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      Structural basis for Smoothened regulation by its extracellular domains

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          Abstract

          Developmental signals of the Hedgehog (Hh) and Wnt families are transduced across the membrane by Frizzled-class G-protein coupled receptors (GPCRs) composed of both a heptahelical transmembrane domain (TMD) and an extracellular cysteine-rich domain (CRD). How such large extracellular domains of GPCRs regulate signalling by the TMD is unknown. We present crystal structures of the Hh signal transducer and oncoprotein Smoothened (SMO), which contains two distinct ligand-binding sites in its TMD and CRD. The CRD is stacked atop the TMD, separated by an intervening wedge-like linker domain (LD). Structure-guided mutations show that the interface between the CRD, LD and TMD stabilises the inactive state of SMO. Unexpectedly, we find a cholesterol molecule bound to SMO in the CRD-binding site. Mutations predicted to prevent cholesterol binding impair the ability of SMO 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-LD-TMD interface. Our work elucidates 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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          Most cited references67

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          Linking crystallographic model and data quality.

          In macromolecular x-ray crystallography, refinement R values measure the agreement between observed and calculated data. Analogously, R(merge) values reporting on the agreement between multiple measurements of a given reflection are used to assess data quality. Here, we show that despite their widespread use, R(merge) values are poorly suited for determining the high-resolution limit and that current standard protocols discard much useful data. We introduce a statistic that estimates the correlation of an observed data set with the underlying (not measurable) true signal; this quantity, CC*, provides a single statistically valid guide for deciding which data are useful. CC* also can be used to assess model and data quality on the same scale, and this reveals when data quality is limiting model improvement.
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            Partitioning of lipid-modified monomeric GFPs into membrane microdomains of live cells.

            Many proteins associated with the plasma membrane are known to partition into submicroscopic sphingolipid- and cholesterol-rich domains called lipid rafts, but the determinants dictating this segregation of proteins in the membrane are poorly understood. We suppressed the tendency of Aequorea fluorescent proteins to dimerize and targeted these variants to the plasma membrane using several different types of lipid anchors. Fluorescence resonance energy transfer measurements in living cells revealed that acyl but not prenyl modifications promote clustering in lipid rafts. Thus the nature of the lipid anchor on a protein is sufficient to determine submicroscopic localization within the plasma membrane.
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              Patched1 regulates hedgehog signaling at the primary cilium.

              Primary cilia are essential for transduction of the Hedgehog (Hh) signal in mammals. We investigated the role of primary cilia in regulation of Patched1 (Ptc1), the receptor for Sonic Hedgehog (Shh). Ptc1 localized to cilia and inhibited Smoothened (Smo) by preventing its accumulation within cilia. When Shh bound to Ptc1, Ptc1 left the cilia, leading to accumulation of Smo and activation of signaling. Thus, primary cilia sense Shh and transduce signals that play critical roles in development, carcinogenesis, and stem cell function.
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                Author and article information

                Journal
                0410462
                6011
                Nature
                Nature
                Nature
                0028-0836
                1476-4687
                21 June 2016
                28 July 2016
                28 January 2017
                : 535
                : 7613
                : 517-522
                Affiliations
                [1 ]Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, UK
                [2 ]Departments of Biochemistry and Medicine, Stanford University School of Medicine, Stanford, California, United States of America
                [3 ]Department of Biochemistry, University of Oxford, Oxford, UK
                [4 ]Diamond Light Source Ltd, Harwell Science &Innovation Campus, Didcot, UK
                [5 ]Department of Developmental Biology, Washington University School of Medicine, St. Louis, Missouri, United States of America
                Author notes
                Article
                NIHMS796201
                10.1038/nature18934
                4970916
                27437577
                1f9ea1b8-e1c9-40c2-a465-4cdd271a8cc2

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