Opposite effects of PSD-95 and MPP3 PDZ proteins on serotonin 5-hydroxytryptamine2C receptor desensitization and membrane stability

PSD-95/Disc large/Zonula occludens 1 (PDZ) domain-containing proteins (PDZ proteins) play an important role in the targeting and the trafficking of transmembrane proteins. Our previous studies identified a set of PDZ proteins that interact with the C terminus of the serotonin 5-hydroxytryptamine (5-HT)(2C) receptor. Here, we show that the prototypic scaffolding protein postsynaptic density-95 (PSD-95) and another membrane-associated guanylate kinase, MAGUK p55 subfamily member 3 (MPP3), oppositely regulate desensitization of the receptor response in both heterologous cells and mice cortical neurons in primary culture. PSD-95 increased desensitization of the 5-HT(2C) receptor-mediated Ca(2+) response, whereas MPP3 prevented desensitization of the Ca(2+) response. The effects of the PDZ proteins on the desensitization of the Ca(2+) response were correlated with a differential regulation of cell surface expression of the receptor. Additional experiments were performed to assess how PDZ proteins globally modulate desensitization of the 5-HT(2C) receptor response in neurons, by using a peptidyl mimetic of the 5-HT(2C) receptor C terminus fused to the human immunodeficiency virus type-1 Tat protein transduction domain, which disrupts interaction between the 5-HT(2C) receptor and PDZ proteins. Transduction of this peptide inhibitor into cultured cortical neurons increased the desensitization of the 5-HT(2C) receptor-mediated Ca(2+) response. This indicates that, overall, interaction of 5-HT(2C) receptors with PDZ proteins inhibits receptor desensitization in cortical neurons.     

Regulation of NR1/NR2C N-methyl-D-aspartate (NMDA) receptors by phosphorylation

NR2C-containing N-methyl-D-aspartate (NMDA) receptors are highly expressed in cerebellar granule cells where they mediate the majority of current in the adult. NMDA receptors composed of NR1/NR2C exhibit a low conductance and reduced sensitivity to Mg(2+), compared with the more commonly studied NR2A- and NR2B-containing receptors. Despite these interesting features, very little is known about the regulation of NR2C function. Here we investigate the role of phosphorylation of NR2C in regulating NMDA receptor trafficking and ion channel properties. We identify a phosphorylation site, serine 1244 (Ser(1244)), near the extreme COOH terminus of NR2C, which is phosphorylated by both cAMP-dependent protein kinase and protein kinase C. This residue is located adjacent to the consensus PDZ ligand, a region that regulates protein-protein interactions and receptor trafficking in NR2A and NR2B. We show that Ser(1244) on NR2C is phosphorylated in vitro, in heterologous cells, and in neurons. Moreover, we demonstrate for the first time that NR2C interacts with the PSD-95 family of PDZ domain-containing proteins but that phosphorylation of Ser(1244) does not influence this PDZ interaction. Furthermore, Ser(1244) phosphorylation does not regulate surface expression of NR1/NR2C receptors. However, we find that this site does regulate the kinetics of the ion channel: a phosphomimetic mutation at Ser(1244) accelerates both the rise and decay of NMDA-evoked currents in excised patches from HEK-293 cells. Therefore, phosphorylation of Ser(1244) does not regulate trafficking but unexpectedly affects ion channel function, suggesting that phosphorylation of Ser(1244) on NR2C may be important in defining the functional properties of NMDA receptor-mediated currents in the cerebellum.     

The PDZ protein mupp1 promotes Gi coupling and signaling of the Mt1 melatonin receptor

Intracellular signaling events are often organized around PDZ (PSD-95/Drosophila Disc large/ZO-1 homology) domain-containing scaffolding proteins. The ubiquitously expressed multi-PDZ protein MUPP1, which is composed of 13 PDZ domains, has been shown to interact with multiple viral and cellular proteins and to play important roles in receptor targeting and trafficking. In this study, we show that MUPP1 binds to the G protein-coupled MT(1) melatonin receptor and directly regulates its G(i)-dependent signal transduction. Structural determinants involved in this interaction are the PDZ10 domain of MUPP1 and the valine of the canonical class III PDZ domain binding motif DSV of the MT(1) carboxyl terminus. This high affinity interaction (K(d) approximately 4 nm), which is independent of MT(1) activation, occurs in the ovine pars tuberalis of the pituitary expressing both proteins endogenously. Although the disruption of the MT(1)/MUPP1 interaction has no effect on the subcellular localization, trafficking, or degradation of MT(1), it destabilizes the interaction between MT(1) and G(i) and abolishes G(i)-mediated signaling of MT(1). Our findings highlight a previously unappreciated role of PDZ proteins in promoting G protein coupling to receptors.     

Trafficking of the NMDAR2B receptor subunit distal cytoplasmic tail from endoplasmic reticulum to the synapse

NMDA receptor NR2A/B subunits have PDZ-binding domains on their extreme C-termini that are known to interact with the PSD-95 family and other PDZ proteins. We explore the interactions between PSD-95 family proteins and the NR2A/B cytoplasmic tails, and the consequences of these interactions, from the endoplasmic reticulum (ER) through delivery to the synapse in primary rat hippocampal and cortical cultured neurons. We find that the NR2A/B cytoplasmic tails cluster very early in the secretory pathway and interact serially with SAP102 beginning at the intermediate compartment, and then PSD-95. We further establish that colocalization of the distal C-terminus of NR2B and PSD-95 begins at the trans-Golgi Network (TGN). Formation of NR2B/PSD-95/SAP102 complexes is dependent on the PDZ binding domain of NR2B subunits, but association with SAP102 and PSD-95 plays no distinguishable role in cluster pre-formation or initial targeting to the vicinity of the synapse. Instead the PDZ binding domain plays a role in restricting cell-surface clusters to postsynaptic targets.     

Flavonoids from Radix Scutellariae as potential stroke therapeutic agents by targeting the second postsynaptic density 95 (PSD-95)/disc large/zonula occludens-1 (PDZ) domain of PSD-95.

Excessive activation of N-methyl-D-aspartate receptors (NMDARs) and subsequent production of nitric oxide by neuronal nitric oxide synthase (nNOS) contribute to neuronal damage resulting from hypoxic and ischemic insults. NMDARs and nNOS are coupled together at the postsynaptic membrane through their interaction with postsynaptic density protein (PSD) 95 via PSD-95/disc large/zonula occludens-1 (PDZ) domains. We used NMR (nuclear magnetic resonance) spectroscopy to screen medicinal herbs used in traditional Chinese medicine (TCM) stroke therapy for compounds binding to the second PDZ domain (PDZ2) of PSD-95, the domain linking nNOS and PSD-95. Aqueous extract of Huangqin, the root of Scutellaria baicalensis Georgi (Labiatae), showed significant binding to PDZ2 of PSD-95. The binding site of the active components in the extract overlapped with the nNOS/NR2B-binding pocket of PDZ2 of PSD-95. Four flavones, baicalin, norwogonoside, oroxylin A-glucuronide (oroxyloside), and wogonoside were isolated and found to account for the PDZ-binding activity of the extract. NMR titration experiments showed that baicalin and norwogonoside displayed the highest PDZ2 binding affinity, while oroxylin A-glucuronide and wogonoside showed 4-5 fold less potency in binding to the PDZ domain. Identification of the PDZ binding activity of these compounds will allow investigating whether or not it contributes to the observed clinical effects of Radix Scutellariae. Furthermore, these molecules might provide leads for the development of drugs targeting the signaling pathways mediated by PDZ domains.     

Direct interaction of post-synaptic density-95/Dlg/ZO-1 domain-containing synaptic molecule Shank3 with GluR1 alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor

A class of scaffolding protein containing the post-synaptic density-95/Dlg/ZO-1 (PDZ) domain is thought to be involved in synaptic trafficking of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors during development. To clarify the molecular mechanism of AMPA receptor trafficking, we performed a yeast two-hybrid screening system using the cytoplasmic tail of the GluR1 subunit of AMPA receptor as a bait and identified a synaptic molecule, Shank3/ProSAP2, as a GluR1 subunit-interacting molecule. Shank3 is a PDZ domain-containing multidomain protein and is predominantly expressed in developing neurons. Using the glutathione S-transferase pull-down assay and immunoprecipitation technique we demonstrated that the GluR1 subunit directly binds to the PDZ domain of Shank3 via its carboxyl terminal PDZ-binding motif. We raised anti-Shank3 antibody to investigate the expression of Shank3 in cortical neurons. The pattern of Shank3 immunoreactivity was strikingly punctate, mainly observed in the spines, and closely matched the pattern of post-synaptic density-95 immunoreactivity, indicating that Shank3 is colocalized with post-synaptic density-95 in the same spines. When Shank3 and the GluR1 subunit were overexpressed in primary cortical neurons, they were also colocalized in the spines. Taken together with the biochemical interaction of Shank3 with the GluR1 subunit, these results suggest that Shank3 is an important molecule that interacts with GluR1 AMPA receptor at synaptic sites of developing neurons.     

The serotonin 5-HT2A and 5-HT2C receptors interact with specific sets of PDZ proteins

The 5-hydroxytryptamine type 2A (5-HT(2A)) receptor and the 5-HT(2C) receptor are closely related members of the G-protein-coupled receptors activated by serotonin that share very similar pharmacological profiles and cellular signaling pathways. These receptors express a canonical class I PDZ ligand (SXV) at their C-terminal extremity. Here, we have identified proteins that interact with the PDZ ligand of the 5-HT(2A) and 5-HT(2C) receptors by a proteomic approach associating affinity chromatography using immobilized synthetic peptides encompassing the PDZ ligand and mass spectrometry. We report that both receptor C termini interact with specific sets of PDZ proteins in vitro. The 5-HT(2C) receptor but not the 5-HT(2A) receptor binds to the Veli-3.CASK.Mint1 ternary complex and to SAP102. In addition, the 5-HT(2C) receptor binds more strongly to PSD-95 and MPP-3 than the 5-HT(2A) receptor. In contrast, a robust interaction between the 5-HT(2A) receptor and the channel-interacting PDZ protein CIPP was found, whereas CIPP did not significantly associate with the 5-HT(2C) receptor. We also show that residues located at the -1 position and upstream the PDZ ligand in the C terminus of the 5-HT(2A) and 5-HT(2C) receptors are major determinants in their interaction with specific PDZ proteins. Immunofluorescence and electron microscopy studies strongly suggested that these specific interactions also take place in living cells and that the 5-HT(2) receptor-PDZ protein complexes occur in intracellular compartments. The interaction of the 5-HT(2A) and the 5-HT(2C) receptor with specific sets of PDZ proteins may contribute to their different signal transduction properties.     

Structural determinants governing β-arrestin2 interaction with PDZ proteins and recruitment to CRFR1

β-Arrestins are multifunctional adaptor proteins best know for their vital role in regulating G protein coupled receptor (GPCR) trafficking and signaling. β-arrestin2 recruitment and receptor internalization of corticotropin-releasing factor receptor 1 (CRFR1), a GPCR whose antagonists have been shown to demonstrate both anxiolytic- and antidepressant-like effects, have previously been shown to be modulated by PDZ proteins. Thus, a structural characterization of the interaction between β-arrestins and PDZ proteins can delineate potential mechanism of PDZ-dependent regulation of GPCR trafficking. Here, we find that the PDZ proteins PSD-95, MAGI1, and PDZK1 interact with β-arrestin2 in a PDZ domain-dependent manner. Further investigation of such interaction using mutational analyses revealed that mutating the alanine residue at 175 residue of β-arrestin2 to phenylalanine impairs interaction with PSD-95. Additionally, A175F mutant of β-arrestin2 shows decreased CRF-stimulated recruitment to CRFR1 and reduced receptor internalization. Thus, our findings show that the interaction between β-arrestins and PDZ proteins is key for CRFR1 trafficking and may be targeted to mitigate impaired CRFR1 signaling in mental and psychiatric disorders.     

beta 1-adrenergic receptor association with PSD-95. Inhibition of receptor internalization and facilitation of beta 1-adrenergic receptor interaction with N-methyl-D-aspartate receptors

The beta(1)-adrenergic receptor (beta(1)AR) is the most abundant subtype of beta-adrenergic receptor in the mammalian brain and is known to potently regulate synaptic plasticity. To search for potential neuronal beta(1)AR-interacting proteins, we screened a rat brain cDNA library using the beta(1)AR carboxyl terminus (beta(1)AR-CT) as bait in the yeast two-hybrid system. These screens identified PSD-95, a multiple PDZ domain-containing scaffolding protein, as a specific binding partner of the beta(1)AR-CT. This interaction was confirmed by in vitro fusion protein pull-down and blot overlay experiments, which demonstrated that the beta(1)AR-CT binds specifically to the third PDZ domain of PSD-95. Furthermore, the full-length beta(1)AR associates with PSD-95 in cells, as determined by co-immunoprecipitation experiments and immunofluorescence co-localization studies. The interaction between beta(1)AR and PSD-95 is mediated by the last few amino acids of the beta(1)AR, and mutation of the beta(1)AR carboxyl terminus eliminated the binding and disrupted the co-localization of the beta(1)AR and PSD-95 in cells. Agonist-induced internalization of the beta(1)AR in HEK-293 cells was markedly attenuated by PSD-95 co-expression, whereas co-expression of PSD-95 has no significant effect on either desensitization of the beta(1)AR or beta(1)AR-induced cAMP accumulation. Furthermore, PSD-95 facilitated the formation of a complex between the beta(1)AR and N-methyl-d-aspartate receptors, as assessed by co-immunoprecipitation. These data reveal that PSD-95 is a specific beta(1)AR binding partner that modulates beta(1)AR function and facilitates physical association of the beta(1)AR with synaptic proteins, such as the N-methyl-d-aspartate receptors, which are known to be regulated by beta(1)AR stimulation.     

The Golgi-associated PDZ Domain Protein PIST/GOPC Stabilizes the β1-Adrenergic Receptor in Intracellular Compartments after Internalization

Many G-protein-coupled receptors carry C-terminal ligand motifs for PSD-95/discs large/ZO-1 (PDZ) domains; via interaction with PDZ domain-containing scaffold proteins, this allows for integration of receptors into signaling complexes. However, the presence of PDZ domain proteins attached to intracellular membranes suggests that PDZ-type interactions may also contribute to subcellular sorting of receptors. The protein interacting specifically with Tc10 (PIST; also known as GOPC) is a trans-Golgi-associated protein that interacts through its single PDZ domain with a variety of cell surface receptors. Here we show that PIST controls trafficking of the interacting β1-adrenergic receptor both in the anterograde, biosynthetic pathway and during postendocytic recycling. Overexpression and knockdown experiments show that PIST leads to retention of the receptor in the trans-Golgi network (TGN), to the effect that overexpressed PIST reduces activation of the MAPK pathway by β1-adrenergic receptor (β1AR) agonists. Receptors can be released from retention in the TGN by coexpression of the plasma membrane-associated scaffold PSD-95, which allows for transport of receptors to the plasma membrane. Stimulation of β1 receptors and activation of the cAMP pathway lead to relocation of PIST from the TGN to an endosome-like compartment. Here PIST colocalizes with SNX1 and the internalized β1AR and protects endocytosed receptors from lysosomal degradation. In agreement, β1AR levels are decreased in hippocampi of PIST-deficient mice. Our data suggest that PIST contributes to the fine-tuning of β1AR sorting both during biosynthetic and postendocytic trafficking.     

A PDZ-binding motif controls basolateral targeting of syndecan-1 along the biosynthetic pathway in polarized epithelial cells

The cell surface proteoglycan, syndecan-1, is essential for normal epithelial morphology and function. Syndecan-1 is selectively localized to the basolateral domain of polarized epithelial cells and interacts with cytosolic PDZ (PSD-95, discs large, ZO-1) domain-containing proteins. Here, we show that the polarity of syndecan-1 is determined by its type II PDZ-binding motif. Mutations within the PDZ-binding motif lead to the mislocalization of syndecan-1 to the apical surface. In contrast to previous examples, however, PDZ-binding motif-dependent polarity is not determined by retention at the basolateral surface but rather by polarized sorting prior to syndecan-1's arrival at the plasma membrane. Although none of the four known PDZ-binding partners of syndecan-1 appears to control basolateral localization, our results show that the PDZ-binding motif of syndecan-1 is decoded along the biosynthetic pathway establishing a potential role for PDZ-mediated interactions in polarized sorting.     

Interaction with cystic fibrosis transmembrane conductance regulator-associated ligand (CAL) inhibits beta1-adrenergic receptor surface expression

G protein-coupled receptors such as the beta1-adrenergic receptor (beta1AR) must be trafficked to the plasma membrane in order to bind with their extracellular ligands and regulate cellular physiology. By using glutathione S-transferase pull-down techniques, we found that the beta1AR carboxyl terminus directly interacts with the cystic fibrosis transmembrane conductance regulator-associated ligand (CAL; also known as PIST, GOPC, and FIG), a protein known to be primarily localized to the Golgi apparatus. CAL contains two predicted coiled-coil domains and one PSD-95/Discs-large/ZO-1 homology (PDZ) domain. The beta1AR carboxyl terminus (CT) binds to the PDZ domain of CAL, with the last few amino acids (ESKV) of the beta1AR-CT being the key determinants for the interaction. Mutation of the terminal valine residue resulted in markedly reduced association of the beta1AR-CT with CAL. Numerous other mutations to the ESKV motif also impaired the beta1AR-CT/CAL interaction, suggesting that this motif is close to optimal for association with the CAL PDZ domain. In cells, full-length beta1AR robustly associates with CAL, and this interaction is abolished by mutation of the terminal valine to alanine of the receptor (V477A), as determined by co-immunoprecipitation experiments and immunofluorescence co-localization studies. Consistent with observations that CAL is a Golgi-associated protein, overexpression of CAL reduces surface expression of beta1AR. Interaction with CAL promotes retention of beta1AR within the cell, whereas PSD-95, another beta1AR-associated PDZ domain-containing protein, competitively blocks beta1AR association with CAL and promotes receptor trafficking to the cell surface. These data reveal that CAL, a novel beta1AR-binding partner, modulates beta1AR intracellular trafficking, thereby revealing a new mechanism of regulation for beta1AR anterograde trafficking through the endoplasmic reticulum-Golgi complex to the plasma membrane.     

Microtubule-associated protein light chain 2 is a stargazin-AMPA receptor complex-interacting protein in vivo

The ataxic mutant mouse stargazer is a null mutant for stargazin, a protein involved in the regulation of cell surface trafficking and synaptic targeting of AMPA receptors. The extreme C terminus of stargazin (sequence, -TTPV), confers high affinity for PDZ domain-containing proteins e.g. PSD-95. Interaction with PDZ proteins enables stargazin to fulfill its role as an AMPA receptor synaptic targeting molecule but is not essential for its ability to influence AMPA receptor trafficking to the neuronal cell surface. Using the yeast-two hybrid approach we screened for proteins that interact with the intracellular C-terminal tail of stargazin. Positive interactors included PDZ domain-containing proteins e.g. SAP97, SAP102, and PIST. Interestingly, light chain 2 of microtubule-associated protein 1 (LC2), which does not contain a PDZ domain, was also a strong interactor. This was shown to be a direct interaction that occurred upstream of the -TTPV sequence of stargazin. Immunoprecipitations of Triton X-100 soluble cerebellar extracts revealed that LC2 is pulled down not only by anti-stargazin antibodies but also anti-GluR2 antibodies suggesting that stargazin and AMPA receptor subunits associate with LC2. Immunopurified full-length, native stargazin was shown to co-associate not only with GluR2 in vivo but also with full-length, native LC2. Indeed, LC2 co-associates with stargazin when part of a tripartite complex comprising LC2-stargazin-GluR2. Since this complex was extracted using Triton X-100 and was devoid of PSD95, SAP97, and actin we postulate that LC2 is involved in trafficking of AMPA receptors in cerebellar neurons before they are anchored at the synapse.     

Deficiency of sorting nexin 27 (SNX27) leads to growth retardation and elevated levels of N-methyl-D-aspartate receptor 2C (NR2C)

Phox (PX) domain-containing sorting nexins (SNXs) are emerging as important regulators of endocytic trafficking. Sorting nexin 27 (SNX27) is unique, as it contains a PDZ (Psd-95/Dlg/ZO1) domain. We show here that SNX27 is primarily targeted to the early endosome by interaction of its PX domain with PtdIns(3)P. Although targeted ablation of the SNX27 gene in mice did not significantly affect growth and survival during embryonic development, SNX27 plays an essential role in postnatal growth and survival. N-Methyl-d-aspartate (NMDA) receptor 2C (NR2C) was identified as a novel SNX27-interacting protein, and this interaction is mediated by the PDZ domain of SNX27 and the C-terminal PDZ-binding motif of NR2C. Increased NR2C expression levels, together with impaired NR2C endocytosis in SNX27(-/-) neurons, indicate that SNX27 may function to regulate endocytosis and/or endosomal sorting of NR2C. This is consistent with a role of SNX27 as a general regulator for sorting of membrane proteins containing a PDZ-binding motif, and its absence may alter the trafficking of these proteins, leading to growth and survival defects.     

Interdomain chaperoning between PSD-95, Dlg, and Zo-1 (PDZ) domains of glutamate receptor-interacting proteins.

The multiple PSD-95, Dlg, and Zo-1 (PDZ) domain protein, glutamate receptor-interacting protein (GRIP), is involved in the clustering and trafficking of the alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor by directly binding to the cytoplasmic tail of the receptor's GluR2 subunit. Both the forth and fifth PDZ domains (PDZ4 and PDZ5) of GRIP are required for effective binding to the receptor. Using NMR and circular dichroism spectroscopic techniques, we show that PDZ5 is completely unstructured in solution. Freshly prepared PDZ4 is largely folded, but the domain can spontaneously unfold. Neither PDZ4 nor PDZ5 binds to GluR2 in solution. Unexpectedly, when PDZ4 and PDZ5 are covalently connected (i.e. PDZ45), both PDZ domains become well folded and stable in solution. The covalent linkage of the two PDZ domains is essential for proper folding of the tandem PDZ domains and its effective binding to GluR2. The interdomain chaperoning effect observed in the PDZ domains of GRIP represents a previously uncharacterized function of PDZ domains.     

Differential modulation of NR1-NR2A and NR1-NR2B subtypes of NMDA receptor by PDZ domain-containing proteins

The PSD-95/Dlg/ZO-1 (PDZ) domain-containing proteins MALS and PSD-95 localize to post-synaptic densities and bind the COOH-termini of NR2 subunits of the NMDA receptor. The effects of MALS-2 and PSD-95 on the channel activity of NMDA receptors were compared using the Xenopus oocyte expression system. Both MALS-2 and PSD-95 increased the current response of the NR1-NR2B receptor to l-glutamate. In contrast, the current response of the NR1-NR2A receptor was increased by PSD-95 but not by MALS-2. MALS-2 had no effect either on the potentiation of NR1-NR2A or NR1-NR2B channel activity by protein kinase C, or on Src-mediated potentiation of NR1-NR2A activity, whereas PSD-95 almost completely inhibited the effects of these protein kinases. Construction of chimeras of MALS-2 and PSD-95 revealed that the first two PDZ domains and two NH(2)-terminal cysteine residues are essential for the inhibitory effects of PSD-95 on protein kinase C-mediated potentiation of NR1-NR2A and NR1-NR2B channel activity, respectively. The second of the three PDZ domains of PSD-95 was required for its inhibition of Src-mediated potentiation of NR1-NR2A activity. These results indicate that the NR1-NR2A and NR1-NR2B receptors are modulated differentially by MALS-2 and PSD-95, and that similar regulatory effects of PSD-95 on these receptors are achieved by distinct mechanisms.     

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.     

Formation of a native-like beta-hairpin finger structure of a peptide from the extended PDZ domain of neuronal nitric oxide synthase in aqueous solution.

Neuronal nitric oxide synthase (nNOS) is targeted to the cell membrane via interactions of its extended PDZ domain with PDZ domains of membrane-associated proteins including PSD-95 and alpha1-syntrophin. The formation of heterodimers between the nNOS PDZ domain and the PDZ domains of nNOS-binding proteins requires a stretch of continuous amino-acid residues C-terminal to the canonical nNOS PDZ domain. In this work, we show that a 27-residue peptide comprising the C-terminal extension of the extended nNOS PDZ domain is capable of binding to PSD-95. The structure of the 27-residue peptide in aqueous solution was determined using multidimensional NMR-spectroscopic techniques. The free peptide adopts a native-like beta-hairpin finger structure in aqueous solution. The results indicate that the C-terminal extension peptide of the nNOS PDZ domain may represent a relatively independent structural unit in the mediation of the interaction between nNOS and PDZ domain-containing proteins including PSD-95 and alpha1-syntrophin.     

Insulin receptor substrate of 53 kDa links postsynaptic shank to PSD-95

The insulin receptor substrate of 53 kDa (IRSp53) is a target of the small GTPase cdc42 which is strongly enriched in the postsynaptic density of excitatory synapses. IRSp53 interacts with the postsynaptic shank1 scaffolding molecule in a cdc42 regulated manner. The functional significance of the cdc42/IRSp53 pathway in postsynaptic sites is however, unclear. Here we identify PSD-95 as a second synaptic interaction partner of IRSp53. Interaction is mediated by a C-terminal PDZ binding motif in IRSp53 and the second PDZ domain of PSD-95. In HEK cells, overexpressed IRSp53 induces filopodia and targets PSD-95 into these processes. Immunoprecipitation and immunocytochemistry experiments demonstrate that the interaction occurs at postsynaptic sites in the brain. By virtue of its PDZ-binding and SH3 domains, IRSp53 is capable of inducing the formation of a triple complex (shank1/IRSp53/PSD-95).     

Proteomic analysis of beta1-adrenergic receptor interactions with PDZ scaffold proteins

Many G protein-coupled receptors possess carboxyl-terminal motifs ideal for interaction with PDZ scaffold proteins, which can control receptor trafficking and signaling in a cell-specific manner. To gain a panoramic view of beta1-adrenergic receptor (beta AR) interactions with PDZ scaffolds, the beta1AR carboxyl terminus was screened against a newly developed proteomic array of PDZ domains. These screens confirmed beta1AR associations with several previously identified PDZ partners, such as PSD-95, MAGI-2, GIPC, and CAL. Moreover, two novel beta1AR-interacting proteins, SAP97 and MAGI-3, were also identified. The beta1AR carboxyl terminus was found to bind specifically to the first PDZ domain of MAGI-3, with the last four amino acids (E-S-K-V) of beta1AR being the key determinants of the interaction. Full-length beta1AR robustly associated with full-length MAGI-3 in cells, and this association was abolished by mutation of the beta1AR terminal valine residue to alanine (V477A), as determined by co-immunoprecipitation experiments and immunofluorescence co-localization studies. MAGI-3 co-expression with beta1AR profoundly impaired beta1AR-mediated ERK1/2 activation but had no apparent effect on beta1AR-mediated cyclic AMP generation or agonist-promoted beta1AR internalization. These findings revealed that the interaction of MAGI-3 with beta1AR can selectively regulate specific aspects of receptor signaling. Moreover, the screens of the PDZ domain proteomic array provide a comprehensive view of beta1AR interactions with PDZ scaffolds, thereby shedding light on the molecular mechanisms by which beta1 AR signaling and trafficking can be regulated in a cell-specific manner.