Binding of PTEN to specific PDZ domains contributes to PTEN protein stability and phosphorylation by microtubule-associated serine/threonine kinases

The tumor suppressor phosphatase PTEN is a key regulator of cell growth and apoptosis that interacts with PDZ domains from regulatory proteins, including MAGI-1/2/3, hDlg, and MAST205. Here we identified novel PTEN-binding PDZ domains within the MAST205-related proteins, syntrophin-associated serine/threonine kinase and MAST3, characterized the regions of PTEN involved in its interaction with distinctive PDZ domains, and analyzed the functional consequences on PTEN of PDZ domain binding. Using a panel of PTEN mutations, as well as PTEN chimeras containing distinct domains of the related protein TPTE, we found that the PTP and C2 domains of PTEN do not affect PDZ domain binding and that the C-terminal tail of PTEN (residues 350-403) provides selectivity to recognize specific PDZ domains from MAGI-2, hDlg, and MAST205. Binding of PTEN to the PDZ-2 domain from MAGI-2 increased PTEN protein stability. Furthermore, binding of PTEN to the PDZ domains from microtubule-associated serine/threonine kinases facilitated PTEN phosphorylation at its C terminus by these kinases. Our results suggest an important role for the C-terminal region of PTEN in the selective association with scaffolding and/or regulatory molecules and provide evidence that PDZ domain binding stabilizes PTEN and targets this tumor suppressor for phosphorylation by microtubule-associated serine/threonine kinases.     

PKC regulates the delta2 glutamate receptor interaction with S-SCAM/MAGI-2 protein

Inside cells, membrane proteins are localized at particular surface domains to perform their precise functions. Various kinds of PDZ domain proteins have been shown to play important roles in the intracellular trafficking and anchoring of membrane proteins. In this study, we show that delta2 glutamate receptor is interacting with S-SCAM/MAGI-2, a PDZ domain protein localized in the perinuclear region and postsynaptic sites of cerebellar Purkinje cells. The binding is regulated by PKC (protein kinase-C) mediated phosphorylation of the receptor with a unique repetitive structure in S-SCAM/MAGI-2. Co-expression of both proteins resulted in drastic changes of the receptor localization in COS7 cells. These results show a novel regulatory mechanism for the binding of PDZ domain proteins and suggest that the interaction between delta2 receptor and S-SCAM/MAGI-2 may be important for intracellular trafficking of the receptor.     

Analysis of PTEN Complex Assembly and Identification of Heterogeneous Nuclear Ribonucleoprotein C as a Component of the PTEN-associated Complex

PTEN (phosphatase and tensin homolog deleted on chromosome 10) is well characterized for its role in antagonizing the phosphoinositide 3-kinase pathway. Previous studies using size-exclusion chromatography demonstrated PTEN recruitment into high molecular mass complexes and hypothesized that PTEN phosphorylation status and PDZ binding domain may be required for such complex formation. In this study, we set out to test the structural requirements for PTEN complex assembly and identify the component(s) of the PTEN complex(es). Our results demonstrated that the PTEN catalytic function and PDZ binding domain are not absolutely required for its complex formation. On the other hand, PTEN phosphorylation status has a significant impact on its complex assembly. Our results further demonstrate enrichment of the PTEN complex in nuclear lysates, suggesting a mechanism through which PTEN phosphorylation may regulate its complex assembly. These results prompted further characterization of other protein components within the PTEN complex(es). Using size-exclusion chromatography and two-dimensional difference gel electrophoresis followed by mass spectrometry analysis, we identified heterogeneous nuclear ribonucleoprotein C (hnRNP C) as a novel protein recruited to higher molecular mass fractions in the presence of PTEN. Further analysis indicates that endogenous hnRNP C and PTEN interact and co-localize within the nucleus, suggesting a potential role for PTEN, alongside hnRNP C, in RNA regulation.Phosphatase and tensin homolog deleted on chromosome 10 (PTEN)⁴ was cloned in 1997 (1–3) and has been well characterized for its tumor-suppressive role by dephosphorylating phosphatidylinositol 3,4,5-trisphosphate to phosphatidylinositol 4,5-bisphosphate and antagonizing the phosphoinositide 3-kinase pathway (4–7). PTEN also regulates cell migration, cell cycle progression, DNA damage response, and chromosome stability independently of its lipid phosphatase activity through its potential protein phosphatase activity and/or protein-protein interaction (8–11) (for recent reviews, see 12–14).PTEN is composed of an N-terminal catalytic domain and a C-terminal regulatory domain. The catalytic domain contains a conserved signature motif (HCXXGXXR) found in dual-specific protein phosphatases, and mutations within this catalytic domain, including the C124S mutation, are known to abrogate PTEN catalytic activity (4). The C terminus of PTEN contains a PDZ (PDS-95/Disc-large/Zo-1) binding domain, which interacts with PDZ-containing proteins such as MAGI-1b, MAGI-2, MAGI-3, hDLG, hMAST and NHERF (15–19). In addition to the PDZ binding domain, several key serine and threonine phosphorylation sites (Ser³⁸⁰, Thr³⁸², Thr³⁸³, and Ser³⁸⁵) at the PTEN C terminus are reported to play an important role in regulating its stability, localization, and activity (20–26).Recent studies suggest that PTEN may function within higher molecular mass complexes. Through size-exclusion chromatography, Vazquez et al. (27) demonstrated that PTEN can be separated into two populations: a monomeric hyperphosphorylated subpopulation and a higher molecular mass hypophosphorylated subpopulation. It was hypothesized that PTEN in its dephosphorylated form can interact with PDZ-containing proteins such as hDLG and be recruited into a higher molecular mass complex. Although the components within PTEN complex(es) have not been systematically studied and purified, MAGI-2, hDLG (27), NHERF2, PDGFR (19), NEP (28), and MVP (29) have been identified as potential components of the PTEN complex using the same size-exclusion chromatography methodology.In this paper, we aim to (i) investigate the essential elements of PTEN required for its complex formation and (ii) dissect the components of the PTEN-associated complex(es). Our results indicate that PTEN catalytic activity or its PDZ binding domain is not absolutely required for complex assembly. PTEN phosphorylation status on amino acids Ser³⁸⁰, Thr³⁸², Thr³⁸³, and Ser³⁸⁵, on the other hand, has a significant role in complex formation. In addition, we demonstrate that the PTEN complex is enriched in nuclear lysates, which suggests a mechanism through which phosphorylation can regulate complex assembly. Using two-dimensional difference gel electrophoresis (DIGE) analysis and comparing proteins present in higher molecular mass fractions in the presence and absence of PTEN followed by mass spectrometry analysis, we have identified heterogeneous nuclear ribonucleoprotein C (hnRNP C) as a potential component within the PTEN complex. PTEN and hnRNP C are shown here to interact and co-localize in the nucleus. We hypothesize that the PTEN and hnRNP C complex may play a role in RNA regulation.     

Defining the minimal interacting regions of the tight junction protein MAGI-1 and HPV16 E6 oncoprotein for solution structure studies

The oncoprotein E6 produced by tumorigenic high-risk genital human papillomaviruses targets a number of cellular proteins containing PDZ domains for proteasome-mediated degradation. In particular, E6 targets the tight junction protein MAGI-1 by binding to its PDZ1 domain. Using light scattering and NMR, we explored different fragments of both the HPV16 E6 and the MAGI-1 PDZ1 domain to define the best-behaving complex for solution structure studies. We showed that the 70-residue HPV16 E6 C-terminal domain (E6C) can be efficiently substituted by a peptide spanning the 11 C-terminal residues of E6. The construct of MAGI-1 PDZ1 best suited for solution structure analysis presents a 14-residue N-terminal extension and a 26-residue C-terminal extension as compared to the construct used for the recently solved X-ray structure of a MAGI-1 PDZ1/HPV18 E6 complex. These data suggest a stabilizing role for the interdomain linker regions which separate the PDZ1 domain from its neighboring domains.     

Par-3-mediated junctional localization of the lipid phosphatase PTEN is required for cell polarity establishment

PDZ domain-containing scaffold protein Par-3 is the central organizer of the evolutionarily conserved cell polarity-regulatory Par-3.Par-6.atypical protein kinase C complex. The PDZ domains of Par-3 have also been implicated as potential phosphoinositide signaling integrators, since its second PDZ domain binds to phosphoinositides, and the third PDZ interacts with phosphoinositide phosphatase PTEN. However, the molecular basis of Par-3/PTEN interaction is still poorly understood. Additionally, it is not known whether the regulatory function of PTEN in cell polarity is specifically mediated by its interaction with Par-3. The structures of Par-3 PDZ3 in both its free and PTEN tail peptide-bound forms determined in this work reveal that Par-3 PDZ3 binds to PTEN with two discrete binding sites: a canonical PDZ-ligand interaction site and a distal, opposite charge-charge interaction site. This distinct target recognition mechanism confers the interaction specificity of the Par-3.PTEN complex. We show that the Par-3 PDZ3-PTEN binding is required for the enrichment of PTEN at the junctional membranes of Madin-Darby canine kidney cells. Finally, we demonstrate that the junctional membrane-localized PTEN is specifically required for the polarization of Madin-Darby canine kidney cells. These results, together with earlier data, firmly establish that Par-3 functions as a scaffold in integrating phosphoinositide signaling events during cellular polarization.     

Role of the PDZ domain-binding motif of the oncoprotein E6 in the pathogenesis of human papillomavirus type 31

A number of PDZ domain-containing proteins have been identified as binding partners for the oncoprotein E6 of the high-risk type human papillomaviruses (HPVs). These include hDlg, hScrib, MAGI-1, MAGI-2, MAGI-3, and MUPP1. The PDZ domain-binding motif (-X-T-X-V) at the carboxy terminus of E6 is essential for targeting PDZ proteins for proteasomal degradation. The presence of this motif only in the high-risk HPVs suggests its possible role in HPV-induced oncogenesis. To investigate the role of the PDZ domain-binding motif of E6 in the HPV life cycle, two mutant HPV31 genomes were constructed: E6ValDelta, with a deletion of the last amino acid residue of E6 (valine), and E6ETQVDelta, with a deletion of the entire PDZ domain-binding motif of E6 (ETQV). Three human foreskin keratinocyte (HFK) cell lines were established which maintained transfected wild-type HPV31 or either of two mutant genomes. Cells containing either of two mutant genomes were significantly retarded in their growth rates and reduced in their viral copy numbers compared to those transfected with wild-type genomes. Western analysis did not reveal any significant changes in the levels of PDZ proteins following stable transfection of any HPV31 genomes into HFKs. Although the E6ETQVDelta-transfected HFKs exhibited a pattern of morphological differentiation that appeared different from the HPV31 wild-type-transfected HFKs in organotypic raft cultures, immunohistochemical analysis failed to identify substantial changes in the differentiation-dependent membrane localization of hDlg proteins. These results suggest that binding of E6 to PDZ proteins modulates the early viral functions such as proliferation and maintenance of the viral copy number in undifferentiated cells.     

A systematic analysis of human papillomavirus (HPV) E6 PDZ substrates identifies MAGI-1 as a major target of HPV type 16 (HPV-16) and HPV-18 whose loss accompanies disruption of tight junctions

The E6 proteins from high-risk, cancer-causing types of human papillomavirus (HPV) are characterized by the presence of a PDZ (PSD95/Dlg/ZO-1) binding motif in their extreme carboxy termini, through which they interact with a number of cellular PDZ domain-containing substrates. In order to ascertain how many of these are degraded by E6 in vivo, we performed an extensive analysis of the effects of E6 ablation on the expression levels of a number of previously reported E6 PDZ substrates. Using HPV type 16 (HPV-16)-positive CaSKi cells and HPV-18-positive HeLa cells, we have found that MAGI-1 is a major degradation target of both HPV-16 and HPV-18 E6. In contrast, hDlg, hScrib, PTPN3, TIP2, FAP1, and PSD95 all exhibit various degrees of susceptibility to E6-induced degradation, and a high degree of HPV type specificity is observed for certain substrates. We also show that E6 preferentially targets MAGI-1 within the nucleus and at membrane sites. One of the direct consequences of MAGI-1 degradation is a loss of tight-junction integrity, as determined by mislocalization of the tight-junction protein ZO-1. Ablation of E6 expression restores tight junctions, and this restoration is dependent on the presence of MAGI-1. These results demonstrate that oncogenic HPV E6 proteins disrupt cellular tight junctions through the degradation of MAGI-1, and they provide further evidence of how the PDZ binding potential of E6 can contribute to HPV-induced malignancy.     

The structural and dynamic response of MAGI-1 PDZ1 with noncanonical domain boundaries to the binding of human papillomavirus E6

PDZ domains are protein interaction domains that are found in cytoplasmic proteins involved in signaling pathways and subcellular transport. Their roles in the control of cell growth, cell polarity, and cell adhesion in response to cell contact render this family of proteins targets during the development of cancer. Targeting of these network hubs by the oncoprotein E6 of “high-risk” human papillomaviruses (HPVs) serves to effect the efficient disruption of cellular processes. Using NMR, we have solved the three-dimensional solution structure of an extended construct of the second PDZ domain of MAGI-1 (MAGI-1 PDZ1) alone and bound to a peptide derived from the C-terminus of HPV16 E6, and we have characterized the changes in backbone dynamics and hydrogen bonding that occur upon binding. The binding event induces quenching of high-frequency motions in the C-terminal tail of the PDZ domain, which contacts the peptide upstream of the canonical X-[T/S]-X-[L/V] binding motif. Mutations designed in the C-terminal flanking region of the PDZ domain resulted in a significant decrease in binding affinity for E6 peptides. This detailed analysis supports the notion of a global response of the PDZ domain to the binding event, with effects propagated to distal sites, and reveals unexpected roles for the sequences flanking the canonical PDZ domain boundaries.     

MAGI-3 regulates LPA-induced activation of Erk and RhoA

Lysophosphatidic acids (LPA) exert multiple biological effects through specific G protein-coupled receptors. The LPA-activated receptor subtype LPA(2) contains a carboxyl-terminal motif that allows interaction with PDZ domain-containing proteins, such as NHERF2 and PDZ-RhoGEF. To identify additional interacting partners of LPA(2), the LPA(2) carboxyl-terminus was used to screen a proteomic array of PDZ domains. In addition to the previously identified NHERF2, several additional LPA(2)-interacting PDZ domains were found. These included MAGI-2, MAGI-3 and neurabin. In the present work, we demonstrate the specific interaction between LPA(2) and MAGI-3, and the effects of MAGI-3 in colon cancer cells using SW480 as a cell model. MAGI-3 specifically bound to LPA(2), but not to LPA(1) and LPA(3). This interaction was mediated via the fifth PDZ domain of MAGI-3 interacting with the carboxyl-terminal 4 amino acids of LPA(2), and mutational alteration of the carboxyl-terminal sequences of LPA(2) severely attenuated its ability to bind MAGI-3. LPA(2) also associated with MAGI-3 in cells as determined by co-affinity purification. Overexpression of MAGI-3 in SW480 cells showed no apparent effect on LPA-induced activation of Erk and Akt. In contrast, silencing of MAGI-3 expression by siRNA drastically inhibited LPA-induced Erk activation, suggesting that the lack of an effect by overexpression was due to the high endogenous MAGI-3 level in these cells. Previous studies have shown that the cellular signaling elicited by LPA results in activation of the small GTPase RhoA by Galpha(12/13) - as well as Galpha(q)-dependent pathways. Overexpression of MAGI-3 stimulated LPA-induced RhoA activation, whereas silencing of MAGI-3 by siRNA resulted in a small but statistically significant decrease in RhoA activation. These results demonstrate that MAGI-3 interacts directly with LPA(2) and regulates the ability of LPA(2) to activate Erk and RhoA.     

抑癌蛋白PTEN及PDZ蛋白对其的调节

pten基因是迄今为止发现的第1个具有双特异性磷酸酶活性的抑癌基因,该基因的编码产物PTEN蛋白,是具有蛋白与脂质磷酸酯酶活性的双特异性磷酸酯酶,作为1种重要的信号分子参与细胞增殖、分化、黏附、迁移、凋亡以及基因转录的调控. 最近,关于PTEN在信号转导中的作用以及细胞内PTEN的调节机制研究较多,尤其是PDZ蛋白对PTEN的调节作用. PTEN蛋白包括1个氨基端（N端）磷酸酯酶区域,1个与脂质结合的C2区域和1个含有PDZ结合序列的羧基端（C端）区域. PDZ结构域通过识别目标蛋白羧基端PDZ结合序列与目标蛋白相互作用,调控多种重要的细胞生理过程和信号传导途径.本文就抑癌基因pten编码产物PTEN蛋白的结构、PTEN的生物学功能和PDZ蛋白对PTEN调节的研究进展进行综述.     

Structures of a human papillomavirus (HPV) E6 polypeptide bound to MAGUK proteins: mechanisms of targeting tumor suppressors by a high-risk HPV oncoprotein

Human papillomavirus (HPV) E6 oncoprotein targets certain tumor suppressors such as MAGI-1 and SAP97/hDlg for degradation. A short peptide at the C terminus of E6 interacts specifically with the PDZ domains of these tumor suppressors, which is a property unique to high-risk HPVs that are associated with cervical cancer. The detailed recognition mechanisms between HPV E6 and PDZ proteins are unclear. To understand the specific binding of cellular PDZ substrates by HPV E6, we have solved the crystal structures of the complexes containing a peptide from HPV18 E6 bound to three PDZ domains from MAGI-1 and SAP97/Dlg. The complex crystal structures reveal novel features of PDZ peptide recognition that explain why high-risk HPV E6 can specifically target these cellular tumor suppressors for destruction. Moreover, a new peptide-binding loop on these PDZs is identified as interacting with the E6 peptide. Furthermore, we have identified an arginine residue, unique to high-risk HPV E6 but outside the canonical core PDZ recognition motif, that plays an important role in the binding of the PDZs of both MAGI-I and SAP97/Dlg, the mutation of which abolishes E6's ability to degrade the two proteins. Finally, we have identified a dimer form of MAGI-1 PDZ domain 1 in the cocrystal structure with E6 peptide, which may have functional relevance for MAGI-1 activity. In addition to its novel insights into the biochemistry of PDZ interactions, this study is important for understanding HPV-induced oncogenesis; this could provide a basis for developing antiviral and anticancer compounds.     

The PDZ3 domain of the cellular scaffolding protein MAGI-1 interacts with the Coxsackievirus and adenovirus receptor (CAR)

The Coxsackievirus and adenovirus receptor (CAR) is an essential cellular protein that is involved in cell–cell adhesion, protein trafficking, and viral infection. The major isoform of CAR is selectively sorted to the basolateral membrane of polarized epithelial cells where it co-localizes with the cellular scaffolding protein membrane-associated guanylate kinase with inverted domain structure-1 (MAGI-1). Previously, we demonstrated CAR interacts with MAGI-1 through a PDZ–domain dependent interaction. Here, we show that the PDZ3 domain of MAGI-1 is exclusively responsible for the high affinity interaction between the seven exon isoform of CAR and MAGI-1 using yeast-two-hybrid analysis and confirming this interaction biochemically and in cellular lysates by in vitro pull down assay and co-immunoprecipitation. The high affinity interaction between the PDZ3 domain and CAR C-terminus was measured by fluorescence resonance energy transfer. Further, we investigated the biological relevance of this high affinity interaction between CAR and the PDZ3 domain of MAGI-1 and found that it does not alter CAR-mediated adenovirus infection. By contrast, interruption of this high affinity interaction altered the localization of MAGI-1 indicating that CAR is able to traffic MAGI-1 to cell junctions. These data deepen the molecular understanding of the interaction between CAR and MAGI-1 and indicate that although CAR plays a role in trafficking PDZ-based scaffolding proteins to cellular junctions, association with a high affinity intracellular binding partner does not significantly alter adenovirus binding and entry via CAR.     

NHERF1/EBP50 head-to-tail intramolecular interaction masks association with PDZ domain ligands

Loss of cell polarity is one of the initial alterations in the development of human epithelial cancers. Na(+)/H(+) exchanger regulatory factor (NHERF) homologous adaptors 1 and 2 are membrane-associated proteins composed of two amino (N)-terminal PDZ domains and an ezrin-radixin-moesin (ERM)-binding (EB) carboxyl (C)-terminal region. We describe here an intramolecular conformation of NHERF1/EBP50 (ERM-binding phosphoprotein 50) in which the C-terminal EB region binds to the PDZ2 domain. This novel head-to-tail conformation masked the interaction of both PDZ domains with PDZ domain-specific ligands, such as PTEN and beta-catenin. An EB region composite structure comprising an alpha-helix ending in a PDZ-binding motif imparted opposite effects to NHERF1 associations, mediating binding to ERM proteins and inhibiting binding of PDZ domain ligands. The PDZ domain inhibition was released by prior association of ezrin with the EB region, a condition that occurs in vivo and likely disrupts NHERF1 head-to-tail interaction. In contrast, NHERF2 did not present a regulatory mechanism for protein complex formation. Functionally, NHERF1 is required to organize complexes at the apical membranes of polarized epithelial cells. The regulation of NHERF1 interactions at the apical membrane thus appears to be a dynamic process that is important for maintaining epithelial-tissue integrity.     

The PDZ1 and PDZ3 Domains of MAGI-1 Regulate the Eight-Exon Isoform of the Coxsackievirus and Adenovirus Receptor

Epithelial integrity is essential for homeostasis and poses a formidable barrier to pathogen entry. Major factors for viral entry into epithelial cells are the localization and abundance of the primary receptor. The coxsackievirus and adenovirus receptor (CAR) is a primary receptor for these two pathogenic groups of viruses. In polarized epithelia, a low-abundance, alternatively spliced eight-exon isoform of CAR, CAR(Ex8), is localized apically where it can support viral infection from the air-exposed surface. Using biochemical, cell biology, genetic, and spectroscopic approaches, we show that the levels of apical CAR(Ex8) are negatively regulated by the PDZ domain-containing protein MAGI-1 (membrane-associated guanylate kinase with inverted orientation protein-1) and that two MAGI-1 PDZ domains, PDZ1 and PDZ3, regulate CAR(Ex8) levels in opposing ways. Similar to full-length MAGI-1, expression of the isolated PDZ3 domain significantly reduces cell surface CAR(Ex8) abundance and adenovirus infection. In contrast, the PDZ1 domain is able to rescue CAR(Ex8) and adenovirus infection from MAGI-1-mediated suppression. These data suggest a novel cell-based strategy to either suppress viral infection or augment adenovirus-based gene therapy.     

MAGI proteins can differentially regulate the signaling pathways of 5-HT2AR by enhancing receptor trafficking and PLC recruitment

MAGI proteins are Membrane-Associated Guanylate Kinase Inverted proteins that belong to the MAGUK family. They are scaffolding proteins that were shown to mediate the trafficking and signaling of various G protein-coupled receptors (GPCRs). They contain PDZ domains in their structure and many GPCRs interact with these proteins via the PDZ motifs on the carboxyl terminal end of a receptor. In a PDZ overlay assay performed with the carboxyl terminal tail of 5-HT_2AR, we were able to detect all three members of the MAGI subfamily, MAGI-1, MAGI-2 and MAGI-3 as interacting PDZ proteins. The PDZ motif of 5-HT_2AR consists of three amino acids; serine (S), cysteine (C) and valine (V). In this study, we characterize these 5-HT_2AR interactions with MAGI proteins. We first confirm the interaction using co-immunopricipitation and illustrate that the interaction is PDZ motif-dependent in human embryonic kidney (HEK 293) cells. We then assess the effects of overexpression and knockdown of the MAGI proteins on the internalization, trafficking and signaling of 5-HT_2AR. We find that knockdown of either MAGI-1 or MAGI-3 using siRNA results in a significant reduction in the internalization of 5-HT_2AR. As for signaling, we report here that MAGI proteins can modulate the signaling via the two transduction pathways that 5-HT_2AR can activate. We illustrate a significant effect of modulating MAGI proteins expression on 5-HT-stimulated IP formation. We demonstrate an enhancement in 5-HT_2AR-stimulated IP formation upon MAGI proteins overexpression. In addition, we show that knockdown of MAGI proteins with siRNA leads to a significant reduction in 5-HT_2AR-stimulated IP formation. Furthermore, we illustrate a significant increase in 5-HT-stimulated ERK1/2 phosphorylation upon MAGI proteins knockdown. Interestingly, this effect on ERK1/2 activation is PDZ motif-independent. We also suggest two possible mechanisms of regulation for the effect of MAGI proteins on 5-HT_2AR function. One mechanism involves the regulation of cell surface expression since we show that both MAGI-2 and MAGI-3 can enhance receptor trafficking to the plasma membrane when overexpressed in HEK 293 cells. The other mechanism points to regulation of second messengers in the signaling pathways. Specifically, we show that overexpression of any of the three MAGI proteins can enhance the recruitment of PLCβ3 to 5-HT_2AR. In addition, we report a negative effect for knocking down MAGI-3 on β-arrestin recruitment to the receptor and this effect is PDZ motif-independent. Taken together, our findings document distinct roles for the three MAGI proteins in regulating 5-HT_2AR trafficking and signaling and emphasize the importance of studying PDZ proteins and their interactions with GPCRs to regulate their function.     

Autoinhibition of X11/Mint scaffold proteins revealed by the closed conformation of the PDZ tandem

Members of the X11/Mint family of multidomain adaptor proteins are composed of a divergent N terminus, a conserved PTB domain and a pair of C-terminal PDZ domains. Many proteins can interact with the PDZ tandem of X11 proteins, although the mechanism of such interactions is unclear. Here we show that the highly conserved C-terminal tail of X11alpha folds back and inserts into the target-binding groove of the first PDZ domain. The binding of this tail occludes the binding of other target peptides. This autoinhibited conformation of X11 requires that the two PDZ domains and the entire C-terminal tail be covalently connected to form an integral structural unit. The autoinhibited conformation of the X11 PDZ tandem provides a mechanistic explanation for the unique target-binding properties of the protein and hints at potential regulatory mechanisms for the X11-target interactions.     

Ggamma13 interacts with PDZ domain-containing proteins

The G protein gamma13 subunit (Ggamma13) is expressed in taste and retinal and neuronal tissues and plays a key role in taste transduction. We identified PSD95, Veli-2, and other PDZ domain-containing proteins as binding partners for Ggamma13 by yeast two-hybrid and pull-down assays. In two-hybrid assays, Ggamma13 interacted specifically with the third PDZ domain of PSD95, the sole PDZ domain of Veli-2, and the third PDZ domain of SAP97, a PSD95-related protein. Ggamma13 did not interact with the other PDZ domains of PSD95. Coexpression of Ggamma13 with its Gbeta1 partner did not interfere with these two-hybrid interactions. The physical interaction of Ggamma13 with PSD95 in the cellular milieu was confirmed in pull-down assays following heterologous expression in HEK293 cells. The interaction of Ggamma13 with the PDZ domain of PSD95 was via the C-terminal CAAX tail of Ggamma13 (where AA indicates the aliphatic amino acid); alanine substitution of the CTAL sequence at the C terminus of Ggamma13 abolished its interactions with PSD95 in two-hybrid and pull-down assays. Veli-2 and SAP97 were identified in taste tissue and in Ggamma13-expressing taste cells. Coimmunoprecipitation of Ggamma13 and PSD95 from brain and of Ggamma13 and SAP97 from taste tissue indicates that Ggamma13 interacts with these proteins endogenously. This is the first demonstration that PDZ domain proteins interact with heterotrimeric G proteins via the CAAX tail of Ggamma subunits. The interaction of Ggamma13 with PDZ domain-containing proteins may provide a means to target particular Gbetagamma subunits to specific subcellular locations and/or macromolecular complexes involved in signaling pathways.     

beta 1-adrenergic receptor association with the synaptic scaffolding protein membrane-associated guanylate kinase inverted-2 (MAGI-2). Differential regulation of receptor internalization by MAGI-2 and PSD-95

The beta1-adrenergic receptor (beta1AR) is known to be localized to synapses and to modulate synaptic plasticity in many brain regions, but the molecular mechanisms determining beta1AR subcellular localization are not fully understood. Using overlay and pull-down techniques, we found that the beta1AR carboxyl terminus associates with MAGI-2 (membrane-associated guanylate kinase inverted-2), a protein also known as S-SCAM (synaptic scaffolding molecule). MAGI-2 is a multidomain scaffolding protein that contains nine potential protein-protein interaction modules, including 6 PDZ domains, 2 WW domains, and a guanylate kinase-like domain. The beta1AR carboxyl terminus binds with high affinity to the first PDZ domain of MAGI-2, with the last few amino acids of the beta1AR carboxyl terminus being the key determinants of the interaction. In cells, the association of full-length beta1AR with MAGI-2 occurs constitutively and is enhanced by agonist stimulation of the receptor, as assessed by both co-immunoprecipitation experiments and immunofluorescence co-localization studies. Agonist-induced internalization of the beta1AR is markedly increased by co-expression with MAGI-2. Strikingly, this result is the opposite of the effect of co-expression with PSD-95, a previously reported binding partner of the beta1AR. Further cellular experiments revealed that MAGI-2 has no effect on beta1AR oligomerization but does promote association of beta1AR with the cytoplasmic signaling protein beta-catenin, a known MAGI-2 binding partner. These data reveal that MAGI-2 is a specific beta1AR binding partner that modulates beta1AR function and facilitates the physical association of the beta1AR with intracellular proteins involved in signal transduction and synaptic regulation.     

Type I PDZ ligands are sufficient to promote rapid recycling of G Protein-coupled receptors independent of binding to N-ethylmaleimide-sensitive factor

Molecular sorting of G protein-coupled receptors (GPCRs) between divergent recycling and lysosomal pathways determines the functional consequences of agonist-induced endocytosis. The carboxyl-terminal cytoplasmic domain of the beta2 adrenergic receptor (beta2AR) mediates both PDZ binding to Na+/H+ exchanger regulatory factor/ezrin/radixin/moesin-binding phosphoprotein of 50 kDa (NHERF/EBP50) family proteins and non-PDZ binding to the N-ethylmaleimide-sensitive factor (NSF). We have investigated whether PDZ interaction(s) are actually sufficient to promote rapid recycling of endocytosed receptors and, if so, whether PDZ-mediated sorting is restricted to the beta2AR tail or to sequences that bind NHERF/EBP50. The trafficking effects of short (10 residue) sequences differing in PDZ and NSF binding properties were examined using chimeric mutant receptors. The recycling activity of the beta2AR-derived tail sequence was not blocked by a point mutation that selectively disrupts binding to NSF, and naturally occurring PDZ ligand sequences were identified that do not bind detectably to NSF yet function as strong recycling signals. The carboxyl-terminal cytoplasmic domain of the beta1-adrenergic receptor, which does not bind either to NSF or NHERF/EBP50 and interacts selectively with a distinct group of PDZ proteins, promoted rapid recycling of chimeric mutant receptors with efficiency similarly high as that of the beta2AR tail. These results indicate that PDZ domain-mediated protein interactions are sufficient to promote rapid recycling of GPCRs, independent of binding to NSF. They also suggest that PDZ-directed recycling is a rather general mechanism of GPCR regulation, which is not restricted to a single GPCR, and may involve additional PDZ domain-containing protein(s) besides NHERF/EBP50.     

Regulation of Interferon-β by MAGI-1 and Its Interaction with Influenza A Virus NS1 Protein with ESEV PBM

The NS1 protein from avian influenza A viruses contains a PDZ binding motif (PBM) at its carboxyl terminus with the consensus sequence ESEV. The ESEV PBM confers binding to several cellular PDZ proteins, including Dlg1, MAGI-1 and Scribble. The interaction between NS1 and Scribble protects infected cells from apoptosis, while the interaction between NS1 and both Dlg1 and Scribble disrupts tight junctions. In this study, we examined the MAGI-1 protein. We made the unexpected observation that siRNA depletion of MAGI-1 activates IRF3 and induces the IFN-β promoter. We found that the ESEV NS1 protein sequesters MAGI-1 away from the plasma membrane in infected cells. Using plasmid vectors to express NS1 proteins, we observed that the ESEV PBM elicits an IFN-β induction signal as indicated by activation of IRF3 and a relative deficiency in NS1 inhibition of induction of the IFN-β promoter by dsRNA or RIG-I. Taken together, our data suggest that disruption of MAGI-1 by the ESEV PBM activates an IFN-β induction signal. During viral infection, however, induction of the IFN-β gene does not occur presumably because other anti-IFN functions dominate over the IFN-activation activity of the ESEV PBM. We postulate that the ESEV PBM's broad binding activity for PDZ proteins may allow NS1 to bind to some PDZ proteins such as MAGI-1 that confer no benefit or may even be detrimental to viral replication. However, the advantage of binding to key PDZ proteins such as Dlg1 and Scribble may dominate and therefore provide an overall benefit for the virus to encode the ESEV PBM.