β-Arrestin 1 mediates non-canonical Wnt pathway to regulate convergent extension movements

https://doi.org/10.1016/j.bbrc.2013.04.088Get rights and content

Highlights

  • β-Arrestin 1 regulates convergent extension (CE) movements in Xenopus.

  • β-Arrestin 1 has no effect on cell differentiation during gastrulation.

  • β-Arrestin 1 mediates the Wnt/PCP pathway to control CE movements.

  • β-Arrestins 1 and 2 perform interchangeable activities in regulating CE.

Abstract

β-Arrestins are multifaceted proteins that play critical roles in termination of G protein-coupled receptor (GPCR) signaling by inducing its desensitization and internalization as well as in facilitation of many intracellular signaling pathways. Here, we examine using Xenopus embryos whether β-arrestin 1 might act as a mediator of β-catenin-independent Wnt (non-canonical) signaling. Xenopus β-arrestin 1 (xβarr1) is expressed in the tissues undergoing extensive cell rearrangements in early development. Gain- and loss-of-function analyses of xβarr1 revealed that it regulates convergent extension (CE) movements of mesodermal tissue with no effect on cell fate specification. In addition, rescue experiments showed that xβarr1 controls CE movements downstream of Wnt11/Fz7 signal and via activation of RhoA and JNK. In line with this, xβarr1 associated with key Wnt components including Ryk, Fz, and Dishevelled. Furthermore, we found that xβarr1 could recover CE movements inhibited by xβarr2 knockdown or its endocytosis defective mutant. Overall, these results suggest that β-arrestin 1 and 2 share interchangeable endocytic activity to regulate CE movements downstream of the non-canonical Wnt pathway.

Introduction

The Wnt family of secreted glycoproteins regulates cell proliferation, differentiation and polarity, and cell fate determination during early embryonic development and later tissue homeostasis [1], [2]. It has also emerged as an essential signal for the self-renewal of stem cells in the hematopoietic system, the skin and the intestinal epithelium [3]. Currently, three different Wnt signaling pathways have been identified which are activated upon Wnt receptor activation: the canonical Wnt/β-catenin pathway, the non-canonical Wnt signaling or planar cell polarity (PCP) pathway, and the Wnt/Ca2+ pathway [2], [4], [5], [6]. In the canonical Wnt pathway, the binding of Wnts to a cell-surface receptor complex consisting of Frizzled and LRP5/6 induces the stabilization of cytoplasmic β-catenin and its entry into the nucleus, leading to the formation of a complex with the DNA-binding factor, Tcf/Lef and subsequent transcriptional activation of tissue-specific target genes. In the absence of Wnt ligands, β-catenin is bound by the destruction complex composed of the scaffolding proteins Axin and APC, and the serine/threonine kinases, CK1 and GSK3. β-catenin is phosphorylated sequentially by CK1 and GSK3, thereby undergoing the βTrCP-dependent ubiquitination and proteasomal degradation. The Wnt/PCP and Wnt/Ca2+ pathways are β-catenin-independent signaling, which regulate convergent extension (CE) movements indispensable for body axis elongation in vertebrates, the orientation of bristles in fly wing, and the induction of epithelial to mesenchymal transition (EMT) in cancer. The Wnt/PCP pathway involves activation of intracellular proteins Dishevelled (Dsh), Daam1, RhoA, Rac1, Rho-associated kinase α, and Jun N-terminal kinase (JNK). The Wnt/Ca2+ pathway functions through the activation of trimeric G proteins, which leads to a transient release of intracellular calcium and subsequent activation of protein kinase Cα (PKCα), Cdc42 GTPase, and calcium calmodulin mediated kinase II (CAMKII). The strength and activity of these Wnt pathways are tightly regulated at the multiple levels in signaling cascade from the secretion and spreading of Wnt ligands to the post-translational modifications of cytoplasmic and nuclear signaling mediators [4], [7]. Recently, cellular trafficking of Wnt components, including the membrane distribution and endocytosis of Frizzled (Fz) and LRP5/6 receptors and the compartment-specific localization of other signaling mediators, has been shown to play crucial roles in controlling the activity of Wnt pathway [8], [9], [10], but the molecular mechanisms underlying this regulation remain to be further investigated.

β-Arrestins (β-arrestin 1 and 2) are multifaceted scaffolds and adaptors that regulate numerous aspects of G protein-coupled receptor (GPCR) functions [11], [12]. Classically, they have been shown to play roles in termination of GPCR signaling by inducing the desensitization and internalization of the receptor. However, recent studies demonstrate that they also serve as signal transducers of a variety of receptor signaling through interaction with various binding partners. β-Arrestins have been implicated as critical mediators of both canonical and non-canonical Wnt signaling [12], [13]. β-Arrestin 1 was found to synergistically enhance Lef-mediated transcriptional activity when overexpressed with Dsh [14]. β-Arrestin 2 mediates not only the Wnt5a-induced internalization of Fz4 by binding to phosphorylated Dsh but also Wnt3a-induced signaling downstream of Dsh and casein kinase, but upstream of β-catenin [15], [16]. Furthermore, β-arrestin 2 has been shown to regulate CE movements in Xenopus by activating RhoA or Rac-1 in a Dsh-dependent manner [17], [18]. Knockout studies show that either β-arrestin 1 or 2-deficient mice develop normally, whereas simultaneous ablation of both isoforms in mice results in embryonic lethality [12], [19]. These phenotypes suggest that each β-arrestin can functionally substitute for the other isoform in mice, but there are several pieces of evidence that β-arrestin 1 and 2 can also regulate differentially GPCR desensitization and internalization [19], [20]. Thus, in this study, we performed the loss-of-function analysis of β-arrestin 1 in Xenopus embryo in order to examine whether both isoforms of β-arrestin have distinct or shared activities in regulation of non-canonical Wnt signaling.

Section snippets

Embryo manipulation and DMZ elongation assay

In vitro fertilization, microinjection and embryo culture were performed as described previously [17]. Developmental stages of embryos were determined according to the Nieuwkoop and Faber’s normal table of development [21]. For elongation assay, dorsal marginal zone (DMZ) explants were dissected at stage 10.25 from the injected embryos and then cultured to stage 18 in 1× Modified Ringer’s (MR) media containing 10 μg/ml of bovine serum albumin, 50 μg/ml of gentamycin and 5 μg/ml of streptomycin.

Plasmid constructs and morpholino oligo

The

Expression pattern of β-arrestin 1 in Xenopus early development

A full-length cDNA of Xenopus β-arrestin 1 (xβarr1) was obtained using its putative sequence in the EST database and a PCR-based method. It encodes a protein of 412 amino acids, which shows 91% identity at the amino acid level with human and mouse homologues (Fig. 1A). We next examined the spatiotemporal expression pattern of xβarr1 in early embryogenesis. First, xβarr1 has both maternal and zygotic transcription throughout early development as analyzed by RT-PCR (Fig. 1B), which is similar to

Acknowledgments

This work was supported by Korea Basic Science Institute (T33414) and the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (2012-0008685).

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  • Cited by (6)

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    These authors contributed equally to this work.

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