GRK6 overexpression mediates βarr2 recruitment to Lgr5 intracellular vesicles.

<p>GFP-tagged βarr2 and HA epitope-tagged Lgr5 was transiently overexpressed in HEK cells (A) without GRK overexpression, or with overexpression of (B) GRK2, (C) GRK4, (D) GRK5, or (E) GRK6. Cells were fixed, permeabilized, and stained with a primary and secondary (568nm) antibody pair to HA. Representative images are presented of native GFP fluorescence (Left Panels, Green), HA-568 (Middle Panels, Red), and merged (Right Panels, yellow denotes colocalization). Arrows point to representative βarr2/Lgr5 colocalization. Inset depicts a 2x magnification. </p

Quantitation of βarr2 recruitment to Lgr5 using an ArrestinZoom Assay (Part 1).

<p>GFP-tagged βarr2 and GRK-6 were transiently co-expressed together with the permutations of Lgr5 previously described for confocal analysis and reviewed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084476#pone-0084476-t001" target="_blank">Table 1</a>. (A) 48-wells of a 96-well plate were imaged for GFP fluorescence. For each well, 32 images were tiled at 108X magnification and subsequently stitched together. Stitched images were imported into ImageJ and converted to a single montage where each column represents a different experimental condition (as labeled) and each row designates a technical replicate (3.34X Magnification). As described in materials and methods, βarr2-GFP aggregates were identified (highlighted in yellow). (B) Areas outlined in (A) for (1) βarr2-GFP + GRK-6, (2) βarr2-GFP + GRK-6 + Wild-type Lgr5, and (3) βarr2-GFP + GRK-6 + S873-5A at 8.9X magnification (C) Areas outlined in B, presented at 108X magnification (Arrow in 2’ denotes βarr2 aggregates, that are absent without Lgr5 and when the “SSS” cluster is mutated to “AAA”). (D) Highlighted βarr2 aggregates in “A” were quantitated, graphed, and tested for statistically significant differences by 1-way ANOVA and <i>post </i><i>hoc</i> Bonferroni correction for multiple comparisons (p<0.05). The results are representative of three independent experiments.</p

GRK6 overexpression stimulates Class B βarr2 translocation to Lgr5 that is dependent upon a structural determinant in the C-tail.

<p>(Left panels) Wild-type HA epitope-tagged Lgr5 or (Right panels) HA epitope-tagged 834del (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084476#pone-0084476-t001" target="_blank">Table 1</a> for truncation site) Lgr5 were cotransfected in HEK293 cells with GFP-tagged βarr2 and (A) GRK2, (B) GRK4, (C) GRK5, or (D) GRK6. Cells were pulsed with an HA antibody on ice, washed, chased, and fixed. Cells were permeabilized and stained with a secondary antibody to the primary HA antibody. Images were collected on a confocal microscope at 100X. (Red:HA and Green:Native GFP were Merged:yellow). Inset depicts 2x magnification.</p

GRK overexpression mediates βarr2 translocation to Lgr5.

<p>GFP tagged βarr2 was transiently transfected in HEK 293 cells alone (Left Panels) or in the presence of overexpressed Lgr5/V2R chimera (Middle Panels) or wild-type HA epitope-tagged Lgr5 (Right Panels). βarr2-GFP translocation was visualized using a confocal microscope at 100X in (A) the absence of overexpressed GRK2 or in the presence of overexpressed (B) GRK2, (C) GRK4, (D) GRK5, or (E) GRK6. Inset depicts 2x magnification of images. </p

Cooperation of Lgr5 internalization motifs is necessary for “SSS” mediated βarr2 translocation.

<p>(A) GFP-tagged βarr2 was expressed in HEK cells without GRK overexpression (Left panel) or co-expresssed with wild-type Lgr5/-GRK or Lgr5/+GRK (Right panel and Middle panel, respectively). (B) GFP-tagged βarr2 was cotransfected with GRK6 and the indicated Lgr5 C-tail mutant. The primary amino acid sequence of mutants are provided in tabular form. (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084476#pone-0084476-t001" target="_blank">Table 1</a> 8.1-12). Native GFP fluorescence was imaged using confocal microscopy at 100X.</p

Conservation of typical GPCR signaling motifs in Lgr4-6.

<p>The primary amino acid sequences of 282 Class 1 rhodopsin-like GPCRs were aligned in ClustalΩ and a subset presented due to space limitations (ClustalX color view based upon degree of conservation; See <a href="http://jalview.org" target="_blank">jalview.org</a> for complete color table, briefly, Acidic (E,D: Magenta); Basic (R, K:Red); (P: Yellow). The entire sequence alignment can be downloaded from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084476#pone.0084476.s003" target="_blank">File S1</a> and opened in Jalview. Lgr1-3 (FSHR, LSHR, and TSHR) and Lgr4-6 are highlighted by hatched and solid boxes. (A) Primary acid alignment from a subset of GPCRs at the “DRY” motif and (B) primary acid alignment from a subset of GPCRs at the NPXXY motif. Arrow in (A) points to the conserved Arginine in the “DRY” motif. Line in (B) highlights the NPXXY motif. The position label at top indicates amino acid position from the start of the alignment. At the bottom, alignment conservation, quality, and consensus for each amino acid (+, indicates significant conservation of the site).</p

Conservation of a high-affinity interaction motif in Lgr5 but not Lgr4 or Lgr6.

<p>The C-terminal of tails of human Lgr4, Lgr5, Lgr6, V2R, and β2AR (Uniprot IDs: Q9BXB1, O75473, Q9HBX8, P30518, and P07550) were aligned in ClustalΩ starting with the conserved NPXXY motif at the end of transmembrane seven (Clustal X color view based upon degree of conservation, See <a href="http://jalview.org" target="_blank">jalview.org</a> for complete color table). Asterisk and arrow, respectively denote the position of the conserved palmitoylated cysteine (absent in Lgr4/5/6) and leucine. Underlined residues identify serine/threonine clusters which are necessary for Class B βarr2 affinity of the V2R. Of particular interest, is the conservation of a similar motif in Lgr5 (aa873-5) but not Lgr4, Lgr6, or β2AR. The boxed residues represent conservation of the motif necessary for Lgr5 internalization that are also seen in the V2R and β2AR.</p

Quantitation of βarr2 recruitment to Lgr5 using an ArrestinZoom Assay (Part 2).

<p>GFP-tagged βarr2 and GRK-6 were transiently co-expressed together with the permutations of Lgr5 previously described for confocal analysis and reviewed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084476#pone-0084476-t001" target="_blank">Table 1</a>. (A) 48-wells of a 96-well plate were imaged for GFP fluorescence. For each well, 32 images were tiled at 108X magnification and subsequently stitched together. Stitched images were imported into ImageJ and converted to a single montage where each column represents a different experimental condition (as labeled) and each row designates a technical replicate (3.34X Magnification). As described in materials and methods, βarr2-GFP aggregates were identified and are shown in yellow. (B) Areas outlined in (A) for (1) βarr2-GFP + GRK-6, (2) βarr2-GFP + GRK-6 + Wild-type Lgr5, and (3) βarr2-GFP + GRK-6 + pDEL 844-864 at 8.9X magnification (C) Areas outlined in B, presented at 108X magnification (Arrow in 2’ denotes βarr2 aggregates, that are absent without Lgr5 and when the phosphor-acceptors between amino acids 844-864 are mutated to alanine; pDEL844-864). (D) Highlighted βarr2 aggregates in “A” were quantitated, graphed, and tested for statistically significant differences by 1-way ANOVA and <i>post </i><i>hoc</i> Bonferroni correction for multiple comparisons (p<0.05). The results are representative of three independent experiments.</p

The Stem Cell-Expressed Receptor Lgr5 Possesses Canonical and Functionally Active Molecular Determinants Critical to β-arrestin-2 Recruitment

<div><p>Lgr5 is a membrane protein related to G protein-coupled receptors (GPCR)s whose expression identifies stem cells in multiple tissues and is strongly correlated with cancer. Despite the recent identification of endogenous ligands for Lgr5, its mode of signaling remains enigmatic. The ability to couple to G proteins and βarrestins are classical molecular behaviors of GPCRs that have yet to be observed for Lgr5. Therefore, the goal of this study was to determine if Lgr5 can engage a classical GPCR behavior and elucidate the molecular determinants of this process. Structural analysis of Lgr5 revealed several motifs consistent with its ability to recruit βarr2. Among them, a “SSS” serine cluster located at amino acid position 873-875 within the C-terminal tail (C-tail), is in a region consistent with other GPCRs that bind βarr2 with high-affinity. To test its functionality, a ligand-independent βarr2 translocation assay was implemented. We show that Lgr5 recruits βarr2 and that the “SSS” amino acids (873-875) are absolutely critical to this process. We also demonstrate that for full efficacy, this cluster requires other Lgr5 C-tail serines that were previously shown to be important for constitutive and βarr2 independent internalization of Lgr5. These data are proof of principle that a classical GPCR behavior can be manifested by Lgr5. The existence of alternative ligands or missing effectors of Lgr5 that scaffold this classical GPCR behavior and the downstream signaling pathways engaged should be considered. Characterizing Lgr5 signaling will be invaluable for assessing its role in tissue maintenance, repair, and disease.</p> </div

The amino acids “SSS” from position 873-875 in Lgr5 mediate βarr2 translocation.

<p>(A) GFP-tagged βarr2 was transiently expressed alone (Left panel) or with wild-type Lgr5/-GRK6 (Middle panel) or Lgr5/+GRK6 (Right panel) in HEK 293 cells. (B) GFP-tagged βarr2 and GRK6 were transiently expressed with the indicated C-tail mutants. The primary acid sequence of the C-tail mutants is provided in tabular form (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084476#pone-0084476-t001" target="_blank">Table 1</a> 7.1-.6). (C) GFP-tagged βarr2 and Lgr4 were transiently expressed in HEK293 cells in the absence of GRK overexpression or with GRK2 or GRK6 (Left, Middle, and Right panels, respectively). Native GFP fluorescence was imaged by confocal at 100X. </p