ABSTRACT

Introduction

Secreted hedgehog (HH) ligands are paracrine signaling factors that mediate communication between cells over distances as large as several hundred microns (Lewis et al., 2001). The first gene encoding a HH ligand was identified genetically through its role in patterning the Drosophila larval epidermis (Nusslein-Volhard and Wieschaus, 1980). Expansion of this gene family has produced three paralogs in amniotes: desert hedgehog (Dhh), Indian hedgehog (Ihh) and sonic hedgehog (Shh). Vertebrate HH ligands, like the Drosophila Hh protein, play roles in patterning multiple tissues including the limb bud, nervous system and skeleton (McMahon et al., 2003). HH ligands can drive proliferation or function as morphogens: secreted from organizing centers, they disperse to form spatial and temporal gradients that provide positional information across a field of progenitor cells to inscribe a pattern of cell fates on a developing tissue (Echelard et al., 1993; Krauss et al., 1993; Riddle et al., 1993; Roelink et al., 1994). HH signaling should be viewed as a system that drives distinct outcomes depending on the strength and duration of signaling activity in target cells, and not a binary ON/OFF switch. Indeed, even modest alterations in HH signaling strength can lead to human birth defects (Nieuwenhuis and Hui, 2005). The capacity for quantitative signaling might be an intrinsic consequence of the evolution of the HH pathway from an ancient system that sensed and regulated cellular metabolite levels (Bazan and de Sauvage, 2009; Hausmann et al., 2009). In addition to their roles during embryogenesis, HH ligands also function in paracrine signaling networks to regulate tissue homeostasis and regenerative responses in adults (Lee et al., 2016). Mutations in HH pathway components that increase signaling strength can drive tumorigenesis, and two HH pathway inhibitors are currently used in the clinic to treat basal cell carcinoma (Raleigh and Reiter, 2019).

The HH pathway (reviewed by Briscoe and Therond, 2013; Lee et al., 2016) is unusual among signaling systems in being composed of a series of inhibitory interactions (Fig. 1A). The main receptor for HH ligands is the 12-pass transmembrane (TM) protein patched (PTCH). In the absence of ligands, PTCH inhibits signaling by suppressing the activity of smoothened (SMO), a heptahelical TM protein that belongs to the G-protein coupled receptor (GPCR) superfamily (Ingham et al., 1991). When SMO is inactive, two inhibitory components – suppressor of fused (SUFU) and protein kinase A (PKA) – restrain the transcriptional activity of the GLI family of transcription factors by direct association and phosphorylation, respectively. Under the influence of SUFU and PKA, the GLI proteins undergo partial proteolysis into repressors (GLI-R) that enter the nucleus and suppress the transcription of target genes. HH ligands trigger serial dis-inhibition of steps in the pathway (Fig. 1A). They bind and inhibit PTCH, thus liberating SMO to adopt an active conformation. Activated SMO transmits the HH signal across the membrane and overcomes the negative influence of PKA and SUFU on GLI proteins. Instead of undergoing proteolytic processing, GLI proteins dissociate from SUFU, enter the nucleus and activate the transcription of target genes.

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Fig. 1.

Overview of HH signaling. (A) HH signaling regulates a bi-functional transcription factor that can repress (GLI-R) or activate (GLI-A) the transcription of target genes. HH ligands bind and inhibit the function of their receptor PTCH, allowing SMO to adopt an active conformation. SMO transmits the HH signal across the membrane and antagonizes the function of two negative regulators, SUFU and PKA, which promote GLI-R formation. Consequently, full-length GLI proteins (GLI-FL) are converted to GLI-A. (B) All HH ligands are modified with a cholesteroyl group at their C termini, attached through an auto-proteolytic reaction catalyzed by the C-terminal domain, and a palmitoyl group at their N termini, attached by a membrane-bound O-acyltransferase. (C) Vertebrate HH signaling is associated with protein trafficking events at primary cilia. When the HH pathway is ‘off’ (left), PTCH is enriched in cilia and inhibits SMO. PKA and SUFU restrain GLI activity and promote its proteolysis into GLI-R. HH signaling is turned on in target cells (right) when HH ligands inhibit PTCH and induce its clearance from primary cilia. As a result, SMO is activated and accumulates in cilia in association with a scaffolding complex, the Ellis van Creveld (EvC) complex. Activated SMO antagonizes the inhibitory effect of PKA on the GLI proteins, leading to the dissociation of SUFU. Now, instead of being converted into GLI-R, GLI-FL can enter the nucleus and activate target gene transcription (GLI-A). The transition zone at the cilia base regulates receptor access to cilia, cilia tips form a compartment (marked by the kinesin KIF7) that regulates the GLI proteins, and the EvC complex scaffolds SMO signaling near the cilia base (Box 1).

HH signaling in vertebrates (but not in Drosophila) depends on primary cilia – solitary microtubule-based organelles that function as signaling hubs in development (Box 1) (Fig. 1C) (Huangfu et al., 2003). The connections between primary cilia and HH signaling are not our primary focus, and we refer the reader to a recent comprehensive review (Bangs and Anderson, 2017). Instead, we analyze recent progress in understanding the series of biochemical reactions that transmit the HH signal from the cell surface to the nucleus. We pay particular attention to recently reported structures of the TM proteins that detect HH signals at the cell surface and transmit them across the membrane to the cytoplasm. A theme that links our discussions is the regulation of signaling strength by HH morphogens. We do not discuss the myriad biological roles of HH signaling in development, cancer and regeneration, which is best left to a dedicated review. Finally, we focus on the vertebrate HH pathway, but note that HH signaling was discovered and elucidated by genetic and cell biological studies in Drosophila (recently recounted by Ingham, 2018).

The biogenesis of HH ligands and their spread through tissues

HH ligands are synthesized as ~45 kDa precursors that undergo an intein-like self-cleavage reaction, liberating an N-terminal signaling domain (HhN) covalently attached to a cholesteroyl moiety at the C terminus (Fig. 1B) (Lee et al., 1994; Porter et al., 1996). A palmitoyl moiety is then added to the N-terminus by the membrane-bound O-acyltransferase HHAT (Buglino and Resh, 2008; Chamoun et al., 2001; Pepinsky et al., 1998), generating the mature, dually lipidated ligand (Fig. 1B). The biogenesis, secretion and dispersal of HH ligands through tissues has been thoroughly reviewed recently (Manikowski et al., 2018; Petrov et al., 2017).

Regulation of secretion and transport is important for shaping temporal and spatial gradients of HH ligands across developing tissues, signaling strength in target cells and consequently tissue patterning outcomes. The dual lipidic modification on HH ligands render them highly hydrophobic and tethered to cell membranes. Short-range signaling between adjacent cells can be mediated by cell-surface-bound HH ligands (Strigini and Cohen, 1997). However, long-range signals require specialized components to release ligands from membranes and shield their lipidic appendages in the aqueous interstitial environment (Caspary et al., 2002; Lewis et al., 2001). The TM protein Dispatched (DISP1) is required exclusively for ligand release and likely functions to isolate HH ligands from the bulk membrane by binding to their cholesteroyl moieties and transferring them to a carrier for transport through tissues (Burke et al., 1999; Caspary et al., 2002; Kawakami et al., 2002; Ma et al., 2002; Tukachinsky et al., 2012). Several types of carriers have been identified, including the Scube family of secreted proteins, lipoproteins, extracellular vesicles and multimers of ligands themselves (summarized by Petrov et al., 2017). A distinct solution to the ligand transport problem is provided by cytonemes – long, actin-based cellular extensions that directly deliver ligands to distant cells without the requirement for ligand release from membranes (Bischoff et al., 2013; Chen et al., 2017; Ramírez-Weber and Kornberg, 1999; Sanders et al., 2013). Finally, there is a large body of work (summarized by Petrov et al., 2017) showing that heparan sulfate proteoglycans (HSPGs) can regulate the release, dispersal and reception of HH ligands.

PTCH is the main receptor for HH ligands

All HH ligands must bind and inhibit the function of PTCH to trigger signaling in target cells (Marigo et al., 1996a; Stone et al., 1996). Vertebrates have two PTCH genes, PTCH1 and PTCH2, but PTCH1 is the major regulator of signaling in vivo (Carpenter et al., 1998; Motoyama et al., 1998; Nieuwenhuis et al., 2006). We use PTCH to refer to both for simplicity. PTCH plays two separate roles in HH signaling: it inhibits SMO and reduces the abundance of HH ligands by promoting their endocytosis and lysosomal degradation (Chen and Struhl, 1996). PTCH gene expression is directly activated by HH signaling, resulting in a negative feedback loop: HH ligands inactivate PTCH and de-repress SMO, causing increased production of PTCH, which then feeds back both to inhibit SMO and reduce the abundance of HH ligands (Goodrich et al., 1996; Marigo et al., 1996b). A consequence of this negative feedback is that different doses of HH ligands are translated into different durations of signaling in target cells, which activates different sets of target genes (Dessaud et al., 2007). A combination of in vitro morphogen modeling in cultured fibroblasts and computational simulations show that several unique properties of PTCH are important for generating stable signaling gradients (Li et al., 2018). Such properties include negative feedback, inhibition of HH ligands and SMO, and involvement in a ‘double-negative’ circuit (in which SHH inhibits PTCH, which inhibits SMO).

PTCH has homology with two membrane transporter types: the resistance-nodulation-division (RND)-family pumps, which use transmembrane proton gradients to efflux toxic molecules out of gram-negative bacteria, and Niemann-Pick C1 (NPC1), which transports cholesterol from the lumen of the lysosome to the cytoplasm (Carstea et al., 1997; Loftus et al., 1997; Pfeffer, 2019; Tseng et al., 1999). Recently published cryo-electron microscopy (cryo-EM) structures reveal that PTCH resembles RND transporters and is composed of two pseudo-symmetrical segments (Fig. 2A) (Gong et al., 2018; Qi et al., 2018a,b; Zhang et al., 2018). Each segment includes a transmembrane domain (TMD) composed of six TM helices and one extracellular domain (ECD) interposed between TM1 and TM2. Likewise, NPC1 is also related to RND transporters, but notably transports cholesterol in the opposite direction to bacterial RND proteins. In PTCH, TM2-TM6 forms a sterol sensing domain (SSD; dark blue in Fig. 2A), which is also found in NPC1 and other proteins that handle cholesterol (Davies and Ioannou, 2000). The similarities between PTCH, NPC1 and RND proteins suggest that PTCH transports a hydrophobic ligand for SMO.

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Fig. 2.

Structures of PTCH. (A) Structure of unliganded PTCH (PDB 6DMB; Gong et al., 2018) showing the transmembrane domain (TMD), which includes a sterol-sensing domain (SSD), and two extracellular domains (ECD1 and ECD2). A possible hydrophobic tunnel (ocher surface) is shown connecting two putative sterol-binding sites (meshed surfaces, asterisks) in ECD1 and the SSD. (B) Structure of the asymmetric 1SHH:2PTCH complex (adapted from PDB 6E1H; Qi et al., 2018b) reveals two distinct SHH-PTCH interfaces. PTCH1 molecule 1 (mol1) binds to SHH at an interface including its calcium- and zinc-binding sites and PTCH1 molecule 2 (mol2) engages the N-terminal palmitoyl and C-terminal cholesteroyl modifications of SHH, which are inserted into the PTCH protein core. The interaction of SHH with mol1 drives PTCH endocytosis and the palmitate-centered interaction with mol2 inactivates the transporter function of PTCH. (C-F) Structures of SHH in complex with the PTCH ECD (C; adapted from PBD 6E1H), CDO (D; PDB 3D1M; McLellan et al., 2008), HHIP (E; PDB 2WFX; Bishop et al., 2009) and heparin (F; PDB 4C4N; Whalen et al., 2013) reveal overlapping interfaces that would prevent simultaneous binding. Binding footprints for each protein on SHH are shown below the corresponding structure, with hydrophilic interactions in pink and hydrophobic interactions in brown.

The interaction of HH ligands with co-receptors and antagonists

A conundrum raised by the SHH:PTCH1-mol1 structure is that the interaction interface between PTCH and SHH overlaps with the interface between SHH and several other cell-surface proteins and glycosaminoglycan chains of HSPGs, all of which are implicated in regulating ligand reception in target cells (Fig. 2C-F) (Bishop et al., 2009; Bosanac et al., 2009; Kavran et al., 2010; McLellan et al., 2008; Whalen et al., 2013). Vertebrate HH ligand co-receptors include three partially redundant proteins: the TM proteins CDO and BOC, and the glycosylphosphatidylinositol (GPI)-anchored protein GAS1. Elimination of all three proteins results in reduced HH signaling (Allen et al., 2011; Izzi et al., 2011). Although the binding of GAS1 to HH ligands is not well defined, it is clear that SHH cannot simultaneously interact with CDO/BOC and PTCH via the protein-based interface seen in the SHH:PTCH1-mol1 structure (Fig. 2C,D). However, the interaction between CDO/BOC and SHH (Fig. 2D) would leave the palmitate of SHH free to inactivate a molecule of PTCH, suggesting the possibility of a CDO/BOC-SHH-PTCH signaling complex, analogous to the PTCH-SHH-PTCH complex (Fig. 2B) (Qi et al., 2018b). Here, SHH could inactivate the biochemical function of PTCH without inducing its endocytosis and degradation. Alternatively, CDO and BOC might increase the local concentration of SHH on the cell surface and indirectly increase the chance of a SHH-PTCH interaction. Finally, HHIP is a secreted antagonist of HH ligands that binds to the same interface of SHH as both PTCH and CDO/BOC with low nanomolar affinity (Fig. 2E) (Chuang and McMahon, 1999). HHIP associates with the cell surface and extracellular matrix by binding to HSPGs (Holtz et al., 2015). An interesting question is why are CDO and BOC ligand agonists whereas HHIP is a ligand antagonist, even though both proteins bind to the same surface of SHH? Perhaps the higher affinity of HHIP for SHH, and its lack of tethering close to the plasma membrane, allow it to purely sequester extracellular HH ligands. In summary, co-receptors play key roles in regulating the availability of HH ligands and their influence on PTCH biochemical activity and PTCH trafficking.

Regulation of SMO

The endogenous ligand for SMO

Whereas HH ligands are received by PTCH and co-receptors, the HH signal is transmitted across the membrane by SMO, which belongs to the family of class F GPCRs, named for the frizzled (FZD) family of receptors for WNT ligands. SMO is composed of a heptahelical TMD, an extracellular cysteine-rich domain (CRD) and a linker domain (LD) that connects the CRD and TMD (Fig. 3A). Finally, SMO has an ~240 amino acid C-terminal cytoplasmic tail domain (CCT) that is required for SMO localization to cilia and to activate downstream signaling, but is removed from all the proteins used for structural studies because of its partially disordered nature (Varjosalo et al., 2006).

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Fig. 3.

Multi-domain structures of SMO bound to agonists and antagonists. (A,B) Human SMO (hSMO, blue) carrying an inactivating V329F mutation in the TMD bound to the agonist cholesterol (A; PDB 5L7D) in the CRD-binding site or to the antagonist vismodegib (B; PBD 5L7I) in the TMD site (Byrne et al., 2016). (C) hSMO bound to the TMD antagonist TC114 (PBD 5V57; Zhang et al., 2017). Arrows show movement of the CRD in the antagonist-bound structures (B,C) relative to the cholesterol bound structure (A). (D,E) Structures of Xenopus SMO (xSMO, green) bound to the agonist cholesterol (D; PDB 6D35; Huang et al., 2018) or the antagonist cyclopamine (E; PDB 6D32) are identical and show a dramatic re-orientation of the CRD relative to the TMD. Dotted circles (D,E) highlight a potential steric clash between the CRD and an N-linked glycan in the third extracellular loop of SMO. The N-linked glycans for Xenopus SMO were modeled because they were removed for crystallization. (F) Overlay of the indicated SMO structures showing rupture of the ionic lock between a tryptophan (W) and an arginine (R) residue resulting in the outward movement of TM6 (solid arrow) and the opening to a hydrophobic channel proposed to run through the xSMO TMD in the activated state (dotted arrow). Asterisks in all structures show the connections to the BRIL or flavodoxin domains interposed between TM5 and TM6 to facilitate crystallization. CRD, cysteine-rich domain; ECL3, extracellular loop 3; LD, linker domain; TMD, transmembrane domain.

Three observations led to the hypothesis that PTCH regulates a small molecule ligand for SMO: the similarity of PTCH to transporter-like proteins described above, the lack of physical interactions between PTCH and SMO, and the observation that each molecule of PTCH can inhibit multiple molecules of SMO (Denef et al., 2000; Ingham et al., 2000; Taipale et al., 2002). Several structures of SMO have been determined by X-ray crystallography, both of the isolated TMD and CRD and of the multi-domain CRD-TMD (Byrne et al., 2016; Huang et al., 2016, 2018; Nachtergaele et al., 2013; Rana et al., 2013; Wang et al., 2013, 2014; Zhang et al., 2017). The structures provide an atomic view of two major ligand-binding sites in SMO: the CRD site and the TMD site (Fig. 3A,B). A number of synthetic and natural small molecules bind to both these sites to positively or negatively regulate SMO activity (Fig. 3A-E). We refer the reader to reviews on small molecule regulation of SMO for a more detailed discussion (Byrne et al., 2018; Sharpe et al., 2015). We focus below on the endogenous ligand that mediates communication between PTCH and SMO in vertebrates.

Several different approaches suggest that the endogenous SMO regulator is a sterol lipid. In addition to the homology of PTCH to NPC1, an early indication came from the discovery that a plant sterol alkaloid cyclopamine binds to and inhibits SMO at the TMD site (Chen et al., 2002a; Taipale et al., 2000), which has since become the target of pharmaceutical SMO inhibitors (Chen et al., 2002b; Frank-Kamenetsky et al., 2002). Second, pharmacological or genetic approaches that reduce cellular cholesterol levels also attenuate HH signaling in target cells, showing that cholesterol plays a second role in signal reception distinct from its role in ligand biogenesis (Blassberg et al., 2016; Cooper et al., 1998, 2003; Incardona and Roelink, 2000). Third, side-chain oxysterols (endogenous metabolites of cholesterol) activate HH signaling at the level of SMO and induce its accumulation in primary cilia – even in the absence of HH ligands (Corcoran and Scott, 2006; Dwyer et al., 2007; Rohatgi et al., 2007). Pharmacological and ligand-affinity studies led to the discovery of a second oxysterol-binding site in SMO that is entirely distinct from the TMD-binding site (Nachtergaele et al., 2012). This second site was later shown to be formed by a shallow hydrophobic groove in the CRD site – the same groove in FZD receptors that binds to the palmitoleyl modification on WNT ligands (Fig. 3A) (Janda et al., 2012; Myers et al., 2013; Nachtergaele et al., 2013; Nedelcu et al., 2013). Though physically separate, the oxysterol-binding site in the CRD is allosterically linked to the TMD-binding site (Nachtergaele et al., 2012). Interestingly, cyclopamine, although initially characterized as a TMD antagonist, can also function as an agonist by binding the CRD site (Huang et al., 2016; Nachtergaele et al., 2013) (Fig. 3E), though its dominant effect on signaling in cells is inhibitory. Recent studies suggest that endogenous oxysterols enriched in the ciliary membrane may activate SMO through the CRD site in specific developmental or oncogenic contexts (Raleigh et al., 2018).

The first multi-domain structure of SMO unexpectedly reveals a cholesterol molecule bound to the CRD site in the same position that oxysterols were predicted to bind, raising the possibility that the endogenous ligand for the CRD is cholesterol itself, rather than oxysterols (Byrne et al., 2016) (Fig. 3A). Mutations in the CRD site that prevent cholesterol binding impair HH signaling in cultured cells (Byrne et al., 2016) and mouse embryos (Xiao et al., 2017). Two independent studies demonstrate that cholesterol is sufficient to activate signaling even in the absence of HH ligands (Huang et al., 2016; Luchetti et al., 2016). Both studies identify mutations in the CRD that could discriminate between cholesterol-activation and oxysterol-activation, and show that mutations that prevent oxysterol activation (but leave cholesterol activation intact) have little effect on SHH-induced signaling. Low doses of cholesterol (but not oxysterols) synergize with SHH in signaling assays (Huang et al., 2016; Luchetti et al., 2016; Nachtergaele et al., 2012). Collectively, these results suggest that cholesterol itself may be the endogenous small molecule SMO agonist regulated by PTCH – the elusive second messenger that communicates the HH signal between its receptor and the TM transducer (Byrne et al., 2016; Huang et al., 2016; Luchetti et al., 2016). The model proposed by these studies provides a unifying explanation for the diverse set of observations made over nearly two decades that link cholesterol to the reception of HH signals.

The mechanism of SMO activation

Although these studies point to a role for cholesterol in the PTCH-SMO interaction, there is uncertainty about which domain of SMO is targeted by the inhibitory effect of PTCH. Complete deletion of the CRD (in SMOΔCRD), or mutations that destabilize the CRD-TMD interface, increase the constitutive (ligand-independent) signaling activity of SMO in cells (Byrne et al., 2016). SMOΔCRD is markedly less sensitive to PTCH, but its activity can be suppressed by PTCH overexpression (Myers et al., 2013). One possibility is that PTCH regulates the access of both the CRD and TMD to cholesterol. Cholesterol is not bound to the TMD in any of the solved SMO structures, even though SMO was crystallized with a high concentration of cholesterol. The classical TMD site (Fig. 3B) is unlikely to mediate the effect of cholesterol because various mutations in this site fail to alter SHH-driven signaling (Dijkgraaf et al., 2011; Myers et al., 2013). However, molecular dynamic simulations identify a potential cholesterol-binding site between the extracellular ends of TM2 and TM3, at the outer leaflet of the plasma membrane (Fig. 4A) (Hedger et al., 2018). Furthermore, mutagenesis and computational docking studies identify a putative oxysterol-binding site at the cytoplasmic end of the TMD (Fig. 4A) (Raleigh et al., 2018). Finally, a recent multi-domain structure of SMO suggests that cholesterol from the inner leaflet of the plasma membrane could gain access to a hydrophobic tunnel in the center of the TMD bundle through a gate at the cytoplasmic end of the TMD (Fig. 3F) (Huang et al., 2018). It will be important to determine whether any of these proposed SMO-sterol interactions can explain the ability of PTCH to inhibit the activity of SMOΔCRD.

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Fig. 4.

Models for how PTCH inhibits SMO. (A) Schematic of PTCH and SMO embedded in a model lipid bilayer. Structurally identified cholesterol molecules bound to PTCH and SMO are depicted as yellow spheres. Two potential sterol-binding sites on SMO identified by computational methods are shown as green surfaces. Three possible sterol transport paths are shown by black arrows. In model 1, PTCH reduces the abundance or accessibility of inner leaflet cholesterol, preventing it from interacting with the hydrophobic channel or the cytoplasmic sterol-binding site of SMO. In model 2, cholesterol moves through PTCH from the outer leaflet of the membrane to the ECD1 and eventually to a protein or membrane acceptor, thereby depleting the membrane of cholesterol. In model 3, PTCH accepts cholesterol from the SMO CRD (or another donor) and transports it to the membrane, thereby turning off SMO activity. (B) Models for how PTCH could deplete cholesterol from the ciliary membrane (thereby reducing its access to SMO) by transporting it between the two closely opposed membranes of the ciliary pocket.

Transmission of the HH signal across the membrane requires a conformational change in the TMD of SMO, so there is considerable interest in understanding the structural transitions that are associated with activation. Interactions between the CRD and the TMD play a key role in stabilizing the inactive state of SMO; mutations that destabilize the linker domain that connects the CRD to TMD or that introduce glycosylation sites at the CRD-TMD interface increase the constitutive activity of SMO (Byrne et al., 2016). Small angle X-ray scattering (SAXS) shows that activation involves the movement of the CRD relative to the TMD (Byrne et al., 2016). However, the precise orientation of the CRD relative to the TMD in active SMO remains uncertain (Fig. 3A-E and Box 2). One of the putative active-state structures highlights a cation-pi bond (the ‘ionic lock’) between an arginine and a tryptophan residue at the cytoplasmic end of the TM bundle (Huang et al., 2018) (Fig. 3F). This ionic lock is broken by known oncogenic mutations that activate SMO and is also conserved in the FZD group of WNT receptors (Wright et al., 2019). SMO activation may result in the rupture of this ionic lock, leading to the outward movement of TM6 and the consequent exposure of a new molecular surface to engage a cytoplasmic effector.

Box 2. Structural models of SMO activation

All of the multi-domain SMO structures described to date, regardless of whether they are presented as active- or inactive-state conformations, are derived from proteins that lack signaling capacity. To enable crystallization, mutations or small molecules are used to stabilize the inactive state and heterologous protein domains inserted to facilitate crystal contacts. One model for activation proposes that the CRD moves away from a long helical extension formed by the third extracellular loop to allow cholesterol binding (Fig. 3A,B) (Byrne et al., 2016). However, this model is based on the structure of SMO carrying a mutation in the TMD that stabilizes the inactive state and so does not provide insights into TMD conformational changes (Fig. 3A,B). A second study proposes a conformational change in the CRD itself that is propagated to the TMD (Huang et al., 2016); however, this conformation is unlikely to be physiological as it is induced by a zinc-promoted crystal contact (discussed by Luchetti et al., 2016). A third structural model (Fig. 3D,E) shows a large reorientation of the CRD relative to the TMD (Huang et al., 2018); however, this active-state conformation would produce a steric clash with N-linked glycans removed for determining the structure (dotted circle in Fig. 3D,E) and thus is unlikely to be adopted by endogenous SMO in cells. In addition, the TMD conformations of the cholesterol-bound and cyclopamine-bound structures are identical (Fig. 3D,E), even though we know that cyclopamine binding to the TMD inhibits SMO signaling. Overall, none of the structures fully explains the difference between active- and inactive-state SMO. Additional active-state structures for SMO, ideally in complex with a downstream effector, are required to resolve the details of the structural changes that drive signal transmission across the membrane.

Signal transmission from SMO

In contrast to the Drosophila pathway, the issue of how vertebrate SMO transmits the HH signal from the cell membrane to the cytoplasm remains unresolved, with multiple implicated components. One important point to remember is that just because a component is required for signaling, it does not mean that its activity is changed by SMO signaling. A change in biochemical activity in response to HH ligands has not been conclusively demonstrated for any of the myriad components (many with strong loss-of-function phenotypes) implicated in SMO signaling to the cytoplasm. Our focus below is on signaling mechanisms that relay signals from SMO to the GLI transcription factors, but we note that SMO can activate other non-transcriptional signaling outputs (Box 3).

Box 3. GLI-independent mechanisms of SMO signaling

SMO regulates several cellular processes independent of GLI proteins. In the nervous system, SMO signaling mediates both attractive and repulsive responses during axon guidance and calcium spiking in the developing spinal cord (Belgacem and Borodinsky, 2011; Charron et al., 2003; Yam et al., 2009). SMO can regulate cytoskeletal responses through the small GTPases RhoA and Rac1 (Chinchilla et al., 2010) and metabolic reprogramming through a calcium-AMPK pathway (Teperino et al., 2012). Although the signaling mechanisms in each case remain to be fully elucidated, they frequently involve activation of the Gαi family of heterotrimeric G-proteins.

In all animals, a central task of SMO is to antagonize the inhibitory effect of PKA, a multifunctional kinase, on the GLI family of transcription factors (Fig. 1A). In principle, SMO could regulate PKA in one of two general ways, although the challenge in both cases is how to selectively inhibit the effects of PKA on the HH pathway, while sparing any effect on non-HH PKA substrates in the cell. The first possibility is that active SMO reduces PKA enzymatic activity in a specific subcellular compartment relevant to HH signaling (such as the primary cilium) or in a HH-specific protein complex. Alternatively, SMO could reduce the access of GLI proteins to PKA by segregating GLI and PKA in different compartments or different complexes.

PKA activity can be increased or decreased by a signaling cascade initiated by GPCRs (Fig. 5). When GPCRs are activated by ligands, they catalyze the exchange of guanosine diphosphate (GDP) for guanosine triphosphate (GTP) on the Gα subunit of heterotrimeric G-proteins, resulting in their activation and dissociation from the Gβ and Gγ subunits. Gαs subunits activate adenylyl cyclases (AC), which synthesize the second messenger cyclic adenosine 3′,5′-monophosphate (cAMP) from ATP; Gαi subunits reduce cAMP levels by inhibiting AC (Fig. 5). cAMP directly binds and activates PKA. The balance of Gαi and Gαs activities, along with the activities of phosphodiesterases (PDEs) that hydrolyze cAMP, sets the cellular cAMP concentration and hence the PKA activity level.

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Fig. 5.

GLI proteins are regulated by PKA. PKA is a conserved inhibitor of GLI proteins, and the strength of HH signaling is inversely correlated with the activity of PKA in cells. The pathway regulating cAMP levels and PKA activity in cells is shown: proteins that increase PKA activity (red background) inhibit HH signaling, whereas those that decrease PKA activity (green background) enhance HH signaling. Among positive regulators of signaling, GPCRs coupled to Gαi reduce cAMP synthesis by inhibiting AC, phosphodiesterases (PDEs) hydrolyze cAMP and ARHGAP36 inhibits PKA. GPCRs coupled to Gαs, such as GPR161, inhibit signaling by increasing cAMP synthesis by AC. The kinases GRK2 and GRK3 are strong positive regulators that may function either by downregulating GPCRs coupled to Gαs or by directly promoting SMO activity. The mechanism by which SMO antagonizes the PKA axis is not clear (dashed arrows with ‘?’), but may involve direct activation of Gαi, inhibition of a Gαs-coupled GPCR or activation of a Gαi-coupled GPCR. PKA is composed of catalytic (C) and regulatory (R) subunits.

The relationship between HH signaling and PKA activity is well established: increasing PKA activity inhibits signaling, whereas decreasing PKA activates signaling (even in the absence of HH ligands) in a variety of systems ranging from cultured cells to vertebrate embryos. This is shown by directly manipulating PKA activity or the upstream ACs, PDEs and Gα proteins (Fig. 5) (Fan et al., 1995; Hammerschmidt et al., 1996; Huang et al., 2002; Hynes et al., 1995; Regard et al., 2013; Tuson et al., 2011; Vuolo et al., 2015; Wechsler-Reya and Scott, 1999; Williams et al., 2015; Yao and Capel, 2002).

Regulation of the GLI transcription factors by a multi-site phosphorylation code

HH signaling converges on a small family of zinc-finger transcription factors encoded by Gli genes (Gli1, Gli2 and Gli3) in vertebrates (Fig. 6A) (Hui et al., 1994). In vertebrates, GLI2 and GLI3 proteins (together referred to as GLI2/3) can exist in at least three states: proteolytically processed transcriptional repressors (GLI2/3R), full-length transcriptionally inactive proteins (GLI2/3FL), and full-length transcriptional activators (GLI2/3A) (Fig. 6B) (reviewed by Hui and Angers, 2011; see also Aza-Blanc et al., 1997; Methot and Basler, 1999; Wang et al., 2000). Mouse genetic studies have shown that the transcriptional activator function is largely allocated to GLI2 and transcriptional repressor function to GLI3 (Litingtung and Chiang, 2000; Matise et al., 1998). GLI1 is a transcriptional target of GLI2 and GLI3, and functions exclusively as a transcriptional activator that amplifies existing HH signaling activity. We will focus our discussion on GLI2/3 below, as they are directly regulated by SMO signaling. GLI1 is regulated by distinct mechanisms, especially important for HH-driven cancers (Atwood et al., 2013; Huntzicker et al., 2006; Mirza et al., 2019).

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Fig. 6.

Regulation of GLI proteins in HH signaling. (A) Domain structures of mouse GLI2 and GLI3 proteins. Regions of the protein involved in various biochemical reactions are annotated. (B) Sequence of reactions that convert full-length GLI2/3 (blue shading) into a transcriptional repressor (red shading) or a transcriptional activator (green shading). Phosphorylation by PKA primes further phosphorylation by GSK3β and CK1 and promotes recognition by βTRCP, the substrate recognition adaptor of the SCF E3 ubiquitin ligase (Tempe et al., 2006; Wang and Li, 2006). SCF^βTRCP-mediated ubiquitination targets GLI2/3 to the proteasome for degradation. Owing to the presence of a processing determinant domain (PDD, see A), GLI3 (and to a lesser extent GLI2) are subject to an unusual partial proteasomal degradation reaction that generates fragments that function as pure transcriptional repressors (Schrader et al., 2011). (C) Multiple states of GLI activity can be encoded by different patterns of GLI phosphorylation. Full phosphorylation of GLI at the PKA phosphorylation sites (red circles) drives proteolytic production of GLI-R and repression of target genes. Graded dephosphorylation at the PKA sites prevents formation of GLIR and increases the ability of GLI to activate transcription. Maximum GLI transcriptional activity is associated with a separate activating hyper-phosphorylation (green circles).

How are the functions of primary cilia, SUFU and PKA coordinated by HH ligands to produce the multiple states of GLI2/3 activity that are required for graded responses? Epistasis experiments show that primary cilia appear to function at a step between PKA and SUFU – the loss of primary cilia blocks signaling that is triggered by PKA inhibition but has no effect on signaling that is triggered by loss of SUFU (Chen et al., 2009; Ocbina and Anderson, 2008). GLI2/3, in association with SUFU, travel through primary cilia both in the absence and presence of HH ligands, though their abundances at the tips of cilia increase in response to SMO activation (Haycraft et al., 2005; Kim et al., 2009; Tukachinsky et al., 2010) (Fig. 1C). The consequences of GLI-SUFU trafficking through cilia is entirely different in the absence and presence of SMO activity (Humke et al., 2010; Tukachinsky et al., 2010; Tuson et al., 2011) (Fig. 1C). When SMO is inactive, ciliary trafficking promotes the partial proteolytic processing of GLI2/3FL into GLI2/3R fragments that dissociate from SUFU and enter the nucleus to repress target genes (Figs 1C and ​and6B).6B). When SMO is active and accumulates in cilia, ciliary trafficking promotes the dissociation of SUFU from full-length GLI2/3, allowing the formation of GLI2/3A proteins that can enter the nucleus and activate target genes (Fig. 1C). The biochemical mechanisms that link cilia trafficking to either GLI2/3 proteolysis or to SUFU dissociation from GLI2/3 remain largely obscure. However, they are likely controlled by the phosphorylation of GLI2/3 by PKA (which in turn is regulated by the activity-state of SMO) and by the atypical kinesin KIF7 (Fig. 1C) (reviewed by Hui and Angers, 2011).

Regulation of the GLI2/3 activity state by PKA is key for graded HH ligand responses to be converted into multiple levels of GLI activity in the nucleus, and ultimately to alternative differentiation outcomes in target cells (Stamataki et al., 2005). Multi-site phosphorylation of the GLI2/3 proteins is used to resolve these graded responses into multiple discrete activity states, referred to as multi-stability (Fig. 6C) (Thomson and Gunawardena, 2009). There is evidence, both from cultured cells and chicken neural tube development, that the transcriptional activity of GLI2/3 is regulated by a multi-site phosphorylation code that is inscribed on two clusters of phosphorylation sites (Fig. 6A) (Niewiadomski et al., 2014). Although the PKA sites were originally thought to only control proteolysis to generate the GLI2/3R fragment (Fig. 6B), more recent mutagenesis and phosphorylation analysis using mass spectrometry shows that increasing stoichiometry of phosphorylation at the PKA-target sites can inhibit the transcriptional activity of full-length GLI2/3 proteins in a graded fashion (Niewiadomski et al., 2014) (Fig. 6C). Notably in Drosophila, multi-site phosphorylation of both SMO and the KIF7 ortholog Cos2 has been implicated in enabling graded signaling responses, suggesting a common regulatory device to encode multistability in morphogen signaling systems (Ranieri et al., 2012; Su et al., 2011).

Mechanisms that regulate target cell sensitivity to HH ligands

Although ligand secretion and distribution play important roles in regulating the strength of HH signaling, more recent work has uncovered signaling mechanisms that tune the sensitivity of target cells to HH ligands. GPCRs can modify target-cell sensitivity to HH ligands, presumably by changing PKA activity (Fig. 5). The best studied of these is GPR161 (described above), which reduces the sensitivity of target cells to HH ligands, likely by activating Gαs and consequently PKA activity (Mukhopadhyay et al., 2013; Pusapati et al., 2018b). GPR161 has been shown to attenuate HH signaling in the developing limb, skeleton and spinal cord, and deletion of GPR161 in neural stem cells can induce cerebellar tumors (Hwang et al., 2018; Mukhopadhyay et al., 2013; Shimada et al., 2018). A ligand that regulates GPR161, positively or negatively, remains to be identified. In the cerebellum, the GPCR ADCYAP1R1 attenuates HH signaling, both in the context of normal cerebellar development and HH-driven medulloblastoma, by increasing PKA activity through Gαs (Niewiadomski et al., 2013). PKA can also be regulated by mechanisms other than GPCRs to influence HH signaling. ARHGAP36, a RHO-GTPase activating protein that is overexpressed in HH-driven medulloblastoma, potentiates HH signaling by both inhibiting PKA enzymatic activity and inducing PKA degradation (Eccles et al., 2016; Rack et al., 2014). Neuropilins are TM receptors that positively regulate signaling by activating PDE4D to induce the hydrolysis of cAMP (Ge et al., 2015) (Fig. 5).

A genome-wide CRISPR screen recently uncovered a set of three proteins that dampen target cell sensitivity to HH ligands by reducing the abundance of SMO on the cell surface and primary cilium (Pusapati et al., 2018a). Two of these proteins, MEGF8 and MOSMO, are TM proteins, and a third (MGRN1) is a RING-family E3 ubiquitin ligase, suggesting that this regulation may involve SMO ubiquitination and that it may be regulated by a yet-undiscovered ligand. Mouse and human phenotypes of MEGF8 mutations are consistent with tissue-specific roles in HH signaling (Twigg et al., 2012; Zhang et al., 2009), but elucidating the developmental and oncogenic roles of these proteins requires further genetic analysis. These findings raise the possibility that membrane protein trafficking events at the cell surface or the primary cilium might play roles in regulating target-cell responses to HH ligands.

We expect that these, and perhaps other undiscovered modules, will play important roles in modifying responses to HH ligands, thus allowing the same core signaling system to be adapted for use in myriad developmental, regenerative and oncogenic contexts.

Conclusions

Research on the HH pathway has uncovered unexpected principles in signal transduction: the covalent modification of ligands by cholesterol, the use of primary cilia to organize signaling, the regulation of a transcription factor by partial proteolysis and the use of cholesterol as a second messenger to regulate a cell-surface receptor. Several mechanistic questions remain to be fully answered, including the questions of how PTCH inhibits SMO activity and how SMO relays the signal to the GLI transcription factors. Further work is necessary to understand how the biochemical mechanisms of HH signaling are integrated with the cell biology of cilia trafficking and cilia localization. For example, why are cilia required for the formation of activator and repressor forms of the GLI proteins? The discoveries of signaling components that modify HH signaling strength in specific tissues are also likely to reveal new mechanistic and biological insights. Finally, the use of in vitro systems to reconstitute various aspects of signaling in both cells and across tissues, in combination with computational analyses, will be necessary to fully understand how the formation and interpretation of the HH signaling gradient can pattern the diverse metazoan body plans that are seen across evolution.

Acknowledgements

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References