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Review
. 2023 Feb 15;150(3):dev201352.
doi: 10.1242/dev.201352. Epub 2023 Feb 10.

The role of Evi/Wntless in exporting Wnt proteins

Lucie Wolf  1 , Michael Boutros  1
Affiliations
Review

The role of Evi/Wntless in exporting Wnt proteins

Lucie Wolf et al. Development. .

Abstract

Intercellular communication by Wnt proteins governs many essential processes during development, tissue homeostasis and disease in all metazoans. Many context-dependent effects are initiated in the Wnt-producing cells and depend on the export of lipidated Wnt proteins. Although much focus has been on understanding intracellular Wnt signal transduction, the cellular machinery responsible for Wnt secretion became better understood only recently. After lipid modification by the acyl-transferase Porcupine, Wnt proteins bind their dedicated cargo protein Evi/Wntless for transport and secretion. Evi/Wntless and Porcupine are conserved transmembrane proteins, and their 3D structures were recently determined. In this Review, we summarise studies and structural data highlighting how Wnts are transported from the ER to the plasma membrane, and the role of SNX3-retromer during the recycling of its cargo receptor Evi/Wntless. We also describe the regulation of Wnt export through a post-translational mechanism and review the importance of Wnt secretion for organ development and cancer, and as a future biomarker.

Keywords: Cancer; Development; Evi; PORCN/Porcupine; Wnt secretion; Wnt signalling; Wntless/Wls.

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Conflict of interest statement

Competing interests The authors declare no competing or financial interests.

Figures

Fig. 1.
Fig. 1.
Wnt signalling pathways. The Wnt signalling pathways can be divided into β-catenin dependent (‘canonical’) and independent (‘non-canonical’), the most prominent of which are the Wnt/planar cell polarity (PCP) and the Wnt/Ca2+ pathways. The β-catenin-dependent pathway is the best studied Wnt signalling branch that activates cell proliferation and differentiation; for example, via the proto-oncogene MYC. β-Catenin is a bifunctional protein involved in cellular adhesion at the plasma membrane and signal transduction in the cytoplasm and the nucleus. The β-catenin-independent pathways are diverse and not extensively studied, mainly due to the lack of well-defined molecular endpoints that are easy to analyse and to pronounced context-dependent effects. They regulate embryonic pattern formation or cell migration, among others. There is considerable overlap between the Wnt pathways, as, for example, the ‘non-canonical’ pathways often inhibit canonical signalling and all Wnt pathways compete for shared proteins, such as Dishevelled (DVL). (A) In the absence of Wnt ligands, β-catenin is phosphorylated by the destruction complex centred on the scaffolding protein AXIN, adenomatous polyposis coli (APC), and the two kinases glycogen synthase kinase 3 (GSK3β) and casein kinase (CK) 1α, allowing its subsequent ubiquitylation by β-transducin repeat-containing protein (β-TrCP) and proteasomal degradation. In the absence of β-catenin, β-catenin-responsive elements in the DNA are bound by transcriptional repressors (TLE). Further regulators of Wnt signalling are the secreted molecules sclerostin (SOST), Dickkopf-related protein (DKK), and the membrane-bound E3 ubiquitin ligases RING finger protein (RNF) 43 and zinc and RING finger 3 (ZNRF3), which target Frizzled receptors for degradation. (B) RNF43 and ZNRF3 can be inhibited by R-Spondin (RSPO), which binds them and leucine-rich repeat-containing G-protein-coupled receptor 4/5 (LGR4/5) to stabilize Frizzled receptors. Further context-dependent regulators of Wnt signalling are Wnt inhibitory factor (WIF) and secreted frizzled-related protein (sFRP). The engagement of ‘canonical’ Wnt ligands, such as human WNT1, WNT3/3A or WNT10A/B, with Frizzled proteins and low-density lipoprotein receptor-related protein 5/6 (LRP5/6) results in the phosphorylation of LRP5/6, e.g. by CK1γ, and in the recruitment of DVL and the destruction complex to the plasma membrane, leading to their inactivation. Stabilized β-catenin translocates into the nucleus to bind T-cell factor (TCF)/lymphoid enhancer factor (LEF) transcription factors and others [e.g. B-cell CLL/lymphoma 9 protein (BCL9)] to activate target gene transcription. Inhibition of the kinase GSK3β also affects many other proteins, an effect that is referred to ‘Wnt/STOP’. (C) The core PCP components [Frizzled, Vang-like protein (VANGL), cadherin EGF LAG seven-pass G-type receptor (CELSR), Prickle-like protein (PK) and DVL] establish an asymmetric network of protein complexes across the membranes of neighbouring cells by inhibiting each other within the same cell and stabilizing each other in bordering cells. Signalling by WNT5A or WNT11, Frizzled and receptor tyrosine kinase-like orphan receptor 1/2 (ROR1/2), receptor-like tyrosine kinase (RYK) or protein tyrosine kinase 7 (PTK7) leads to recruitment and activation of DVL and DVL-associated activator of morphogenesis 1 (DAAM1), and others, and several members of the Ras homolog family (especially RHOA and RAC1), resulting in ROCK-dependent rearrangements of the cytoskeleton or activation of Jun N-terminal kinase (JNK) and transcriptional regulation, e.g. by cAMP-dependent transcription factor 2 (ATF2) and JUN phosphorylation. This is essential to break symmetry during embryonic development and to pattern functional complex organ structures. (D) Wnt/Ca2+ signalling is initiated by the interaction of Wnt with Frizzled and RYK or ROR1/2, which results in the intracellular activation of a G protein and cleavage of phosphatidylinositol-4,5-bisphosphate (PIP2) by phospholipase C (PLC) into inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). Binding of IP3 with its receptor at the ER membrane triggers the release of Ca2+ from the endoplasmic reticulum (ER) and activation of the Ca2+-dependent enzymes protein kinase C (PKC) and calmodulin (CAM), as well as downstream effectors calcineurin and Ca2+/CAM-dependent kinase II (CAMKII), and leads to actin rearrangements by CDC42 and the activation of transcriptional programs via nuclear factor of activated T-cells (NFAT) and nemo-like kinase (NLK), an inhibitor of TCF (reviewed by Acebron and Niehrs, 2016; Albrecht et al., 2021; Humphries and Mlodzik, 2018; Niehrs, 2012; Rim et al., 2022; Söderholm and Cantù, 2021; Zhan et al., 2017).
Fig. 2.
Fig. 2.
The Evi/Wntless (Wls) structure determines its function as a Wnt cargo protein. (A) The structure of human Evi/Wls (pink) in complex with human WNT3A [green; protein data bank identifier (PDB) ID, 7DRT] and human Evi/Wls (blue) and WNT8A (orange; PDB ID 7KC4) reveals the large and complex interaction surface between Evi/Wls and its cargo proteins. Wnts can only bind Evi/Wls when modified with palmitoleate. This long unsaturated fatty acid in the Evi/Wls-Wnt complex is buried in the Evi/Wls transmembrane domain. It is conceivable that lipid-modified Wnts are transferred to Evi/Wls from the acyl-transferase Porcupine through the large lateral opening in the transmembrane domain of Evi/Wls, which leads to the large cavity visible in B. (B) Overlay of the two Evi/Wls structures [Evi/Wls-WNT3A (pink, PDB ID 7DRT) and Evi/Wls-WNT8A (blue, PDB ID 7KC4)] shows that they are very similar, overall, with eight membrane-spanning helices and a luminal or extracellular domain consisting of eight β-strands. Structural figures were generated using UCSF ChimeraX (Version 1.2).
Fig. 3.
Fig. 3.
Molecular mechanisms of Wnt export. (A) Wnts are post-translationally modified with palmitoleate in the endoplasmic reticulum (ER) by the acyl-transferase Porcupine (Porcn) before engaging with their specific transport protein Evi/Wntless (Wls). (B) Wnt-Evi/Wls complexes are then packaged into COPII vesicles. (C) The Evi/Wls-Wnt complex then traverses the Golgi and reaches the plasma membrane, from where Wnts can be secreted. (D) Several different routes of intercellular Wnt transport have been described and potentially occur in a context-dependent manner. These routes include transport on cellular protrusions (cytonemes), glypicans or secretion on exosomes. (E) For efficient Wnt secretion, Evi/Wls needs to be endocytosed in a clathrin-dependent manner. (F) Via early endosomes, Evi/Wls can undergo retrograde transport back to the trans-Golgi network and the ER with the help of retromer, where it can associate with another Wnt molecule. (G) When retromer-dependent retrieval of Evi/Wls is inhibited, Evi/Wls is trafficked along the degradative endolysosomal route to lysosomes for degradation. (H) Evi/Wls is transported back to the ER via the RKEAQE C-terminal sequence. (I-K) Alternative routes of Wnt secretion include the packaging of the Evi/Wls-Wnt complex into intraluminal vesicles of multivesicular bodies (MVBs) and their secretion on exosomes (I) and basolateral (J) or apical (K) re-secretion of Wnts after trafficking through the endo-lysosomal system. Although depicted here in one summarising figure, the processes of Wnt export are likely context-, tissue and cell-type dependent. Refer to the main text for further molecular details.
Fig. 4.
Fig. 4.
Evi/Wls is degraded by the proteasome in the absence of Wnt ligands. Evi/Wls is destined for proteasomal degradation in Wnt-producing cells when no Wnts are being secreted. In the endoplasmic reticulum (ER) membrane, Evi/Wls binds ER lipid raft-associated protein 2 (ERLIN2) before being ubiquitinylated by the E2 ubiquitin-conjugating enzymes UBE2J2, UBE2K and UBE2N (not shown), and the E3 ubiquitin ligase cell growth regulator with RING finger domain protein 1 (CGRRF1). Ubiquitylated Evi/Wls is removed from the ER membrane and delivered to the proteasome with the help of the AAA ATPase valosin-containing protein (VCP; also known as p97 or Cdc48), which is recruited to the ER membrane through Fas-associated factor 2 (FAF2; also known as UBXD8) and UBXN4. Once Wnt production is activated, lipid-modified Wnts are transferred from the acyl transferase Porcupine to their cargo protein Evi/Wls and the Evi/Wls-Wnt complex travels to the Golgi and the plasma membrane.
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