Abstract
Leucine-rich repeat-containing G protein–coupled receptor 5 (LGR5) is a bona fide marker of adult stem cells in several epithelial tissues, most notably in the intestinal crypts, and is highly up-regulated in many colorectal, hepatocellular, and ovarian cancers. LGR5 activation by R-spondin (RSPO) ligands potentiates Wnt/β-catenin signaling in vitro; however, deletion of LGR5 in stem cells has little or no effect on Wnt/β-catenin signaling or cell proliferation in vivo. Remarkably, modulation of LGR5 expression has a major impact on the actin cytoskeletal structure and cell adhesion in the absence of RSPO stimulation, but the molecular mechanism is unclear. Here, we show that LGR5 interacts with IQ motif-containing GTPase-activating protein 1 (IQGAP1), an effector of Rac1/CDC42 GTPases, in the regulation of actin cytoskeleton dynamics and cell–cell adhesion. Specifically, LGR5 decreased levels of IQGAP1 phosphorylation at Ser-1441/1443, leading to increased binding of Rac1 to IQGAP1 and thus higher levels of cortical F-actin and enhanced cell–cell adhesion. LGR5 ablation in colon cancer cells and crypt stem cells resulted in loss of cortical F-actin, reduced cell–cell adhesion, and disrupted localization of adhesion-associated proteins. No evidence of LGR5 coupling to any of the four major subtypes of heterotrimeric G proteins was found. These findings suggest that LGR5 primarily functions via the IQGAP1–Rac1 pathway to strengthen cell–cell adhesion in normal adult crypt stem cells and colon cancer cells.
Highlights
Leucine-rich repeat-containing G protein– coupled receptor 5 (LGR5) is a bona fide marker of adult stem cells in several epithelial tissues, most notably in the intestinal crypts, and is highly up-regulated in many colorectal, hepatocellular, and ovarian cancers
No evidence of LGR5 coupling to any of the four major subtypes of heterotrimeric G proteins was found. These findings suggest that LGR5 primarily functions via the IQ motif-containing GTPase-activating protein 1 (IQGAP1)–Rac1 pathway to strengthen cell– cell adhesion in normal adult crypt stem cells and colon cancer cells
LGR5 expression is restricted to the crypt stem cells in the intestine, but its conditional knock-out was found to have no obvious effect on the proliferation and differentiation of the stem cells both in vivo and ex vivo [6, 28]
Summary
Knock-out of LGR5 disrupts the cytoskeletal architecture of intestinal crypt organoids. RT-PCR analysis showed that parental and CHO-LGR5 cell lines did not express any of the four RSPOs (supplemental Fig. S1), indicating that LGR5 has constitutive or RSPO-independent activity in regulating the actin cytoskeleton and cell morphology. These results suggest that the LGR5-induced decrease in IQGAP1 phosphorylation at Ser-1441/1443 was probably due to increased dephosphorylation. Together with the LGR5 overexpression data, these findings coherently suggest a mechanism whereby LGR5 reduces phosphorylation of IQGAP1 at Ser-1441/1443 to increase the IQGAP1–Rac interaction and enhance cell– cell adhesion via regulating the actin cytoskeleton
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