptors function as ionotropic receptors domains, and these receptors often have auto-proteolytic activity and are not bona fide GPCRs (Krasnoperov et al., 1997). Their ligands include components of the There are 60 odorant receptors encoded in the Drosophila genome extracellular matrix, such as collagen (Luo et al., 2011). and, based on our Clustal Omega analysis, they fall into four major clades (A–D) (supplementary material Fig. S1; Table S1). The Ligand identification odorant receptor A group includes 22 genes. The odorant receptor The most common way to identify the upstream and downstream B and odorant receptor C group include 17 genes each, whereas components of a GPCR pathway is through heterologous expression odorant receptor D, the smallest clade, includes only four genes. Journal of Cell Science

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Table 1. Drosophila G-proteins Symbol Name(s) CG Mammalian output Drosophila expression pattern Gαi G-iα65A, Gi, CG10060 Inhibition of AC, ↓cAMP Embryonic ubiquitious, embryonic brain, embryonic midgut, embryonic Gai CNS, embryonic gonad, nurse cell, oocyte, maxillary palps Gαo G-oα47A, Go, CG2204 Inhibition of AC, ↓cAMP Maternally deposited, embryonic dorsal vessel primordium, visceral Goα muscle primordium, embryonic brain, nurse cell, oocyte, maxillary palpus Gαs G-sα60A, Gs, CG2835 Activator of AC, ↑cAMP Ubiquitous embryonic early, embryonic midgut primordium, embryonic GSα hindgut primordium, embryonic visceral embryonic muscle primordium, adult brain cell body, maxillary palpus α α ↑ G q dgq, G 49b, CG17759 Activator of PLC, IP3 Maternally deposited, embryonic head mesoderm, embryonic gut Gqα and DAG, ↑Ca2+ primordium, embryonic brain, embryonic Bolwig organ, adult eye, adult testis, maxillary palpus GαfGα73B, CG12232 Embryonic midgut, oocyte Gα73B, Gfα Gαq CG17760 Not expressed in embryos Gαq CG30054 Not expressed in embryos Gα CG40005 Pseudogene cta Concertina, CG17678 Activator of Rho via Embryonic ubiquitous, oocyte, maxillary palpus conc RhoGEFs Gβ76C Gβe, Gβ76C CG8770 Embryonic Bolwig organ, adult eye, adult Johnston organ Gβ13F Gβ13F CG10545 Maternally deposited, embryonic early ubiquitous, embryonic brain, embryonic midgut, embryonic hindgut, embryonic ventral nerve cord, adult ovary, adult brain cell, maxillary palpus Gβ5Gβ5 CG10763 Embryonic brain, embryonic midgut, embryonic ventral nerve cord, adult maxillary palpus Gγ30A Gγ30A, Gγ, CG3694 Adult eye, maxillary palpus Gγe Gγ1Gγ1, Gγ CG8261 Embryonic ubiquitous, embryonic brain, adult thorax, adult abdomen, ovary, oocyte, maxillary palpus Information presented here has been compiled from Flybase. AC, adenylate cyclase; CNS, central nervous system; PNS, peripheral nervous system.

Vertebrate odorant receptors are bona fide GPCRs; they are clades A, B and C based on a Clustal Omega analysis of all members seven-transmembrane-pass proteins that are members of the (supplementary material Fig. S2; Table S2). The large A clade, large rhodopsin class of GPCRs. The vertebrate odorant receptors which includes 45 of the known gustatory receptors, can be further are coupled to Gα proteins that activate an adenylate cyclase, divided into two subordinate clades, I and II. Clade A-I includes the leading to increased levels of cAMP, which eventually leads to eight related sweet-taste receptors (Gr5a, Gr61a, Gr64a Gr64b, cell depolarization through the sequential activation of cyclic- Gr64c, Gr64d, Gr64e and Gr64f) and the CO2 receptors (Gr21a and nucleotide- and ion-gated channels. Insect odorant receptors, by Gr63a), which form a functional heterodimer. Gustatory neurons contrast, do not appear to function as classical GPCRs. The first hint that respond to sweet taste are also known to express γ-aminobutyric that they could be different was the discovery that these proteins acid (GABA) receptors, and the loss of these receptors influences have an inverse topology from typical GPCRs: the N-terminus is the perception of sugar (Chu et al., 2014). The GABA receptors are intracellular, whereas the C-terminus is extracellular (Benton et al., proposed to be important for increasing the dynamic range of sweet 2006; Lundin et al., 2007). This would suggest that the domains perception and for suppressing output when bitter tastes are also that normally directly interact with the Gα subunit are outside the present. The two receptors for a female mating pheromone (Gr68a cell. Indeed, insect odorant receptors are not metabotropic receptors. and Gr32a) cluster together in the small C clade. Unlike the Upon ligand binding, insect odorant receptors dimerize with a gustatory receptors of vertebrates, which, upon ligand binding, co-receptor protein known as Orco, an odorant receptor found undergo conformational changes that initiate G-protein signaling to in the odorant receptor B clade. The activated heterodimer activate specific downstream enzymes, the Gr proteins of (or possibly heterotetramer) of an odorant-specific odorant Drosophila and other insects might function as ligand-gated ion receptor subunit and Orco then functions directly as a non- channels. Based entirely on sequence analyses, the gustatory selective cation channel (Wicher et al., 2008), allowing for a much receptors of Drosophila and other insects, like the odorant quicker response to perceived odorants, which has been suggested receptors, appear to be inserted into the plasma membrane with a to be a particular advantage for insects that fly and need to process reverse topology relative to that of the vertebrate gustatory receptors, information quickly (Wicher, 2015). Thus, as ionotropic receptors, which are bona fide GPCRs (Kent and Robertson, 2009). Indeed, the Drosophila seven-transmembrane-domain olfactory receptors many Gr proteins are predicted to have more than seven are not true GPCRs; they do not function as GEFs for G-proteins. transmembrane domains, further separating them from classical GPCRs. Thus, the Drosophila gustatory receptors might also Insect gustatory receptors might also function as ionotropic function as ionotropic receptors, although direct experimental receptors evidence for this is lacking. Overall, it appears that insects sense Gustatory receptors are located on the antenna, labellum, wing hairs large parts of their chemical worlds by linking ligand binding and leg hairs, and sense a diverse array of compounds including directly to the opening of ion channels rather than using G-proteins sugars, bitter compounds, amino acids and sex pheromones. The 60 to indirectly produce second messengers, which then open ion identified Drosophila gustatory receptors separate into three major channels (Silbering and Benton, 2010). Journal of Cell Science

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Phylogenetic analysis of classical Drosophila GPCRs fide GPCRs, we see a very different tree. The classical GPCRs Having removed the odorant and gustatory receptors from the list of separate into three major clades, A, B and C. The large clade A GPCRs, we are left with 116 presumed GPCRs encoded by the includes many members of the classical rhodopsin-like family but Drosophila genome. Using Clustal Omega with five combined also includes all of the other previously categorized families: iterations, we created a phylogenetic tree using these sequences secretin-like, mGlu-like, and Frizzled and atypical. Clade B (Fig. 2, supplementary material Table S3). We termed this GPCR includes the receptors for the biogenic amines, receptors that are set the classical GPCRs. The classical data set comprises 116 bound and activated by molecules such as serotonin, dopamine, proteins, whose sequences were obtained from Flybase (www. adrenaline and epinephrine. This clade includes three proteins flybase.org); for proteins with more than one isoform, only the first identified only by CG number (CG18208, CG12796 and CG13579) isoform listed was used. Historically, Drosophila GPCRs have been that, based on this classification, might also bind biogenic amines. classified into four separate families: rhodopsin-like, secretin-like, Clade C includes a subset of rhodopsin-like receptors that bind mGlu-like and atypical, which included the Frizzled group. Based small peptides that often function as neuropeptides, including the on our analysis, which includes only the Drosophila seven- allostatin, tachykinin and pyrokinin receptors; we refer to this clade transmembrane-pass proteins that are likely to function as bona as peptide A.

Rhodopsin-like/Class A Biogenic Amine

PeptidePeptide A PeptidePeptide C

Glycoproteintein

B Opsin C A

Glutamate/ Family C Orphan

Peptideeptide B MeMethuselahthuselah

SSecretinecretin Secretin-like/ClassSecretin-like B Frizzled Atypical Cell Adhesion

Frizzled/Class F

Fig. 2. Rooted phylogenetic tree of classical Drosophila GPCRs. Full-length sequences of GPCRs obtained from Flybase (www.flybase.org) were used to construct the tree. The names correspond with the colored clades. Colors also correspond to those used in supplementary material Table S3. Journal of Cell Science

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Within clade A, the remaining rhodopsin-like receptors fall into A five different groups: the peptide B (eight proteins) and peptide C Apical constriction during Mist Fog (three proteins) groups bind different flavors of small peptides. Two gastrulation proteins with only CG numbers share a common branch with the α peptide B group of proteins, indicating that they might have related Cta β ligands. The glycoprotein group (four proteins) includes a single γ uncharacterized CG protein (CG34411), Rk, and the Lgr1 and Lgr2 Rho1 proteins; these receptors have large leucine-rich repeat domains. The opsins fall into a separate group that includes all seven of the Pre-mesodermal cells known rhodopsin vision receptors. Finally, two CG proteins (CG30340 and CG13995) cluster with the ‘orphan’ receptors, B Tre1 ? receptors for which the ligands remain completely unknown. Also included in the large A clade are GPCRs known as the ‘glutamate’ or ‘Class C’ family; these include two glutamate and three GABA α Germ cell migration β13f receptors. The Frizzled and Smoothened receptors, which have been γ 1 shown to function as bona fide GPCRs with GEF activity (Riobo Rho1 et al., 2006; Koval and Katanaev, 2011; Shen et al., 2013), fall into a Germ cells single cluster, which was previously known as the ‘Frizzled’ or ECad ‘Class F’ GPCRs. The Frizzled proteins are closely grouped with the cell adhesion, cysteine-rich and secretin-like proteins. The Lateral view Lateral view Tre1 ? Methuselah-like proteins, which have been described as being Neuroblasts secretin-like, share a common upstream branch, but this branch is αo also shared with the glutamate or Class C group. Only two of the 16 Polarized division of Pins β γ Methuselah-like proteins have been characterized, one is involved in neuroblasts Mitotic aging and another involved in gastrulation (see below). Two spindle additional groups in clade A fall outside the previously categorized GPCRs, including one with two proteins, Boss and CG32547, and another with six proteins, including Pog and five genes that are only known by their CG numbers. C Moody Further supporting our phylogenetic analysis is the finding that Establishment of glial cell genes of known related functions cluster into single groups. blood–brain barrier γ 1 For example, all of the visual (rhodopsin) receptors cluster into a β 1 3f Actin single group, as do all Methuselah-like proteins. Likewise, α o,i neurotransmitter receptors, including those for dopamine, Septate junction octopamine, histamine, serotonin and acetylcholine, group together ? proteins into the biogenic amine clade. view Ventral Midline glia CNS

GPCRs in Drosophila development Many of the characterized Drosophila GPCRs regulate adult D Unknown GPCR behavior, but considerably less is known about the role of GPCRs Cardial and pericardial in development. Before cellularization occurs, embryonic cell attachment Pericardial cell development in Drosophila is largely dictated by the action of diffusible gradients. Because the early embryo is essentially one cell Septate junction proteins with many nuclei, cell-to-cell communication is not necessary. γ β 1 However, as development progresses, the activities of newly formed Lateral view 13f α tissues and organs must be coordinated to form a viable organism. ? o Cardial Pericardial Although many GPCRs are expressed during embryonic stages, cells cells ? very little is known with regard to their roles in specific Cardial cell developmental processes. Interestingly, only six Gα, two Gβ (Gβ5 and Gβ13f) and one Gγ (Gγ1) genes are expressed in early embryos Fig. 3. GPCRs in Drosophila development. (A) The GPCR Mist binds to its (Table 1). Here, we discuss the only known roles of GPCRs in ligand Fog in the early embryo; this activates the Gα Concertina (Cta) and its Drosophila development. downstream effector Rho1 to mediate apical constriction of mesodermal cells during gastrulation. (B) Tre1 has two roles in the developing embryo. In germ cells One of the first major events of embryonic development is β γ α (teal), Tre1 activates G 13f and G 1 to relocalize Rho1 and E-cadherin (ECad) to gastrulation. Concertina (a G ) and Fog (a secreted protein) were the rear of the migrating cells. In neuroblast stem cells (green), Tre1 interacts with known to be necessary for this process by the early 1990s, but the Gα(o), which binds to Pins. Pins interacts with proteins that orient the mitotic GPCR that linked these two molecules remained undiscovered for spindle in such a way that correct cell fates are established. (C) Moody activates over 20 years (Parks and Wieschaus, 1991; Costa et al., 1994). In Gα(i), Gα(o), Gβ13f, and Gγ1 to alter the accumulation of actin in the surface glia, addition, the GEF Ric8a was also identified as a crucial component and affecting the organization of septate junction proteins. (D) An unknown α β γ for gastrulation (Peters and Rogers, 2013), further supporting the GPCR is activated in cardial cells, which activates G (o), G 13f, and G 1. These proteins influence the localization of septate junction proteins in cardial cells, idea that a GPCR was involved in this process. Recently, Mist has which interact in trans with septate junction proteins on pericardial cells, thereby been identified as a GPCR that coordinates these events and triggers facilitating adhesion between these two types of cells to ensure proper the initial cell shape changes (Fig. 3A; Manning et al., 2013). The cardiogenesis. PNS, peripheral nervous system; CNS, central nervous system. Journal of Cell Science

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GPCR kinase Gprk2 acts downstream of Fog signaling and limits adult organisms as responding to behavior inputs such as odorants the cell-shape changes to ventral mesodermal cells (Fuse et al., or mating pheromones. More nuanced analysis is needed to 2013), potentially by limiting Mist activity. understand the role of GPCRs in developmental processes, where The role of the atypical GPCRs of the Frizzled family in both components are genetically encoded and must be expressed at establishing segment polarity through the Wnt signaling pathway the right time and place to interact with one another. Bioinformatic has been well characterized. Frizzled family members can also act as analysis can point to potential shared or related ligands in clades canonical GPCRs by signaling through Gα(o) (Katanaev et al., where well-characterized and uncharacterized receptors cluster 2005), which in turn recruits Rab5, an important regulator of together. Our analysis suggests that there are roles for several endocytosis, to the plasma membrane (Purvanov et al., 2010). uncharacterized Drosophila GPCRs, which will hopefully spur Endocytosis and morphogenesis are tightly linked, so this link further research about the exact ligand, pathway and expression between Gα(o) and Rab5 has potentially important implications for profile for each of these genes. embryonic development. Frizzled and other proteins involved in planar polarity have also proven roles in tracheal development Acknowledgements (Chung et al., 2009; Warrington et al., 2013), but a specific role We would like to thank members of the Andrew laboratory for helpful comments and suggestions. of Frizzled in GPCR signaling within the context of this developmental process has not been examined. Competing interests As in other organisms, embryonic germ cell migration is also The authors declare no competing or financial interests. directed by GPCR signaling in the fly. Loss of Tre1 severely affects germ cell migration in the embryo (Fig. 3B; Kunwar et al., 2003). Funding Tre1 was named for its phenotype: instead of migrating to the Our own work in this area has been funded by the Ruth l. Kirschstein national individual research award [grant number 5F31DE022233] and the National gonad, the germ cells remain trapped in the endoderm. The arginine Institutes of Health [grant number RO1 DE013899]. Deposited in PMC for release in the DRY motif is necessary for Tre1 function (Kamps et al., after 12 months. 2010). Tre1 has been shown to influence the distribution of Rho1 and E-cadherin (known as Shotgun in Drosophila) in the germ cells, Supplementary material although the mechanism for this interaction remains unknown Supplementary material available online at http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.175158/-/DC1 (Kunwar et al., 2008). Tre1 also influences embryonic neuroblast stem cell divisions (Yoshiura et al., 2012). Through interactions References with its partners Gα(o) and Pins (a GDI protein), Tre1 has been Allen, J. A., Halverson-Tamboli, R. A. and Rasenick, M. M. (2007). Lipid raft shown to play a key role in the oriented cell divisions that establish microdomains and neurotransmitter signalling. Nat. Rev. Neurosci. 8, 128-140. Anantharaman, V., Abhiman, S., de Souza, R. F. and Aravind, L. (2011). neuroblast cell polarity and cell fate. Despite these known roles for Comparative genomics uncovers novel structural and functional features of the Tre1 in controlling individual cell behavior, Tre1 remains an orphan heterotrimeric GTPase signaling system. Gene 475, 63-78. receptor as its ligand is unknown. Bayliss, W. M. and Starling, E. H. (1902). The mechanism of pancreatic secretion. There are also some insights into the role of Drosophila GPCRs J. Physiol. 28, 325-353. Becker, E. L. Kanaho, Y. and Kermode, J. C. (1987). Nature and functioning of the in cell adhesion and boundary integrity. The Gα(o)–Gβ13f–Gγ1 pertussis toxin-sensitive G protein of neutorphils. Biomed. Pharmacother. 41, complex mediates cardiomyocyte adhesion, and loss of Gγ1 causes 289-297. mislocalization of septate junction proteins in the embryonic heart Benton, R., Sachse, S., Michnick, S. W. and Vosshall, L. B. (2006). Atypical membrane topology and heteromeric function of Drosophila odorant receptors in (Fig. 3D; Yi et al., 2008). Septate junctions are also compromised in vivo. PLoS Biol. 4, e20. moody mutants, which functions in the late embryonic surface glia Bessis, A.-S., Rondard, P., Gaven, F., Brabet, I., Triballeau, N., Prezeau, L., to form the blood–brain barrier that insulates the nerve cord Acher, F. and Pin, J.-P. (2002). Closure of the Venus flytrap module of mGlu8 α α receptor and the activation process: insights from mutations converting (Fig. 3C; Schwabe et al., 2005). G (i), G (o) and Loco likely antagonists into agonists. Proc. Natl. Acad. Sci. USA 99, 11097-11102. function in the same pathway, which is thought to directly affect the Bhola, N. E. and Grandis, J. R. (2008). Crosstalk between G-protein-coupled actin organization required for septate junction assembly. Like Tre1, receptors and epidermal growth factor receptor in cancer. Front. Biosci. 13, Moody is also an orphan receptor, and these proteins belong to the 1857-1865. Bornert, O., Møller, T. C., Boeuf, J., Candusso, M.-P., Wagner, R., Martinez, K. L. same group within clade A in our phylogenetic analysis. As many and Simonin, F. (2013). Identification of a novel protein-protein interaction motif Drosophila GPCRs remain uncharacterized, exciting work remains mediating interaction of GPCR-associated sorting proteins with G protein-coupled to fully understand their potential roles in developmental processes. receptors. PLoS ONE 8, e56336. Brody, T. and Cravchik, A. (2000). Drosophila melanogaster G protein – coupled receptors. J. Cell Biol. 150, F83-F88. Conclusions Caers, J., Peymen, K., Suetens, N., Temmerman, L., Janssen, T., Schoofs, L. In conclusion, GPCR signaling is vital to life. GPCRs are able to and Beets, I. (2014). Characterizatino of G protein-coupled receptors by a translate an outside stimulus into a cellular response on a fluorescence-based calcium mobilization assay. J. Vis. Exp. 89, e51516. Cerione, R. A., Staniszewski, C., Benovic, J. L., Lefkowitz, R. J., Caron, M. G., millisecond timescale, thus connecting the behavior of a cell to its Gierschik, P. Somers, R. Spiegel, A. M., Codina, J. and Birnbaumer, L. (1985). outer environment. GPCRs are involved in many key developmental Specificity of the functional interactions of the beta-adrenergic receptor and events, but they also participate in more refined events, such as rhodopsin with guanine nucleotide regulatory proteins reconstituted in phospholipid vesicles. J. Biol. Chem. 260, 1493-1500. discerning the difference between closely related neuropeptides. Chey, W. Y. and Chang, T. M. (2003). Neural control of the release and action of Ligands for GPCRs are immensely diverse, and many GPCRs secretin. J. Physiol. Pharmacol. 54, 105-112. remain orphans. Through phylogenetic analysis of the bona fide Chini, B. and Parenti, M. (2004). G-protein coupled receptors in lipid rafts and caveolae: how, when, and why do they go there? J. Mol. Endocrinol. 32, 325-338. GPCRs encoded by the Drosophila genome, we hope to add new Chu, B., Chui, V., Mann, K. and Gordon, M. D. (2014). Presynaptic gain control insights into the activities of this important family. Heterologous drives sweet and bitter taste integration in Drosophila. Curr. Biol. 24, 1978-1984. expression systems have historically been used to identify GPCR Chun, L., Zhang, W.-H. and Liu, J.-F. (2012). Structure and ligand recognition of ligands. However, this method has limitations. Most notably, this class C GPCRs. Acta Pharmacol. Sin. 33, 312-323. Chung, S., Vining, M. S., Bradley, P. L., Chan, C.-C., Wharton, K. A., Jr and method ignores spatiotemporal information, an aspect that is vital in Andrew, D. J. (2009). Serrano (Sano) functions with the planar cell polarity genes living organisms. Most ligand–receptor pairs have been identified in to control tracheal tube length. PLoS Genet. 5, e1000746. Journal of Cell Science

3540 COMMENTARY Journal of Cell Science (2015) 128, 3533-3542 doi:10.1242/jcs.175158

Costa, M., Wilson, E. T. and Wieschaus, E. (1994). A putative cell signal encoded Lin, C., Koval, A., Tishchenko, S., Gabdulkhakov, A., Tin, U., Solis, G. P. and by the folded gastrulation gene coordinates cell shape changes during Drosophila Katanaev, V. L. (2014). Double suppression of the Gα protein activity by RGS gastrulation. Cell 76, 1075-1089. proteins. Mol. Cell 53, 663-671. Curtis, D. R. and Watkins, J. C. (1965). The pharmacology of amino acids related to Lundin, C., Käll, L., Kreher, S. A., Kapp, K., Sonnhammer, E. L., Carlson, J. R., gamma-aminobutyric acid. Pharmacol. Rev. 17, 347-391. von Heijne, G. and Nilsson, I. (2007). Membrane topology of the Drosophila De Vries, L., Zheng, B., Fischer, T., Elenko, E. and Farquhar, M. G. (2000). The OR83b odorant receptor. FEBS Lett. 581, 5601-5604. regulator of G protein signaling family. Annu. Rev. Pharmacol. Toxicol. 40, Luo, R., Jeong, S.-J., Jin, Z., Strokes, N., Li, S. and Piao, X. (2011). G protein- 235-271. coupled receptor 56 and collagen III, a receptor-ligand pair, regulates cortical DeMaria, S. and Ngai, J. (2010). The cell biology of smell. J. Cell Biol. 191, 443-452. development and lamination. Proc. Natl. Acad. Sci. USA 108, 12925-12930. Dingus, J., Wells, C. A., Campbell, L., Cleator, J. H., Robinson, K. and Luttrell, L. M., Ferguson, S. S. G., Daaka, Y., Miller, W. E., Maudsley, S., Della Hildebrandt, J. D. (2005). G protein βγ dimer formation: Gβ and Gγ differentially Rocca, G. J., Lin, F.-T., Kawakatsu, H., Owada, K., Luttrell, D. K. et al. (1999). determine efficiency of in vitro dimer formation. Biochemistry 44, 11882-11890. Β-arrestin-dependent formation of the β2 adrenergic receptor-Src protein kinase Doitsidou, M., Reichman-Fried, M., Stebler, J., Köprunner, M., Dörries, J., complexes. Science 283, 655-661. Meyer, D. Esguerra, C. V., Leung, T. and Raz, E. (2002). Guidance of primordial Manning, A. J., Peters, K. A., Peifer, M. and Rogers, S. L. (2013). Regulation of germ cell migration by the chemokine SDF-1. Cell 111, 647-659. epithelial morphogenesis by the G protein-coupled receptor mist and its ligand Drake, M. T., Shenoy, S. K. and Lefkowitz, R. J. (2006). Trafficking of G protein- fog. Sci. Signal. 6, ra98. coupled receptors. Circ. Res. 99, 570-582. Marchese, A., and Paing, M. M., Temple, B. R. S. and Trejo, J. (2008). G protein- Ferré, S., Casadó, V., Devi, L. A., Filizola, M., Jockers, R., Lohse, M. J., Milligan, coupled receptor sorting to endosomes and lysosomes. Annu. Rev. Pharmacol. G. Pin, J.-P. and Guitart, X. (2014). G protein-coupled receptor oligomerization Toxicol. 48, 601-629. revisited: functional and pharmacological perspectives. Pharmacol. Rev. 66, McCusker, E. C., Bane, S. E., O’Malley, M. A. and Robinson, A. S. (2007). 413-434. Heterologous GPCR expression: a bottleneck to obtaining crystal structures. Fredriksson, R. and Schioth, H. B. (2005). The repertoire of G-protein-coupled Biotechnol. Prog. 23, 540-547. receptors in fully sequenced genomes. Mol. Pharmacol. 67, 1414-1425. Mertens, I., Vandingenen, A., Johnson, E. C., Shafer, O. T., Li, W., Trigg, J. S., Fuse, N., Yu, F. and Hirose, S. (2013). Gprk2 adjusts Fog signaling to organize cell De Loof, A., Schoofs, L. and Taghert, P. H. (2005). PDF receptor signaling in movements in Drosophila gastrulation. Development 140, 4246-4255. Drosophila contributes to both circadian and geotactic behaviors. Neuron 48, Garcia-Marcos, M., Ghosh, P. and Farquhar, M. G. (2015). GIV/Girdin transmits 213-219. signals from multiple receptors by triggering trimeric G protein activation. J. Biol. Molyneaux, K. A. (2003). The chemokine SDF1/CXCL12 and its receptor CXCR4 Chem. 290, 6697-6704. regulate mouse germ cell migration and survival. Development 130, 4279-4286. Ha, T. S. and Smith, D. P. (2008). Insect odorant receptors: channeling scent. Cell 5, Mullershausen, F., Zecri, F., Cetin, C., Billich, A., Guerini, D. and Seuwen, K. 761-763. (2009). Persistent signaling induced by FTY720-phosphate is mediated by Higgins, J. B. and Caseys, P. J. (1994). In vitro processing of recombinant G internalized S1P1 receptors. Nat. Chem. Biol. 5, 428-434. protein γ subunits. J. Biol. Chem. 269, 9067-9073. Nathans, J. and Hogness, D. S. (1983). Isolation, sequence analysis, and intron- Hyun, S., Lee, Y., Hong, S.-T., Bang, S., Paik, D., Kang, J., Shin, J., Lee, J., Jeon, exon arrangement of the gene encoding bovine rhodopsin. Cell 34, 807-814. K., Hwang, S. et al. (2005). Drosophila GPCR Han is a receptor for the circadian Nichols, A. S., Floyd, D. H., Bruinsma, S. P., Narzinski, K. and Baranski, T. J. clock neuropeptide PDF. Neuron 48, 267-278. (2013). Frizzled receptors signal through G proteins. Cell Signal. 25, 1468-1475. Jensen, R. T. and Gardner, J. D. (1981). Identification and characterization of Oakley, R. H., Laporte, S. A., Holt, J. A., Caron, M. G. and Barak, L. S. (2000). receptors for secretagogues on pancreatic acinar cells. Fed. Proc. 40, 2486-2496. Differential affinities of visual arrestin, beta arrestin1, and beta arrestin2 for G Kamps, A. R., Pruitt, M. M., Herriges, J. C. and Coffman, C. R. (2010). An protein-coupled receptors delineate two major classes of receptors. J. Biol. Chem. evolutionarily conserved arginine is essential for Tre1 G protein-coupled receptor 275, 17201-17210. function during germ cell migration in Drosophila melanogaster. PLoS ONE 5, Palczewskiss, K., Buczylkost, J., Kaplans, M. W., Polanss, A. S. and Crabb, e11839. J. W. (1991). Mechanism of rhodopsin kinase activation. J. Biol. Chem. 266, Katanaev, V. L., Ponzielli, R., Sémériva, M. and Tomlinson, A. (2005). Trimeric G 12949-12955. protein-dependent frizzled signaling in Drosophila. Cell 120, 111-122. Parks, S. and Wieschaus, E. (1991). The Drosophila gastrulation gene concertina Katanayeva, N., Kopein, D., Portmann, R., Hess, D. and Katanaev, V. L. (2010). encodes a Ga-like protein. Cell 64, 447-458. Competing activities of heterotrimeric G proteins in Drosophila wing maturation. Peters, K. A. and Rogers, S. L. (2013). Drosophila Ric-8 interacts with the Ga12/13 PLoS ONE 5, e12331. subunit, Concertina, during activation of the Folded gastrulation pathway. Mol. Kent, L. B. and Robertson, H. M. (2009). Evolution of the sugar receptors in Biol. Cell 24, 3460-3471. insects. BMC Evol. Biol. 9, 41. Pitcher, J. A., Freedman, N. J. and Lefkowitz, R. J. (1998). G protein-coupled Kleuss, C., Raw, A. S., Lee, E., Sprang, S. R. and Gilman, A. G. (1994). receptor kinases. Annu. Rev. Biochem. 67, 653-692. Mechanism of GTP hydrolysis by G-protein alpha subunits. Proc. Natl. Acad. Sci. Prabhu, Y. and Eichinger, L. (2006). The Dictyostelium repertoire of seven USA 91, 9828-9831. transmembrane domain receptors. Eur. J. Cell Biol. 85, 937-946. Kobilka, B. K., Kobilka, T. S., Daniel, K., Regan, J. W., Caron, M. G. and Purvanov, V., Koval, A. and Katanaev, V. L. (2010). A direct and functional Lefkowitz, R. J. (1988). Chimeric alpha 2-, beta 2-adrenergic receptors: interaction between go and rab5 during g protein coupled receptor signaling. Sci. delineation of domains involved in effector coupling and ligand binding Signal. 3, ra65. specificity. Science 240, 1310-1316. Riobo, N. A., Lu, K. and Emerson, C. P., Jr. (2006). Hedgehog signal transduction: Koval, A. and Katanaev, V. L. (2011). Wnt3a stimulation elicits G-protein-coupled signal integration and crosstalk in development and cancer. Cell Cycle 5, receptor properties of mammalian Frizzled proteins. Biochem. J. 433, 435-440. 1612-1615. Krasnoperov, V. G., Bittner, M. A., Beavis, R., Kuang, Y., Salnikow, K. V., Rovati, G. E., Capra, V. and Neubig, R. R. (2007). The highly conserved DRY motif Chepurny, O. G., Little, A. R., Plotnikov, A. N., Wu, D., Holz, R. W. et al. (1997). of class A G protein-coupled receptors: beyond the ground state. Mol. Pharmacol. α-latrotoxin stimulates exocytosis by the interaction with a neuronal G-protein- 71, 959-964. coupled receptor. Neuron 18, 925-937. Schöneberg, T., Schulz, A., Biebermann, H., Hermsdorf, T., Römpler, H. and Kühne, W. (1877). Zur Photochemie der Netzhaut. Untersuch. Physiol. Instit. Univ. Sangkuhl, K. (2004). Mutant G-protein-coupled receptors as a cause of human Heidelberg 1, 1-14. diseases. Pharmacol. Ther. 104, 173-206. Kunwar, P. S., Starz-Gaiano, M., Bainton, R. J., Heberlein, U. and Lehmann, R. Schulte, G. and Bryja, V. (2007). The Frizzled family of unconventional G-protein- (2003). Tre1, a G protein-coupled receptor, directs transepithelial migration of coupled receptors. Trends Pharmacol. Sci. 28, 518-525. Drosophila germ cells. PLoS Biol. 1, e80. Schwabe, T., Bainton, R. J., Fetter, R. D., Heberlein, U. and Gaul, U. (2005). Kunwar, P. S., Sano, H., Renault, A. D., Barbosa, V., Fuse, N. and Lehmann, R. GPCR signaling is required for blood-brain barrier formation in Drosophila. Cell (2008). Tre1 GPCR initiates germ cell transepithelial migration by regulating 123, 133-144. Drosophila melanogaster E-cadherin. J. Cell Biol. 183, 157-168. Shen, F., Cheng, L., Douglas, A. E., Riobo, N. A. and Manning, D. R. (2013). Lappano, R. and Maggiolini, M. (2011). G protein-coupled receptors: novel targets Smoothened is a fully competent activator of the heterotrimeric G protein Gi. Mol. for drug discovery in cancer. Nat. Rev. Drug Discov. 10, 47-60. Pharmacol. 83, 691-697. Lau, W. W. I., Chan, A. S. L., Poon, L. S. W., Zhu, J. and Wong, Y. H. (2013). Gβγ- Silbering, A. F. and Benton, R. (2010). Ionotropic and metabotropic mechanisms in mediated activation of protein kinase D exhibits subunit specificity and requires chemoreception: ‘chance or design’? EMBO Rep. 11, 173-179. Gβγ-responsive phospholipase C β isoforms. Cell Commun. Signal. 11, 1-17. Simonin, F., Karcher, P., Boeuf, J. J.-M., Matifas, A. and Kieffer, B. L. (2004). Leal, W. S. (2013). Odorant reception in insects: roles of receptors, binding proteins, Identification of a novel family of G protein-coupled receptor associated sorting and degrading enzymes. Annu. Rev. Entomol. 58, 373-391. proteins. J. Neurochem. 89, 766-775. Lefkowitz, R. J. (2013). A brief history of G-protein coupled receptors (Nobel Sondek, J., Bohm, A., Lambright, D. G., Hamm, H. E. and Sigler, P. B. (1996).

Lecture). Angew. Chem. Int. Ed. Engl. 52, 6366-6378. Crystal structure of a Gα protein βγ dimer at 2.1A resolution. Nature 379, 369-374. Journal of Cell Science

3541 COMMENTARY Journal of Cell Science (2015) 128, 3533-3542 doi:10.1242/jcs.175158

Taddese, B., Upton, G. J. G., Bailey, G. R., Jordan, S. R. D., Abdulla, N. Y., Watkins, H. A., Au, M. and Hay, D. L. (2012). The structure of secretin family GPCR Reeves, P. J. and Reynolds, C. A. (2014). Do plants contain g protein-coupled peptide ligands: implications for receptor pharmacology and drug development. receptors? Plant Physiol. 164, 287-307. Drug Discov. Today 17, 1006-1014. Tang, X.-L., Wang, Y., Li, D.-L., Luo, J. and Liu, M.-Y. (2012). Orphan G protein- Wettschureck, N. and Offermanns, S. (2005). Mammalian G proteins and their cell coupled receptors (GPCRs): biological functions and potential drug targets. Acta type specific functions. Physiol. Rev. 85, 1159-1204. Pharmacol. Sin. 33, 363-371. Wicher, D. (2015). Olfactory signaling in insects. Prog. Mol. Biol. Transl. Sci. 130, Tsvetanova, N. G. and von Zastrow, M. (2014). Spatial encoding of cyclic AMP 37-54. signaling specificity by GPCR endocytosis. Nat. Chem. Biol. 10, 1061-1065. Wicher, D., Schäfer, R., Bauernfeind, R., Stensmyr, M. C., Heller, R., Urano, D. and Jones, A. M. (2014). Heterotrimeric G protein-coupled signaling in Heinemann, S. H. and Hansson, B. S. (2008). Drosophila odorant receptors plants. Annu. Rev. Plant Biol. 65, 365-384. are both ligand-gated and cyclic-nucleotide-activated cation channels. Nature Urano, D., Jones, J. C., Wang, H., Matthews, M., Bradford, W., Bennetzen, J. L. 452, 1007-1011. and Jones, A. M. (2012). G protein activation without a GEF in the plant kingdom. Witherow, D. S. and Slepak, V. Z. (2003). A novel kind of G protein heterodimer: the PLoS Genet. 8, e1002756. Venkatakrishnan, A. J., Deupi, X., Lebon, G., Tate, C. G., Schertler, G. F. and G beta5-RGS complex. Receptors Channels 9, 205-212. Babu, M. M. (2013). Molecular signatures of G-protein-coupled receptors. Nature Yang-Snyder, J., Miller, J. R., Brown, J. D., Lai, C.-J. and Moon, R. T. (1996). A 494, 185-194. frizzled homolog functions in a vertebrate Wnt signaling pathway. Curr. Biol. 6, Vögler, O., Barceló, J. M., Ribas, C. and Escribá,P.V.(2008). Membrane 1302-1306. interactions of G proteins and other related proteins. Biochim. Biophys. Acta 1778, Yi, P., Johnson, A. N., Han, Z., Wu, J. and Olson, E. N. (2008). Heterotrimeric G 1640-1652. proteins regulate a non-canonical function of septate junction proteins to maintain Warrington, S. J., Strutt, H. and Strutt, D. (2013). The Frizzled-dependent planar cardiac integrity in Drosophila. Dev. Cell. 15, 704-713. polarity pathway locally promotes E-Cadherin turnover via recruitment of Yoshiura, S., Ohta, N. and Matsuzaki, F. (2012). Tre1 GPCR signaling orients stem RhoGEF2. Development 140, 1045-1054. cell divisions in the Drosophila central nervous system. Dev. Cell 22, 79-91. Journal of Cell Science

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