title: Inhibiting clathrin-mediated endocytosis of the leucine-rich G protein-coupled receptor-5 diminishes cell fitness.
abstract: The leucine-rich G protein-coupled receptor-5 (LGR5) is expressed in adult tissue stem cells of many epithelia, and its overexpression is negatively correlated with cancer prognosis. LGR5 potentiates WNT/β-catenin signaling through its unique constitutive internalization property that clears negative regulators of the WNT-receptor complex from the membrane. However, both the mechanism and physiological relevance of LGR5 internalization are unclear. Therefore, a natural product library was screened to discover LGR5 internalization inhibitors and gain mechanistic insight into LGR5 internalization. The plant lignan justicidin B blocked the constitutive internalization of LGR5. Justicidin B is structurally similar to more potent vacuolar-type H+-ATPase inhibitors, which all inhibited LGR5 internalization by blocking clathrin-mediated endocytosis. We then tested the physiological relevance of LGR5 internalization blockadein vivoA LGR5-rainbow (LBOW) mouse line was engineered to express three different LGR5 isoforms along with unique fluorescent protein lineage reporters in the same mouse. In this manner, the effects of each isoform on cell fate can be simultaneously assessed through simple fluorescent imaging for each lineage reporter. LBOW mice express three different forms of LGR5, a wild-type form that constitutively internalizes and two mutant forms whose internalization properties have been compromised by genetic perturbations within the carboxyl-terminal tail. LBOW was activated in the intestinal epithelium, and a year-long lineage-tracing course revealed that genetic blockade of LGR5 internalization diminished cell fitness. Together these data provide proof-of-concept genetic evidence that blocking the clathrin-mediated endocytosis of LGR5 could be used to pharmacologically control cell behavior.
authors.affiliation: From the Departments of Cell Biology, joshua.snyder@duke.edu.|Surgery, and.
authors.forename: Joshua C
authors.initials: JC
authors.lastname: Snyder
authors.affiliation: From the Departments of Cell Biology.
authors.forename: Lauren K
authors.initials: LK
authors.lastname: Rochelle
authors.affiliation: From the Departments of Cell Biology.
authors.forename: Caroline
authors.initials: C
authors.lastname: Ray
authors.affiliation: Pharmacology and Cancer Biology and.
authors.forename: Thomas F
authors.initials: TF
authors.lastname: Pack
authors.affiliation: Duke Cancer Institute Transgenic Core, Duke University Medical Center, Durham, North Carolina 27712 and.
authors.forename: Cheryl B
authors.initials: CB
authors.lastname: Bock
authors.affiliation: Surgery, and.
authors.forename: Veronica
authors.initials: V
authors.lastname: Lubkov
authors.affiliation: Surgery, and.
authors.forename: H Kim
authors.initials: HK
authors.lastname: Lyerly
authors.affiliation: Department of Biological Sciences and Molecular Biosensor and Imaging Center, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213.
authors.forename: Alan S
authors.initials: AS
authors.lastname: Waggoner
authors.affiliation: From the Departments of Cell Biology.
authors.forename: Larry S
authors.initials: LS
authors.lastname: Barak
authors.affiliation: From the Departments of Cell Biology, marc.caron@duke.edu.
authors.forename: Marc G
authors.initials: MG
authors.lastname: Caron
mesh_terms: D000251:Adenosine Triphosphatases / Q000737:chemistry; D000818:Animals; D045744:Cell Line, Tumor; D019070:Cell Lineage; D049109:Cell Proliferation; D002966:Clathrin / Q000737:chemistry*; D004148:Dioxolanes / Q000737:chemistry; D004705:Endocytosis; D004848:Epithelium / Q000378:metabolism; D005260:Female; D006706:Homeostasis; D006801:Humans; D007930:Leucine / Q000737:chemistry*; D017705:Lignans / Q000737:chemistry; D051379:Mice; D008810:Mice, Inbred C57BL; D020033:Protein Isoforms; D051381:Rats; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry*; D013234:Stem Cells / Q000166:cytology; D013269:Stochastic Processes; D060449:Wnt Signaling Pathway
chemical_list: D002966:Clathrin; D004148:Dioxolanes; C116884:LGR5 protein, human; D017705:Lignans; D020033:Protein Isoforms; D043562:Receptors, G-Protein-Coupled; C049719:justicidins; D000251:Adenosine Triphosphatases; D007930:Leucine
keywords: G protein-coupled receptor (GPCR); LGR5; clathrin; drug screening; intestinal epithelium; natural product; receptor internalization; stem cells; trafficking; vacuolar ATPase