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 blockade in vivo A 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.
Highlights
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
We described a high-throughput screen (HTS) based upon an infrared fluorogen-activating protein (IRFAP) that can be cloned onto the amino terminus of GPCRs [24]
Recent observations suggest that LGR5 and Rspondins act together to tune WNT/catenin signaling by controlling the plasma membrane expression of the WNT receptor negative regulators RNF43/ZNRF3 [49, 50]
Summary
We described a high-throughput screen (HTS) based upon an infrared fluorogen-activating protein (IRFAP) that can be cloned onto the amino terminus of GPCRs [24]. Similar to justicidin B, IRFAP live cell trafficking revealed that both BafA1 and diphyllin inhibit LGR5 internalization and result in accumulation of fluorescent punctae at the cell surface (Fig. 4, D and E). Two distinct subcellular localizations of each fluorescent protein can be observed (Fig. 8F, mCherry cytosolic signal with dark nuclei denoted by arrowheads versus E2-Crimson signal with uniform expression throughout the cell denoted by asterisks) Their staining intensity markedly differed, which enabled discrimination of both lineages by compartmentalization and signal intensity thresholding. Both the mCherry (834del LGR5-positive) and E2-Crimson (LGR5/V2R-positive) clones are at a competitive disadvantage, resulting in a decreased fractional contribution over time (Fig. 10C) Together these data demonstrate that the clathrin-mediated internalization of LGR5 is critical to regulating cell behavior in vivo
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