Multiple ligand binding sites regulate the Hedgehog signal transducer Smoothened in vertebrates
Introduction
The Hedgehog (Hh) signalling pathway is essential for embryogenesis and adult stem cell homeostasis in all bilaterians [1, 2, 3]. Its dysregulation is linked to developmental abnormalities and various types of cancers, including basal cell carcinoma (BCC) and medulloblastoma. Many tissues in the early embryo are patterned by gradients of morphogens, exemplified by ligands such as Hh in Drosophila and Sonic Hedgehog (SHH) in vertebrates. Local concentrations of such morphogens are interpreted by target cells to drive cell fate decisions, ultimately forming the basis for a body plan.
In Hh-producing cells, a precursor form of a secreted Hh ligand (e.g. SHH) is expressed and auto-catalytically cleaved into a doubly lipidated N-terminal domain to produce the mature morphogen [1, 2, 3]. Hh ligands are received on target cells by the twelve-pass transmembrane (TM) protein Patched 1 (PTC1), in cooperation with other co-receptors that can modify ligand reception (for details see [4, 5, 6]). A unique feature of the Hh signalling cascade is that ligand reception and signal transduction across the plasma membrane have been assigned to two different membrane proteins, PTC1 and Smoothened (SMO). SHH binding inactivatesPTC1, thereby relieving its constitutive inhibition of the G-protein coupled receptor (GPCR) SMO (Figure 1). SMO activation is the critical step that transmits the Hh signal across the membrane to the cytoplasm, ultimately resulting in the activation of the glioma-associated oncogene family members (GLI) transcription factors [1, 2]. This separation of function requires that the Hh signal must be relayed from PTC1 to SMO, a mysterious step in signalling that is thought to be mediated by a small molecule second messenger [7, 8, 9] (Figure 1).
SMO has been the focus of intense study because it is required for transmembrane signalling in all animals and because it has become a validated drug target for Hh-driven human cancers [3, 10]. This review will discuss recent advances and enduring puzzles related to the mechanisms of SMO signal transduction, with a focus on the recent structural characterization of SMO and its regulation by various small molecules.
Section snippets
Architecture of Smoothened
SMO is a Frizzled-class GPCR. Unlike the ‘classical’ Class A GPCRs, SMO contains not just the stereotypical seven-pass α-helical transmembrane bundle (TMD), but also sizeable extracellular and intracellular domains (Figure 2, left panel). The extracellular region of SMO consists primarily of a cysteine-rich domain (CRD), named for its conserved disulphide bonding architecture, which has homologs in the Frizzled receptors (Fzd), Niemann–Pick type-C protein 1 (NPC1), and riboflavin-binding
Small molecule modulators of Smoothened
Unlike many GPCRs, SMO can be activated or inhibited through at least two ligand-binding sites (reviewed in [15]). The first binding site (hereafter the ‘TMD site’), which corresponds to the canonical ‘orthosteric’ ligand-binding site in many GPCRs, is located at the extracellular end of the transmembrane bundle (Figure 2, right panel). The TMD site was first shown to engage the SMO antagonist cyclopamine [20] and subsequently shown to bind to multiple small molecule agonists and antagonists,
Ligand-binding sites that regulate endogenous SMO signalling
Much of our understanding of SMO function described above comes from studies of signalling in response to exogenously added ligands. But what are the endogenous ligands that regulate SMO activity and do they act through the CRD site, the TMD site or a yet undiscovered third site? Both the CRD and TMD sites have been subjected to mutagenesis to address this question. Mutations in the CRD site can impair signalling, not just in response to oxysterols and cholesterol, but also in response to SHH
Cholesterol as an endogenous regulator of SMO signalling
Cholesterol, an abundant component of vertebrate cell membranes, is both necessary and sufficient to activate SMO signalling (summarized in [32••]). A permissive function for cholesterol was first suggested by the observation that cellular cholesterol depletion or drugs that impair intracellular cholesterol transport reduce Hh signalling [39, 40, 41, 42]). In addition, humans with mutations in genes encoding enzymes of distal cholesterol biosynthesis, such as Smith-Lemli-Opitz syndrome (SLOS),
Conclusions and perspectives
The crucial processes of embryonic development and regenerative responses depend upon proper functioning of the Hh signalling pathway. Recent multi-domain structures of SMO have cast a new light on the role of the extracellular domains of SMO and the allosteric interaction between its two defined ligand-binding sites. Functional studies have nominated cholesterol as an endogenous instructive modulator of SMO, mediating the critical regulatory interaction between SMO and the receptor for Hh
References and recommended reading
Papers of particular interest, published within the period of review, have been highlighted as:
• of special interest
•• of outstanding interest
Acknowledgements
This work was supported by Cancer Research UK (C20724/A14414), the European Research Council under the European Union's Horizon 2020 Research and Innovation Programme Grant 647278, the US National Institutes of Health (GM106078, GM105448 and GM118082) and Wellcome Trust (102890/Z/13/Z, 092970/Z/10/Z and 090532/Z/09/Z). Further support by NDM Oxford (E.F.X.B.) and the Ford Foundation (G.L.) is acknowledged.
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2022, Biochimica et Biophysica Acta - BiomembranesCitation Excerpt :Analysis of these structures, and computational investigations, searching for sterol binding sites reveal essentially two distinct clusters of sites where cholesterol and/or other sterols can potentially interact or bind. One of them is in its extracellular CRD, and another is in the TMD [20,25,27,33,34] (Table S2). A crystal structure of human SMO unexpectedly revealed a cholesterol bound to a hydrophobic groove in the CRD (PDB ID: 5L7D; hereafter called the CRD_5L7D) [25].
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2022, Current Topics in Developmental BiologyThe 3-beta-hydroxysteroid-Delta(8), Delta(7)-isomerase EBP inhibits cholesterylation of Smoothened
2021, Biochimica et Biophysica Acta - Molecular and Cell Biology of LipidsCitation Excerpt :The endogenous Gli1 mRNA level was increased as well (Fig. 4F), demonstrating that EBP negatively regulates Hh pathway. Because SMO possesses multiple sterol-binding sites in extracellular cysteine-rich domain (CRD) and the transmembrane domain (TMD) [45–50], we then asked whether the inhibition of EBP on SMO was resulted from the accumulation of sterol intermediates downstream of EBP, which might engage the cholesterol-binding sites and compete off cholesterol to inactivate SMO [27,51]. Several mutations have been reported to abolish the catalytic activity of EBP [42,52].
Hedgehog signaling promotes lipolysis in adipose tissue through directly regulating Bmm/ATGL lipase
2020, Developmental BiologyCitation Excerpt :The Hh protein locally expressed in the fat body and remotely circulated from the gut may contribute to high levels, but might not be all parts, of the Hh signaling activation. Recent structural studies have shown that Smo can be activated by cholesterol interaction with both the cysteine rich and the transmembrane domains of Smo (Byrne et al., 2017; Huang et al., 2016, 2018; Rana et al., 2013). Interestingly, extracellular sodium and the transmembrane sodium gradient may also regulate Smo activation (Blassberg and Briscoe, 2018; Myers et al., 2017).