Interaction of Class Frizzled Receptors with G Proteins and Dishevelled

Home , Dishevelled, Frizzled, FZD7, TSPAN12

Interaction of Class Frizzled receptors with G proteins and Dishevelled

Thesis for doctoral degree

Kateřina Straková Brno 2017 MASARYK UNIVERSITY FACULTY OF SCIENCE Department of Experimental Biology

Interaction of Class Frizzled receptors with G proteins and Dishevelled

Kateřina Straková

Thesis for doctoral degree

Supervisor: Professor Gunnar Schulte Brno 2017

BIBLIOGRAPHIC ENTRY

Author Mgr. Kateřina Straková Faculty of Science, Masaryk University Department of Experimental Biology Title of Dissertation Interaction of Class Frizzled receptors with G proteins and Dishevelled Degree Programme Biology Field of Study Animal Physiology Supervisor Professor Gunnar Schulte Karolinska Institutet Department of Physiology and Pharmacology Consultant Associate Professor Vítězslav Bryja, PhD Masaryk University Department of experimental biology Academic Year 2017/2018 Number of Pages 146

Keywords Frizzled; FZD6; FZD4; Dishevelled; DVL; G protein; GNA12; GNA13; WNT signalling; phosphorylation

BIBLIOGRAFICKÝ ZÁZNAM

Autor Mgr. Kateřina Straková Přírodovědecká fakulta, Masarykova univerzita Ústav experimentální biologie Název práce Interakce receptorů třídy Frizzled s G proteiny a Dishevelled Studijní program Biologie Studijní obor Fyziologie živočichů Školitel Professor Gunnar Schulte Karolinska Institutet Ústav fyziologie a farmakologie Konzultant doc. Mgr. Vítězslav Bryja, Ph.D. Masarykova univerzita Ústav experimentální biologie Akademický rok 2017/2018 Počet stran 146

Klíčová slova Frizzled; FZD6; FZD4; Dishevelled; DVL; G protein; GNA12; GNA13; signální dráha WNT; fosforylace

PERMISSIONS

All previously published articles were reproduced with permission from the publisher. Cover: The artwork by Ryan G. Coleman showing a G protein-coupled receptor was modified and reproduced with kind permission from the author. All the pictures in the introduction to the thesis were designed by Jan Kučera. © Kateřina Straková, Masarykova univerzita, 2017

ABSTRACT

The developmental processes driving embryogenesis have to be carefully regulated in order to achieve proper development of the embryo. One of the main signalling pathways responsible for the synchronization of these processes is the WNT signalling network. Through ligands of the WNT family, their receptors Frizzleds (FZDs) and various downstream pathway effectors, key developmental events such as cellular proliferation, polarization, migration, cell fate specification or body axis patterning are controlled. In this thesis, I summarize the results of me and my colleagues obtained during the investigation of the most proximal events in WNT signalling pathway following WNT/FZD binding. The interaction of FZDs with their immediate binding partners Dishevelled (DVL) and heterotrimeric G proteins is scrutinized. I introduce and discuss novel aspects of FZD/G protein and FZD/DVL interaction with regard to the structural features, post-translational modifications, binding selectivity determinants and downstream pathways associated with these interactions.

Our results advocate the formation of WNT-sensitive FZD4/Gα12/13 inactive state complexes and therefore support the hypothesis of GPCR/Gα inactive state precoupling, at least within the Class F. Two novel connections linking FZD4/Gα12/13 with RHO GTPases and effectors of the YAP/TAZ pathway are described, shedding light on the molecular basis of FZD4 function in vasculogenesis and proposing partial redundancy of

FZD4- and FZD10-dependent signalling. We report the formation of a conserved intramolecular network between transmembrane helices 2 and 4 of FZDs, disruption of which results in decreased DVL-binding capacity of FZD4 and impairs DVL-dependent WNT signalling. Further, we show that conformation stabilized by this intra-molecular network as well as the C-terminus of FZD4 is dispensable for the assembly of the

FZD4/Gα12/13 complex. The complex also forms independently of DVL, suggesting a FZD- specific DVL involvement in FZD/Gα-dependent signalling. Our results indicate that

FZD4-mediated recruitment of DVL is enhanced by the multimerization of DVL. Lastly, we report that FZD phosphorylation might not be a prerequisite for FZD/DVL interaction but instead a consequence of such interaction induced by kinases such as CK1δ/ε or GRK2. G protein-coupled receptors (GPCRs) such as FZDs are promising candidates for drug design. The goal in such case is to target a specific signal transduction cascade without affecting the other pathways. Our results help to understand the selectivity of signal transduction induced by WNT/FZD/DVL and WNT/FZD/G protein complexes and therefore can contribute to developing drugs with higher efficacy and fewer side effects.

ABSTRAKT

Aby byl zajištěn správný vývoj embrya, vývojové procesy řídící embryogenezi musí být pečlivě regulovány. Jedna z hlavních signálních drah zodpovědných za synchronizaci těchto procesů je signální dráha WNT. Skrze ligandy z rodiny WNT, jejich receptory Frizzled (FZD) a různé navazující efektory jsou regulovány klíčové procesy, jako například buněčná proliferace, polarizace, migrace, diferenciace a formování tělní osy. V této práci shrnuji výsledky, které byly získány v rámci výzkumu raných událostí nastávajících v signální dráze WNT po vazbě WNT na FZD. Jsou zde detailně popsány interakce receptorů třídy FZD s jejich přímými vazebnými partnery Dishevelled (DVL) a heterotrimerickými G proteiny. Představuji zde a diskutuji nové aspekty vazby FZD/DVL a FZD/G proteinů týkající se struktury, post-translačních modifikací, faktorů určujících jejich vazebnou selektivitu a drah asociovaných s těmito interakcemi.

Naše výsledky poukazují na formování komplexů FZD4 a Gα12/13 v inaktivním stavu a podporují tak hypotézu, že receptory spřažené s G proteiny (GPCR) se mohou párovat s Gα podjednotkami v inaktivním stavu, alespoň v rámci třídy F. Popisujme nově objevené souvislosti propojující FZD4/Gα12/13 s malými GTPázami z rodiny RHO a s efektory dráhy

YAP/TAZ. Tyto souvislosti pomáhají objasnit molekulární podstati funkce FZD4 při tvorbě nových cév a naznačují částečnou redundanci v signalizaci závislé na FZD4 a

FZD10. Popisujeme intra-molekulární interakční síť mezi transmembránovými helixy 2 a

4, jejíž narušení vede ke snížené schopnosti FZD4 vázat DVL a zabraňuje WNT signalizaci závislé na DVL. Dále ukazujeme, že konformace FZD stabilizovaná touto intra- molekulární interakcí a také C-terminus nejsou důležité pro vznik komplexu

FZD4/Gα12/13. Tento komplex také vzniká nezávisle na DVL, což naznačuje, že DVL ovlivňuje signalizaci závislou na FZD/Gα pouze specificky u určitých receptorů třídy F.

Naše výsledky dále naznačují, že translokace DVL na membránu zprostředkovaná FZD4 je zvýšena, pokud je DVL přítomen ve formě multimerů. V neposlední řadě, ukazujeme, že fosforylace FZD nemusí být vyžadována pro vazbu DVL, ale že může být naopak důsledkem takové vazby, a může být indukovaná kinázami CK1δ/ε nebo GRK2. Receptory spřažené s G proteiny, které zahrnují i receptory třídy Frizzled, jsou slibnými kandidáty pro design nových léčiv. Obvykle se snažíme zacílit na specifickou signální kaskádu, aniž by byly ovlivněny ostatní signální dráhy. V tomto kontextu pomáhají výsledky našich experimentů porozumět selektivitě v signální transdukci indukované WNT/FZD/DVL a WNT/FZD/G proteinovými komplexy, a proto mohou přispět k vývoji léčiv s vyšší účinností a menším množstvím vedlejších efektů.

LIST OF PUBLICATIONS

(I) Arthofer E, Hot B, Petersen J, Strakova K, Jäger S, Grundmann M, Kostenis E, Gutkind JS, Schulte G. (2016) WNT Stimulation Dissociates a Frizzled 4 Inactive‐State Complex with Gα12/13. Mol Pharmacol. 90(4):447‐59. PMID 27458145

I have contributed to the FRAP measurements determining the WNT-selectivity of

FZD4/Gα dissociation and analyzed my data (total contribution estimated as 5 %)

(II) Strakova K, Matricon P, Yokota C, Bernatik O, Rodriguez D, Arenas E, Carlsson J, Bryja V, Schulte G. (2017) The tyrosine Y2502.39 in Frizzled 4 defines a conserved motif important for recruitment of DVL and activation of downstream signaling. Cell Signal. Oct;38:85-96. Epub 2017 Jun 29. PMID: 28668722

I have designed, performed and analysed most of the experiments and wrote most of the paper (total contribution estimated as 75 %)

(III) Strakova K, Gybel T, Dhople VM, Bernatik O, Sedova K, Harnos J, Bryja V, Schulte G. Frizzled 6 is phosphorylated in a CK1ε-, GRK- and DVL-dependent manner. Manuscript for J Biol Chem

I have designed, performed and analysed most of the experiments and wrote the paper (total contribution estimated as 90 %)

CONTENTS

1. INTRODUCTION ...... 17

1.1 WNT signalling pathway ...... 17

1.1.1 WNT ...... 19

1.1.2 FRIZZLED ...... 20

1.1.3 DISHEVELLED ...... 21

1.1.4 β-catenin-dependent pathway ...... 22

1.1.5 Planar cell polarity pathway ...... 23

1.1.6 FZD4 and FZD6 ...... 24

1.2 G protein-coupled receptors ...... 24

1.2.1 Heterotrimeric G proteins ...... 25 2+ 1.2.2 WNT/Ca and other G protein-dependent pathways ...... 27

1.3 Crosstalk between G protein-dependent and DVL-dependent signalling ...... 27

1.4 WNT/FZD/pathway selectivity ...... 28

1.5 FZD-DVL interaction ...... 29

1.6 FZD-G protein interaction ...... 30

1.7 GPCR/FZD phosphorylation ...... 31

2. AIMS ...... 33

3. RESULTS AND DISCUSSION ...... 35

3.1 FZD4 interacts with Gα12 and Gα13 heterotrimeric G proteins ...... 36

3.2 FZD4 and Gα12/13 mediate signalling to small GTPase RHO and

to YAP/TAZ pathway ...... 36

3.3 Tyrosine 250 in FZD4 forms an intramolecular bond with TM4 ...... 37

3.4 Structural requirements for FZD4- Gα12/13 inactive state preassembly ...... 39 3.5 Intact IL1/TM2 and the Y250/H348/W352 network is required

for FZD4-DVL interaction ...... 39

3.6 FZD4 more effectively recruits DVL present in signalosomes ...... 41

3.7 DVL does not affect formation of a FZD4/Gα12/13 inactive state complex ...... 42

3.8 Phosphorylatable residues in FZD4 as a potential requirement

for the recruitment of DVL ...... 43

3.9 FZD6 can be phosphorylated by CK1ε and GRKs ...... 44

3.10 DVL mediates hyper-phosphorylation of FZD6 ...... 45

4. CONCLUSIONS ...... 49

5. ACKNOWLEDGEMENTS ...... 51

6. REFERENCES ...... 53

List of abbreviations ...... 66

1. INTRODUCTION

The development of multicellular organisms is a very complex procedure where numerous processes have to be carefully orchestrated and strictly regulated. The developmental processes are governed by several main developmental pathways – Notch, Hedgehog, MAPK/ERK (mitogen-activated protein kinases/extracellular signal- regulated kinases), TGFβ (transforming growth factor β), and WNT (Wingless/Integrated1) – each pathway performs its unique role in the development but there is also a substantial amount of crosstalk and mutual regulation among these pathways.

1.1 WNT SIGNALLING PATHWAY The WNT signalling pathway, whose exploration and characterization began in eighties with the discovery of WNT proteins, takes part in many fundamental developmental processes such as cellular proliferation, migration, cell fate specification or body axis patterning. It regulates cellular polarity and shape and is important also in adult organism for maintaining homeostasis. When WNT signalling is malfunctioning during development, it leads to abortions of the foetus or more or less severe congenital malformations. Deregulated WNT signalling in adults contributes to multiple diseases with cancer in the lead, but including also neurodegenerative disorders or type II diabetes. The WNT signalling sensu lato is historically seen as a number of parallel sub-pathways with distinct cellular functions. However, all the specific sub-pathways share some common transduction steps. All the pathways are activated by WNT proteins binding to receptors from the Frizzled (FZD) family. Then the scaffold protein Dishevelled (DVL) is recruited and the signal is transmitted to different downstream components of the pathways and different final effectors [1] (Figure 1). The originally studied and probably best described branch of WNT signalling regulates the intracellular level of transcriptional co-activator β-catenin, and is thus called the β- catenin-dependent pathway. The planar cell polarity pathway, as the name indicates, commands the polarity of cells within a two-dimensional cell layer. WNT/calcium pathway is transmitted via calcium and is well known for the dependence on heterotrimeric G proteins. Gradually, even more branches of WNT signalling are emerging and evidence is piling for more and more crosstalk between all the branches. Thus, an integrative model of WNT signalling pathway was proposed, merging all the distinct branches into one complex signalling network initiated by WNT-FZD interaction, taking different complicated routes and executed into many different outcomes [2].

For the sake of simplicity, most of the signalling molecules and events described in this thesis are related to the WNT signalling as we know it in mammals. However, there are great differences between WNT signalling in mammals and other classes of the animal kingdom and when I find it relevant, the differences are discussed.

1.1.1 WNT WNTs are glycolipoproteins functioning as extracellular signalling molecules in all the branches of WNT signalling pathway. There are 19 mammalian members of the WNT family divided into 12 subfamilies and they are remarkably phylogenetically conserved [3]. Already Cnidaria contain members of 11 WNT subfamilies [4]; at least some members of the WNT family are present in all metazoans in contrast to their absence in unicellular organisms, which only underlines the fundamental role of WNTs in the development of multicellular organisms. Further, aberrant expression of WNTs during development leads to many different (and only partially overlapping) phenotypes, suggesting rather unique developmental roles for all 19 WNTs. The mouse phenotypes are well described at the Roel Nusse lab webpage: http://web.stanford.edu/group/nusselab/cgi-bin/wnt/mouse After translation, WNTs go through a complex process involving post-translational modifications, transport to the membrane and final secretion into the extracellular space [5]. In the endoplasmic reticulum they are lipidated by acyltransferase called Porcupine, and this acylation is important for their further transfer to the plasma membrane and outside the cell; later also for their binding to FZDs [6,7]. WNTs are also glycosylated which might also be required for their secretion [8,9] though other studies indicate otherwise [10,11]. The acylation makes WNTs hydrophobic and thus poorly soluble; that is the reason why purification of active WNTs was impossible for quite some time which slowed down the progress of the whole WNT field considerably. Lipid-modified WNTs are then transported to the plasma membrane with the assistance of the transmembrane protein Wntless (WLS) [12]. Whether WNTs work as long-distance or rather short-distance morphogens and how they are transported extracellularly to reach recipient cells, is still a matter of debate. There are many studies showing involvement of exosomes [13,14], some groups advocate coupling of WNTs with lipoproteins [15,16] or other carriers such as Swim (secreted Wingless-interacting molecule) or afamin [17,18]. Recently, transport of WNTs attached to cytonemes, long-range membrane protrusions of cells, has been proposed [19].

Figure 1. WNT signalling network is simplified into three main pathways – the WNT/Ca2+ pathway (left, yellow), WNT/β-catenin-dependent pathway (middle, red) and the planar cell polarity (PCP) pathway. Typical pathway progression is indicated by black arrows, selected crosstalks between pathways are indicated by grey arrows. References can be found in the text. Modified from von Maltzahn et al. [20].

1.1.2 FRIZZLED The WNT receptors Frizzleds are seven-transmembrane receptors which are present in mammals in 10 distinct paralogs (FZD1-10) [21] (Figure 2). The close homologue of FZDs, Smoothened (SMO), an important receptor of the Hedgehog pathway, has been recently successfully crystallized which allowed some extrapolation even for FZDs [22]. The schematic representation of Frizzled is shown in Figure 3.

Figure 2. The phylogenetic tree of Class F receptors. Modified from Schulte et al. 2010 [21].

Structurally, Frizzleds harbour an N-terminal signal sequence required for membrane targeting, then, again extracellularly, a predominantly α-helical large cysteine-rich domain (CRD) of about 120 amino acids (aa), where 10 conserved cysteines form disulfide bridges collectively forming a well defined 3D structure implicated in WNT binding [23,24]. Janda et al. have shown, that the WNT/CRD structure resembles the form of a “hand” which holds the CRD of Frizzled by its “thumb” and acylated “index finger” at two distinct sites [25]. However, the importance of the CRD for WNT binding has been questioned, since Drosophila Fz and Dfz2 with deleted CRDs kept the ability to induce downstream WNT signalling [26]. Two asparagines in the CRD are predicted to be N-glycosylated. By preventing this glycosylation, a protein called Shisa can inhibit the expression of functional WNT pathway receptors and thus downregulate the whole signalling pathway [27]. Immediately after the CRD follows a linker domain, which connects CRD to the transmembrane core. The core of the receptor is made of 7 transmembrane α-helices (TM1-7) flanked by three extracellular (EL1-3) and three intracellular (IL1-3) loops. The shape of extracellular domains is most likely also ordered to some extent by disulfide bonds between conserved cysteines [22]. C-terminally from the last transmembrane helix, there is a short eighth α-helix (TM8) which runs parallel with the membrane and contains the conserved KTxxxW motif associated with binding DVL [28].

Figure 3. Simplified scheme of Frizzled structure. Modified from Schulte et al. 2010 [21].

Intracellularly, FZDs contain a C-terminal domain which varies in length among different

FZDs, with FZD3/6/SMO having the longest C-termini (cca 150-230 aa) and the shortest ones being found in FZD1/2/7 (only 17 aa).

1.1.3 DISHEVELLED Dishevelled is a scaffold protein which plays a central role in the WNT signalling pathway, as it lies together with WNT and FZD at the point of divergence between different downstream signalling events. In mammals, Dishevelled exists as three paralogs (DVL1, 2, 3) with partially redundant and partially unique functions [29]. In human, mutations in DVL1 were found in a subset of Robinow syndrome cases [30]. When DVLs are absent during the development of a mouse, it leads to more or less severe congenital defects or socio-neurological impairments. When DVL1 is missing, the mice exhibit social and sensomotoric abnormalities [31]; mice deficient in DVL2 die perinatally or display defective skeleton, cardiac outflow tract and neural tube closure [32]; DVL3 knockout mice mostly die perinatally also due to cardiac morphogenesis defects [33]. Moreover, the neural tube defects and cardiac defects are more pronounced when more DVLs are missing [32,33]. When all three DVL proteins are missing, the embryo is aborted early even before the beginning of gastrulation, further demonstrating the overlapping functions of DVL [34]. It was shown that DVL2 is the most abundantly expressed DVL protein in a cell, however changing the expression level of DVL1 has the largest effect on the activation of β- catenin-dependent pathway [35,36]. Also, DVL2 and DVL3 share higher sequence homology and DVL2/3 were found to form complexes detected in tandem affinity purification/mass spectrometry experiments and DVL1 was not found in these complexes [37].

From the structural point of view, all DVLs contain three conserved functional domains connected by flexible unstructured regions (see Figure 4) – the N-terminal DIX domain (Dishevelled/Axin), then a basic region, followed by a region of multiple serine/threonine sites. Then, the centrally positioned PDZ domain (Post-synaptic density protein-95/Disc large tumour suppressor/Zonula occludens-1), a proline-rich region, DEP domain (Dishevelled/Egl-10/Pleckstrin) and a C-terminus highly conserved among all three DVLs. The unstructured regions between DIX, PDZ and DEP domains grant Dishevelled the flexibility which a multi-functional scaffold proteins need.

Figure 4. Simplified schematic picture of DVL structural regions.

The predominantly helical DIX domain is required for DVL polymerization with axin and other DVL molecules [38–40]. The compact globular structure of the PDZ domain is formed by six β-strands and two α-helices and allows DVL to bind PDZ ligands [28,41]. The DEP domain, important for membrane recruitment of DVL, is centered around a complex of three helices stabilized by hydrophobic interactions, it contains a β-hairpin 'arm' composed of two β-strands and C-terminally are positioned two anti-parallel β- sheets which cover the hydrophobic core of DEP domain [42].

1.1.4 β-CATENIN-DEPENDENT PATHWAY The β-catenin-dependent pathway is centered around β-catenin, the transcriptional co- activator of genes regulated by TCF/LEF (T-cell factor/Lymphoid enhancer-binding factor) transcription factors [43]. The main processive power lies within the destruction complex, a composite of following proteins: two scaffold proteins - APC (adenomatous polyposis coli) and axin (axis inhibition protein) - which keep the structure together, two serine/threonine kinases - GSK3β and CK1α - which can phosphorylate bound β- catenin, and an E3 ubiquitin ligase β-TrCP (β-transducin repeat containing E3 ubiquitin protein ligase) which ubiquitinates phosphorylated β-catenin (Figure 1). When the pathway is inactive, β-catenin bound to destruction complex is being subjected to phosphorylation, subsequent ubiquitination and degradation by the proteasome [44]. In the nucleus, TCF/LEF transcription factors bind the transcriptional repressor Groucho and transcription of target genes is downregulated [45]. Upon WNT stimulation, the ligand binds to FZD and LRP (low-density lipoprotein receptor-related protein 5 or 6), a pathway-specific co-receptor, creating a trimeric complex [46]. DVL gets recruited to FZD and polymerizes with axin through their DIX

22 domains [38]. LRP5/6 gets phosphorylated, binds axin/DVL polymers and thus recruits the destruction complex, which becomes inactive by a not fully understood mechanism [47]. The levels of β-catenin rise and as a result, the protein translocates to the nucleus where it binds the enhancer regions of target genes and transcription is upregulated. The target genes of TCF/LEF transcription factors include c-myc or cyclin D, generally mostly genes driving cellular proliferation or regulating cell fate [48,49].

1.1.5 PLANAR CELL POLARITY PATHWAY The planar cell polarity pathway, probably the most ancient WNT pathway [3] controls the orientation of cells within a specific structure. Originally it was studied in Drosophila, where it organizes for example cells in the epithelial planes of the wings or cells comprising ommatidia (the functional units of the compound insect eye) [50]. In mammals, a very similar process operates in the oriented development of inner-ear sensory hair cells or hair follicles in the skin [51]. Only later it became apparent that homologs of the genes implicated in Drosophila PCP pathway play a role also in more complex mammalian developmental processes, such as convergent extension movements where cells directionally migrate and intercalate between their neighbours to elongate the body axis or close the neural tube [52,53]. In Drosophila, the PCP pathway seemingly operates without the need for a ligand from the WNT family [54]; the planar polarity is established by directional arrangement of signalling components in the cooperating cells. At the distal side of a cell in the plane, the complex of proteins consisting of Frizzled (Fz), Flamingo (Fmi), Dishevelled (Dsh) and Diego (Dgo) is formed. At the proximal side, Fmi complexes with Van Gogh (Vang) and Prickle (Pk). The neighbouring cells interact extracellularly through Fmi, Fz and Vang [50]. The transducers of the pathway include small GTPases RHO and RAC which transfer the signal to downstream components leading to reorganization of the cytoskeleton or activation of JNK-depedent transcription, respectively [55,56] (Figure 1). In convergent extension movements in mammals, the core PCP components are conserved – mammalian Celsr (cadherin EGF LAG seven-pass G-type receptor 1/2/3) corresponds to Fmi, Vangl (vang-like protein 1/2) corresponds to Vang and there are two Prickle-like proteins [57,58]. However, many components of the pathway are different and perhaps the biggest difference is the responsiveness to a ligand similar to other WNT pathways [59]. The pathway employs also other receptors, such as tyrosine kinases ROR (RTK-like orphan receptor 1/2) [60]. Same as in Drosophila, the downstream signalling is directed towards Rac/RHO-dependent signalling events.

1.1.6 FZD4 and FZD6

In adult mammals, FZD4 is expressed ubiquitously with higher levels in endothelial cells and adipose tissue [61]. Mouse knockout phenotypes indicate that FZD4 function is required for proper vascular development [62–64] and the development of cerebellum, esophagus, ocular and auditory system [65]. In cancer patients, FZD4 is often found

23 amplified (tumours of the prostate, breast, ovary or lung), mutated (uterus, skin) or overexpressed (cross-cancer) [66,67].

In addition to WNTs, FZD4 can be activated by a FZD4-specific ligand, Norrin [62]. The

Norrin/FZD4/LRP complex activates the standard β-catenin-dependent pathway, the deregulation of which leads to various pathologies of retinal vascularization – for example the familial exudative vitreoretinopathy or the Norrie disease [68–70].

FZD4 and Norrin can employ a co-receptor with chaperone activity called Tspan12 (tetraspanin-12) which stabilizes the Norrin/FZD4 complex [71]. Tspan 12 is required for Norrin-induced, but not WNT-induced, β-catenin-dependent signalling pathway.

FZD6 is highly expressed in olfactory epithelium, thyroid and parathyroid glands, lung, and uterus of adult mice [61]. When mutated or missing during embryogenesis, FZD6 can cause PCP-associated phenotypes, such as hereditary form of nail dysplasia in humans or hair patterning and tissue polarity defects in mice [72,73]. FZD6 is amplified or overexpressed in many tumour samples originating from patients with breast, prostate or ovarian cancer, it is also found mutated in desmoplastic small-round-cell tumours [66,67].

The function of FZD6 is partially overlapping with another PCP-associated Frizzled, FZD3

[74,75], since combined loss of FZD3 and FZD6 leads to much more severe congenital defects than the individual loss of each of the receptors [73,76]. When both FZD3 and

FZD6 are absent during the development of mouse brain, it results in the midbrain morphogenesis defects and failure of neural tube closure, which suggests involvement of

FZD3 and FZD6 in these processes [74,75].

FZD6 is associated almost exclusively with the β-catenin-independent signalling pathways and it was shown to even downregulate β-catenin-dependent signalling through the activation of the Nemo-like kinase pathway [77].

1.2 G PROTEIN-COUPLED RECEPTORs G protein-coupled receptors (GPCRs) comprise a huge superfamily of eukaryotic cell surface receptors which respond to a great variety of signals spanning from photons, ions, amino acids and short peptides to large protein macromolecules. They regulate physiological functions as diverse as sensory perception, cell growth or hormonal regulation and stand literally at the gates of distinct cellular signalling pathways, which makes them one of the most pharmaceutically interesting targets [78]. From the structural point of view, the receptors can exist in a number of different conformations – which are more or less active with regard to induced downstream signalling – and the receptors are constantly shifting their shape, trying to reach energetic equilibrium. Different ligands can bind to one receptor and depending on the shape of the ligand and the place it binds, the receptor will change its form, shift its equilibrium and occupy its cognate conformations with altered frequency; usually binding of the ligand leads to stabilization of the receptor. Thus, binding of a ligand

24 changes the shape of the receptor, and intracellularly, new binding space opens for downstream signalling molecules [79]. The immediate binding partners of GPCRs are heterotrimeric G proteins, which consist of three subunits – Gα, Gβ and Gγ. When the receptor is inactive, the Gα subunit binds guanosine diphosphate (GDP). Upon ligand binding, the receptor acts as a guanine nucleotide exchange factor (GEF) and removes GDP from Gα, which is in turn passively exchanged for the higher-energy molecule GTP via concentration gradient. The GTP- bound Gα subunit dissociates from the Gβγ complex, and both Gα and Gβγ can activate downstream pathways (Table 1). The Gα has intrinsic GTPase activity and when the energy bound in GTP is spent, Gα-GDP re-associates with Gβγ and the whole complex is prepared for the next signal. To fine-tune the GPCR-dependent signalling, another group of proteins called GAP (GTPase-activating proteins) or RGS (regulators of G protein signalling) exists – their purpose is to accelerate the rate of GTP to GDP hydrolysis and thus terminate the Gα-dependent signalling.

1.2.1 HETEROTRIMERIC G PROTEINS

There are four different families of Gα subunits - Gαi, Gαs, Gαq and Gα12/13 – and each receptor displays a variable level of selectivity towards the G proteins with specific Gα subunits [79–81]. Each Gα subunit activates distinct downstream signalling pathways and the complex of Gβγ subunit can activate further signalling events (Table 1). Thus, the signalling outcome induced in a cell depends on the induction by a specific ligand, the expression of a responsive receptor and its associated G proteins and the availability of downstream signalling components. Structurally, the Gα subunit is composed of two well conserved domains – the GTPase catalytic domain responsible for GTP hydrolysis which is homologous to Ras-like small GTPases and a helical domain specific for heterotrimeric G proteins; both domains are connected by short linkers [82]. The Gβ is an elliptical structure formed mainly of β- sheets, Gγ is composed of two helices connected by a loop; both subunits interact mainly throuth their N-terminal helices which form a coiled-coil [83,84]. Gα binds the high- affinity complex of Gβγ mainly through Gβ and due to Gαβγ interaction, the effector contact sites in both Gα and Gβγ are masked, disabling downstream signalling events [85,86]. In the inactive state, GDP is kept in place by the helical domain which forms something like a lid over the bound nucleotide [81]. Upon receptor activation by a ligand, the TM6 moves outwards to create binding pocket for the G protein, which binds with several loops of the GTPase domain and the C-terminal α-helix of Gα [87]. Eventually, the structure of the Gαβγ complex is rearranged in a complicated, not fully understood way. Seemingly the activated receptor displaces the helical domain due to the interaction with the C-terminal α-helix and GDP escapes [79,88,89]. That creates space for GTP and once GTP is bound, the structure of Gα changes one more time affecting also the structure of regions where effector molecules bind [82,90]. The extra phosphate in GTP

25 interferes with the Gα-Gβ interaction and the whole complex dissociates [85] to activate downstream targets summarized in Table 1.

Family Variant Expression Effectors Adenylyl cyclase Gα Ubiquitous s(s, l) ↑ Olfactory, Gα s epithelium, Adenylyl cyclase Gα olf brain, testes, ↑ pancreas Retinal rods, Gα cGMP-PDE ↑ t(r, c) cones, taste cells Mostly ubiquitous Adenylyl cyclase Gα Gα i i(1, 2, 3) (isoform- ↓ specific) Neuronal, Adenylyl cyclase Gα o(1, 2, 3) neuroendocrine ↓

Gαq Ubiquitous PLCβ ↑ Gαq Gα11 Ubiquitous PLCβ ↑ Rho-GEF ↑, Btk ↑, Gα Ubiquitous 12 cadherin Gα 12 Rho-GEF ↑, Gα Ubiquitous 13 radixin Adenylyl cyclase

Gβ Gβ(1, 2, 3, 4, 5) Ubiquitous ↑↓, PLCβ ↑, Ion channels Adenylyl cyclase

Gγ Gγ(1- 5, 7 - 12, c) Ubiquitous ↑↓, PLCβ ↑, Ion channels

Table 1. Selected heterotrimeric G proteins, their expression patterns and downstream targets. Simplified from Ellis 2004 [80].

The N-terminus of Gα and C-terminus of Gγ are lipid-modified which is required for Gα- Gβγ interaction as well as for the interaction with the receptor and keeps the proteins membrane-anchored [91,92]. 1.2.2 WNT/Ca2+ and other G protein-dependent pathways It has been a matter of debate for quite some time, whether the seven-transmembrane receptors Frizzleds are genuinely G protein-coupled receptors, that is whether they truly

26 bind the heterotrimeric G proteins and whether they can trigger G-protein dependent pathways. Some evidence has been collected over the past twenty years and as a consequence, FZDs have been recently included into the GPCR superfamily as Class F receptors [93]. In the context of WNT signalling, there is a well established WNT/calcium pathway employing WNTs as ligands and heterotrimeric G proteins as FZD-dependent signal transducers [94,95]. The consequence is activation of a standard Gαq/11-induced pathway, where phospholipase C (PLC) is activated, leading to the cleavage of phosphatidylinositol 4,5-biphosphate (PIP2) into the second messengers inositol (1,4,5) trisphosphate (IP3) and diacylglycerol (DAG). DAG activates calcium-dependent protein kinase (PKC) and IP3 triggers release of calcium from endoplasmatic reticulum (ER).

Another FZD-G-protein-dependent pathway operates through transducin (Gαt)- dependent activation of phosphodiesterase 6 (PDE6), which results in the decline of cyclic guanosine monophosphate (cGMP) and subsequently also in the release of Ca2+ from ER [96] (Figure 1). Calcium is a multi-functional cellular transducer which further activates calcium- dependent kinases such as PKC or Ca2+/calmodulin-dependent kinase (CAMK) or a phosphatase called calcineurin (Cn), ultimately leading to transcription dependent on nuclear factor of activated T cells (NFAT), nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) or cAMP responsive element binding protein (CREB) [95]. However, G protein-dependent WNT signalling is not limited to the two pathways which have just been described. In mouse microglia cells, WNT-5A can induce Gαi/o/PLC/PKC- dependent activation of ERK1/2 [97]. Recently, overexpression of FZD10 has been shown to Gα13-dependently increase the transcriptional activity dependent on Yes Associated Protein 1 (YAP) and Tafazzin (TAZ), the downstream effectors molecules of the Hippo pathway implicated in the development [98].

1.3 Crosstalk between G protein-dependent and DVL-dependent signalling From the integrative point of view, there are many studies showing the complex nature of the WNT signalling network. For instance, WNT/FZD-induced cellular response cannot be clearly cut into DVL-dependent and G protein-dependent pathways, as I will discuss in the following section. DVL was reported to be dispensable for WNT/Ca2+ signalling mediated by PDE6 [99] whereas in other experiments DVL behaved as an inductor of Ca2+ signalling [100]. Also, physiological levels of DVL were required for maintaining the FZD6-Gαi/q precoupling and WNT-5A-induced Gαi/o–mediated phosphorylation of ERK1/2 [97,101]. On the other hand, G proteins have been implicated in the classical DVL-dependent WNT signalling events. It was reported that WNT-3A/FZD1-induced Gαo activation can result in a DVL-dependent activation of JNK, and thus positively interfere with the PCP pathway [102]. Furthermore, G proteins were shown to positively affect also the β- catenin-dependent signalling. In primary mouse microglia cells, WNT-3A-induced

27 phosphorylation of LRP6 and stabilization of β-catenin was sensitive to pertussis toxin

(PTX), an inhibitor of Gαi/o-mediated signalling [103]. In another study, Gαi/o/q were required for the WNT-induced disruption of destruction complex and stabilization of β- catenin [104]. Gαi/o/q induced stabilization of β-catenin also in Xenopus egg extracts and Gβγ further promoted activation of the pathway by recruiting GSK3β to the membrane and facilitating the phosphorylation of LRP6 [105]. Another study found that Gβγ recruits to the plasma membrane also DVL [106]. The multitude of positive effects the G proteins exert on the β-catenin-dependent pathway has even lead to the proposal of a convergent pathway merging G protein-dependent and DVL-dependent positive effects on the β-catenin-dependent transcription [107]. In conflict with the previous results, Gβγ was also shown to induce degradation of DVL via the WNT/calcium pathway and thus inhibit β-catenin-dependent pathway [108]. Even though it cannot be clearly dissected whether G proteins play a positive or negative role in WNT signalling pathways, they are clearly involved. The involvement of Gβγ in DVL-dependent WNT signalling events is further supported by the detection of Gβγ in complex with proteins such as DVL2, CK1ε or Vangl1/2 [109]. And the crosstalk between different branches of WNT signalling is not limited to the level of G proteins and DVL. Multiple further connections within the WNT signalling cascades have been reported and they are not the focus of my work, so I at least outlined some of them in the Figure 1 (grey arrows) [110,111]. The integrated view of WNT signalling functioning as a network will need more time and supporting evidence to find its way into the textbooks, however the simplistic view of several distinct WNT signalling pathways is for sure challenged, if not outdated [2].

1.4 WNT/FZD/pathway selectivity Another intriguing question in the WNT field concerns the selectivity of induced signalling pathways. In other words, what factor does determine which effectors will be activated by a WNT? To answer this question we have to inspect and understand the most proximal signalling events in the pathway determining signal initiation and specification. The first obvious candidates are the ligands of the pathway – WNT proteins. In the past, mammalian WNTs were considered to stimulate either the β-catenin-dependent pathway (for example WNT-1 or WNT-3A) or the other pathways (e.g. WNT-5A or WNT- 11) [2,112,113]. However, later it was shown that the β-catenin-pathway-related WNTs can induce also events traditionally associated with the β-catenin-independent signalling [37,103,114] and vice versa [115–117], so the two groups themselves cannot explain the signalling selectivity. Perhaps the presence of multiple WNT proteins early in metazoan phylogenesis does not mean that their variety is needed for conferring signalling specificity; it could just mean that in this way it is easier to regulate WNT spatio-temporal expression during the development [3]. If not WNTs, then perhaps their receptors Frizzleds could be the factors bestowing selectivity upon downstream signalling pathways. However, most Frizzleds do not show

28 much bias towards specific downstream signalling pathways [118]. The WNT receptor displaying the highest level of pathway selectivity is probably FZD6 which was shown to induce mostly β-catenin-independent signalling [77,119,120]; but even this receptor was implicated in the β-catenin-dependent pathway [72]. On the other hand, pairing of WNTs with FZDs shows certain specificity and even selectivity towards downstream signalling and that could perhaps partially explain how signalling selectivity can be achieved [113,118,121,122]. Also, FZDs are not the only receptors for WNTs and seemingly, employing different co- receptors of the WNT pathway could represent another means of pathway selectivity. It was shown that WNT-5A can stabilize β-catenin when coexpressed with FZD4 and LRP5 or inhibit the stabilization of β-catenin through ROR2, both in vitro [123] and in the developing mouse [124]. In this model, only WNTs capable of binding LRP6, such as WNT-3A, could recruit and activate the cellular machinery specific for β-catenin- dependent pathway, whereas WNTs capable of binding ROR1/2, such as WNT-5A, would elicit β-catenin-independent response (on its own or in concert with specific Frizzleds). In accordance, Sato et al. have shown that it is the expression of co-receptors which matters for the signal specification, as knockdown of ROR1 and ROR2 abolished WNT- 5A-induced RAC activation and correspondingly, knockdown of LRP6 suppressed the WNT-3A-dependent stabilization of β-catenin [125]. In summary, it seems that specificity in WNT signalling might be achieved by a combination of factors including WNT/receptor selectivity and subsequent pathway bias as well as meticulous spatio-temporal expression – and thus the availability - of WNTs, receptors and other downstream components of the pathway during the development of metazoans.

1.5 FZD-DVL interaction Although the FZD/DVL interaction lies at the centre of WNT signalling pathways, it is still not fully understood how the receptor and DVL interact and how their interaction is regulated within the specific WNT signalling events. Originally, DVL and FZD were reported to interact through a PDZ domain-PDZ ligand interaction [28]. PDZ domains are abundant modules with diverse functions including organization of protein scaffolds or attachment of proteins to the cytoskeleton [126]. PDZ domains often bind to an utmost C-terminal PDZ ligand sequence and FZDs harbour such PDZ sequences at their C-termini [28]. Thus, the finding that DVL PDZ domain does not bind to such C-terminal PDZ ligand sequence, but instead to an unusual internal PDZ ligand sequence, was indeed surprising. The internal PDZ ligand sequence of FZD was mapped to the helix 8 and consists of a conserved KTxxxW motif, where lysine, threonine and tryptophan are conserved, whereas x can be any amino acid. The conserved KTxxxW motif was shown to be required for WNT/ β-catenin-dependent pathway which further confirms previous results from structural studies [127]. Even though the DEP domain of DVL was predicted to bind upstream WNT signalling components already in 2000 [42], it lasted 12 years until the counterpart binding

29 interface in FZD was discovered [128]. From a peptide-binding array it was deducted, that DVL binds via its DEP domain and C-terminus to FZD sequence contained within the IL3 and C-terminus. The DEP domain binds mostly the C-terminus of FZD - TM8 and its KTxxxW motif seems to be involved in both PDZ and DEP binding, on top of that the DEP domain binds another 5 amino acids C-terminal from KTxxxW. On the other hand, DVL C-terminus binds regions of FZD IL3 proximal to the membrane (around 9 amino acids at each 'side' of IL3) which are well conserved in FZDs with the exception of FZD3 and

FZD6. The 3 amino acids directly following the KTxxxW motif were later reported to be also required for DVL recruitment by FZD and for the activation of β-catenin-dependent pathway [129]. From the DVL point of view, at the tip of the β-hairpin 'arm' of DEP domain, a well- conserved lysine and two aspartates create an electric dipole involved in the interaction with FZD [42]. Mutation in the aforementioned lysine (K438M in hDVL1/K446M in hDVL2/K435M in hDVL3/K417M in fly Dsh) creates a PCP-deficient Drosophila mutant [130] and generally a DVL unable to get recruited to the membrane by FZD [131]. DEP domain of DVL also bears several basic residues distinct from the FZD-DVL binding site which promote binding of DVL to the negatively charged phospholipids of the plasma membrane [42]; this interaction of DVL-DEP with the membrane is stabilizing the FZD-DVL interaction [132]. Finally, several additional residues in FZD IL1 and IL2 (corresponding to R247, R249,

Y250, F328 and A339 in human FZD4) have been shown to complement the FZD-DVL interface at the side of FZD [133–135], which basically implicates that DVL binds through its PDZ, DEP and C-terminus to a large and dispersed interaction interface in FZD.

1.6 FZD-G protein interaction From the structural point of view, we don’t know much about the interaction of FZDs and G proteins, since no crystal structure of a Class Frizzled member bound to a G protein was published. When we compare different families of GPCRs with regard to the intracellular interface required for G protein binding, we can assume that the interaction usually requires intact TM/cytoplasmic borders between TM3/IL2, TM5/IL3, IL3/TM6 and the C-terminus [136]. However, the contribution of residues in other TMs and IL1 has also been reported [88,137–139] in some receptors and generally, there is a considerable variability in GPCR-G protein interaction interfaces throughout the GPCR superfamily which makes any predictions cumbersome [140]. When we look into the dynamics of GPCR/G protein interaction, there are currently two possible models explaining the immediate events happening upon ligand binding. In the collision coupling model, the receptor and the GDP-bound G protein exist separately [141–143]. When a ligand binds, the receptor changes its conformation which allows for G protein binding, receptor facilitates displacement of GDP and GTP-bound G protein exits the interaction with the receptor. Thus, receptor in an activated state can activate tens to hundreds G proteins before being desensitized [144]. In a

30 precoupling/preassembly model, the receptor and an inactive G protein form a coherent unit even before the activation of the receptor [145–147]. Upon receptor activation, GDP escapes the G protein with the aid of a receptor, and GTP-bound G protein exits the binding of the receptor similarly as in the previous model. The first model better explains how a signal transduced by receptors gets amplified, on the other hand the second model justifies the ultra-quick GDP/GTP exchange which is sometimes detected in the orders of milliseconds [81]. Most likely, both models coexist and different GPCRs can utilize collision coupling or precoupling or even switch between the two modes of interaction; the same could be true also for Frizzleds. Regarding the FZD/G protein selectivity issue, limited information is available as well.

Based on the in silico predictive analysis, Frizzleds were expected to bind Gαi/o and Gαq subunits [148] which was later confirmed [37,97,149]. However, the detailed FZD/G protein selectivity map is still missing due to the fact, that in all the assays assessing FZD/G protein signalling, more Frizzled proteins are endogenously expressed. A recent contribution to solving this question is brought by FRAP (fluorescence recovery after photobleaching) experiments which show that FZD6 can be precoupled to Gαi1 and Gαq but not to GαoA, Gαs and Gα12 and that the FZD/Gα complexes dissociate upon WNT-5A stimulation [101]. Similarly, FZD10 is shown to be precoupled to Gα13 [98].

1.7 GPCR/FZD phosphorylation The function of GPCRs is often regulated by phosphorylation of their intracellular parts [150]. The phosphorylation sites are mostly comprised within the receptor C-terminus and IL3, however even phosphorylation events in the IL1 and IL2 have been reported [150–153]. The phosphorylation can be mediated by a number of kinases including GRKs (G protein-coupled receptor kinases), PKA (cAMP-dependent protein kinase), PKC, CK1, CK2 (casein kinase 1/2) or Src [154–159]. Not much is known about phosphorylation of Frizzleds, except for few studies linking

FZD1 and FZD3 phosphorylation to the β-catenin-independent planar cell polarity (PCP) signalling [160–163]. First, Djiane et al. reported that the phosphorylation of the Drosophila Frizzled-1 C-terminal tail by atypical Ca2+-dependent protein kinases (aPKC) doesn't affect receptor localization but leads to an inhibitory effect on the downstream PCP pathway [160]. Later, it was shown that Xenopus Frizzled 3 (Fz3) can be phosphorylated at S576 in the presence of DVL by an unknown kinase [161]. Further results suggested that phosphorylation of S576 could be a result of activated WNT signalling and an instrument of Fz3 desensitization rather than activation. During PCP- regulated commissural axon guidance, FZD3 was reported to be phosphorylated in a

DVL1-dependent manner [162]. FZD3 phosphorylated in this way was more membranous and less active with regard to the observed PCP signalling. Lastly, it was discovered that the DVL1-induced phosphorylation of FZD3 can be inhibited by WNT-5A stimulation through the action of DVL2 and aPKC [163]. Subsequently, FZD3 is internalized and FZD3-dependent PCP signalling is re-activated [163].

The best described member of the Class F family with regard to phosphorylation is SMO [164]. SMO was shown to be phosphorylated by CK2, PKA, CK1 and GRK2 with different outcomes including stabilization of the active conformation of the receptor, relocalization to the membrane, internalization, activation or termination of downstream signalling.

2. AIMS

Despite FZD/DVL/Gαβγ being at the centre of signal specification in WNT signalling, the interaction interface and binding selectivity of FZD/DVL and FZD/G proteins is still a matter of debate. Also, the phosphorylation of FZDs is largely unexplored.

My specific aims were defined as follows:

 to determine FZD4/G protein binding selectivity and to find the downstream

pathways mediated by FZD4 and G proteins

 to explore the involvement of DVL in FZD4/G protein interaction

 to investigate the significance of FZD4 IL1/TM2 in the FZD/DVL and FZD/G protein binding interface

 to find out whether phosphorylation of FZD4 is required for DVL binding

 to examine whether FZD6 is phosphorylated similarly to GPCRs and explore the circumstances and function of such phosphorylation

5. ACKNOWLEDGEMENTS

Firstly, I would like to express deep gratitude to my supervisor, Prof. Gunnar Schulte. Thank you for taking me on board of the Schultelab and introducing me to Karolinska Institutet. Thank you for your scientific enthusiasm, for all the ideas that brought my projects into life. Thank you for all the support you gave me through my whole PhD and for teaching me how to write a scientific text. For cheering me up (many times!) when nothing worked and laughing through all my 'sub-optimal' experimental setups. Your kind and generous attitude was highly appreciated and I hope to visit Sweden from time to time to share a fika. Also, a big Thank You goes to my co-supervisor, doc. Vítězslav Bryja. Your careful systematic analysis of results, planning and prioritizing the experiments as well as the management of a big research group was a big inspiration. Also, thank you for most of the financial support that made my PhD possible and for the opportunity to be part of the KI-MU project and live in Stockholm for a while. Lastly, thank you for creating an amazing working environment called Bryjalab and for being such wonderful person to work with. I would like to thank also the administration of Masaryk University, the Department of Experimental Biology and OFIŽ for creating a smooth and pleasurable working environment. Especially, I would like to thank Bára for the KI-MU project, Andrejka and Nicole at the study department for the benevolence with which they handled us, unruly students, and Naďa and Lucka for the general administrative support. The work presented in this thesis was supported by grants from the Czech Science Foundation, European Social Fund and the state budget of the Czech Republic and I am grateful to the tax-payers who made my education and research possible. I would like to thank all the members of Schultelab and Bryjalab and all the other people at OFIŽ and FyFa for being such great people. Thank you, Ondro, Pájo, Jamesi, Ranjani, Igorko, Marki, Vendy, Tomku, Zankruti, Peťo, Marku, Aničko, Jožo, Katy, Julí, Terko, Tome, Míšo... and all the other kids  Thank you, Jančo (for all the fun and sarcasm), Julian, Belma, Shane, Elisa, Jacomijn, Eva, Mika, Phil, Tibo, Lei, Karuna, Anthi… The KI-MI crew from the Kista house has a special place in my heart – thank you, Karolko, Zuzi, Simčo for all the wonderful days we spent together. I thank my family for their love and support: To my father for first bringing me to the idea of studying molecular biology, to my mother for taking care of everything, to my brother for being a good companion in making fun of our crazy family and to my grandma for being the most nice person in the world. I would like to give special thanks to my best of bests - to Mája, Bla and Čva for all the laughs and griefs we shared and to Miška for being herself. Last but not least, my very special thanks belong to Jakub for keeping me company when going through the deep dark valleys of my PhD and to Honza for bringing sun into my life. Let's go with the flow.

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LIST OF ABBREVIATIONS aa amino acid APC adenomatous polyposis coli aPKC atypical Ca2+-dependent protein kinases Axin axis inhibition protein β-TrCP β-transducin repeat containing E3 ubiquitin protein ligase CAMK Ca2+/calmodulin-dependent kinase Celsr cadherin EGF LAG seven-pass G-type receptor 1/2/3 cGMP cyclic guanosine monophosphate CK casein kinase CLR calcitonin receptor-like receptor Cn calcineurin CRD cysteine-rich domain CREB cAMP responsive element binding protein Daam dishevelled-associated activator of morphogenesis DAG diacylglycerol DEP dishevelled/Egl-10/pleckstrin Dgo diego DIX dishevelled/axin DMR dynamic mass redistribution Dsh dishevelled (usually non-mammalian) DVL dishevelled (usually mammalian) EL extracellular loop ER endoplasmatic reticulum ERK extracellular signal-regulated kinase Fmi flamingo FRAP fluorescence recovery after photobleaching FRET Förster resonance energy transfer Fz frizzled (usually non-mammalian) FZD frizzled (usually mammalian) GAP GTPase-activating protein GDP guanosine diphosphate GEF guanine nucleotide exchange factor GPCR G protein-coupled receptor GRK G protein-coupled receptor kinase IL intracellular loop IP3 inositol (1,4,5) trisphosphate LEF lymphoid enhancer-binding factor LRP low-density lipoprotein receptor-related protein MAPK mitogen-activated protein kinase MD molecular dynamics NFAT nuclear factor of activated T cells NFκB nuclear factor kappa-light-chain-enhancer of activated B cells

PCP planar cell polarity PDE phosphodiesterase PDZ post-synaptic density protein-95/disc large tumour suppressor/zonula occludens-1 PIP2 phosphatidylinositol 4,5-biphosphate Pk prickle PKA cAMP-dependent protein kinase PKC calcium-dependent protein kinase PLC phospholipase C PTX pertussis toxin RGS regulator of G protein signalling ROCK RHO-associated kinase ROR RTK-like orphan receptor SMO smoothened Swim secreted Wingless-interacting molecule TAZ tafazzin TCF T-cell factor TGFβ transforming growth factor β TM transmembrane helix Tspan12 tetraspanin-12 Vang Van Gogh Vangl vang-like protein WLS wntless WNT wingless/integrated1 YAP yes associated protein