Signaling pathways that mediate cell-cell communication are essential for collective cell behaviors in multicellular systems. The hedgehog (HH) pathway, first discovered and elucidated in
, is one of ...these iconic signaling systems that plays many roles during embryogenesis and in adults; abnormal HH signaling can lead to birth defects and cancer. We review recent structural and biochemical studies that have advanced our understanding of the vertebrate HH pathway, focusing on the mechanisms by which the HH signal is received by patched on target cells, transduced across the cell membrane by smoothened, and transmitted to the nucleus by GLI proteins to influence gene-expression programs.
Developmental signals of the Hedgehog (Hh) and Wnt families are transduced across the membrane by Frizzledclass G-protein-coupled receptors (GPCRs) composed of both a heptahelical transmembrane ...domain (TMD) and an extracellular cysteine-rich domain (CRD). How the large extracellular domains of GPCRs regulate signalling by the TMD is unknown. We present crystal structures of the Hh signal transducer and oncoprotein Smoothened, a GPCR that contains two distinct ligand-binding sites: one in its TMD and one in the CRD. The CRD is stacked a top the TMD, separated by an intervening wedge-like linker domain. Structure-guided mutations show that the interface between the CRD, linker domain and TMD stabilizes the inactive state of Smoothened. Unexpectedly, we find a cholesterol molecule bound to Smoothened in the CRD binding site. Mutations predicted to prevent cholesterol binding impair the ability of Smoothened to transmit native Hh signals. Binding of a clinically used antagonist, vismodegib, to the TMD induces a conformational change that is propagated to the CRD, resulting in loss of cholesterol from the CRD-linker domain-TMD interface. Our results clarify the structural mechanism by which the activity of a GPCR is controlled by ligand-regulated interactions between its extracellular and transmembrane domains.
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IJS, KISLJ, NUK, SBMB, UL, UM, UPUK
Although originally discovered as neuronal growth cone-collapsing factors, repulsive guidance molecules (RGMs) are now known as key players in many fundamental processes, such as cell migration, ...differentiation, iron homeostasis, and apoptosis, during the development and homeostasis of many tissues and organs, including the nervous, skeletal, and immune systems. Furthermore, three RGMs (RGMa, RGMb/DRAGON, and RGMc/hemojuvelin) have been linked to the pathogenesis of various disorders ranging from multiple sclerosis (MS) to cancer and juvenile hemochromatosis (JHH). While the molecular details of these (patho)biological effects and signaling modes have long remained unknown, recent studies unveil several exciting and novel aspects of RGM processing, ligand–receptor interactions, and downstream signaling. In this review, we highlight recent advances in the mechanisms-of-action and function of RGM proteins.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UL, UM, UPCLJ, UPUK, ZRSKP
Structural, biochemical and biophysical studies of eukaryotic soluble and membrane proteins require their production in milligram quantities. Although large-scale protein expression strategies based ...on transient or stable transfection of mammalian cells are well established, they are associated with high consumable costs, limited transfection efficiency or long and tedious selection of clonal cell lines. Lentiviral transduction is an efficient method for the delivery of transgenes to mammalian cells and unifies the ease of use and speed of transient transfection with the robust expression of stable cell lines. In this protocol, we describe the design and step-by-step application of a lentiviral plasmid suite, termed pHR-CMV-TetO
, for the constitutive or inducible large-scale production of soluble and membrane proteins in HEK293 cell lines. Optional features include bicistronic co-expression of fluorescent marker proteins for enrichment of co-transduced cells using cell sorting and of biotin ligase for in vivo biotinylation. We demonstrate the efficacy of the method for a set of soluble proteins and for the G-protein-coupled receptor (GPCR) Smoothened (SMO). We further compare this method with baculovirus transduction of mammalian cells (BacMam), using the type-A γ-aminobutyric acid receptor (GABA
R) β3 homopentamer as a test case. The protocols described here are optimized for simplicity, speed and affordability; lead to a stable polyclonal cell line and milligram-scale amounts of protein in 3-4 weeks; and routinely achieve an approximately three- to tenfold improvement in protein production yield per cell as compared to transient transduction or transfection.
Cholesterol is necessary for the function of many G-protein coupled receptors (GPCRs). We find that cholesterol is not just necessary but also sufficient to activate signaling by the Hedgehog (Hh) ...pathway, a prominent cell-cell communication system in development. Cholesterol influences Hh signaling by directly activating Smoothened (SMO), an orphan GPCR that transmits the Hh signal across the membrane in all animals. Unlike many GPCRs, which are regulated by cholesterol through their heptahelical transmembrane domains, SMO is activated by cholesterol through its extracellular cysteine-rich domain (CRD). Residues shown to mediate cholesterol binding to the CRD in a recent structural analysis also dictate SMO activation, both in response to cholesterol and to native Hh ligands. Our results show that cholesterol can initiate signaling from the cell surface by engaging the extracellular domain of a GPCR and suggest that SMO activity may be regulated by local changes in cholesterol abundance or accessibility.
Primary cilia are required for Smoothened to transduce vertebrate Hedgehog signals, but how Smoothened accumulates in cilia and is activated is incompletely understood. Here, we identify ...cilia-associated oxysterols that promote Smoothened accumulation in cilia and activate the Hedgehog pathway. Our data reveal that cilia-associated oxysterols bind to two distinct Smoothened domains to modulate Smoothened accumulation in cilia and tune the intensity of Hedgehog pathway activation. We find that the oxysterol synthase HSD11β2 participates in the production of Smoothened-activating oxysterols and promotes Hedgehog pathway activity. Inhibiting oxysterol biosynthesis impedes oncogenic Hedgehog pathway activation and attenuates the growth of Hedgehog pathway-associated medulloblastoma, suggesting that targeted inhibition of Smoothened-activating oxysterol production may be therapeutically useful for patients with Hedgehog-associated cancers.
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•Primary cilia from diverse organisms and tissues contain oxysterol lipids•Cilia-associated oxysterols bind and activate Smoothened through multiple domains•The oxysterol synthase HSD11β2 generates Smoothened-activating oxysterols•HSD11β2 is enriched in medulloblastoma, and blocking HSD11β2 inhibits cancer growth
How Smoothened localizes to primary cilia and is activated is poorly understood. Raleigh et al. find that cilia contain oxysterol lipids that bind to Smoothened and activate the Hedgehog pathway. Moreover, they identify an enzyme involved in cilia-associated oxysterol biosynthesis that is enriched in Hedgehog pathway-associated medulloblastoma and that blocking this enzyme using a compound in black licorice inhibits tumor growth.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
MHC class II molecules on the surface of antigen-presenting cells display a range of peptides for recognition by the T-cell receptors of CD4+ T helper cells. Therefore, MHC class II molecules are ...central to effective adaptive immune responses, but conversely, genetic and epidemiological data have implicated these molecules in the pathogenesis of autoimmune diseases. Indeed, the strength of the associations between particular MHC class II alleles and disease render them the main genetic risk factors for autoimmune disorders such as type 1 diabetes. Here, we discuss the insights that the crystal structures of MHC class II molecules provide into the molecular mechanisms by which sequence polymorphisms might contribute to disease susceptibility.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Previously we proposed that transmission of the hedgehog signal across the plasma membrane by Smoothened is triggered by its interaction with cholesterol (Luchetti et al., 2016). But how is ...cholesterol, an abundant lipid, regulated tightly enough to control a signaling system that can cause birth defects and cancer? Using toxin-based sensors that distinguish between distinct pools of cholesterol, we find that Smoothened activation and Hedgehog signaling are driven by a biochemically-defined, small fraction of membrane cholesterol, termed accessible cholesterol. Increasing cholesterol accessibility by depletion of sphingomyelin, which sequesters cholesterol in complexes, amplifies Hedgehog signaling. Hedgehog ligands increase cholesterol accessibility in the membrane of the primary cilium by inactivating the transporter-like protein Patched 1. Trapping this accessible cholesterol blocks Hedgehog signal transmission across the membrane. Our work shows that the organization of cholesterol in the ciliary membrane can be modified by extracellular ligands to control the activity of cilia-localized signaling proteins.
Ionotropic glutamate receptor (iGIuR) family members are integrated into supramolecular complexes that modulate their location and function at excitatory synapses. However, a lack of structural ...information beyond isolated receptors or fragments thereof currently limits the mechanistic understanding of physiological iGIuR signaling. Here, we report structural and functional analyses of the prototypical molecular bridge linking postsynaptic iGIuR δ2 (GIuD2) and presynaptic β-neurexin 1 (β-NRX1) via CbIn1, a C1q-like synaptic organizer. We show how CbIn1 hexamers "anchor" GIuD2 amino-terminal domain dimers to monomeric β-NRX1. This arrangement promotes synaptogenesis and is essential for D-serine–dependent GIuD2 signaling in vivo, which underlies long-term depression of cerebellar parallel fiber–Purkinje cell (PF-PC) synapses and motor coordination in developing mice. These results lead to a model where protein and small-molecule ligands synergistically control synaptic iGIuR function.
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BFBNIB, NMLJ, NUK, ODKLJ, PNG, SAZU, UL, UM, UPUK
Wnt morphogens control embryonic development and homeostasis in adult tissues. In vertebrates the N-terminal WIF domain (WIF-1(WD)) of Wnt inhibitory factor 1 (WIF-1) binds Wnt ligands. Our crystal ...structure of WIF-1(WD) reveals a previously unidentified binding site for phospholipid; two acyl chains extend deep into the domain, and the head group is exposed to the surface. Biophysical and cellular assays indicate that there is a WIF-1(WD) Wnt-binding surface proximal to the lipid head group but also implicate the five epidermal growth factor (EGF)-like domains (EGFs I-V) in Wnt binding. The six-domain WIF-1 crystal structure shows that EGFs I-V are wrapped back, interfacing with WIF-1(WD) at EGF III. EGFs II-V contain a heparan sulfate proteoglycan (HSPG)-binding site, consistent with conserved positively charged residues on EGF IV. This combination of HSPG- and Wnt-binding properties suggests a modular model for the localization of WIF-1 and for signal inhibition within morphogen gradients.
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DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
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