Multiple ligand binding sites regulate the Hedgehog signal transducer Smoothened in vertebrates

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The Hedgehog (Hh) pathway plays a central role in the development of multicellular organisms, guiding cell differentiation, proliferation and survival. While many components of the vertebrate pathway were discovered two decades ago, the mechanism by which the Hh signal is transmitted across the plasma membrane remains mysterious. This fundamental task in signalling is carried out by Smoothened (SMO), a human oncoprotein and validated cancer drug target that is a member of the G-protein coupled receptor protein family. Recent structural and functional studies have advanced our mechanistic understanding of SMO activation, revealing its unique regulation by two separable but allosterically-linked ligand-binding sites. Unexpectedly, these studies have nominated cellular cholesterol as having an instructive role in SMO signalling.

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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