Abstract
LGR5 is a Wnt pathway associated G protein-coupled receptor (GPCR) that serves as a molecular determinant of stem cells in numerous tissues including the intestine, stomach, hair follicle, eye, and mammary gland. Despite its importance as a marker for this critical niche, little is known about LGR5 signaling nor the biochemical mechanisms and receptor determinants that regulate LGR5 membrane expression and intracellular trafficking. Most importantly, in cells LGR5 is predominantly intracellular, yet the mechanisms underlying this behavior have not been determined. In this work we elucidate a precise trafficking program for LGR5 and identify the motif at its C terminus that is responsible for the observed constitutive internalization. We show that this process is dependent upon dynamin GTPase activity and find that wild-type full-length LGR5 rapidly internalizes into EEA1- and Rab5-positive endosomes. However, LGR5 fails to rapidly recycle to the plasmid membrane through Rab4-positive vesicles, as is common for other GPCRs. Rather, internalized LGR5 transits through Rab7- and Rab9-positive vesicles, co-localizes in vesicles with Vps26, a retromer complex component that regulates retrograde trafficking to the trans-Golgi network (TGN) and reaches a steady-state distribution in the TGN within 2 h. Using mutagenesis, particularly of putative phosphorylation sites, we show that the amino acid pair, serine 861 and 864, is the principal C-tail determinant that mediates LGR5 constitutive internalization. The constitutive internalization of LGR5 to the TGN suggests the existence of novel biochemical roles for its Wnt pathway related, but ill defined signaling program.