Structure and Function of the Fgd Family of Divergent FYVE Domain Proteins

Biochemistry and Cell Biology

Journal: Biochemistry and Cell Biology

Manuscript ID bcb-2018-0185.R1

Manuscript Type: Mini Review

Date Submitted by the 03-Aug-2018 Author:

Complete List of Authors: Eitzen, Gary; University of Alberta Faculty of Medicine and Dentistry Smithers, Cameron C.; University of Alberta, Biochemistry Murray, Allan; University of Alberta Faculty of Medicine and Dentistry Overduin, Michael; University of Alberta Faculty of Medicine and Dentistry Draft

Fgd, Pleckstrin Homology domain, FYVE domain, Dbl Homology Domain, Keyword: Rho GEF

Is the invited manuscript for consideration in a Special CSMB Special Issue Issue? :

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Title:

Structure and Function of the Fgd Family of Divergent FYVE Domain Proteins

Authors:

Gary Eitzen1, Cameron C. Smithers2, Allan G Murray3 and Michael Overduin2* Draft

1Department of Cell Biology, 2Department of Biochemistry, 3Department of Medicine, University of Alberta, Edmonton, Alberta, Canada

*Corresponding author. Michael Overduin Telephone: +1 780 492 3518 Fax: +1 780 492-0886 E-mail: [email protected]

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Abstract

FYVE domains are highly conserved protein modules that typically bind phosphatidylinositol 3-phosphate (PI3P) on the surface of early endosomes. Along with pleckstrin homology (PH) and phox homology (PX) domains, FYVE domains are the principal readers of the phosphoinositide (PI) code that mediate specific recognition of eukaryotic organelles. Of all the human FYVE domain-containing proteins, those within the Faciogenital dysplasia (Fgd) subfamily are particularly divergent, and couple with GTPases to exert unique cellular functions. The subcellular distributions and functions of these evolutionarily conserved signal transducers, which also include Dbl homology (DH) and two PH domains, are discussed here in order to better understand the biological range of processes that such multidomain proteins engage in. Determinants of their various functions include specific multidomain architectures, post-translational modifications including PIP stops that have been discovered in sorting nexins, PI recognition motifs and phospholipid binding surfaces as defined by the Membrane Optimal Docking Area (MODA) program. How these orchestrate Fgd function remains unclearDraft but has implications for developmental diseases including Aarskog-Scott syndrome, which is also known as faciogenital dysplasia, and forms of cancer that are associated with mutations and amplifications of Fgd genes.

Keywords:

Cdc42, Dbl, FYVE, Fgd, GEF, GTPase, PH, phosphoinositide, pleckstrin, cancer, faciogenital dysplasia, Aarskog-Scott syndrome , lipid signaling, membrane trafficking, MODA, PIP, Rho.

Text:

Phosphoinositide Code

Stimulation and differentiation of cells involve dramatic alterations of phosphorylation patterns in proteins, lipids and nucleotides. Defining how these molecular events converge to change cellular behaviour remains a central challenge that, if achieved, promises to unlock novel targets for drug discovery. The molecules that transduce biological signals undergo reversible addition of phosphates, thus mediating the flow of cellular information. In the case of protein modifications, these signals are binary.

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That is, amino acid residues are either phosphorylated or unphosphorylated. Mononucleotides are represented by multiple forms such as the cGMP, GMP, GDP and GTP derivatives of guanosine present in eukaryotes, and regulate the activities of proteins such as GTPases. The phosphatidylinositol headgroup is differentially phosphorylated at the 3-, 4- and/or 5- positions to yield 7 distinct PI lipids, which are distributed to various organelles where they are recognized by FYVE, PH, PX and other domains. These modules serve as principal readers of the PI code and direct membrane-dependent signaling and trafficking in eukaryotes (Overduin, Cheever, and Kutateladze 2001). Together, these protein, nucleotide and lipid modifications offer a rich tapestry of signaling states that encodes biological information to determine cellular fate. Our understanding of how this actually works is in its infancy, as illustrated here by the complexity of Fgd proteins. This family is unique in terms of its architecture, integrating strands of information through multiple domains that recognize specific phospholipids and proteins including GTPases to drive cellular development. Here we review what is known about the family and explore the roles of the component domains and family members.

Architecture of the Fgd Family Draft

The human genome encodes six Fgd genes (Fig 1). Homologues are present in most metazoans, and diverged early in chordate evolution while maintaining the same modular architecture. They act as Rho guanine-nucleotide exchange factors (Rho GEFs) that are thought to be guided to membranes by PI- binding FYVE and PH domains. They contain the typical Rho GEF subdomain structure of a DH domain adjacent to the first PH domain (PH1). The DH and PH1 domains function together to mediate the regulated activation of Rho proteins by binding to and catalyzing the exchange of GDP for GTP nucleotides. All Fgd proteins contain a divergent FYVE domain followed by a second PH domain (PH2) at their C-termini. The long N-termini of Fgd proteins are poorly conserved and apparently disordered. Fgd1 contains an N- terminal proline-rich element which is phosphorylated (Hornbeck et al. 2015), influences peripheral membrane localization dynamics (T Oshima et al. 2010), and harbours a critical point mutation linked to Aarskog-Scott syndrome (Orrico et al. 2004). Fgd4 interacts with F-actin via its N-terminal 150 residues (Obaishi et al. 1998). The structural domains of Fgd proteins are discussed individually below, including descriptions of structural and regulatory features.

FYVE Domains

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FYVE domains are found in 29 human proteins, which exhibit a wide range of architectures, with that of the Fgd subfamily (Fig 1) being most recurrent. This short module is named after four proteins in which its 70 residue sequence was first identified: Fab1p, YOTB, Vac1p and EEA1. The fold of the FYVE domain consists of two double-stranded antiparallel β sheets and a C-terminal α-helix (T. G. Kutateladze and Overduin 2004). Two zinc ions are gripped tightly by four cysteines in a tetrahedral arrangement to provide essential structural stabilization (T. G. Kutateladze et al. 1999). FYVE domains represent the most tightly conserved lipid recognition module, and are thought to almost always recognize PI3P as their cognate ligand (T. Kutateladze and Overduin 2001). Organelle recognition involves electrostatic approach and dipping of a membrane insertion loop (MIL) into a bed of PI3P, phosphatidylserine (PS) and phosphatidycholine (PC) molecules for firm anchoring and stereospecific lipid headgroup recognition (T. G. Kutateladze et al. 2004). This bilayer binding is strengthened by the acidic microenvironments found along the endocytic route, thus keeping proteins more stably tethered to low pH endosomal membranes (Lee et al. 2005) rather than the other PI3P pools found in the cell .

Almost all FYVE domains contain three functionallyDraft critical signatures, all of which diverge in the Fgd family (Fig. 2). The aspartic acid in the N-terminal motif WxxD (where x is any residue) excludes binding of PIs that bear phosphates at the 4- or 5-positions, as seen in EEA1, while the tryptophan underpins the binding site (Dumas et al. 2001; T. Kutateladze and Overduin 2001). This highly conserved motif is replaced with PIRE, TEED and LVPV sequences in Fgd1, Fgd3 and Fgd5, respectively, suggesting different ligand specificity profiles and altered accommodation of the inositol ring. The central R+HHCR motif (where + is Arg or Lys) is the core PI3P code reader, and provides direct recognition of the 3-phosphate as well as ensuring pH-dependent ligand recognition. The last arginine of the reader motif mediates a pair of hydrogen bonds to coordinate this phosphate, but is replaced by a lysine in Fgd1 and Fgd3 and a histidine in Fgd5, reinforcing the notion that these Fgd proteins possess altered PI selectivity. The arginine of the C-terminal RVC motif provides electrostatic interactions with the bilayer, but is replaced with a lysine in the case of Fgd4 and Fgd5. The substitution for arginine for lysine is relatively conservative as both have bulky and cationic side chains. Based on analysis of experimentally mapped peripheral membrane protein interfaces (Kufareva et al. 2014), arginines are more common in membrane binding surfaces than lysines, reflecting their more stable and specific bidentate hydrogen bonding of phospholipid ligands.

Anomalies in PI binding motifs could be expected to influence the ligand specificities of the Fgd family of FYVE domains. The FYVE domain of Fgd1 binds not only to PI3P but also to PI5P, albeit more weakly, in a

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dot-blot lipid interaction assay, as performed at pH 8.0 (Sankaran et al. 2001). Both Fgd3 and Fgd4, as expressed in colon cancer cells, are pulled down by PI3P bait molecules. Moreover, Fgd4 binds preferentially to immobilized PI3P with an affinity of 195 nM as measured at pH 7.4, although PI4P and PI5P were not tested (Catimel et al. 2013). Thus it is possible that Fgd FYVE domains still recognize PI3P but are broader in specificity and less dependent on acidic pH values when binding PI-containing bilayer surfaces.

While no structures have been determined of any Fgd FYVE domain, other atypical FYVE domains have been resolved. The FYVE domain of the Protrudin protein also lacks the canonical PI binding motifs, and prefers to bind doubly and triply phosphorylated PIs, as measured at pH 7.4. This interaction depends on the integrity of its PI reader element which contains a novel KKRRSCS sequence in place of the central canonical motif. This sequence lacks the pH-switch represented by the pair of conserved histidines, and

directs protrudin to different vesicles and plasma membrane compartments that contain PI(4,5)P2 (Gil et al. 2012). These results are consistent with Fgd FYVE domains possessing broader PI binding profiles while retaining pH dependent PI3P recognition. Draft

The regulation of the Fgd family may depend not just on lipid levels, but also on protein phosphorylation. In several cancer cells Fgd3 is hyperphosphorylated on a pair of conserved serine and threonine residues within its membrane insertion loop (MIL) (Figs. 1&2) (Hornbeck et al. 2015). This feature is analogous to the PIP-stop, a phosphorylated switch residue which toggles endosomal localization and PI3P binding as discovered in PX domain-containing Snx proteins (Lenoir et al. 2018). Unlike the EEA1 protein which dimerizes through a coiled coil to increase membrane avidity, Fgd family members lack obvious multimerization motifs, and may instead rely on its multiple membrane binding domains. These modules are connected via variable, unstructured linkers that are susceptible to phosphorylation (Fig. 2), which could regulate multi-domain membrane binding.

PH Domains

PH domains were the first module that was found to recognize a PI (Sankaran et al. 2001). They are present in 285 human proteins, nine per cent of which contain a pair of PH domains as in Fgd proteins (Lenoir, Kufareva, et al. 2015). The PH fold consists of approximately 120 amino acids that fold into a seven stranded antiparallel β-sheet capped by a C-terminal α-helix. Analysis of the surface properties of PH

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domain structures indicates that the majority interact with membranes, typically inserting the hairpin loop connecting the β1 and β2 strands (Lenoir, Kufareva, et al. 2015), although the yeast PH proteome appears to bind PIs less tightly, commonly and selectively (J. W. Yu et al. 2004). Experimental studies have shown that PI(3,4,5)P3 and PI(4,5)P2 are the most common PH ligands. Unusually, PEPP1 binds PI3P spotted on nitrocellulose membranes through its PH domain (DOWLER et al. 2000), although these results have been questioned (Lemmon 2007).

The structures of the Fgd PH1 and PH2 domains (PDB: 1wgq, 2coc, 3mpx) possess the expected folds, with that of PH1 packing against the last helix of the DH domain (Fig. 3). They display membrane binding propensities within their exposed β1-β2 loops and proximal residues based on MODA analysis (Kufareva et al. 2014). Additional positively charged sidechains that are absolutely conserved and exposed in the β1- β2 sheet of PH1 may be also involved in lipid binding, although they lack discernable electron density in the Fgd5 crystal (PDB: 3mpx). The PH2 domain of Fgd6 displays an extensive membrane binding surface that is rich in lysines and aromatic residues (PDB: 1wgq) (Fig. 3C). In contrast, the solution structure of human Fgd3’s PH2 domain (PDB: 2coc) displaysDraft little membrane binding propensity based on MODA analysis (Lenoir, Kufareva, et al. 2015). The PH1 domain of Fgd4 binds preferentially to surfaces presenting biotin-conjugated PI3P molecules, and also weakly to PI(4,5)P2, although the full breadth of the PI specificity is unclear as PI4P and PI5P were not tested (Catimel et al. 2013). Phosphorylation events in Fgd PH1 domains include recurrent phosphorylation of a conserved tyrosine in the β3 strand near the membrane insertion loop, suggesting another PIP stop (Lenoir, M.; Ustunel, C.; Rajesh, S.; Kaur, J.; Moreau, D.; Gruenberg, J.; Overduin 2018), as well as in the C-terminal helix which could affect interdomain interactions. Together this suggests that Fgd PH1 and PH2 domains can mediate regulated PI interactions that could synergize with those of the FYVE domain and could offer a wider breadth of lipid specificities and membrane affinities.

Analogies can be drawn to other PH domains. The FAPP1 trafficking protein is recruited to trans-Golgi network membranes via its N-terminal PH domain. The action involves stereospecific recognition of PI4P, along with complementary interactions with PS and PC lipids (Lenoir et al. 2010), and simultaneous interaction with the GTP-loaded form of Arf1 (He et al. 2011). The lipids must be in a disordered phase for FAPP1’s PH domain to insert into the bilayer (Lenoir, Grzybek, et al. 2015). It is possible that Fgd’s PH domains also prefer to bind disordered phase lipids, although their membrane insertion loops are shorter and hence could individually bind more superficially. Coincident binding of Fgd proteins to lipid and

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protein partners is also conceivable. The PH domain of the AKAP13 protein doesn’t bind membranes (Lenoir, Kufareva, et al. 2015) but does structurally stabilize the adjacent DH domain and negatively regulates its GEF activity, while also apparently providing a secondary docking site for its GTPase partner RhoA, as seen in an unpublished structure (PDB: 6bca) and references (Abdul Azeez et al. 2014; Lenoir et al. 2014). This illustrates the complexity of such multidomain proteins, and underscores the need for determination of multiple structures including of complexed states of Fgd members.

Localization of Fgd proteins

domain interaction with membrane ruffles, which are enriched in PI(4,5)P2 and PI(3,4,5)P3, while the full- length protein also binds PI5P (Huber et al. 2008). Through these actions, Fgd2 may be positioned to influence cell polarization and migration orDraft micropinocytosis. Fgd3 induces and localizes to lamellipodia (Hayakawa et al. 2008) and, when expressed as a tandem DH and PH1 construct, induces formation of filopodial extensions (Pasteris et al. 2000). FGD4 is predominantly recruited to the actin cytoskeleton through its N-terminal region to mediate formation of filopodia, with its PI recognition domains serving as a bridge to the membrane, including its FYVE domain’s interaction with membrane ruffles (Kim et al.

2002). A Fgd6 construct missing the N-terminal region binds to PI3P, PI(3,4)P2, PI(4,5)P2 and PI(3,4,5)P3 (Steenblock et al. 2014). These N-terminal interactions localize Fgd6 to the plasma membrane of osteoclasts, where it couples cell adhesion and actin dynamics in order to regulate podosome formation through Cdc42 activation, as well as to endosomes to regulate retromer-dependent membrane recycling through the actin nucleation-promoting factor WASH. Thus, Fgd proteins localize to multiple membrane environments within endosomes and the cell surface as mediated by their multiple PI-specific domains.

The various interactions of Fgd proteins allow membrane remodeling and receptor trafficking. This is exemplified by Fgd5, the subcellular distribution and signaling of which has been studied in detail in endothelial cells. It associates with peripheral membrane cytoskeletal structures in cultured endothelial cells (Kurogane et al. 2012; Nakhaei-Nejad et al. 2012), suggesting a role in regulating cytsoskeletal structure remodelling. Consistent with this idea, Fgd5 loss-of-function by knockdown both decreased endothelial cell motility and monolayer wound repair in vitro (Kurogane et al. 2012), and assembly of

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matrix cytoskeletal adhesion structures in cultured endothelial cells (Nakhaei-Nejad et al. 2012). Remodelling of the endothelial cytoskeleton occurs after vascular endothelial growth factor (VEGF) stimulation of the vascular endothelial receptor-2 (VEGFR2) receptor tyrosine kinase. Activated VEGFR2 translocates to the endosome, where it forms an assembly with mTOR complex-2 and Akt to enable sustained cytoskeletal remodelling (Farhan et al. 2015). Fgd5 localizes to actin-rich lamellipodia and cytoplasmic structures, and specifically to the migratory leading edge (Farhan et al. 2017). Remarkably, Fgd5 loss-of-function perturbs the trafficking of VEGFR2, and assembly of this complex on the early endosome. It both associates with VEGFR2, and restrains transfer of VEGFR2 from the early endosome to the Rab7-positive late endosome degradation pathway (Farhan et al. 2017). Fgd5 loss further decreases stable association of mTORC2/Akt with activated VEGFR2 at the endosome. The exact mechanisms of the molecular interactions have not yet been defined, but may involve Fgd GEF, scaffolding or membrane binding functions.

GEF Activities of the Fgd Family Draft

Various intramolecular interactions regulate the activities of diverse Rho GEFs including Vav, Trio and Tim (Bustelo 2014; Das et al. 2000; Schiller et al. 2006; Yohe et al. 2007). The Fgd5 structure (PDB: 3mpx) includes the expected helical bundle for the DH domain, with its last helix being uniquely kinked and packed against PH1 (Fig. 3A) and provides a context for exploring its Rho specificity and regulation. In the case of Vav1, the tandem DH-PH domain is autoinhibited by intramolecular interactions with immediately adjacent upstream acidic domains that are stabilized by the downstream PH domain (Aghazadeh et al. 2000; B. Yu et al. 2010), with autoinhibition being relieved by phosphorylation of tyrosines in the acidic domain. Some Fgd proteins also contain acidic clusters immediately upstream of the DH domain and therefore we can speculate that they may have a similar autoinhibitory mechanism. Fgd6 is hyperphosphorylated on tyrosines 748, 754 and 760, and Fgd1 is hyperphosphorylated on Ser365 (Hornbeck et al. 2015), all of which are near acidic stretches of residues that precede DH domains. Thus, by analogy, these Fgd proteins may be activated by kinases, although additional relief mechanisms are also possible.

As is the case for all GEFs that contain tandem DH-PH domains, affinity for specific Rho proteins and subsequent activation of their downstream signaling is driven by interactions with Rho specificity patches. Key features for ligand specificity including the “QR” element are largely found within the DH domain

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(Snyder et al. 2002), but are divergent in Fgd proteins. While some Rho GEFs contain a specificity patch with low binding preference and interact with numerous Rho proteins, others are highly specific (Cook, Rossman, and Der 2014). Pharmacological inhibitors have been designed to disrupt this interface, which optimally can produce highly selective effects (Y. Gao et al. 2004; Montalvo-Ortiz et al. 2012; Smithers and Overduin 2016).

The specific partner of Fgd1–4 proteins and GEF activity is Cdc42 (Huber et al. 2008; Ono et al. 2000; Pasteris et al. 2000; Zheng et al. 1996). Amino acids upstream of the QR element for these family members are nearly identical. Apart from implications based on sequence homology, the GEF activity of Fgd5 and Fgd6 are so far uncharacterized. The specificity patch of Fgd5 and Fgd6 are quite dissimilar to the other Fgd family members and therefore distinct binding preferences are likely. The uniqueness of the Fgd5 and Fgd6 specificity patch also favours the development of highly specific pharmacological inhibitors (Nassar et al. 2006).

Effects Downstream of Fgd Proteins Draft

Rho GEFs catalyze the exchange of GDP for GTP which results in the activation of Rho proteins and subsequent downstream signaling events. There are several emerging roles for Fgd proteins in controlling such developmental programs. Fgd1, the founding member of the Fgd family, was originally identified as the gene defective in the Aarskog-Scott syndrome in which patients present multiple developmental defects, including bone malformations (Pasteris et al. 1994). Fgd1 promotes directional cell migration, and this function likely involves the stimulation of podosome formation and extracellular matrix remodeling (Daubon, Buccione, and Génot 2011; Toshiyuki Oshima et al. 2011). The formation of podosomes by osteoclasts is important for proper bone formation and resorption (Kylmaoja, Nakamura, and Tuukkanen 2016). Mutations in Fgd4 cause Charcot-Marie-Tooth disease, a neurological disorder that is characterized by progress sensory nerve loss likely due to defects in growth of neuron microspikes, which are membrane protrusions that require Cdc42 activation for formation (Boubaker et al. 2013; Delague et al. 2007; Hyun et al. 2015).

Disease-associated mutations have not been found for the other Fgd family members; however, roles in cellular processes are beginning to be defined. The Fgd2 gene is highly expressed in antigen presenting cells and has a role in endosomal-plasma membrane vesicle trafficking (Huber et al. 2008). Several studies

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indicate that Fgd5 contributes to pro-angiogenesis processes in endothelial cells (Farhan et al. 2017; Heldin et al. 2017). The Fgd5 gene is selectively expressed by endothelial cells (while Fgd2, 3 and 4 are not) (Ho et al. 2003), where it is upregulated upon stimulation by VEGF (Hernández-García et al. 2015). Fgd5 is involved in the regulation of endothelial adhesion, survival, and angiogenesis by modulating phosphatidylinositol 3-kinase signaling (Nakhaei-Nejad et al. 2012). Highly vascularized organs, such as lung and kidney in the adult mouse, also express Fgd5 (Kurogane et al. 2012). It plays an essential role in embryogenic development and vascular patterning, and is expressed in early hematopoietic stem cells and in a broad range of endothelial cells (Gazit et al. 2014). However it appears that expression of Fgd5 in endothelial cells must be tightly controlled, since Fgd5 transgene overexpression resulted in disordered retinal vascular development in the mouse post-natal retina (Cheng et al. 2012). Active pruning of the developing microvascular structures may depend on Fgd5, although its role in the stable mature vasculature, and during injury or repair, remains largely unknown (Cheng et al. 2012). Fgd6 is expressed in osteoclasts and was found to coordinately regulate multiple intracellular trafficking steps to maintain cell polarity (Steenblock et al. 2014). Collectively, these studies suggest the Fgd family of proteins are important regulators of cellular events thatDraft require signals to be transmitted from intracellular sites, such as endosome, to the cell periphery.

Cancer studies have identified Fgd proteins as potential biomarkers and drivers for cancer development. Elevated levels of expression of Fgd1 influence the aggressiveness of infiltrating and poorly differentiated breast and invasive prostate tumor cells (Ayala et al. 2009), consistent with its role in activation of Cdc42 (Smithers and Overduin 2016). Analysis of metastatic prostate tumors revealed Fgd1 amplification (Robinson et al. 2015). The FGD5 gene is frequently inactivated by methylation or deletions in renal cell and squamous cell carcinomas (Dmitriev et al. 2014). It is amplified in luminal breast cancer (Gatza et al. 2014), suggesting a role in carcinogenesis and utility as a prognostic marker (Valla et al. 2017). Sequencing studies have identified hundreds of mutations in Fgd genes and a wide variety of cancers (Cerami et al. 2012; J. Gao et al. 2013; Sjoblom et al. 2006), although their specific consequences have yet to be elucidated. Further analysis is clearly needed in order to understand the detailed binding mechanisms of Fgd proteins and exploit their signaling pathways for development of novel diagnostics or therapeutics. How the Fgd domains insert into lipid bilayers and recognize multiple PIs in a regulated manner remains of fundamental interest and could shed light on how the phosphoinositide code allows pairs of membranes to be recognized, linked and moved.

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Conclusion The multi-domain architecture of the Fgd family confers a platform for highly specific and regulated signal integration. The tandem DH and PH1 domains provide the GEF activity necessary for signal propagation to the appropriate downstream effectors. Spatial regulation of Fgd proteins appear to be mediated by multiple potential membrane interaction surfaces offered by the FYVE and PH2 domain. Analysis of all human FYVE sequences indicates a notable divergence of the Fgd family from the canonical PI3P binders. Divergence in the classical motifs known to be essential for PI head group stabilization suggest that the Fgd FYVE domains may accommodate different headgroup phosphorylation patterns, thereby providing unique membrane binding specificity. Detailed structural and functional studies are required to elucidate the Fgd FYVE-PI binding modes. Although the Fgd PH domains have not been studied in detail, inferences from other PH containing proteins suggest that PH2 may improve membrane avidity by interacting directly with lipids. While the MIL enhances bilayer affinity through non-specific protein- lipid interactions, phosphorylation of residues within this region may provide sufficient electrostatic repulsion to dissociate Fgd proteins from membranes. The exact processes by which the FYVE and PH2 domains interact individually or synergisticallyDraft with membranes remains unclear but is essential for both signal modulation, and targeting for the Fgd protein to the subcellular membrane surfaces. With many different possibilities for regulation, the spatial orientation and interactions of these domains appears to be critical for correct propagation of intracellular signaling. Early indications show the Fgd family are integral to physiological processes that require alterations to dynamic alterations cell morphology. Aberrant cell signaling caused by point mutations and upregulation leads to developmental defects and cancer progression. Identifying the specific roles that Fgd proteins play in their respective processes will provide insight into the molecular abnormalities that occur in diseased states. Understanding the mechanisms by which the Fgd family performs cellular functions may yet open new avenues for targeted invention.

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Hyun, Young Se et al. 2015. “Charcot-Marie-Tooth Disease Type 4H Resulting from Compound Heterozygous Mutations in FGD4 from Nonconsanguineous Korean Families.” Ann Hum Gen 79(6): 460–69. Kim, Yongman et al. 2002. “Association of Frabin with Specific Actin and Membrane Structures.” Genes Cells 7(4): 413–20. Kufareva, I. et al. 2014. “Discovery of Novel Membrane Binding Structures and Functions.” Biochem Cell Biol 92(6): 555–63. Kurogane, Yusuke et al. 2012. “FGD5 Mediates Proangiogenic Action of Vascular Endothelial Growth Factor in Human Vascular Endothelial Cells.” Arterioscler Thromb Vasc Biol 32(4): 988–96. Kutateladze, T., and M. Overduin. 2001. “Structural Mechanism of Endosome Docking by the FYVE Domain.” Science 291(5509). Kutateladze, T.G. et al. 1999. “Phosphatidylinositol 3-Phosphate Recognition by the FYVE Domain.” Molecular Cell 3(6). Kutateladze, T.G., and M. Overduin. 2004. “FYVE Domain.” In Handbook of Metalloproteins, eds. A. Messerschmidt, W. Bode, and M. Cygler.Draft Chichester: John Wiley & Sons, 390–99. Kylmaoja, Elina, Miho Nakamura, and Juha Tuukkanen. 2016. “Osteoclasts and Remodeling Based Bone Formation.” Curr Stem Cell Res Ther11(8): 626–33. Lee, S.A. et al. 2005. “Targeting of the FYVE Domain to Endosomal Membranes Is Regulated by a Histidine Switch.” Proceedings of the National Academy of Sciences of the United States of America 102(37). Lemmon, M.A. 2007. “Pleckstrin Homology (PH) Domains and Phosphoinositides.” Biochem Soc Symp 93(74): 81–93. Lenoir, M.; Ustunel, C.; Rajesh, S.; Kaur, J.; Moreau, D.; Gruenberg, J.; Overduin, M. 2018. “Phosphorylation of Conserved Phosphoinositide Binding Pocket Regulates Sorting Nexin Membrane Targeting.” Nature Commun 9(1):993. Lenoir, M. et al. 2010. “Structural Basis of Wedging the Golgi Membrane by FAPP Pleckstrin Homology Domains.” EMBO Reports 11(4). Lenoir M, Sugawara M, Kaur J, Ball LJ, Overduin M. 2004. “Multivalent Mechanism of Membrane Insertion by the FYVE Domain.” J Biol Chem 279(4): 3050–57. Lenoir, M., M. Grzybek, et al. 2015. “Structural Basis of Dynamic Membrane Recognition by Trans-Golgi Network Specific FAPP Proteins.” J Mol Biol 427(4): 966–81. Lenoir, M. et al. 2018. “Phosphorylation of Conserved Phosphoinositide Binding Pocket Regulates Sorting Nexin Membrane Targeting.” Nature Commun 9(1).

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Lenoir, M., I. Kufareva, R. Abagyan, and M. Overduin. 2015. “Membrane and Protein Interactions of the Pleckstrin Homology Domain Superfamily.” Membranes 5(4). Montalvo-Ortiz, Brenda L et al. 2012. “Characterization of EHop-016, Novel Small Molecule Inhibitor of Rac GTPase.” J Biol Chem 287(16): 13228–38. Nakhaei-Nejad, Maryam, George Haddad, Qiu-Xia Zhang, and Allan G Murray. 2012. “Facio-Genital Dysplasia-5 Regulates Matrix Adhesion and Survival of Human Endothelial Cells.” Arterioscler Thromb Vasc Biol 32(11): 2694–2701. Nassar, Nicolas et al. 2006. “Structure-Function Based Design of Small Molecule Inhibitors Targeting Rho Family GTPases.” Curr Top Med Chem 6(11): 1109–16. Obaishi, Hiroshi et al. 1998. “Frabin, a Novel FGD1-Related Actin Filament-Binding Protein Capable of Changing Cell Shape and Activating c-Jun N-Terminal Kinase.” J Biol Chem 273(30): 18697–700. Ono, Yuichi et al. 2000. “Two Actions of Frabin: Direct Activation of Cdc42 and Indirect Activation of Rac.” Oncogene 19(27): 3050–58. Orrico, Alfredo et al. 2004. “Phenotypic and Molecular Characterisation of the Aarskog-Scott Syndrome: A Survey of the Clinical Variability inDraft Light of FGD1 Mutation Analysis in 46 Patients.” Eur J Hum Genet 12(1): 16–23. Oshima, T, T Fujino, K Ando, and M Hayakawa. 2010. “Proline-Rich Domain Plays a Crucial Role in Extracellular Stimuli-Responsive Translocation of a Cdc42 Guanine Nucleotide Exchange Factor, FGD1.” Biol Pharm Bull 33(1): 35–39. Oshima, Toshiyuki, Tomofumi Fujino, Ken Ando, and Makio Hayakawa. 2011. “Role of FGD1, a Cdc42 Guanine Nucleotide Exchange Factor, in Epidermal Growth Factor-Stimulated c-Jun NH2-Terminal Kinase Activation and Cell Migration.” Biol Pharm Bull 34(1): 54–60. Overduin, M., M.L. Cheever, and T.G. Kutateladze. 2001. “Signaling with Phosphoinositides: Better than Binary.” Mol Interv 1(3). Pasteris, N G et al. 1994. “Isolation and Characterization of the Faciogenital Dysplasia (Aarskog-Scott Syndrome) Gene: A Putative Rho/Rac Guanine Nucleotide Exchange Factor.” Cell 79(4): 669–78. Pasteris, N G, K Nagata, A Hall, and J L Gorski. 2000. “Isolation, Characterization, and Mapping of the Mouse Fgd3 Gene, a New Faciogenital Dysplasia (FGD1; Aarskog Syndrome) Gene Homologue.” Gene 242(1–2): 237–47. Robinson, Dan et al. 2015. “Integrative Clinical Genomics of Advanced Prostate Cancer.” Cell 161(5): 1215– 28.

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Sankaran, V. G., D. E. Klein, M. M. Sachdeva, and M. A. Lemmon. 2001. “High-Affinity Binding of a FYVE Domain to Phosphatidylinositol 3-Phosphate Requires Intact Phospholipid but Not FYVE Domain Oligomerization.” Biochemistry 40(29): 8581–87. Schiller, Martin R. et al. 2006. “Regulation of RhoGEF Activity by Intramolecular and Intermolecular SH3 Domain Interactions.” J Biol Chem 281(27): 18774–86. Sievers, Fabian et al. 2011. “Fast, Scalable Generation of High-Quality Protein Multiple Sequence Alignments Using Clustal Omega.” Molecular Systems Biology 7. Sjoblom, T. et al. 2006. “The Consensus Coding Sequences of Human Breast and Colorectal Cancers.” Science 314(5797): 268–74. Smithers, C.C., and M. Overduin. 2016. “Structural Mechanisms and Drug Discovery Prospects of Rho GTpases.” Cells 5(2). Snyder, Jason T et al. 2002. “Structural Basis for the Selective Activation of Rho GTPases by Dbl Exchange Factors.” Nature structural biology 9(6): 468–75. Steenblock, Charlotte et al. 2014. “The Cdc42 Guanine Nucleotide Exchange Factor FGD6 Coordinates Cell Polarity and Endosomal Membrane RecyclingDraft in Osteoclasts.” J Biol Chem 289(26): 18347–59. Valla, Marit et al. 2017. “FGD5 Amplification in Breast Cancer Patients Is Associated with Tumour Proliferation and a Poorer Prognosis.” Breast Cancer Research and Treatment 162(2): 243–53. Waterhouse, A M et al. 2009. “Jalview Version 2: A Multiple Sequence Alignment and Analysis Workbench.” Bioinformatics 25(9): 1189–91. Yohe, Marielle E. et al. 2007. “Auto-Inhibition of the Dbl Family Protein Tim by an N-Terminal Helical Motif.” J Biol Chem 282(18): 13813–23. Yu, Bingke et al. 2010. “Structural and Energetic Mechanisms of Cooperative Autoinhibition and Activation of Vav1.” Cell 140(2): 246–56. Yu, Jong W. et al. 2004. “Genome-Wide Analysis of Membrane Targeting by S. Cerevisiae Pleckstrin Homology Domains.” Molecular Cell 13(5): 677–88. Zheng, Y et al. 1996. “The Faciogenital Dysplasia Gene Product FGD1 Functions as a Cdc42Hs-Specific Guanine-Nucleotide Exchange Factor.” J Biol Chem 271(52): 33169–72.

Figures

Figure 1. Modular architectures of human Fgd proteins. The DH, FYVE and PH domains are shown for Fgd1 (also known as FgdY, ZFYVE3), Fgd2 (ZFYVE4), Fgd3 (ZFYVE5), Fgd4 (Frabin), Fgd5 (ZFYVE23), and Fgd6

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(ZFYVE24). The number of amino acids in the main isoform is shown. Positions of frequent phosphorylation sites (Hornbeck et al. 2015) and disease-linked substitutions are indicated by green and red labels, respectively.

Figure 2. Structure-aided sequence alignment of DH, FYVE and PH domains of the six human Fgd proteins. The domain boundaries, variable linkers and secondary structures from PDB files shown in Fig. 3 are indicated above the alignments, with strand and helix names alongside. The sequences were aligned with Clustal Omega (Sievers et al. 2011) and coloured with Jalview (Waterhouse et al. 2009). FYVE domain motifs which typically bind membranes are indicated. Each residue which is phosphorylated (Hornbeck et al. 2015) or that is positioned to interact with lipid bilayers is indicated with an asterisk.

Figure 3. Structures of Fgd protein domains. A) The backbone trace of the tandem DH and PH1 domain structure of human Fgd5 (3mpx) was rendered with PyMOL (DeLano 2014). The domains, key residues and the N- and C-termini are labelled. The sidechains of basic residues which are predicted by MODA to have significant membrane binding propensitiesDraft or are positioned nearby are shown in red, as are nearby polar, aliphatic and aromatic residues. Y1142 represents a potential PIP-stop residue where phosphorylation could interfere with membrane binding (Lenoir et al, 2018) and Y1199 phosphorylation could conceivably alter interdomain dynamics. B) The backbone trace of the EEA1 FYVE domain (PDB: 1joc). The PI3P headgroup (1,3-diphosphate) is indicated in yellow. The sidechains of key stabilizing residues are shown as sticks, with negatively charged groups in cyan, negatively charged groups in red and aromatic groups in magenta. The MIL is indicated in blue. Zinc ions are denoted as orange spheres. C) The murine Fgd6 PH2 domain (1wgq) structure is shown, which is similar to human Fgd3 PH2 domain (2coc) but has stronger membrane binding propensity. The basic, aliphatic and aromatic residues predicted by MODA (Kufareva et al. 2014) analysis are coloured red, orange and magenta, respectively and labelled.

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Title:

Structure and Function of the Fgd Family of Divergent FYVE Domain Proteins

Authors:

Gary Eitzen1, Cameron C. Smithers2, Allan G Murray3 and Michael Overduin2* Draft

1Department of Cell Biology, 2Department of Biochemistry, 3Department of Medicine, University of Alberta, Edmonton, Alberta, Canada

*Corresponding author. Michael Overduin Telephone: +1 780 492 3518 Fax: +1 780 492-0886 E-mail: [email protected]

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Abstract

FYVE domains are highly conserved protein modules that typically bind phosphatidylinositol 3-phosphate (PI3P) on the surface of early endosomes. Along with pleckstrin homology (PH) and phox homology (PX) domains, FYVE domains are the principal readers of the phosphoinositide (PI) code that mediate specific recognition of eukaryotic organelles. Of all the human FYVE domain-containing proteins, those within the Facio-genittial dysplasia (Fgd) subfamily are particularly divergent, and couple with GTPases to exert unique cellular functions. The subcellular distributions and functions of these evolutionarily conserved signal transducers, which also include Dbl homology (DH) and two PH domains, are discussed here in order to better understand the biological range of processes that such multidomain proteins engage in. Determinants of their various functions include specific multidomain architectures, post-translational modifications including PIP stops that have been discovered in sorting nexins, PI recognition motifs and phospholipid binding surfaces as defined by the Membrane Optimal Docking Area (MODA) program. How these orchestrate Fgd function remains unclearDraft but has implications for developmental diseases including Aarskog-Scott syndrome, which is also known as faciogenital dysplasia, and forms of cancer that are associated with mutations and amplifications of Fgd genes.

Keywords:

Cdc432, Dbl, FYVE, Fgd, GEF, GTPase, PH, phosphoinositide, pleckstrin, cancer, faciogenital dysplasia, Aarskog-Scott syndrome , lipid signaling, membrane trafficking, MODA, PIP, Rho.

Text:

Phosphoinositide Code

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That is, amino acid residues are either phosphorylated or unphosphorylated. Mononucleotides are represented by multiple forms such as the cGMP, GMP, GDP and GTP derivatives of guanosine present in eukaryotes, and regulate the activities of proteins such as GTPases. The phosphatidylinositol headgroup is differentially phosphorylated at the 3-, 4- and/or 5- positions to yield 7 distinct PI lipids, which are distributed to various organelles where they are recognized by FYVE, PH, PX and other domains. These modules serve as principal readers of the PI code and direct membrane-dependent signaling and trafficking in eukaryotes (Overduin, Cheever, and Kutateladze 2001). Together, these protein, nucleotide and lipid modifications offers a rich tapestry of signaling states that encodes biological information to determine cellular fate. Our understanding of how this actually works is in its infancy, as is illustrated here by the complexity of Fgd proteins. This family is unique in terms of its architecture, integrating strands of information through multiple domains that recognize specific phospholipids and proteins including GTPases to drive cellular development. Here we review what is known about the family and explore the roles of the component domains and family members.

Architecture of the Fgd Family Draft

The human genome encodes six Fgd genes (Fig 1). Homologues are present in most metazoans, and diverged early in chordate evolution while maintaining the same modular architecture. They act as Rho guanine-nucleotide exchange factors (Rho GEFs) that are thought to be guided to membranes by PI- binding FYVE and PH domains. They contain the typical Rho GEF subdomain structure of a DH domain adjacent to the first PH domain (PH1). The DH and PH1 domains function , which together to mediate the regulated activation of Rho proteins that by binding to and catalyzinge the exchange of GDP for GTP nucleotides. All Fgd proteins contain a divergent FYVE domain followed by a second PH domain (PH2) at their C-termini. The long N-termini of Fgd proteins are poorly conserved and apparently disordered. Fgd1 contains an N-terminal proline-rich element which is phosphorylated (Hornbeck et al. 2015), influences in dynamic peripheral membrane localization dynamics (T Oshima et al. 2010), and harbours a critical point mutation linked to Aarskog-Scott syndrome (Orrico et al. 2004). The Fgd4 protein interacts with F-actin via its N-terminal 150 residues (Obaishi et al. 1998). The structural domains of Fgd proteins are discussed individually below, including descriptions of structural and regulatory features.

FYVE Domains

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FYVE domains are found in 29 human proteins, which exhibit a wide range of architectures, with that of the Fgd subfamily (Fig 1) being most recurrent (Fig 1). This short module is named after four proteins in which its 70 residue sequence was first identified: Fab1p, YOTB, Vac1p and EEA1. The fold of the FYVE domain consists of two double-stranded antiparallel β sheets and a C-terminal α-helix (T. G. Kutateladze and Overduin 2004). Two zinc ions are gripped tightly by four cysteines in a tetrahedral arrangement to provide essential structural stabilization (T. G. Kutateladze et al. 1999). FYVE domains represent the most tightly conserved lipid recognition module, and are thought to almost always recognize PI3P as their cognate ligand (T. Kutateladze and Overduin 2001). Organelle recognition involves electrostatic approach and dipping of a membrane insertion loop (MIL) into a bed of PI3P, phosphatidylserine (PS) and phosphatidycholine (PC) molecules for firm anchoring and stereospecific lipid headgroup recognition (T. G. Kutateladze et al. 2004). This bilayer binding is strengthened by the acidic microenvironments found along the endocytic route, thus keeping proteins more stably tethered to low pH endosomal membranes (Lee et al. 2005) rather than the other PI3P pools found in the cell .

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While no structures have been determined of any Fgd FYVE domain, other atypical FYVE domains have been resolved. The portrudin FYVE domain of the Protrudin protein also lacks the canonical PI binding motifs, and prefers to bind doubly and triply phosphorylated PIs, as measured at pH 7.4. This interaction depends on the integrity of its PI reader element which contains a novel KKRRSCS sequence in place of the central canonical motif. This sequence lacks the pH-switch represented by the pair of conserved histidines, and directs protrudin to different vesicles and plasma membrane compartments that contain

PI(4,5)P2 (Gil et al. 2012). These results are consistent with Fgd FYVE domains possessing broader PI binding profiles while retaining pH dependentDraft PI3P recognition.

The regulation of the Fgd family may depend not just on lipid levels, but also on protein phosphorylation. In several cancer cells Fgd3 is hyperphosphorylated on a pair of conserved serine and threonine residues within its membrane insertion loop (MIL) (Figs. 1&2) (Hornbeck et al. 2015). This feature is analogous to the PIP-stop, a phosphorylated switch residue which toggles endosomal localization and PI3P binding as discovered in PX domain-containing Snx proteins (Lenoir et al. 2018). Unlike the EEA1 protein which dimerizes through a coiled coil to increase membrane avidity, Fgd family members lack obvious multimerization motifs, and may instead rely on its neighbouring its multiple membrane binding domains. These modules are connected via variable, unstructured linkers that are vulnerable susceptible to phosphorylation (Fig. 2), which could regulate multi-domain membrane binding.

PH Domains

PH domains are were the first module that was found to recognize a PI (Sankaran et al. 2001). They are present in 285 human proteins, nine per cent of which contain a pair of PH domains as in Fgd proteins (Lenoir, Kufareva, et al. 2015). The PH fold consists of approximately 120 amino acids that fold into a seven stranded antiparallel β-sheet capped by a C-terminal α-helix. Analysis of the surface properties of PH

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that PI(3,4,5)P3 and PI(4,5)P2 are the most common PH ligands. Unusually, PEPP1 binds PI3P spotted on nitrocellulose membranes through its PH domain (DOWLER et al. 2000), although these results have been questioned (Lemmon 2007).

The structures of the Fgd PH1 and PH2 domains (PDB: 1wgq, 2coc, 3mpx) possess the expected folds, with that of PH1 packing against the last helix of the DH domain (Fig. 3). They display membrane binding propensities within their exposed β1-β2 loops and proximal residues based on MODA analysis (Kufareva et al. 2014). Additional positively charged sidechains that are absolutely conserved and exposed in the β1- β2 sheet of PH1 may be also involved in lipid binding, although they lack discernable electron density in the Fgd5 crystal (PDB: 3mpx). The PH2 domain of Fgd6 displays an extensive membrane binding surface that is rich in lysines and aromatic residues (PDB: 1wgq) (Fig. 3C). In contrast, the solution structure of human Fgd3’s PH2 domain (PDB: 2coc) displaysDraft little membrane binding propensity based on MODA analysis (Lenoir, Kufareva, et al. 2015). The PH1 domain of Fgd4 binds preferentially to surfaces presenting biotin-conjugated PI3P molecules, and also weakly to PI(4,5)P2, although the full breadth of the PI specificity is unclear as PI4P and PI5P were not tested (Catimel et al. 2013). Phosphorylation events in Fgd PH1 domains include recurrent phosphorylation of a conserved tyrosine in the β3 strand near the membrane insertion loop, suggesting another PIP stop (Lenoir, M.; Ustunel, C.; Rajesh, S.; Kaur, J.; Moreau, D.; Gruenberg, J.; Overduin 2018), as well as in the C-terminal helix which could affect interdomain interactions. Together this suggests that Fgd PH1 and PH2 domains can mediate regulated PI interactions that could synergize with those of the FYVE domain, and could offer a wider breadth of lipid specificities and membrane affinities.

Analogies can be drawn to other PH domains. The FAPP1 trafficking protein is recruited to trans-Golgi network membranes via its N-terminal PH domain. The action involves stereospecific recognition of PI4P, along with complementary interactions with PS and PC lipids (Lenoir et al. 2010), and simultaneously interaction with the GTP-loaded form of Arf1 (He et al. 2011). The lipids must be in a disordered phase for FAPP1’s PH domain to insert into the bilayer (Lenoir, Grzybek, et al. 2015). It is possible that Fgd’s PH domains also prefer to bind disordered phase lipids, although their membrane insertion loops are shorter and hence could individually bind more superficially. Coincident binding of Fgd proteins to lipid and

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Localization of Fgd proteins

FGD1 associates with the subcortical actin cytoskeleton through interactions of its N-terminal region with cortactin and actin-binding protein 1 (Hou et al. 2003). Fgd2 is directed to endosomal membranes through FYVE domain-mediated recognition of PI3P. In addition, it is recruited to actin-rich lamellipodia via PH2 domain interaction with membrane ruffles, which are enriched in PI(4,5)P2 and PI(3,4,5)P3, while the full- length protein also binds PI5P (Huber et al. 2008). Through these actions, Fgd2 may be positioned to influence cell polarization and migration orDraft micropinocytosis. Fgd3 induces and localizes to lamellipodia (Hayakawa et al. 2008), and, when expressed as a tandem DH and PH1 construct, induces formation of filopodial extensions (Pasteris et al. 2000). FGD4 is predominantly recruited to the actin cytoskeleton through its N-terminal region to mediate formation of filopodia, with its PI recognition domains serving as a bridge to the membrane, including its FYVE domain’s interaction with membrane ruffles (Kim et al.

2002). A Fgd6 construct missing the N-terminal region binds to PI3P, PI(3,4)P2, PI(4,5)P2 and PI(3,4,5)P3 (Steenblock et al. 2014). These N-terminal interactions localizes Fgd6 to the plasma membrane of osteoclasts, where it couples cell adhesion and actin dynamics in order to regulate podosome formation through Cdc42 activation, as well as to endosomes to regulate retromer-dependent membrane recycling through the actin nucleation-promoting factor WASH. ThusThus, Fgd proteins localize to multiple membrane environments within endosomes and the cell surface as mediated by their multiple PI-specific domains.

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endothelial cell motility and monolayer wound repair in vitro (Kurogane et al. 2012), and assembly of matrix cytoskeletal adhesion structures in cultured endothelial cells (Nakhaei-Nejad et al. 2012). Remodelling of the endothelial cytoskeleton occurs after vascular endothelial growth factor (VEGF) stimulation of the vascular endothelial receptor-2 (VEGFR2) receptor tyrosine kinase. Activated VEGFR2 translocates to the endosome, and where it forms an assembly with mTOR complex-2 and Akt to enable sustained cytoskeletal remodelling (Farhan et al. 2015). Fgd5 localizes to actin-rich lamellipodia and cytoplasmic structures, and specifically to the migratory leading edge (Farhan et al. 2017). Remarkably, Fgd5 loss-of-function perturbs the trafficking of the VEGFR2, and assembly of this complex on the early endosome. It both associates with VEGFR2, and restrains transfer of VEGFR2 from the early endosome to the Rab7-positive+ late endosome degradation pathway (Farhan et al. 2017). Fgd5 loss further decreases stable association of mTORC2/Akt with activated VEGFR2 at the endosome. The exact mechanisms of the molecular interactions have not yet been defined, but may involve Fgd GEF, scaffolding or membrane binding functions.

GEF Activities of the Fgd Family Draft

Various intramolecular interactions regulate the activities of diverse Rho GEFs including Vav, Trio and Tim (Bustelo 2014; Das et al. 2000; Schiller et al. 2006; Yohe et al. 2007). The Fgd5 structure (PDB: 3mpx) includes the expected helical bundle for the DH domain, with its last helix being uniquely kinked and packed against PH1 (Fig. 3A) and provides a context for exploring its Rho specificity and regulation. In the case of Vav1, the tandem DH-PH domain is autoinhibited by intramolecular interactions with immediately adjacent upstream acidic domains that are stabilized by the downstream PH domain (Aghazadeh et al. 2000; B. Yu et al. 2010), with autoinhibition being relieved by phosphorylation of tyrosines in the acidic domain. Some Fgd proteins also contain acidic clusters immediately upstream of the DH domain and therefore we can speculate that they may have a similar autoinhibitory mechanism. Fgd6 is hyperphosphorylated on tyrosines 748, 754 and 760, and Fgd1 is hyperphosphorylated on Ser365 (Hornbeck et al. 2015), all of which are near acidic stretches of residues that precede DH domains. ThusThus, by analogy, these Fgd proteins may be activated by kinases, although additional relief mechanisms are also possible.

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Key features for ligand specificity including the “QR” element are largely found within the DH domain (Snyder et al. 2002), but are divergent in Fgd proteins. While some Rho GEFs contain a specificity patch with low binding preference and interact with numerous Rho proteins, others are highly specific (Cook, Rossman, and Der 2014). Pharmacological inhibitors have been designed to disrupt this interface, which optimally can produce highly selective effects (Y. Gao et al. 2004; Montalvo-Ortiz et al. 2012; Smithers and Overduin 2016).

The specific partner of Fgd1 – 4 proteins and GEF activity is Cdc42 (Huber et al. 2008; Ono et al. 2000; Pasteris et al. 2000; Zheng et al. 1996). Amino acids upstream of the QR element for these family members are nearly identical. Apart from implications based on sequence homology, the GEF activity of Fgd5 and Fgd6 are so far uncharacterized. The specificity patch of Fgd5 and Fgd6 are quite dissimilar to the other Fgd family members and therefore distinct binding preferences are likely. The uniqueness of the Fgd5 and Fgd6 specificity patch also favours the development of highly specific pharmacological inhibitors (Nassar et al. 2006). Draft Effects Downstream of Fgd Proteins

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cells and has a role in endosomal-plasma membrane vesicle trafficking (Huber et al. 2008). Several studies indicate that Fgd5 contributes to pro-angiogenesis processes in endothelial cells (Farhan et al. 2017; Heldin et al. 2017). The Fgd5 gene is selectively expressed by endothelial cells (while Fgd2, 3 and 4 are not) (Ho et al. 2003), where it is upregulated upon stimulation by VEGF (Hernández-García et al. 2015). The Fgd5 protein is involved in the regulation of endothelial adhesion, survival, and angiogenesis by modulating phosphatidylinositol 3-kinase signaling (Nakhaei-Nejad et al. 2012). Highly vascularized organs, such as lung and kidney in the adult mouse, also express Fgd5 (Kurogane et al. 2012). It plays an essential role in embryogenic development and vascular patterning, and is expressed in early hematopoietic stem cells and in a broad range of endothelial cells (Gazit et al. 2014). However it appears that expression of Fgd5 in endothelial cells must be tightly controlled, since Fgd5 transgene overexpression also resulted in disordered retinal vascular development in the mouse post-natal retina (Cheng et al. 2012). Active pruning of the developing microvascular structures may depend on Fgd5, although its role in the stable mature vasculature, and during injury or repair, remains largely unknown (Cheng et al. 2012). Fgd6 is expressed in osteoclasts and was found to coordinately regulate multiple intracellular trafficking steps to maintain cellDraft polarity (Steenblock et al. 2014). Collectively, these studies suggest the Fgd family of proteins are important regulators of cellular events that require signals to be transmitted from intracellular sites, such as endosome, to the cell periphery.

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Conclusion The multi-domain architecture of the Fgd family confers a platform for highly specific and regulated signal integration. The tandem DH and PH1 domains provide the GEF activity necessary for signal propagation to the appropriate downstream effectors. Spatial regulation of Fgd proteins appear to be mediated by multiple potential membrane interaction surfaces offered by the FYVE and PH2 domain. Analysis of all human FYVE sequences indicates a notable divergence of the Fgd family from the canonical PI3P binders. Divergence in the classical motifs known to be essential for PI head group stabilization suggest that the Fgd FYVE domains may accommodate different headgroup phosphorylation patterns, thereby providing unique membrane binding specificity. Detailed structural and functional studies are required to elucidate the Fgd FYVE-PI binding modes. Although the Fgd PH domains have not been studied in detail, inferences from other PH containing proteins suggest that PH2 may improve membrane avidity by interacting directly with lipids. While the MIL enhances bilayer affinity through non-specific protein- lipid interactions, phosphorylation of residues within this region may provide sufficient electrostatic repulsion to dissociate Fgd proteins from membranes.Draft The exact processes by which the FYVE and PH2 domains interact individually or synergistically with membranes remains unclear but is essential for both signal modulation, and targeting for the Fgd protein to the subcellular membrane surfaces. With many different possibilities for regulation, the spatial orientation and interactions of these domains appears to be critical for correct propagation of intracellular signaling. Early indications show the Fgd family are integral to physiological processes that require alterations to dynamic alterations cell morphology. Aberrant cell signaling caused by point mutations and upregulation leads to developmental defects and cancer progression. Identifying the specific roles that Fgd proteins play in their respective processes will provide insight into the molecular abnormalities that occur in diseased states. Understanding the mechanisms by which the Fgd family performs cellular functions may yet open new avenues for targeted invention.

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Figures

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Fgd1 R402W N424D S48 S116 S135 S205 S365 T711 S715 DH PH1 FYVE PH2 961 S205I P312L E380A R443H R522H R610Q Fgd2 S11 S48 S654 DH PH1 FYVE PH2 655

Fgd3 S446 S547 T549 S128 Draft 725 DH PH1 FYVE PH2

Fgd4 R442H M566I S702 Y36 DH PH1 FYVE PH2 766

M298R/T M345T R435Q R468Q Fgd5 S744 Y820 Y1142 Y1199 DH PH1 FYVE PH2 1462

Fgd6 S515 S605 S721 Y754 S1197 DH PH1 FYVE PH2 1430 S554 S692 Y748 Y760

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Draft

Figure 2 as CMYK

296x226mm (300 x 300 DPI)

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Draft

FIGURE 3 CMYK

251x172mm (300 x 300 DPI)

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