Hedgehog induces opposite changes in turnover and subcellular localization of patched and smoothened

Secreted signaling proteins of the Hedgehog family organize spatial pattern during animal development. Two integral membrane proteins have been identified with distinct roles in Hedgehog signaling. Patched functions in Hedgehog binding, and Smoothened functions in transducing the signal. Current models view Patched and Smoothened as a preformed receptor complex that is activated by Hedgehog binding. Here we present evidence that Patched destabilizes Smoothened in the absence of Hedgehog. Hedgehog binding causes removal of Patched from the cell surface. In contrast, Hedgehog causes phosphorylation, stabilization, and accumulation of Smoothened at the cell surface. Comparable effects can be produced by removing Patched from cells by RNA-mediated interference. These findings raise the possibility that Patched acts indirectly to regulate Smoothened activity.     

Hedgehog signaling: a smoothened conformational switch

An intracellular conformational switch in the serpentine transmembrane protein Smoothened appears to underlie Hedgehog pathway activation. The switch is gated by electrostatic interactions that are regulated by multiple phosphorylations, potentially endowing a dose-dependent response.     

Sonic hedgehog induces the segregation of patched and smoothened in endosomes

BACKGROUND: Sonic hedgehog (Shh) signal transduction involves the ligand binding Patched1 (Ptc1) protein and a signaling component, Smoothened (Smo). A select group of compounds inhibits both Shh signaling, regulated by Ptc1, and late endosomal lipid sorting, regulated by the Ptc-related Niemann-Pick C1 (NPC1) protein. This suggests that Ptc1 regulates Smo activity through a common late endosomal sorting pathway also utilized by NPC1. During signaling, Ptc accumulates in endosomal compartments, but it is unclear if Smo follows Ptc into the endocytic pathway.RESULTS: We characterized the dynamic subcellular distributions of Ptc1, Smo, and activated Smo mutants individually and in combination. Ptc1 and Smo colocalize extensively in the absence of ligand and are internalized together after ligand binding, but Smo becomes segregated from Ptc1/Shh complexes destined for lysosomal degradation. In contrast, activated Smo mutants do not colocalize with nor are cotransported with Ptc1. Agents that block late endosomal transport and protein sorting inhibit the ligand-induced segregation of Ptc1 and Smo. We show that, like NPC1-regulated lipid sorting, Shh signal transduction is blocked by antibodies that specifically disrupt the internal membranes of late endosomes, which provide a platform for protein and lipid sorting.CONCLUSIONS: These data support a model in which Ptc1 inhibits Smo only when in the same compartment. Ligand-induced segregation allows Smo to signal independently of Ptc1 after becoming sorted from Ptc1/Shh complexes in the late endocytic pathway.     

Pulling together and pulling apart: collective cargo movement in eukaryotic cells

To establish and maintain their internal organization, living cells must move molecules to their correct locations. Long-range intracellular movements are often driven by motor molecules moving along microtubules, similarly to trucks driving along a highway. Recent work demonstrates that some randomly dispersed cargos can generate actin filaments that form a connected network whose contraction drives collective cargo movement.     

Glypican-3 inhibits Hedgehog signaling during development by competing with patched for Hedgehog binding

Loss-of-function mutations in glypican-3 (GPC3), one of the six mammalian glypicans, causes the Simpson-Golabi-Behmel overgrowth syndrome (SGBS), and GPC3 null mice display developmental overgrowth. Because the Hedgehog signaling pathway positively regulates body size, we hypothesized that GPC3 acts as an inhibitor of Hedgehog activity during development. Here, we show that GPC3 null embryos display increased Hedgehog signaling and that GPC3 inhibits Hedgehog activity in cultured mouse embryonic fibroblasts. In addition, we report that GPC3 interacts with high affinity with Hedgehog but not with its receptor, Patched, and that GPC3 competes with Patched for Hedgehog binding. Furthermore, GPC3 induces Hedgehog endocytosis and degradation. Surprisingly, the heparan sulfate chains of GPC3 are not required for its interaction with Hedgehog. We conclude that GPC3 acts as a negative regulator of Hedgehog signaling during mammalian development and that the overgrowth observed in SGBS patients is, at least in part, the consequence of hyperactivation of the Hedgehog signaling pathway.     

Hedgehog signalling: off the shelf modulation

Many cell signalling pathways are modulated in important ways by general cellular machineries, such as those mediating protein degradation and translocation. Two recent studies have revealed roles for such mechanisms in the Hedgehog signalling pathway in Drosophila.     

Human receptors patched and smoothened partially transduce hedgehog signal when expressed in Drosophila cells

In humans, dysfunctions of the Hedgehog receptors Patched and Smoothened are responsible for numerous pathologies. However, signaling mechanisms involving these receptors are less well characterized in mammals than in Drosophila. To obtain structure-function relationship information on human Patched and Smoothened, we expressed these human receptors in Drosophila Schneider 2 cells. We show here that, as its Drosophila counterpart, human Patched is able to repress the signaling pathway in the absence of Hedgehog ligand. In response to Hedgehog, human Patched is able to release Drosophila Smoothened inhibition, suggesting that human Patched is expressed in a functional state in Drosophila cells. We also provide experiments showing that human Smo, when expressed in Schneider cells, is able to bind the alkaloid cyclopamine, suggesting that it is expressed in a native conformational state. Furthermore, contrary to Drosophila Smoothened, human Smoothened does not interact with the kinesin Costal 2 and thus is unable to transduce the Hedgehog signal. Moreover, cell surface fluorescent labeling suggest that human Smoothened is enriched at the Schneider 2 plasma membrane in response to Hedgehog. These results suggest that human Smoothened is expressed in a functional state in Drosophila cells, where it undergoes a regulation of its localization comparable with its Drosophila homologue. Thus, we propose that the upstream part of the Hedgehog pathway involving Hedgehog interaction with Patched, regulation of Smoothened by Patched, and Smoothened enrichment at the plasma membrane is highly conserved between Drosophila and humans; in contrast, signaling downstream of Smoothened is different.     

New roles for Galpha and RGS proteins: communication continues despite pulling sisters apart

Large G protein alpha subunits and their attendant regulators of G-protein signaling (RGS) proteins control both intercellular signaling and asymmetric cell divisions by distinct pathways. The classical pathway, found throughout higher eukaryotic organisms, mediates intercellular communication via hormone binding to G-protein-coupled receptors (GPCRs). Recent studies have led to the discovery of GPCR-independent activation of Galpha subunits by the guanine nucleotide exchange factor RIC-8 in both asymmetric cell division and synaptic vesicle priming in metazoan organisms. Protein-protein interactions and protein function in each pathway are driven through the cycle of GTP binding and hydrolysis by the Galpha subunit. This review builds a conceptual framework for understanding RIC-8-mediated pathways by comparison with the mechanism of classical G-protein activation and inhibition in GPCR signaling.     

Patching the gaps in Hedgehog signalling

The Hedgehog (Hh) pathway plays central roles in animal development and stem-cell function. Defects in Hh signalling lead to birth defects and cancer in humans. The first and often genetically damaged step in this pathway is the interaction between two membrane proteins - Patched (Ptc), encoded by a tumour suppressor gene, and Smoothened (Smo), encoded by a proto-oncogene. Recent work linking Hh signalling to sterol metabolites and protein-trafficking events at the primary cilium promises to shed light on the biochemical basis of how Patched inhibits Smoothened, and to provide new avenues for cancer treatment.     

Opposing roles for glypicans in Hedgehog signalling

Glypican members of the heparan sulphate proteoglycan family regulate several developmental signalling pathways, such as those involving Hedgehog, Wnt, BMP and FGF. Two studies reveal opposite effects of glypicans on Hedgehog signalling and show how their core protein domains and GPI anchor contribute to their versatile biological functions.     

Mechanisms and functions of Hedgehog signalling across the metazoa

Hedgehog proteins constitute one of a small number of families of secreted signals that have a central role in the development of metazoans. Genetic analyses in flies, fish and mice have uncovered the major components of the pathway that transduces Hedgehog signals, and recent genome sequence projects have provided clues about its evolutionary origins. In this Review we provide an updated overview of the mechanisms and functions of this signalling pathway, highlighting the conserved and divergent features of the pathway, as well as some of the common themes in its deployment that have emerged from recent studies.     

Hedgehog signaling in the Drosophila eye and head: an analysis of the effects of different patched trans-heterozygotes

Characterization of different alleles of the Hedgehog receptor patched (ptc) indicates that they can be grouped into several classes. Most mutations result in complete loss of Ptc function. However, missense mutations located within the putative sterol-sensing domain (SSD) or C terminus of ptc encode antimorphic proteins that are unable to repress Smo activity and inhibit wild-type Ptc from doing so, but retain the ability to bind and sequester Hh. Analysis of the eye and head phenotypes of Drosophila melanogaster in various ptc/ptc(tuf1) heteroallelic combinations shows that these two classes of ptc allele can be easily distinguished by their eye phenotype, but not by their head phenotype. Adult eye size is inversely correlated with head vertex size, suggesting an alteration of cell fate within the eye-antennal disc. A balance between excess cell division and cell death in the mutant eye discs may also contribute to final eye size. In addition, contrary to results reported recently, the role of Hh signaling in the Drosophila head vertex appears to be primarily in patterning rather than in proliferation, with Ptc and Smo having opposing effects on formation of medial structures.