SAP97 binds two other mLIN-7 binding MAGUK proteins. One of these MAGUK proteins, DLG3, coimmunoprecipitates with SAP97 in lysates from rat brain and transfected MDCK cells. This interaction requires the MRE (MAGUK recruitment) domain of SAP97 and surprisingly, both the L27N and L27 carboxyl-terminal (L27C) domains of DLG3. SAP97 can interact with the MAGUK protein, DLG2, but not the highly related protein, PALS2. The ability of SAP97 to interact with multiple MAGUK proteins is likely to be important for the targeting of specific protein complexes in polarized cells (Karnak et al., 2002).
The kinesin-3 motor protein, GAKIN, is regulated by the direct binding of its protein cargo hDlg. Direct binding of the SH3-I3-GUK module of hDlg to the MAGUK Binding Stalk domain of GAKIN activates the microtubule-stimulated ATPase activity of GAKIN (Hanada et al., 2000; Yamada et al., 2007; Unno et al., 2008).
Using the yeast two-hybrid screening a novel protein from a human cDNA library was isolated as a binding partner of DLG1. This protein is a component of TJs rather than AJs (where DLG1 is normally found), even if it is incorporated into TJs after TJ strands are formed, and therefore it is named Pilt (protein incorporated later into TJs) (Kawabe et al., 2001).
DLG1 is known to interact also with several human virus oncoproteins : HPV E6 (Lee et al., 1997; Kiyono et al.,1997, Gardiol et al., 1999) through its C-terminus and DLG1 PDZ2 domain and as result is subjected to proteasome mediated degradation; HTLV-1 TAX (Suzuki et al., 1999), via the C-terminus of Tax and the PDZ domain of hDLG. Tax prevents the binding of hDLG to APC tumor suppressor gene product, suggesting the mechanism for inhibition of hDLG function; Adenovirus type 9 E4-ORF1 specifically requires endogenous DLG1 to provoke oncogenic activation of phosphatidylinositol 3-kinase (PI3K) in cells. E4-ORF1 binding to Dlg1 on ts PDZ domain triggers the resulting complex to translocate to the plasma membrane and, at this site, to promote Ras-mediated PI3K activation, suggesting a surprising oncogenic function for DLG1 in virus-mediated cellular transformation (Frese et al., 2006; Chung et al., 2007). Moreover, phosphatidylinositol 3-kinases are one class of signaling molecules reported to regulate adherens junction and to be activated by adherens junction assembly . While the exact molecular mechanisms involved are not clear, data indicate that one of the earliest events likely involves c-Src which is rapidly activated by E-cadherin-mediated cellular aggregation and may facilitate the recruitment and activation of PI3K to E-cadherin-containing complexes. Beta-catenin, gamma-catenin and hDlg which are present at cell-cell adhesions can act as docking proteins for PI3K (Rivard N. 2009).
hDlg also binds the tumor endothelial marker 5 (TEM5), a seven-pass transmembrane protein that is homologous to the B family of G-protein-coupled receptors (GPCRs). The PDZ domains of hDlg bound the C-terminal PDZ-binding motif of TEM5. DLG1 is furthermore able to interact with a novel seven-pass transmembrane protein, which was homologous to TEM5, and was named here a TEM5-like protein (TEM5-like) (Yamamoto et al., 2004).
SAP97/hDlg as a scaffold protein is also targeted to the cytoskeleton by its association with the protein guanylate kinase-associated protein (GKAP), which is part of the postsynaptic scaffold in neuronal cells (Sabio et al., 2005). Moreover, hDlg is believed to associate with AMPA receptors (AMPARs) containing the GluR1 subunit, but the functional significance of these interactions is partially unclear, even if this interaction seems to be occur early in the secretory pathway, while the receptors are in the endoplasmic reticulum or cis-Golgi (Sans et al., 2001). In light membrane fractions prepared from rat brain, myosin VI and SAP97 form a trimeric complex with the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor subunit, GluR1. It is possible that SAP97 may serve as a molecular link between GluR1 and the actin-dependent motor protein myosin VI during the dynamic translocation of AMPA receptors to and from the postsynaptic plasma membrane (Wu et al., 2002).
DLG1 is also able to translocate to the immune synapse and lipid rafts in response to T cell receptor (TCR)/CD28 engagement and LckSH3-mediated interactions with DLG1 control its membrane targeting. TCR/CD28 engagement induces the formation of endogenous Lck-DLG1-Zap70-Wiskott-Aldrich syndrome protein (WASp) complexes in which DLG1 acts to facilitate interactions of Lck with Zap70 and WASp (Round et al., 2005).
Delta 1 acts as a membrane-bound ligand that interacts with the Notch receptor and plays a critical role in cell fate specification. DLG1 binds the Delta 1 C-terminal region, in a PDZ dependent manner. Delta 4 also interacts with DLG1, whereas Jagged1, another Notch ligand, does not (Six et al., 2004).
MARCH 2, which is part of the MARCH family ubiquitin ligases and is implicated in the endosomal trafficking interacts with full-length DLG1 in a PDZ domain dependent manner. Furthermore, MARCH2 co-localized with DLG1 at sites of cell-cell contact (Cao et al., 2008).
SAP97 is a binding partner of the cytoplasmic domain of TACE, which is the Tumour necrosis factor alpha converting enzyme and is the metalloprotease-disintegrin responsible for the ectodomain shedding of several proteins, including tumour necrosis factor alpha. The interaction involved the PDZ3 domain of SAP97 and the extreme C-terminal amino-acid sequence of TACE (Peiretti et al., 2003).
DLG1 is able to interact also with Net 1 which is a nuclear RhoA-specific guanine nucleotide exchange factor. The binding is through the PDZ-binding motif. The ability of oncogenic Net1 to transform cells may be in part related to its ability to sequester tumour suppressor proteins like DLG1 in the cytosol, thereby interfering with their normal cellular function (Garcia-Mata et al., 2007).
DLG1 interacts with the tSNARE syntaxin 4 which is involved in vesicle transport, and this binding may contribute to the correct colocaliation of the other proteins of the Scrib complex: hScribble and Hugl-1 (Massimi et al., 2008).
Osmotic stress triggers hDlg degradation through a mechanism different from the one mediated by proteasomes, and hDlg is also a caspase substrate during the apoptotic process, although its proteolysis may not be implicated in the progression of early apoptosis (Inesta-Vaquera FA et al. 2009).
In response to hyperosmotic stress, p38? also regulates formation of complexes between hDlg and the nuclear protein polypyrimidine tract-binding protein-associated-splicing factor (PSF). Following osmotic shock, p38? in the cell nucleus increases its association with nuclear hDlg, thereby causing dissociation of hDlg-PSF complexes. Moreover, hDlg and PSF bind different RNAs; in response to osmotic shock, p38? causes hDlg-PSF and hDlg-RNA dissociation independently of its kinase activity, affecting mRNA processing and/or gene transcription (Sabio et al. 2010). Moreover, the exposure of cells to osmotic shock induces the hyperphosphorylation of Dlg and its concomitant accumulation within the cell membrane at sites of cell contact, a process that requires an intact actin filament network. In addition, hyperphosphorylation of Dlg also renders it more susceptible to degradation induced by the HPV-18 E6 oncoprotein (Massimi et al.2006).
Also the ERK5 pathway is reported to mediate hDlg cell cycle dependent phosphorylation. This is likely to have important implications in the correct timely mitotic entry and mitosis progression ( Inesta- Vaquera et al. 2010).
Using the yeast two-hybrid system to screen a human aorta cDNA library, mitogen-activated protein/extracellular signal-responsive kinase (ERK) kinase (MEK)2, a member of the ERK cascade it was identified as an hDlg binding partner. Site-directed mutagenesis showed a major involvement of the PSD-95, disc-large, ZO-1 domain-2 of hDlg and the C-terminal sequence RTAV of MEK2 in this interaction. hDlg acts as a MEK2-specific scaffold protein for the ERK signaling pathway differentially tuning MEK1/MEK2 signaling and cell responses ( Maiga et al. 2011). Both proteins localize also to a sub-structure of the midbody, the midbody ring (Massimi et al. 2003; Gaudet et al. 2011).
Using a proteomic approach it has been shown that a strong interacting partner of hDlg is the RhoG-specific guanine nucleotide exchange factor SGEF. The interaction between hDlg1 and SGEF involves both PDZ and SH3 domain recognition, and directly contributes to the regulation of SGEF's cellular localization and activity. Consistent with this, hDlg is a strong enhancer of RhoG activity, which occurs in an SGEF-dependent manner and directly contributes to the invasive capacity of HPV-16 and HPV-18 transformed tumour cells displaying a distinct oncogenic function in the context of HPV induced malignancy (Krishna Subbaiah et al. 2012).
Loss of gap junctional communication correlatedswith relocalization of Cx43 to the cytoplasm late in tumorigenesis. A similar pattern of altered expression for the hDlg was found in cervical tumour cells, with partial co-localization of Cx43 and hDlg in an endosomal/lysosomal compartment. Relocalization of these proteins is not due to a general disruption of cell membrane integrity or Cx targeting. Cx43 (via its C-terminus) and hDlg interact directly in vitro and can form a complex in cells. This novel interaction requires the N- and C-termini of hDlg (Macdonald et al. 2012).
The components of the Scrib/Dlg tumour suppressor complex have complementary roles in Drosophila and loss of both proteins is a common event in many different human tumours. In human keratinocytes the removal of hScrib greatly reduces cell-cell contact and cell-matrix interactions, and promotes an invasive phenotype. Conversely, in cells lacking hDlg1 cell-cell contacts are maintained and there are decreases in both cell growth and invasion (Massimi et al. 2012).