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• W4377023941 abstract "Group I metabotropic glutamate receptors (mGluRs) play important roles in many neuronal processes and are believed to be involved in synaptic plasticity underlying the encoding of experience, including classic paradigms of learning and memory. These receptors have also been implicated in various neurodevelopmental disorders, such as Fragile X syndrome and autism. Internalization and recycling of these receptors in the neuron are important mechanisms to regulate the activity of the receptor and control the precise spatiotemporal localization of these receptors. Applying a “molecular replacement” approach in hippocampal neurons derived from mice, we demonstrate a critical role for protein interacting with C kinase 1 (PICK1) in regulating the agonist-induced internalization of mGluR1. We show that PICK1 specifically regulates the internalization of mGluR1, but it does not play any role in the internalization of the other member of group I mGluR family, mGluR5. Various regions of PICK1 viz., the N-terminal acidic motif, PDZ domain, and BAR domain play important roles in the agonist-mediated internalization of mGluR1. Finally, we demonstrate that PICK1-mediated internalization of mGluR1 is critical for the resensitization of the receptor. Upon knockdown of endogenous PICK1, mGluR1s stayed on the cell membrane as inactive receptors, incapable of triggering the MAP kinase signaling. They also could not induce AMPAR endocytosis, a cellular correlate for mGluR-dependent synaptic plasticity. Thus, this study unravels a novel role for PICK1 in the agonist-mediated internalization of mGluR1 and mGluR1-mediated AMPAR endocytosis that might contribute to the function of mGluR1 in neuropsychiatric disorders. Group I metabotropic glutamate receptors (mGluRs) play important roles in many neuronal processes and are believed to be involved in synaptic plasticity underlying the encoding of experience, including classic paradigms of learning and memory. These receptors have also been implicated in various neurodevelopmental disorders, such as Fragile X syndrome and autism. Internalization and recycling of these receptors in the neuron are important mechanisms to regulate the activity of the receptor and control the precise spatiotemporal localization of these receptors. Applying a “molecular replacement” approach in hippocampal neurons derived from mice, we demonstrate a critical role for protein interacting with C kinase 1 (PICK1) in regulating the agonist-induced internalization of mGluR1. We show that PICK1 specifically regulates the internalization of mGluR1, but it does not play any role in the internalization of the other member of group I mGluR family, mGluR5. Various regions of PICK1 viz., the N-terminal acidic motif, PDZ domain, and BAR domain play important roles in the agonist-mediated internalization of mGluR1. Finally, we demonstrate that PICK1-mediated internalization of mGluR1 is critical for the resensitization of the receptor. Upon knockdown of endogenous PICK1, mGluR1s stayed on the cell membrane as inactive receptors, incapable of triggering the MAP kinase signaling. They also could not induce AMPAR endocytosis, a cellular correlate for mGluR-dependent synaptic plasticity. Thus, this study unravels a novel role for PICK1 in the agonist-mediated internalization of mGluR1 and mGluR1-mediated AMPAR endocytosis that might contribute to the function of mGluR1 in neuropsychiatric disorders. Glutamate is a major excitatory neurotransmitter in the central nervous system, which transduces its signal through ionotropic and metabotropic glutamate receptors (mGluRs) (1Pin J.P. Duvoisin R. The metabotropic glutamate receptors: structure and functions.Neuropharmacology. 1995; 34: 1-26Google Scholar, 2Dhami G.K. Ferguson S.S. Regulation of metabotropic glutamate receptor signaling, desensitization and endocytosis.Pharmacol. Ther. 2006; 111: 260-271Google Scholar). Group I mGluRs comprising of mGluR1 and mGluR5 are localized at the perisynaptic region of the postsynaptic neuron. They are thought to play an important role in multiple forms of experience-dependent synaptic plasticity, including learning and memory (3Citri A. Malenka R.C. Synaptic plasticity: multiple forms, functions, and mechanisms.Neuropsychopharmacology. 2008; 33: 18-41Google Scholar, 4Bhattacharyya S. Inside story of group I metabotropic glutamate receptors (mGluRs).Int. J. Biochem. Cell Biol. 2016; 77: 205-212Google Scholar). Activity-dependent changes in the strength of excitatory synapses result from the mGluR-regulated trafficking of AMPARs to and away from the synapses (3Citri A. Malenka R.C. Synaptic plasticity: multiple forms, functions, and mechanisms.Neuropsychopharmacology. 2008; 33: 18-41Google Scholar, 4Bhattacharyya S. Inside story of group I metabotropic glutamate receptors (mGluRs).Int. J. Biochem. Cell Biol. 2016; 77: 205-212Google Scholar, 5Malenka R.C. Synaptic plasticity and AMPA receptor trafficking.Ann. N. Y Acad. Sci. 2003; 1003: 1-11Google Scholar, 6Snyder E.M. Philpot B.D. Huber K.M. Dong X. Fallon J.R. Bear M.F. Internalization of ionotropic glutamate receptors in response to mGluR activation.Nat. Neurosci. 2001; 4: 1079-1085Google Scholar). These receptors have also been implicated in various neuropsychiatric disorders such as schizophrenia, fragile X syndrome, and autism (3Citri A. Malenka R.C. Synaptic plasticity: multiple forms, functions, and mechanisms.Neuropsychopharmacology. 2008; 33: 18-41Google Scholar, 7Huber K.M. Gallagher S.M. Warren S.T. Bear M.F. Altered synaptic plasticity in a mouse model of fragile X mental retardation.Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7746-7750Google Scholar, 8Bear M.F. Huber K.M. Warren S.T. The mGluR theory of fragile X mental retardation.Trends Neurosci. 2004; 27: 370-377Google Scholar, 9Dolen G. Osterweil E. Rao B.S. Smith G.B. Auerbach B.D. Chattarji S. et al.Correction of fragile X syndrome in mice.Neuron. 2007; 56: 955-962Google Scholar). Similar to many other GPCRs, trafficking plays a crucial role in controlling the spatiotemporal localization and activity of these receptors. Following agonist binding, group I mGluRs undergo desensitization followed by rapid internalization (2Dhami G.K. Ferguson S.S. Regulation of metabotropic glutamate receptor signaling, desensitization and endocytosis.Pharmacol. Ther. 2006; 111: 260-271Google Scholar, 4Bhattacharyya S. Inside story of group I metabotropic glutamate receptors (mGluRs).Int. J. Biochem. Cell Biol. 2016; 77: 205-212Google Scholar, 10Sallese M. Salvatore L. D'Urbano E. Sala G. Storto M. Launey T. et al.The G-protein-coupled receptor kinase GRK4 mediates homologous desensitization of metabotropic glutamate receptor 1.FASEB J. 2000; 14: 2569-2580Google Scholar, 11Dale L.B. Babwah A.V. Ferguson S.S. Mechanisms of metabotropic glutamate receptor desensitization: role in the patterning of effector enzyme activation.Neurochem. Int. 2002; 41: 319-326Google Scholar, 12Francesconi A. Duvoisin R.M. Opposing effects of protein kinase C and protein kinase A on metabotropic glutamate receptor signaling: selective desensitization of the inositol trisphosphate/Ca2+ pathway by phosphorylation of the receptor-G protein-coupling domain.Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6185-6190Google Scholar). Subsequently, they recycle back to the cell surface, which is the mechanism for “resensitization” of these receptors (13Pandey S. Mahato P.K. Bhattacharyya S. Metabotropic glutamate receptor 1 recycles to the cell surface in protein phosphatase 2A-dependent manner in non-neuronal and neuronal cell lines.J. Neurochem. 2014; 131: 602-614Google Scholar, 14Mahato P.K. Pandey S. Bhattacharyya S. Differential effects of protein phosphatases in the recycling of metabotropic glutamate receptor 5.Neuroscience. 2015; 306: 138-150Google Scholar, 15Sharma R. Gulia R. Bhattacharyya S. A critical role for sorting nexin 1 in the trafficking of metabotropic glutamate receptors.J. Neurosci. 2018; 38: 8605-8620Google Scholar). Despite the obvious significance of this trafficking for receptor function, the protein machineries that control group I mGluR internalization and mGluR-mediated AMPAR endocytosis are not fully characterized. Protein interacting with C kinase 1 (PICK1) is a small protein (416 aa) localized in the postsynaptic density (PSD) and containing an N-terminal PSD-95/DLG/ZO1 (PDZ) domain and a C-terminal Bin/amphiphysin/Rvs (BAR) domain (16Xu J. Xia J. Structure and function of PICK1.Neurosignals. 2006; 15: 190-201Google Scholar). PICK1 interacts with most of its partner proteins via its PDZ domain (17Staudinger J. Lu J. Olson E.N. Specific interaction of the PDZ domain protein PICK1 with the COOH terminus of protein kinase C-alpha.J. Biol. Chem. 1997; 272: 32019-32024Google Scholar, 18Dev K.K. Nakanishi S. Henley J.M. The PDZ domain of PICK1 differentially accepts protein kinase C-alpha and GluR2 as interacting ligands.J. Biol. Chem. 2004; 279: 41393-41397Google Scholar, 19Xia J. Zhang X. Staudinger J. Huganir R.L. Clustering of AMPA receptors by the synaptic PDZ domain-containing protein PICK1.Neuron. 1999; 22: 179-187Google Scholar, 20Hanley J.G. Khatri L. Hanson P.I. Ziff E.B. NSF ATPase and alpha-/beta-SNAPs disassemble the AMPA receptor-PICK1 complex.Neuron. 2002; 34: 53-67Google Scholar, 21Dev K.K. PDZ domain protein-protein interactions: a case study with PICK1.Curr. Top Med. Chem. 2007; 7: 3-20Google Scholar). The BAR domain of PICK1 dimerizes and forms a crescent shaped structure that binds and facilitates emergence of membrane curvatures which helps in the formation of vesicles (22Perez J.L. Khatri L. Chang C. Srivastava S. Osten P. Ziff E.B. PICK1 targets activated protein kinase Calpha to AMPA receptor clusters in spines of hippocampal neurons and reduces surface levels of the AMPA-type glutamate receptor subunit 2.J. Neurosci. 2001; 21: 5417-5428Google Scholar, 23Peter B.J. Kent H.M. Mills I.G. Vallis Y. Butler P.J. Evans P.R. et al.BAR domains as sensors of membrane curvature: the amphiphysin BAR structure.Science. 2004; 303: 495-499Google Scholar). Through its BAR domain, PICK1 also interacts with members of the SNARE-dependent membrane fusion machinery, actin, GRIP1, and charged membrane lipids (20Hanley J.G. Khatri L. Hanson P.I. Ziff E.B. NSF ATPase and alpha-/beta-SNAPs disassemble the AMPA receptor-PICK1 complex.Neuron. 2002; 34: 53-67Google Scholar, 24Rocca D.L. Martin S. Jenkins E.L. Hanley J.G. Inhibition of Arp2/3-mediated actin polymerization by PICK1 regulates neuronal morphology and AMPA receptor endocytosis.Nat. Cell Biol. 2008; 10: 259-271Google Scholar, 25Lu W. Ziff E.B. PICK1 interacts with ABP/GRIP to regulate AMPA receptor trafficking.Neuron. 2005; 47: 407-421Google Scholar, 26Jin W. Ge W.P. Xu J. Cao M. Peng L. Yung W. et al.Lipid binding regulates synaptic targeting of PICK1, AMPA receptor trafficking, and synaptic plasticity.J. Neurosci. 2006; 26: 2380-2390Google Scholar). PICK1 contains two acidic stretches, one at the N terminus and another at the C terminus of the protein, which bind calcium (27Hanley J.G. Henley J.M. PICK1 is a calcium-sensor for NMDA-induced AMPA receptor trafficking.EMBO J. 2005; 24: 3266-3278Google Scholar, 28Citri A. Bhattacharyya S. Ma C. Morishita W. Fang S. Rizo J. et al.Calcium binding to PICK1 is essential for the intracellular retention of AMPA receptors underlying long-term depression.J. Neurosci. 2010; 30: 16437-16452Google Scholar). Furthermore, PICK1 plays crucial roles in AMPAR trafficking and synaptic plasticity (22Perez J.L. Khatri L. Chang C. Srivastava S. Osten P. Ziff E.B. PICK1 targets activated protein kinase Calpha to AMPA receptor clusters in spines of hippocampal neurons and reduces surface levels of the AMPA-type glutamate receptor subunit 2.J. Neurosci. 2001; 21: 5417-5428Google Scholar, 26Jin W. Ge W.P. Xu J. Cao M. Peng L. Yung W. et al.Lipid binding regulates synaptic targeting of PICK1, AMPA receptor trafficking, and synaptic plasticity.J. Neurosci. 2006; 26: 2380-2390Google Scholar, 27Hanley J.G. Henley J.M. PICK1 is a calcium-sensor for NMDA-induced AMPA receptor trafficking.EMBO J. 2005; 24: 3266-3278Google Scholar, 28Citri A. Bhattacharyya S. Ma C. Morishita W. Fang S. Rizo J. et al.Calcium binding to PICK1 is essential for the intracellular retention of AMPA receptors underlying long-term depression.J. Neurosci. 2010; 30: 16437-16452Google Scholar, 29Kim C.H. Chung H.J. Lee H.K. Huganir R.L. Interaction of the AMPA receptor subunit GluR2/3 with PDZ domains regulates hippocampal long-term depression.Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 11725-11730Google Scholar). Thus, considering the binding partners with which PICK1 interacts and its role in synaptic plasticity, we hypothesized that PICK1 might play an important role in group I mGluR internalization, as well as in mGluR-mediated AMPAR endocytosis. We performed our study using a molecular replacement strategy that allowed shRNA-mediated acute knockdown of endogenous PICK1 and replacement of the endogenous form with various mutant forms in primary hippocampal neurons. We show here that PICK1 plays a contrasting role in the agonist-mediated internalization of mGluR1 and mGluR5. Acute knockdown of endogenous PICK1 decreased the surface expression and inhibited the agonist-induced endocytosis of mGluR1. PICK1 was observed to interact with mGluR1. On the other hand, although knockdown of PICK1 decreased the surface expression of mGluR5, it did not inhibit the agonist-mediated endocytosis of mGluR5. The PDZ domain and BAR domain of PICK1 were critical for the internalization of mGluR1. We also found that orientation of the amino acid sequence at the N-terminal acidic region of PICK1 was important for the agonist-mediated endocytosis of mGluR1. Finally, we show that PICK1 specifically regulates mGluR1-mediated signaling and mGluR1-mediated AMPAR internalization, the cellular correlate of mGluR1-dependent synaptic plasticity. Thus, our study unravels a previously uncharacterized role for PICK1 in the internalization of mGluR1 and mGluR1-mediated AMPAR endocytosis, with anticipated clinical relevance to the function of mGluR1 in neuropsychiatric disorders. Similar to many other GPCRs, group I mGluRs also undergo rapid internalization following agonist exposure (13Pandey S. Mahato P.K. Bhattacharyya S. Metabotropic glutamate receptor 1 recycles to the cell surface in protein phosphatase 2A-dependent manner in non-neuronal and neuronal cell lines.J. Neurochem. 2014; 131: 602-614Google Scholar, 30Gulia R. Sharma R. Bhattacharyya S. A critical role for ubiquitination in the endocytosis of glutamate receptors.J. Biol. Chem. 2017; 292: 1426-1437Google Scholar, 31Iacovelli L. Salvatore L. Capobianco L. Picascia A. Barletta E. Storto M. et al.Role of G protein-coupled receptor kinase 4 and beta-arrestin 1 in agonist-stimulated metabotropic glutamate receptor 1 internalization and activation of mitogen-activated protein kinases.J. Biol. Chem. 2003; 278: 12433-12442Google Scholar, 32Pandey S. Ramsakha N. Sharma R. Gulia R. Ojha P. Lu W. et al.The post-synaptic scaffolding protein tamalin regulates ligand-mediated trafficking of metabotropic glutamate receptors.J. Biol. Chem. 2020; 295: 8575-8588Google Scholar, 33Ojha P. Pal S. Bhattacharyya S. Regulation of metabotropic glutamate receptor internalization and synaptic AMPA receptor endocytosis by the Postsynaptic protein Norbin.J. Neurosci. 2022; 42: 731-748Google Scholar). The cellular and molecular mechanisms that govern the agonist-mediated endocytosis of group I mGluRs are poorly understood. Like many other GPCRs, group I mGluRs are also tightly regulated by a macromolecular protein complex at the postsynaptic membrane, known as PSD. One member of the PSD is PICK1. It acts like an adapter molecule as it binds to a variety of membrane proteins and organizes the subcellular localization and surface expression of many proteins (16Xu J. Xia J. Structure and function of PICK1.Neurosignals. 2006; 15: 190-201Google Scholar, 18Dev K.K. Nakanishi S. Henley J.M. The PDZ domain of PICK1 differentially accepts protein kinase C-alpha and GluR2 as interacting ligands.J. Biol. Chem. 2004; 279: 41393-41397Google Scholar, 20Hanley J.G. Khatri L. Hanson P.I. Ziff E.B. NSF ATPase and alpha-/beta-SNAPs disassemble the AMPA receptor-PICK1 complex.Neuron. 2002; 34: 53-67Google Scholar, 25Lu W. Ziff E.B. PICK1 interacts with ABP/GRIP to regulate AMPA receptor trafficking.Neuron. 2005; 47: 407-421Google Scholar, 26Jin W. Ge W.P. Xu J. Cao M. Peng L. Yung W. et al.Lipid binding regulates synaptic targeting of PICK1, AMPA receptor trafficking, and synaptic plasticity.J. Neurosci. 2006; 26: 2380-2390Google Scholar). In order to investigate whether PICK1 could play any role in the agonist-mediated internalization of group I mGluRs, we studied the agonist-induced internalization of myc-mGluR1 by using a “molecular replacement” strategy that allows simultaneous shRNA-mediated acute knockdown of endogenous PICK1 and expression of various mutant forms of recombinant PICK1 in primary hippocampal neurons. This approach offers two important advantages. First, the possibility of compensatory adaptations during synaptogenesis and synapse maturation due to the absence of the protein of interest are minimal. Second, the function of the protein of interest can be studied without the necessity to obtain a dominant effect as required by a standard overexpression approach. We used an shRNA against PICK1 that has been reported to efficiently knock down endogenous PICK1 in primary hippocampal neurons and has been employed successfully in an earlier study (28Citri A. Bhattacharyya S. Ma C. Morishita W. Fang S. Rizo J. et al.Calcium binding to PICK1 is essential for the intracellular retention of AMPA receptors underlying long-term depression.J. Neurosci. 2010; 30: 16437-16452Google Scholar). The rescue of the effect of shPICK1 knockdown by expression of the WT PICK1 replacement construct ascertained the specificity of the shRNA (Fig. 1A). In agreement with the previous report, we also found that shPICK1 was efficient in knocking down the endogenous PICK1 in primary neurons as measured through western blots (Fig. S1). In order to investigate whether knockdown of endogenous PICK1 had any effect on the surface expression of mGluR1, primary hippocampal neurons were cotransfected with myc-mGluR1 and shPICK1 or shPICK1:PICK1 (WT PICK1 replacement) constructs. Subsequently, surface myc-mGluR1 were labeled by treating live cells expressing myc-mGluR1 with anti-myc primary antibody followed by fixation of cells with 4% paraformaldehyde (PFA) and staining with goat anti-mouse Alexa-568–conjugated secondary antibody. In all our assays, surface expression of the receptors were quantified by measuring the surface fluorescence and normalizing that with the cell area (see experimental procedures section for details). The transfected cells were identified with the GFP fluorescence. The shRNA-mediated acute knockdown of endogenous PICK1 reduced the surface expression of myc-mGluR1, whereas simultaneously expressing both shPICK1 and WT PICK1 (shPICK1:PICK1) rescued the surface expression of the receptor (control: 1.0 ± 0.09; shPICK1: 0.8 ± 0.09; shPICK1:PICK1: 1 ± 0.12) (Fig. 1, B and C). These results confirmed the efficacy of the shPICK1 construct and the molecular replacement approach in primary hippocampal neurons. However, since knockdown of PICK1 resulted in decrease in the surface expression of myc-mGluR1, for studying the role of PICK1 in the agonist-mediated endocytosis of group I mGluRs, we implemented a procedure that allowed staining of both internalized and remaining surface myc-mGluR1 with different secondary antibodies and measured the proportion of surface receptors that endocytosed following ligand application (see experimental procedures section). Briefly, primary hippocampal neurons were cotransfected with myc-mGluR1 and shPICK1 or shPICK1:PICK1 constructs. Live cells expressing myc-mGluR1 were labeled with anti-myc primary antibody. Subsequent application of 100 μM R,S-3, 5-dihydroxyphenylglycine (DHPG), an agonist of group I mGluRs, resulted in the internalization of myc-mGluR1 within 30 min in control cells. This time point was selected since our earlier studies suggested that R,S-DHPG–mediated internalization of myc-mGluR1 reaches maximum level at 30 min post agonist application (13Pandey S. Mahato P.K. Bhattacharyya S. Metabotropic glutamate receptor 1 recycles to the cell surface in protein phosphatase 2A-dependent manner in non-neuronal and neuronal cell lines.J. Neurochem. 2014; 131: 602-614Google Scholar, 15Sharma R. Gulia R. Bhattacharyya S. A critical role for sorting nexin 1 in the trafficking of metabotropic glutamate receptors.J. Neurosci. 2018; 38: 8605-8620Google Scholar). Subsequently, cells were fixed without permeabilization using ice cold 4% PFA for 15 min on ice. Surface receptors were labeled with a saturating concentration of goat anti-mouse Alexa-568–conjugated secondary antibody. Cells were then permeabilized with 0.1% Triton X-100 for 30 min at room temperature. The endocytosed receptors were then labeled by the application of goat anti-mouse Alexa-647–conjugated secondary antibody. The internalization index was then calculated by normalizing the internal fluorescence with the total fluorescence (surface + internal) as described in detail in the experimental procedures section. The transfected cells were identified with the GFP fluorescence. Importantly, acute knockdown of the endogenous PICK1 inhibited the R,S-DHPG–mediated endocytosis of myc-mGluR1, and the majority of the receptors were observed to be localized at the cell surface in shPICK1-expressing cells (control: 1 ± 0.25; control + DHPG: 1.66 ± 0.39; shPICK1 + DHPG: 1.04 ± 0.31) (Fig. 1, D and E). The inhibition of the internalization of myc-mGluR1 due to the knockdown of the endogenous PICK1 was rescued by expression of the recombinant WT PICK1 (shPICK1:PICK1 + DHPG: 1.63 ± 0.44) (Fig. 1, D and E). Thus, these results suggest that acute knockdown of endogenous PICK1 reduced the surface expression of mGluR1 and also inhibited the agonist-mediated endocytosis of the receptor in primary hippocampal neurons. In order to investigate whether PICK1 interacts with mGluR1 and whether this interaction is dependent on the activation of the receptor by the agonist, we studied the interaction of PICK1 with mGluR1 in the absence and presence of 100 μM R,S-DHPG. Briefly, primary neurons were transfected with FLAG-mGluR1. Seventy two hours post-transfection, 100 μM R,S-DHPG was applied for 5 min. Subsequently, immunoprecipitation assay and western blots were performed following the procedure described in the experimental procedure section. PICK1 showed interaction with FLAG-mGluR1, but application of R,S-DHPG did not increase the interaction of PICK1 with the receptor (control: 1 ± 0.07; DHPG: 1.02 ± 0.07) (Fig. 1, F and G). These results are consistent with an interaction of PICK1 and mGluR1, but further experiments need to be performed to strengthen these results and to find out whether the interaction is direct or indirect between the two. PICK1 contains a BAR domain that is critical for the self-association of the protein (22Perez J.L. Khatri L. Chang C. Srivastava S. Osten P. Ziff E.B. PICK1 targets activated protein kinase Calpha to AMPA receptor clusters in spines of hippocampal neurons and reduces surface levels of the AMPA-type glutamate receptor subunit 2.J. Neurosci. 2001; 21: 5417-5428Google Scholar). This dimerization is thought to create a large crescent shape structure through which the protein interacts with a defined membrane curvature and controls subcellular trafficking (23Peter B.J. Kent H.M. Mills I.G. Vallis Y. Butler P.J. Evans P.R. et al.BAR domains as sensors of membrane curvature: the amphiphysin BAR structure.Science. 2004; 303: 495-499Google Scholar). This domain contains positively charged lysine residues that mediate binding to the negatively charged head groups of membrane phosphoinositides (25Lu W. Ziff E.B. PICK1 interacts with ABP/GRIP to regulate AMPA receptor trafficking.Neuron. 2005; 47: 407-421Google Scholar, 26Jin W. Ge W.P. Xu J. Cao M. Peng L. Yung W. et al.Lipid binding regulates synaptic targeting of PICK1, AMPA receptor trafficking, and synaptic plasticity.J. Neurosci. 2006; 26: 2380-2390Google Scholar). To investigate the function of the BAR domain of PICK1, in the agonist-mediated internalization of mGluR1, we used a replacement construct in which five lysine residues within the BAR domain were mutated to glutamic acid [shPICK1:PICK1(5KE); K251E, K252E, K257E, K266E, K268E] (Fig. 2A). Mutation of these residues has been shown to reduce the lipid-binding capacity, as well as the actin-binding capacity of PICK1 (24Rocca D.L. Martin S. Jenkins E.L. Hanley J.G. Inhibition of Arp2/3-mediated actin polymerization by PICK1 regulates neuronal morphology and AMPA receptor endocytosis.Nat. Cell Biol. 2008; 10: 259-271Google Scholar, 26Jin W. Ge W.P. Xu J. Cao M. Peng L. Yung W. et al.Lipid binding regulates synaptic targeting of PICK1, AMPA receptor trafficking, and synaptic plasticity.J. Neurosci. 2006; 26: 2380-2390Google Scholar). We first examined the effect of replacing endogenous PICK1 with PICK1(5KE) on the surface expression of myc-mGluR1 using the procedure described before. Knockdown of endogenous PICK1 decreased the surface expression of myc-mGluR1 (control: 1.0 ± 0.06; shPICK1: 0.73 ± 0.2) (Fig. 2, B and C). However, unlike WT PICK1, PICK1(5KE) did not rescue the surface expression of myc-mGluR1 (shPICK1:PICK1(5KE): 0.75 ± 0.2) (Fig. 2, B and C). Subsequently, we investigated the role of PICK1(5KE) in the agonist-mediated endocytosis of myc-mGluR1 using the same endocytosis assay that has been described before. In control cells, application of 100 μM R,S-DHPG increased the internalization of myc-mGluR1 at 30 min. Similar to our previous observations, shPICK1-expressing cells showed block in the myc-mGluR1 endocytosis (control: 1 ± 0.19; control + DHPG: 1.59 ± 0.26; shPICK1 + DHPG: 1.02 ± 0.24) (Fig. 2, D and E). Importantly, unlike WT PICK1, PICK1(5KE) replacement construct did not rescue the inhibition of R,S-DHPG–mediated myc-mGluR1 endocytosis caused by the knockdown of endogenous PICK1 (shPICK1:PICK1(5KE) + DHPG: 0.99 ± 0.27) (Fig. 2, D and E). These results suggest that the positively charged residues within the BAR domain of PICK1 play a critical role in the surface expression and agonist-mediated internalization of mGluR1. Previous reports have suggested that the N-terminal acidic motif of PICK1 is important for the Ca2+ binding by the protein (27Hanley J.G. Henley J.M. PICK1 is a calcium-sensor for NMDA-induced AMPA receptor trafficking.EMBO J. 2005; 24: 3266-3278Google Scholar, 28Citri A. Bhattacharyya S. Ma C. Morishita W. Fang S. Rizo J. et al.Calcium binding to PICK1 is essential for the intracellular retention of AMPA receptors underlying long-term depression.J. Neurosci. 2010; 30: 16437-16452Google Scholar). Ca2+ binding at this region promotes structural changes in PICK1 necessary for its function in regulating the AMPAR trafficking (27Hanley J.G. Henley J.M. PICK1 is a calcium-sensor for NMDA-induced AMPA receptor trafficking.EMBO J. 2005; 24: 3266-3278Google Scholar). In order to study the function of this calcium-binding N-terminal acidic region of PICK1 in the agonist-mediated internalization of mGluR1, we utilized a replacement construct in which the N-terminal acidic stretch of PICK1 was deleted [shPICK1:PICK1(Δ9)] (Fig. 3A). Expression of this replacement construct was unable to rescue the decrease in the surface myc-mGluR1 expression due to the knockdown of the endogenous PICK1, suggesting that the N-terminal acidic motif of PICK1 plays an important role in the surface expression of mGluR1 (control: 1.0 ± 0.08; shPICK1: 0.84 ± 0.11; shPICK1:PICK1(Δ9): 0.72 ± 0.13) (Fig. 3, B and C). We subsequently investigated whether this N-terminal acidic region of PICK1 is critical for the agonist-mediated endocytosis of mGluR1. As before, knockdown of endogenous PICK1 with shPICK1 inhibited the myc-mGluR1 endocytosis (control: 1 ± 0.13; control + DHPG: 1.99 ± 0.3; shPICK1 + DHPG: 1.09 ± 0.28) (Fig. 3, D and E). However, the PICK1(Δ9) replacement construct could not rescue the agonist-mediated endocytosis of myc-mGluR1 (shPICK1:PICK1(Δ9) + DHPG: 1.24 ± 0.28) (Fig. 3, D and E). These results suggest that the N-terminal acidic region of PICK1 plays a critical role in the surface localization and agonist-mediated internalization of mGluR1. Further, we wanted to investigate whether the total charge at the N-terminal region of PICK1 is the only important factor or the spatial arrangement of amino acids also plays a crucial role in regulating the endocytosis of mGluR1. We therefore used a PICK1 mutant in which the sequence of the N-terminal acidic motif was inverted [PICK1(FLIP)] (Fig. 4A). In this mutant, the total charge of the N-terminal region of PICK1 was maintained, but the spatial arrangement of amino acids was disrupted. It has been reported that inversion of this calcium-binding motif of PICK1 at the N terminus disrupts the spatial organization of amino acids necessary for calcium binding with the protein but maintains the electrostatic interactions that are required for PICK1 intramolecular folding (28Citri A. Bhattacharyya S. Ma C. Morishita W. Fang S. Rizo J. et al.Calcium binding to PICK1 is essential for the intracellular retention of AMPA receptors underlying long-term depression.J. Neurosci. 2010; 30: 16437-16452Google Scholar). Expression of this replacement construct was unable to rescue the decrease in the surface myc-mGluR1 expression due to the knockdown of the endogenous PICK1 (control: 1.0 ± 0.09; shPICK1: 0.73 ± 0.13; shPICK1:PICK1(FLIP): 0.62 ± 0.17) (Fig. 4, B and C). Furthermore," @default.
• W4377023941 created "2023-05-19" @default.
• W4377023941 creator A5048298854 @default.
• W4377023941 creator A5051259293 @default.
• W4377023941 creator A5067630240 @default.
• W4377023941 creator A5072101615 @default.
• W4377023941 creator A5073605420 @default.
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• W4377023941 date "2023-06-01" @default.
• W4377023941 modified "2023-10-14" @default.
• W4377023941 title "A vital role for PICK1 in the differential regulation of metabotropic glutamate receptor internalization and synaptic AMPA receptor endocytosis" @default.
• W4377023941 cites W1439811294 @default.
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