Constitutive internalization of the leucine-rich G protein-coupled receptor-5 (LGR5) to the trans-Golgi network

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.

FIGURE 1.

The C-terminal tail of LGR5 regulates its constitutive internalization. Shown are primary amino acid sequences of the C-terminal tail for each construct (canonical GPCR NPXXY domain in gray and V2R tail in bold). HEK 293T cells were transiently transfected with the indicated 3×HA N-terminally epitope-tagged constructs: FL-WT LGR5 full-length (A), FL-LGR5 + dynamin K44A (B), WT/V2R tail (C), or Lgr5 with a truncation at amino acid position 834 (D). A, inset depicts a 3×HA FL-WT LGR5-EGFP fusion and imaged for native EGFP fluorescence. A–D, cells were pulsed with a MαHA antibody for 45 min on ice, washed, chased for 0, 5, 15, 30, or 120 min at 37 °C, fixed, permeabilized, and stained with a GαM-568 antibody (gray scale). 100× confocal images are presented.

FIGURE 2.

LGR5 rapidly internalizes into early endosomes and transits through late endosomes and recycling endosomes. HEK 293T cells were transiently transfected with a 3×HA N-terminally epitope WT-LGR5 and stained for EEA1 (A, green) or co-transfected with EGFP (green)-tagged Rab5 (B), Rab4 (C), Rab7 (D), Rab9 (E), or Rab11 (F). Cells were pulsed with a MαHA antibody for 45 min on ice, washed, chased for 0, 5, 15, 30, or 120 min at 37 °C, fixed, permeabilized, and stained with a GαM568 antibody (red). Merged 100× confocal images are presented.

FIGURE 3.

Constitutively internalized LGR5 internalizes into VPS26-positive endosomes and is deposited to the TGN. A–C, HEK 293T cells were transiently transfected with a 3×HA N-terminally epitope-tagged WT-LGR5. Cells were pulsed with an HA antibody for 45 min on ice, washed, chased for 0, 5, 15, 30, or 120 min at 37 °C, fixed, permeabilized, and stained with appropriate primary and secondary antibodies to visualize HA (A–C, red), VPS26 (A, green), M6PR (B, green), or Trip230 (C, green). D–F, HEK cells were transfected with a 3×HA N-terminally epitope-tagged human V2R, pulsed with an αHA antibody for 45 min on ice, washed, chased for 0, 5, 15, 30, or 120 min at 37 °C in the absence (−) or presence (+) of arginine vasopressin (0.1 IU/ml), fixed, permeabilized, and stained with appropriate primary and secondary antibodies to visualize HA (D–F, red), M6PR (D and E, green), or Vps26 (F, green). Merged 100× confocal images are presented.

FIGURE 4.

Internalization of LGR5 is regulated by a motif between positions 834 and 869. Shown are primary amino acid sequences of the C-terminal tail for each construct (canonical GPCR NPXXY domain in gray). HEK 293T cells were transiently transfected with the indicated 3×HA N-terminally (red) and C-terminally EGFP (green)-tagged Lgr5 constructs: FL-LGR5 (A), 869 (B), 874 (C), or 902 (D). Cells were pulsed with a MαHA antibody for 45 min on ice, washed, chased for 0, 7.5, 15, 30, or 120 min at 37 °C, fixed, permeabilized, and stained with a GαM568 antibody (red). Merged 100× confocal images are presented (blue, nuclear counterstain).

FIGURE 5.

Truncation analysis identifies a putative region regulating LGR5 internalization. Shown are primary amino acid sequences of the C-terminal tail for each construct (canonical GPCR NPXXY domain in gray). HEK 293T cells were transiently transfected with the indicated 3×HA N-terminally (red) and C-terminally EGFP (green)-tagged constructs: FL-LGR5 (A), 839 (B), 844 (C), 849 (D), 854 (E), 859 (F), or 864 (G). Cells were pulsed with a MαHA antibody for 45 min on ice, washed, chased for 0, 7.5, 15, 30, or 120 min at 37 °C, fixed, permeabilized, and stained with a GαM568 antibody (red). Merged 100× confocal images are presented (blue, nuclear counterstain).

FIGURE 6.

Mutating putative phosphorylation sites inhibits internalization. Shown are primary amino acid sequences of the C-terminal tail for each construct (canonical GPCR NPXXY domain in gray). HEK 293T cells were transiently transfected with the indicated 3×HA N-terminally (red) and C-terminally EGFP (green)-tagged constructs: FL-LGR5 (A), pDel 833–907 (B), pDel 833–865 (C), and pDel 866–907 (D). Cells were pulsed with a MαHA antibody for 45 min on ice, washed, chased for 0, 7.5, 15, 30, or 120 min at 37 °C, fixed, permeabilized, and stained with a GαM568 antibody (red). Merged 100× confocal images are presented (blue, nuclear counterstain).

FIGURE 7.

Identification of the primary motif responsible for internalization of LGR5. Shown are the primary amino acid sequences of the C-terminal tail for each construct (canonical GPCR NPXXY domain in gray). HEK 293T cells were transiently transfected with the indicated 3×HA N-terminally (red) and C-terminally EGFP (green)-tagged constructs: FL-LGR5 (A), pDel 844–864 (B), +A844S (C), +A848S (D), +A851S (E), +A854S (F), +A861S/A864S (G), or S861A/S864A (H). Cells were pulsed with a MαHA antibody for 45 min on ice, washed, chased for 0, 7.5, 15, 30, or 120 min at 37 °C, fixed, permeabilized, and stained with a GαM568 antibody (red). Merged 100× confocal images are presented (blue, nuclear counterstain).

FIGURE 8.

Unbiased quantitative analysis of LGR5 internalization reveals the C-terminal motif responsible for internalization. HEK cells were transfected with the constructs utilized according to Fig. 1 (A), Fig. 4 (B), Fig. 5 (C), Fig. 6 (D), and Fig. 7 (E). Cells were pulse-chased at 37 °C with primary MSαHA antibody fixed and then stained with a GαM680 without permeabilization to assess the fraction of the receptor pulsed that remained on the surface following the chase. Cells were chased for (A) 0, 3.75, 7.5, 15, 30, or 120 min or (B–E) 0, 7.5, 15, 30, or 120 min. Cells were imaged on a LiCOR Odyssey and data normalized to the receptor on the cell surface at time 0 for each construct. F, data from each receptor construct were log2-transformed and normalized to the geometric average of the FL-LGR5 construct and presented as a heat map over the internalization time course (0, 7.5, 15, 30, and 120 min) where bright magenta indicates 100% cell surface expression and bright yellow indicates 8.4% cell surface expression. Reference values for cell surface expression and their correlation to color are indicated on the map.