As already mentioned, a variety of proteins have been described to bind to syndecan extra- and intracellular domains. The abundance of extracellular ligands makes it impossible to mention all of them here. Besides, the intracellular binding partners are of special interest as they might be involved in signal transduction pathways and their analysis might reveal common mechanisms.
Therefore these proteins or protein families will be presented in the following chapter.
1.7.1. PDZ proteins
These proteins take their name from three proteins: PSD-95 (Postsynaptic Density-95), Discs-large (Dlg) and Zonula Occludens-1 (ZO-1). PSD-95 is a 95 kDa protein of the postsynaptic density; Dlg is the product of Drosophila discs large-1 tumor suppressor gene, and ZO-1 is a vertebrate tight-junction protein (Cohen et al., 1998). These proteins bind specifically to C-terminal sequences at the inner surface and are thought to link membrane components to the underlying actin-containing cytoskeleton (Bernfield et al., 1999).
The common feature of these proteins is the PDZ-domains, which are domains (with about 80 amino acids) composed of compact α- and β-modules, containing 5-6 β-strands and two α-helices. The binding peptide fits into a hydrophobic pocket. PDZ domains are selective (Grootjans et al., 2000). There are two classes of PDZ domains: Type I binds to peptides with a terminal (S/T)XV consensus motif and Type II selects peptides with hydrophobic or aromatic side chains at position –2 relative to the C-terminal (Cohen et al., 1998; Grootjans et al., 2000).
Among the syndecan-binding proteins, many are members of the PDZ-family such as syntenin, synectin and MAGUKs (membrane-associated guanylate kinases), which can bind to the last four amino acids (EFYA) of the syndecan cytoplasmic tail.
1.7.1.1. Syntenin
Syntenin (33 kDa) presents two (tandem) class II PDZ domains. It was shown to interact with syndecan-2 by co-localization, two-hybrid system and surface resonance experiments, as well as overlay assays. Although these results were obtained for syndecan-2, it seems that syntenin does not discriminate between syndecans, as it binds to the motif EFYA that is identical in all syndecans. Syntenin is highly expressed in cell surface projections, cell adhesion sites, FAs, SFs and in the nucleus. Over-expression provokes flattened cell morphology and enhances plasma extensions, which can be reverted by active Rho A. Its function might be that of an adaptor, coupling syndecan to the cytoskeleton (Grootjans et al., 1997). In detail, both PDZ domains can interact with syndecan by cooperative binding and the stoichiometry between syndecan: syntenin is 2:1. Syntenin also binds to neurexins, B-class ephrins, and ephrin receptors (Grootjans et al., 2000; Zimmermann et al., 2001). The PDZ domains are also required for PM localization of syntenin.
1.7.1.2. Synectin
Synectin also belongs to the Type II class of PDZ proteins and its over-expression inhibits migration in ECV304 cells in a dose-dependent manner without affecting adhesion or growth rate of cells. Its molecular mass is 36.1 kDa and it is highly expressed in brain and spleen. It forms homodimers and heterodimers with syntenin and might be involved in the assembly of signaling complexes by serving as a scaffold. Synectin binds exclusively to syndecan-4. This
1. Introduction
22
binding requires additional sequences to the PDZ domain. This does not rule out the possibility that other similar proteins might also bind to the other syndecans (Gao et al., 2000).
1.7.1.3. MAGUKs
MAGUKs are important kinases in the organization of membrane signaling. They possess an src homology 3 (SH3) domain, a domain with homology to guanylate kinase (GUK) without enzymatic activity, a PDZ domain and an N-terminal Ca2+-calmodulin dependent protein kinase- (CAMK-) like domain. They might operate as scaffolding proteins that recruit or organize other proteins at the PM to coordinate signal transduction within the cortical cytoskeleton.
h-CASK is the human homologue of LIN-2A, a Caenorhabditis elegans scaffolding protein.
Mutations in the lin-2 gene inactivate the LET-23 receptor tyrosine kinase/Ras/Map kinase pathway necessary for vulval cell differentiation in C. elegans. h-CASK is ubiquitously expressed, localizes to the PM of epithelial cells and was found to bind to syndecan-2 and protein 4.1 by two-hybrid experiments (Cohen et al., 1998; Craven and Bredt, 1998; Hsueh et al., 1998). This led to the theory that h-CASK might mediate a link between ECM, syndecans and actin via protein 4.1 (Cohen et al., 1998). Protein 4.1 belongs to the ERM (Ezrin/Radixin/Moesin) family, which will be discussed later (see 1.7.4).
hCASK binds to all four syndecans, although with higher affinity to -2 and -4, and to neurexins, TM proteins that are localized near synapses and thought to play role in axon guidance or adhesion (Cohen et al., 1998; Hsueh et al., 1998; Hsueh and Sheng, 1999).
The interactions of syndecans with CASK gained importance when it was discovered, that CASK could translocate to the nucleus. It was shown to regulate gene transcription by interacting with Tbr-1, a T-box transcription factor and to induce for example the expression of reelin, which is essential for cerebrocortical development (Bredt, 2000; Hsueh et al., 2000). By co-expressing syndecan-3 with CASK, CASK translocation to the nucleus was inhibited. This offers an exciting possible explanation of how syndecans might regulate gene expression.
1.7.2. Synbindin
Synbindin is a 24-kDa protein that presents homology to yeast proteins participating in vesicle transport. It was isolated by two-hybrid system assays using the cytoplasmic domains of syndecan-2 as bait. Although it binds to the EFYA motif of syndecan-2, it has no classical PDZ domains. It is mainly found in dendritic cells, especially in the post-synaptic density, co-localizing with syndecan-2 (Ethell et al., 2000). These authors suggest that synbindin clustering induced by syndecan-2 might facilitate local synthesis and transport of neurotransmitter receptors (see 1.5.2 and fig. 1.11). No association of synbindin to other syndecans has been described.
1.7.3. Syndesmos
This protein specifically interacts with parts of the C1- and the V-region of syndecan-4. This 40-kDa protein is ubiquitously expressed and can be myristylated. It co-localizes with syndecan-4 in cells plated on FN and its over-expression leads to cell spreading and FA contact formation in a serum-dependent manner. At mRNA level, syndecan-4 and syndesmos are expressed in the same tissues (Baciu et al., 2000). These authors suggest an involvement of syndesmos in FA formation, in cooperation with syndecan-4. This theory was supported by recent findings that syndesmos also binds to the focal adhesion adaptor protein, paxilin, and to the paxilin homologue hic-5 (Denhez et al., 2002).
1. Introduction
1.7.4. The ERM family
The ERM family is named after the first three members to be isolated. Ezrin was first discovered as a structural component of microvilli, radixin as an F-actin capping protein from adherens junctions, and moesin as heparin-binding molecule. They belong to the superfamily of the 4.1 proteins, which also comprises the protein 4.1 and merlin/schwannomin (among others).
Suppression of ezrin, radixin and moesin proteins led to the destruction of microvilli, cell-cell and cell-substrate adhesion, suggesting a role of Erm family members in these processes. They have been proposed as cross-linkers between transmembrane proteins and the actin cytoskeleton (Bretscher, 1999; Bretscher et al., 2000; Mangeat et al., 1999; Tsukita and Yonemura, 1997; Tsukita and Yonemura, 1999; Vaheri et al., 1997). In cells, they are present in an inactive form, with the N-terminal domain interacting with the C-terminal domain. They can be activated by Rho A and PIP2, see fig. 1.13.
Granes (Granes et al., 2000) showed that ezrin interacts with syndecan-2 in vivo by co-localization and co-immunoprecipitation from COS-1 cells. This interaction was resistant to 0.2%
Triton X-100, indicating that ezrin interacted with the cytoskeleton. Rho A or lysophosphatidic acid (LPA) treatment increased both syndecan-2 insolubility and syndecan-2/ezrin interactions.
This indicated that the ERM protein was implicated in the signal transduction from syndecan-2.
Figure 1.13: Scheme of ERM family activation. ERM proteins (here represented by ezrin), are present in an inactive form due to intramolecular interactions. Rho A activates ERMs by increasing PIP2 via PIP4K and by activating ROCK thereby unfolding the protein, enabling it to bind directly or indirectly via adaptor proteins to transmembrane receptor. By head-to-tail binding, they can convey signal from those receptors to the actin cytoskeleton. PIP2 = Phosphatidyl inositol phosphate 2, CFTR = Cystic fibrosis transmembrane conductance regulator, ICAM = Intercellular adhesion molecule-1, ROCK = Rho-associated kinase, EBP 50 = (ERM)-binding phosphoprotein-50, ERM = Ezrin-Radixin-Moesin, β2-AdrR = β2-Adrenalin-R, NHE-3 = Na+/H+-exchanger-3, E3KARP = NHE3 kinase A regulatory protein, PKA = Protein kinase A, RhoGDI = Rho GDP dissociation inhibitor.
The ERM-Family
Family members
Ezrin Radixin Moesin
(Merli n/ Schwannomin) (Protein 4.1)
N
N
PDZ1 PDZ2 EBP50
Inactive Ezri n
F-Actin
Activation
C N
C N
N N
CFTR CFTR
N
CD44, ICAM-1,-2, syndecan-2; RhoGDI CFTR, β2-AdrR via EBP50, E3KARP
RII-PKA F-Actin
PIP2 RhoA
PDZ1 PDZ2 EBP50
ROCK
The ERM-Family
Family members
Ezrin Radixin Moesin
(Merli n/ Schwannomin) (Protein 4.1)
N
N
PDZ1 PDZ2 EBP50 PDZ1 PDZ2 EBP50
Inactive Ezri n
F-Actin
Activation
C N
C N
N N
CFTR CFTR
N
CD44, ICAM-1,-2, syndecan-2; RhoGDI CFTR, β2-AdrR via EBP50, E3KARP
RII-PKA F-Actin
PIP2 RhoA
PDZ1 PDZ2 EBP50 PDZ1 PDZ2 EBP50
ROCK
1. Introduction
24
1.7.5. Neurofibromin
Neurofibromin is a large tumor-suppressor protein expressed from the neurofibromatosis type 1 (NFT1) gene. Its mutation leads to benign or malignant tumors of the nervous system. It binds to all syndecans shown by two-hybrid experiments. For interaction, the TM and the TM proximal sequences of syndecans are necessary. Neurofibromin, syndecan-2 and CASK have overlapping distribution, as shown by co-IP from rat brain and immunostaining (Hsueh et al., 2001).
1.7.6. Others
The binding of PKC and PIP2, specific for syndecan-4, as well as the specific binding of src, fyn, cortactin and tubulin for syndecan-3, have already been discussed earlier. The association with microfilament has also been mentioned briefly in the text and will not be discussed further.