Protein interacting with C kinase 1 (PICK1) reduces reinsertion rates of interaction partners sorted to Rab11-dependent slow recycling pathway

The scaffolding protein PICK1 (protein interacting with C kinase 1) contains an N-terminal PSD-95/Discs large/ZO-1 (PDZ) domain and a central lipid-binding Bin/amphiphysin/Rvs (BAR) domain. PICK1 is thought to regulate trafficking of its PDZ binding partners but different and even opposing functions have been suggested. Here, we apply ELISA-based assays and confocal microscopy in HEK293 cells with inducible PICK1 expression to assess in an isolated system the ability of PICK1 to regulate trafficking of natural and engineered PDZ binding partners. The dopamine transporter (DAT), which primarily sorts to degradation upon internalization, did not form perinuclear clusters with PICK1, and PICK1 did not affect DAT internalization/recycling. However, transfer of the PICK1-binding DAT C terminus to the β(2)-adrenergic receptor, which sorts to recycling upon internalization, led to formation of PICK1 co-clusters in Rab11-positive compartments. Furthermore, PICK1 inhibited Rab11-mediated recycling of the receptor in a BAR and PDZ domain-dependent manner. In contrast, transfer of the DAT C terminus to the δ-opioid receptor, which sorts to degradation, did not result in PICK1 co-clusters or any change in internalization/recycling. Further support for a role of PICK1 determined by its PDZ cargo was obtained for the PICK1 interaction partner prolactin-releasing peptide receptor (GPR10). GPR10 co-localized with Rab11 and clustered with PICK1 upon constitutive internalization but co-localized with the late endosomal marker Rab7 and did not cluster with PICK1 upon agonist-induced internalization. Our data suggest a selective role of PICK1 in clustering and reducing the recycling rates of PDZ domain binding partners sorted to the Rab11-dependent recycling pathway.     

PICK1 interacts with ABP/GRIP to regulate AMPA receptor trafficking

PICK1 and ABP/GRIP bind to the AMPA receptor (AMPAR) GluR2 subunit C terminus. Transfer of the receptor from ABP/GRIP to PICK1, facilitated by GluR2 S880 phosphorylation, may initiate receptor trafficking. Here we report protein interactions that regulate these steps. The PICK1 BAR domain interacts intermolecularly with the ABP/GRIP linker II region and intramolecularly with the PICK1 PDZ domain. Binding of PKCalpha or GluR2 to the PICK1 PDZ domain disrupts the intramolecular interaction and facilitates the PICK1 BAR domain association with ABP/GRIP. Interference with the PICK1-ABP/GRIP interaction impairs S880 phosphorylation of GluR2 by PKC and decreases the constitutive surface expression of GluR2, the NMDA-induced endocytosis of GluR2, and recycling of internalized GluR2. We suggest that the PICK1 interaction with ABP/GRIP is a critical step in controlling GluR2 trafficking.     

Serine 77 in the PDZ domain of PICK1 is a protein kinase Cα phosphorylation site regulated by lipid membrane binding

PICK1 (protein interacting with C kinase 1) contains an N-terminal protein binding PDZ domain and a C-terminal lipid binding BAR domain. PICK1 plays a key role in several physiological processes, including synaptic plasticity. However, little is known about the cellular mechanisms governing the activity of PICK1 itself. Here we show that PICK1 is a substrate in vitro both for PKCα (protein kinase Cα), as previously shown, and for CaMKIIα (Ca(2+)-calmodulin-dependent protein kinase IIα). By mutation of predicted phosphorylation sites, we identify Ser77 in the PDZ domain as a major phosphorylation site for PKCα. Mutation of Ser77 reduced the level of PKCα-mediated phosphorylation ~50%, whereas no reduction was observed upon mutation of seven other predicted sites. Addition of lipid vesicles increased the level of phosphorylation of Ser77 10-fold, indicating that lipid binding is critical for optimal phosphorylation. Binding of PKCα to the PICK1 PDZ domain was not required for phosphorylation, but a PDZ domain peptide ligand reduced the overall level of phosphorylation ~30%. The phosphomimic S77D reduced the extent of cytosolic clustering of eYFP-PICK1 in COS7 cells and thereby conceivably its lipid binding and/or polymerization capacity. We propose that PICK1 is phosphorylated at Ser77 by PKCα preferentially when bound to membrane vesicles and that this phosphorylation in turn modulates its cellular distribution.     

Targeted in vivo mutations of the AMPA receptor subunit GluR2 and its interacting protein PICK1 eliminate cerebellar long-term depression

Cerebellar long-term depression (LTD) is a major form of synaptic plasticity that is thought to be critical for certain types of motor learning. Phosphorylation of the AMPA receptor subunit GluR2 on serine-880 as well as interaction of GluR2 with PICK1 have been suggested to contribute to the endocytic removal of postsynaptic AMPA receptors during LTD. Here, we show that targeted mutation of PICK1, the GluR2 C-terminal PDZ ligand, or the GluR2 PKC phosphorylation site eliminates cerebellar LTD in mice. LTD can be rescued in cerebellar cultures from mice lacking PICK1 by transfection of wild-type PICK1 but not by a PDZ mutant or a BAR domain mutant deficient in lipid binding, indicating the importance of these domains in PICK1 function. These results demonstrate that PICK1-GluR2 PDZ-based interactions and GluR2 phosphorylation are required for LTD expression in the cerebellum.     

PICK1蛋白C端酸性区域对其功能的调节

蛋白激酶C相互作用蛋白1(protein interacting with Ckinase1,PICK1)是调节AMPA(alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid)受体在细胞膜上的数量与分布,引起LTP与LTD现象的重要蛋白.本文利用基因克隆、荧光光谱以及免疫分析等方法,分析了PICK1蛋白C末端酸性区对BAR结构域与膜脂结合能力以及PICK1分子内BAR(Bin/amphiphysin/RVS)结构域与PDZ结构域相互作用的影响,研究了钙离子结合C末端酸性区后对上述相互作用的调节.结果显示,C末端酸性区的存在使BAR结构域与膜脂的结合能力减弱大约10倍,但PICK1分子内的BAR与PDZ结构域的相互作用与不含C末端的酸性区相比增强了大约4倍.另一方面,C末端酸性区的存在,伴随钙离子浓度的提高,有助于增强BAR与膜脂的结合,却削弱了PDZ和BAR结构域的作用.当钙离子浓度增加到500μmol/L时,BARC的脂质结合能力以及和PDZ的亲和力与不含酸性区相当.     

Structure and function of PICK1

PICK1 is a peripheral membrane protein conserved from Caenorhabditis elegans to the human. It is expressed in many tissues with high levels in brain and testis. Inside cells, PICK1 is localized at the perinuclear region as well as specialized structures such as synapses of neurons. PICK1 contains a PDZ domain and a BAR domain. The PDZ domain of PICK1 binds to a large number of membrane proteins, especially proteins with C-terminal type II PDZ-binding motifs. The BAR domain of PICK1 binds to lipid molecules, mainly phosphoinositides. While the PDZ domain and the linker region of PICK1 enhance BAR domain's lipid binding, the C-terminal region of PICK1 inhibits its lipid binding. PICK1 regulates the subcellular localization and surface expression of its PDZ-binding partners. Lipid binding of PICK1's BAR domain is important for this regulation. With its PDZ domain interacting with membrane proteins and its BAR domain binding to lipids, the unique structure of PICK1 enables it to couple membrane proteins to protein-trafficking machinery.     

Zinc binding site in PICK1 is dominantly located at the CPC motif of its PDZ domain

PICK1 ( p rotein i nteracting with C k inase 1) containing a PDZ domain, a BAR domain, and two short acidic regions is as an adaptor protein that plays an important role in α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid receptor trafficking, cell morphology and migration, as well as in some diseases such as cancer, schizophrenia and pain. To better understand the physiological function of PICK1, we expressed the recombinant PICK1 and its truncated mutants in E.coli, and measured their zinc binding properties by fluorescence and competition assay. It is shown that PICK1 has one Zn²⁺-binding site. The Zn²⁺-binding properties of PICK1 are not appreciably affected after the removal of BARC domain (involving BAR domain and C-terminal acidic region). Deleting the N-terminal acidic region of NPDZ domain (involving PDZ domain and N-terminal acidic region) in PICK1 impairs its Zn²⁺-binding capacity.The mutation of the CPC (Cys-Pro-Cys) motif in the PDZ domain of PICK1 abolishes the ability of Zn²⁺-binding. In addition, Zn²⁺ can enhance the lipid-binding ability of PDZ domain as observed in both protein-lipid overlay assay and fluorescence analysis. The results presented in this report suggested that Zn²⁺ plays a regulatory role in the trafficking of PICK1 from the cytoplasm to cell membrane.     

The PDZ domain of PICK1 differentially accepts protein kinase C-alpha and GluR2 as interacting ligands

The C terminus (ct) of protein kinase C-alpha (PKCalpha) has a type I PDZ binding motif, whereas GluR2 has a type II PDZ binding motif. Both motifs are recognized by the PDZ domain of protein interacting with protein kinase C (PICK1), and PICK1-PKCalpha-controlled phosphorylation regulates the synaptic expression and function of GluR2. Here, we show that a specific mutation within the carboxylate-binding loop of the PDZ domain of PICK1 (K27E; PICK1-KE) results in a loss of interaction with GluR2 but not with PKCalpha. In GST pull-down studies, PICK1-WT (wild type) but not PICK1-KE was retained by GST-ct-GluR2. Furthermore, PICK1-WT co-immunoprecipitated both PKCalpha and GluR2, whereas PICK1-KE only co-immunoprecipitated PKCalpha. In heterologous cells, PICK1-WT, but not PICK1-KE, clustered GluR2 and also clustered GluR1 in a GluR2-dependent manner. However, neither PICK1-WT nor PICK1-KE altered the distribution of PKCalpha, even after phorbol ester-induced redistribution of PKCalpha to the membrane. Finally, PICK1-KE showed no mislocalization when compared with PICK1-WT in neurons. Taken together, it appears that the PDZ domain of PICK1 is less sensitive to mutations for PKCalpha when compared with GluR2 binding. These results suggest that the PDZ domain of PICK1 has distinct PKCalpha and GluR2 binding subsite(s).     

PICK1 is implicated in organelle motility in an Arp2/3 complex–independent manner

PICK1 is a modular scaffold implicated in synaptic receptor trafficking. It features a PDZ domain, a BAR domain, and an acidic C-terminal tail (ACT). Analysis by small- angle x-ray scattering suggests a structural model that places the receptor-binding site of the PDZ domain and membrane-binding surfaces of the BAR and PDZ domains adjacent to each other on the concave side of the banana-shaped PICK1 dimer. In the model, the ACT of one subunit of the dimer interacts with the PDZ and BAR domains of the other subunit, possibly accounting for autoinhibition. Consistently, full-length PICK1 shows diffuse cytoplasmic localization, but it clusters on vesicle-like structures that colocalize with the trans-Golgi network marker TGN38 upon deletion of either the ACT or PDZ domain. This localization is driven by the BAR domain. Live-cell imaging further reveals that PICK1-associated vesicles undergo fast, nondirectional motility in an F-actin–dependent manner, but deleting the ACT dramatically reduces vesicle speed. Thus the ACT links PICK1-associated vesicles to a motility factor, likely myosin, but, contrary to previous reports, PICK1 neither binds nor inhibits Arp2/3 complex.     

Clustering and synaptic targeting of PICK1 requires direct interaction between the PDZ domain and lipid membranes

Protein interacting with c kinase 1 (PICK1) regulates the trafficking of receptors and ion-channels such as AMPA receptors. Traditionally, the PICK1 PDZ domain is regarded as an adaptor capable of binding to receptors trafficked by PICK1, and the lipid-binding BAR domain functions to tether PICK1 directly to membranes. Here, we show that the PICK1 PDZ domain can directly interact with lipid membranes. The PDZ domain and lipid membrane interaction is mediated by both a polybasic amino-acid cluster and a conserved 'Cys-Pro-Cys' motif located away from the peptide ligand-binding groove. Disruption of the PDZ and lipid membrane interaction totally abolished synaptic targeting of PICK1. Although mutation of the CPC motif did not affect the interaction between PICK1 and AMPA receptors, the mutant PICK1 was unable to cluster the GluR2 subunit of the receptor. In neurons, PICK1 containing the same mutation displayed dramatically compromised capacity in the trafficking of AMPA receptors. Taken together, our findings not only uncovered the novel lipid membrane-binding property of the PICK1 PDZ domain, but also provided direct evidence supporting the functional relevance of the PDZ-lipid interaction.     

PDZ binding to the BAR domain of PICK1 is elucidated by coarse-grained molecular dynamics

A key regulator of α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor traffic, PICK1 is known to interact with over 40 other proteins, including receptors, transporters and ionic channels, and to be active mostly as a homodimer. The current lack of a complete PICK1 structure determined at atomic resolution hinders the elucidation of its functional mechanisms. Here, we identify interactions between the component PDZ and BAR domains of PICK1 by calculating possible binding sites for the PDZ domain of PICK1 (PICK1-PDZ) to the homology-modeled, crescent-shaped dimer of the PICK1-BAR domain using multiplexed replica-exchange molecular dynamics (MREMD) and canonical molecular dynamics simulations with the coarse-grained UNRES force field. The MREMD results show that the preferred binding site for the single PDZ domain is the concave cavity of the BAR dimer. A second possible binding site is near the N-terminus of the BAR domain that is linked directly to the PDZ domain. Subsequent short canonical molecular dynamics simulations used to determine how the PICK1-PDZ domain moves to the preferred binding site on the BAR domain of PICK1 revealed that initial hydrophobic interactions drive the progress of the simulated binding. Thus, the concave face of the BAR dimer accommodates the PDZ domain first by weak hydrophobic interactions and then the PDZ domain slides to the center of the concave face, where more favorable hydrophobic interactions take over.     

Cerebellar long-term depression requires PKC-regulated interactions between GluR2/3 and PDZ domain-containing proteins

Cerebellar LTD requires activation of PKC and is expressed, at least in part, as postsynaptic AMPA receptor internalization. Recently, it was shown that AMPA receptor internalization requires clathrin-mediated endocytosis and depends upon the carboxy-terminal region of GluR2/3. Phosphorylation of Ser-880 in this region by PKC differentially regulates the binding of the PDZ domain-containing proteins GRIP/ABP and PICK1. Peptides, corresponding to the phosphorylated and dephosphorylated GluR2 carboxy-terminal PDZ binding motif, were perfused in cerebellar Purkinje cells grown in culture. Both the dephospho form (which blocks binding of GRIP/ABP and PICK1) and the phospho form (which selectively blocks PICK1) attenuated LTD induction by glutamate/depolarization pairing, as did antibodies directed against the PDZ domain of PICK1. These findings indicate that expression of cerebellar LTD requires PKC-regulated interactions between the carboxy-terminal of GluR2/3 and PDZ domain-containing proteins.     

Mutagenesis reveals a role for ABP/GRIP binding to GluR2 in synaptic surface accumulation of the AMPA receptor

We studied the role of PDZ proteins GRIP, ABP, and PICK1 in GluR2 AMPA receptor trafficking. An epitope-tagged MycGluR2 subunit, when expressed in hippocampal cultured neurons, was specifically targeted to the synaptic surface. With the mutant MycGluR2delta1-10, which lacks the PDZ binding site, the overall dendritic intracellular transport and the synaptic surface targeting were not affected. However, over time, Myc-GluR2delta1-10 accumulated at synapses significantly less than MycGluR2. Notably, a single residue substitution, S880A, which blocks binding to ABP/GRIP but not to PICK1, reduced synaptic accumulation to the same extent as the PDZ site truncation. We conclude that the association of GluR2 with ABP and/or GRIP but not PICK1 is essential for maintaining the synaptic surface accumulation of the receptor, possibly by limiting its endocytotic rate.     

Interaction of the PDZ domain of human PICK1 with class I ADP-ribosylation factors

We have cloned the cDNA encoding human PICK1 (protein interacting with C kinase 1), a PDZ domain-containing protein of 415 amino acids, and also identified the Drosophila homologue by search of the databank. Northern blot analysis shows a single mRNA of about 2.0 kb ubiquitously expressed in human tissues. Although PICK1 proteins harbor a region homologous to arfaptin1 and arfaptin2, two proteins that bind to the ARF (ADP-ribosylation factor), this region of PICK1 does not interact with ARFs in the yeast two-hybrid system. On the other hand, the PDZ domain of PICK1 is capable of interacting with constitutively active, GTP-bound forms of ARF1 and ARF3, but neither with those of ARF5/6 nor with the GDP-bound ARFs. The PICK1-ARF interaction is abrogated by introduction of mutations in the PDZ domain or by deletion of the extreme C-terminus of ARF1. Thus, PICK1 specifically interacts with ARF1/3 in the GTP-bound state, suggesting that PICK1 participates in ARF1/3-mediated cellular processes.     

The BAR domain protein PICK1 regulates cell recognition and morphogenesis by interacting with Neph proteins

Neph proteins are evolutionarily conserved membrane proteins of the immunoglobulin superfamily that control the formation of specific intercellular contacts. Cell recognition through these proteins is essential in diverse cellular contexts such as patterning of the compound eye in Drosophila melanogaster, neuronal connectivity in Caenorhabditis elegans, and the formation of the kidney filtration barrier in mammals. Here we identify the PDZ and BAR domain protein PICK1 (protein interacting with C-kinase 1) as a Neph-interacting protein. Binding required dimerization of PICK1, was dependent on PDZ domain protein interactions, and mediated stabilization of Neph1 at the plasma membrane. Moreover, protein kinase C (PKCα) activity facilitated the interaction through releasing Neph proteins from their binding to the multidomain scaffolding protein zonula occludens 1 (ZO-1), another PDZ domain protein. In Drosophila, the Neph homologue Roughest is essential for sorting of interommatidial precursor cells and patterning of the compound eye. RNA interference-mediated knockdown of PICK1 in the Drosophila eye imaginal disc caused a Roughest destabilization at the plasma membrane and a phenotype that resembled rst mutation. These data indicate that Neph proteins and PICK1 synergistically regulate cell recognition and contact formation.     

An essential role for PICK1 in NMDA receptor-dependent bidirectional synaptic plasticity

PICK1 is a calcium-sensing, PDZ domain-containing protein that interacts with GluR2 and GluR3 AMPA receptor (AMPAR) subunits and regulates their trafficking. Although PICK1 has been principally implicated in long-term depression (LTD), PICK1 overexpression in CA1 pyramidal neurons causes a CaMK- and PKC-dependent potentiation of AMPAR-mediated transmission and an increase in synaptic GluR2-lacking AMPARs, mechanisms associated with NMDA receptor (NMDAR)-dependent long-term potentiation (LTP). Here, we directly tested whether PICK1 participates in both hippocampal NMDAR-dependent LTP and LTD. We show that the PICK1 potentiation of AMPAR-mediated transmission is NMDAR dependent and fully occludes LTP. Conversely, blockade of PICK1 PDZ interactions or lack of PICK1 prevents LTP. These observations demonstrate an important role for PICK1 in LTP. In addition, deletion of PICK1 or blockade of PICK1 PDZ binding prevented NMDAR-dependent LTD. Thus, PICK1 plays a critical role in bidirectional NMDAR-dependent long-term synaptic plasticity in the hippocampus.     

Molecular determinants for PICK1 synaptic aggregation and mGluR7a receptor coclustering: role of the PDZ, coiled-coil, and acidic domains

PSD-95/Disc-large/ZO-1 (PDZ) domain-containing proteins play a central role in synaptic organization by their involvement in neurotransmitter receptor clustering and signaling complex assembly. The protein interacting with protein kinase C (PICK1), a synaptic PDZ domain protein that also contains a coiled-coil and acidic domain, binds to several synaptic components including the metabotropic glutamate receptor mGluR7a. Coexpression of PICK1 and mGluR7a in heterologous cells induces coclustering of these two proteins. To examine the role of the different structural motifs of PICK1 in synaptic aggregation of PICK1 and mGluR7a coclustering, several PICK1 mutants were generated to analyze their distribution in transfected hippocampal cultured neurons and to test their ability to induce coclusters with mGluR7a when coexpressed in fibroblast cells. The PDZ and coiled-coil domains are both required, whereas the acidic region plays an inhibitory role in these processes. Our data suggest that synaptic aggregation and receptor coclustering depend on PICK1 binding to a target membrane receptor, e.g. mGluR7a, by a PDZ-mediated interaction and on PICK1 oligomerization through the coiled-coil domain. This study defined three structural signals within PICK1 regulating its synaptic localization and receptor coclustering activity, which could represent molecular substrates involved in synaptic development and plasticity.     

PICK1中N端酸性区域对其PDZ结构域脂质结合能力的调节

蛋白激酶Cα相互作用蛋白1(protein interacting with Cα kinase 1, PICK1)是衔接膜上受体和蛋白激酶Cα的重要蛋白.利用荧光光谱结合定点突变技术 、蛋白与脂质覆盖法等方法,分析了PICK1蛋白N末端区域几个酸性氨基酸残基对PDZ 结构域与膜脂结合的影响,以及钙离子结合N末端酸性区域对PDZ脂结合能力的调节. 结果显示, 带有上游酸性区域的PDZ结构域（NPDZ）的脂质结合能力仅相当PDZ结构 域的15%,相比单独的PDZ结构域与脂质的解离常数Kd(PDZ)为1.58×103 μg·L-1, NPDZ与脂质解离常数Kd(NPDZ)为3.3×104μg·L-1,其中在N末端酸性残基中D8与 D12两个天冬氨酸是影响脂质结合能力减弱的关键残基,若将二者分别突变为丙氨酸 后,NPDZ与脂质的解离常数分别为：Kd (D8/A)=4.42×103μg·L-1;Kd (D12/A) =1.73×103μg·L-1接近于PDZ结构域与脂质结合能力;钙离子会增强NPDZ脂结合能力,当钙离子浓度达到30 μmol/L时,NPDZ的脂结合能力提高2.3倍,但只相当于PDZ的50% 的结合能力.     

Molecular determinants for the complex binding specificity of the PDZ domain in PICK1

PICK1 (protein interacting with C kinase 1) contains a single PDZ domain known to mediate interaction with the C termini of several receptors, transporters, ion channels, and kinases. In contrast to most PDZ domains, the PICK1 PDZ domain interacts with binding sequences classifiable as type I (terminating in (S/T)XPhi; X, any residue) as well as type II (PhiXPhi; Phi, any hydrophobic residue). To enable direct assessment of the affinity of the PICK1 PDZ domain for its binding partners we developed a purification scheme for PICK1 and a novel quantitative binding assay based on fluorescence polarization. Our results showed that the PICK1 PDZ domain binds the type II sequence presented by the human dopamine transporter (-WLKV) with an almost 15-fold and >100-fold higher affinity than the type I sequences presented by protein kinase Calpha (-QSAV) and the beta(2)-adrenergic receptor (-DSLL), respectively. Mutational analysis of Lys(83) in the alphaB1 position of the PDZ domain suggested that this residue mimics the function of hydrophobic residues present in this position in regular type II PDZ domains. The PICK1 PDZ domain was moreover found to prefer small hydrophobic residues in the C-terminal P(0) position of the ligand. Molecular modeling predicted a rank order of (Val > Ile > Leu) that was verified experimentally with up to a approximately 16-fold difference in binding affinity between a valine and a leucine in P(0). The results define the structural basis for the unusual binding pattern of the PICK1 PDZ domain by substantiating the critical role of the alphaB1 position (Lys(83)) and of discrete side chain differences in position P(0) of the ligands.     

Tandem PDZ repeats in glutamate receptor-interacting proteins have a novel mode of PDZ domain-mediated target binding

The interaction of the glutamate receptor subunits 2 and 3 (GluR2/3) with multi-PDZ domain glutamate receptor-interacting protein (GRIP) is important for the synaptic trafficking and clustering of the receptors. Binding of GluR2/3 to GRIP requires both the fourth and fifth PDZ domains (PDZ4 and PDZ5) to be covalently linked, although only one PDZ domain is directly involved in binding to the receptor tail. To elucidate the molecular basis of this mode of PDZ domain-mediated target recognition, we solved the solution structures of the PDZ45 tandem and the isolated PDZ4 of GRIP. The two PDZ domains form a compact structure with a fixed interdomain orientation. The interdomain packing and the stable folding of both PDZ domains require a short stretch of amino acids N-terminal to PDZ4 and a conserved linker connecting PDZ4 and PDZ5. PDZ4 contains a deformed aB-bB groove that is unlikely to bind to carboxyl peptides. Instead, the domain stabilizes the structure of PDZ5.