A vital role for PICK1 in the differential regulation of metabotropic glutamate receptor internalization and synaptic AMPA receptor endocytosis.

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.

Regulation of Metabotropic Glutamate Receptor Internalization and Synaptic AMPA Receptor Endocytosis by the Postsynaptic Protein Norbin.

Group I mGluRs have diverse functions in some fundamental neuronal processes, including modulation of synaptic plasticity; and dysregulation of these receptors could lead to various neuropsychiatric disorders. Trafficking of Group I mGluRs plays critical roles in controlling the precise spatiotemporal localization and activity of these receptors, both of which contribute to proper downstream signaling. Using "molecular replacement" approach in hippocampal neurons derived from mice of both sexes, we demonstrate a critical role for the postsynaptic density protein Norbin in regulating the ligand-induced internalization of Group I mGluRs. We show that Norbin associates with protein kinase A (PKA) through its N-terminus and anchors mGluR5 through its C-terminus, both of which are necessary for the ligand-mediated endocytosis of mGluR5, a member of the Group I mGluR family. A point mutation (A687G) at the C-terminus of Norbin inhibits the binding of Norbin to mGluR5 and blocks mGluR5 endocytosis. Finally, we demonstrate an important mechanism by which Norbin regulates mGluR-mediated AMPAR endocytosis in hippocampal neurons, a cellular correlate for mGluR-dependent synaptic plasticity. Norbin, through its PKA-binding regions, recruits PKA to AMPARs on activation of mGluRs; and deletion of the PKA-binding regions of Norbin inhibits mGluR-triggered AMPAR endocytosis. We further report that Norbin is important specifically for the mGluR-mediated AMPAR endocytosis, but not for NMDAR-dependent AMPAR endocytosis. Thus, this study unravels a novel role for Norbin in the internalization of mGluRs and mGluR-mediated AMPAR endocytosis that can have clinical relevance to the function of Group I mGluRs in pathologic processes.SIGNIFICANCE STATEMENT The postsynaptic protein Norbin interacts with mGluR5, and both of them have been implicated in disorders, such as schizophrenia. However, the mechanistic basis underlying the regulation of mGluRs by Norbin remains elusive. We have identified Norbin as an essential mediator of ligand-mediated endocytosis of Group I mGluRs. Mechanistically, Norbin N-terminus associates with protein kinase-A (PKA) and C-terminus binds to mGluR5 to coordinate receptor internalization. A point mutation NorA687G inhibits endocytosis by disrupting this interaction. Additionally, Norbin is critical for the recruitment of PKA to AMPARs on activation of Group I mGluRs that assists in mGluR-mediated AMPAR endocytosis. Thus, Norbin has a dual function in the hippocampus: regulation of mGluR internalization and PKA-dependent modulation of mGluR-mediated AMPAR endocytosis, a prerequisite for mGluR-mediated synaptic plasticity.

The post-synaptic scaffolding protein tamalin regulates ligand-mediated trafficking of metabotropic glutamate receptors.

Group I metabotropic glutamate receptors (mGluRs) play important roles in various neuronal functions and have also been implicated in multiple neuropsychiatric disorders like fragile X syndrome, autism, and others. mGluR trafficking not only plays important roles in controlling the spatiotemporal localization of these receptors in the cell but also regulates the activity of these receptors. Despite this obvious significance, the cellular machineries that control the trafficking of group I metabotropic glutamate receptors in the central nervous system have not been studied in detail. The post-synaptic scaffolding protein tamalin has been shown to interact with group I mGluRs and also with many other proteins involved in protein trafficking in neurons. Using a molecular replacement approach in mouse hippocampal neurons, we show here that tamalin plays a critical role in the ligand-dependent internalization of mGluR1 and mGluR5, members of the group I mGluR family. Specifically, knockdown of endogenous tamalin inhibited the ligand-dependent internalization of these two receptors. Both N-terminal and C-terminal regions of tamalin played critical roles in mGluR1 endocytosis. Furthermore, we found that tamalin regulates mGluR1 internalization by interacting with S-SCAM, a protein that has been implicated in vesicular trafficking. Finally, we demonstrate that tamalin plays a critical role in mGluR-mediated internalization of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors, a process believed to be the cellular correlate for mGluR-dependent synaptic plasticity. Taken together, these findings reveal a mechanistic role of tamalin in the trafficking of group I mGluRs and suggest its physiological implications in the brain.

Analysis of ubiquitination and ligand-dependent trafficking of group I mGluRs.

Group I metabotropic glutamate receptors (mGluRs) are G-protein coupled receptors (GPCRs). They have been implicated in multiple forms of synaptic plasticity, as well as in various neuropsychiatric disorders. The signaling of these receptors is governed by the mechanisms of desensitization, internalization and resensitization of these receptors. Various post-translational modifications determine the signaling as well as trafficking of these receptors. Ubiquitination is a post-translational modification that has emerged as an essential regulatory process which governs group I mGluR trafficking. In this chapter, we have discussed the strategies to investigate the ubiquitination and the ligand-mediated trafficking of group I mGluRs in HEK293T cells and in primary hippocampal neurons, respectively. We have illustrated the protocols of (i) maintenance and transient transfection in HEK293T cells and primary hippocampal neurons, (ii) immunoprecipitation and western blot analysis to identify the ubiquitination of group I mGluRs, (iii) endocytosis and recycling assay and (iv) image acquisition and analysis.

Revisiting the role of cholesterol in regulating the pore-formation mechanism of Vibrio cholerae cytolysin, a membrane-damaging ß-barrel pore-forming toxin.

Vibrio cholerae cytolysin (VCC) is a ß-barrel pore-forming toxin with potent membrane-damaging cell-killing activity. Previous studies employing the model membranes of lipid vesicles (liposomes) have shown that pore formation by VCC requires the presence of cholesterol in the liposome membranes. However, the exact role of cholesterol in the mode of action of VCC still remains unclear. Most importantly, implication of cholesterol, if any, in regulating the pore-formation mechanism of VCC in the biomembranes of eukaryotic cells remains unexplored. Here, we show that the presence of cholesterol promotes the interaction of VCC with the membrane lipid bilayer, when non-lipid-dependent interactions are absent. However, in the case of biomembranes of human erythrocytes, where accessory interactions are available, cholesterol appears to play a less critical role in the binding step. Nevertheless, in the absence of an optimal level of membrane cholesterol in the human erythrocytes, membrane-bound fraction of the toxin remains trapped in the form of abortive oligomeric assembly, devoid of functional pore-forming activity. Our study also shows that VCC exhibits a prominent propensity to associate with the cholesterol-rich membrane micro-domains of human erythrocytes. Interestingly, mutation of the cholesterol-binding ability of VCC does not block association with the cholesterol-rich membrane micro-domains on human erythrocytes. Based on these results, we propose that the specific cholesterol-binding ability of VCC does not appear to dictate its association with the cholesterol-rich micro-domains on human erythrocytes. Rather, targeting of VCC toward the membrane micro-domains of human erythrocytes possibly acts to facilitate the cholesterol-dependent pore-formation mechanism of the toxin.

A Critical Role for Sorting Nexin 1 in the Trafficking of Metabotropic Glutamate Receptors.

Group I metabotropic glutamate receptors (mGluRs) function as modulators of neuronal physiology and they have also been implicated in various neuropsychiatric disorders. Trafficking of mGluRs plays important roles in controlling the precise localization of these receptors at specific region of the cell, as well as it regulates the activity of these receptors. Despite this obvious significance, we know very little about the cellular machineries that control the trafficking of these receptors in the CNS. Sorting nexin 1 (SNX1) has been shown to regulate the endosomal sorting of few cell surface receptors either to lysosomes where they are downregulated or back to the cell surface. Using "molecular replacement" approach in hippocampal neurons derived from mice of both sexes, we show here that SNX1 plays critical role in the trafficking of mGluR1, a member of the group I mGluR family. Overexpression of dominant-negative SNX1 or knockdown of endogenous SNX1 resulted in the rapid recycling of the receptor. Importantly, recycling via the rapid recycling route, did not allow the resensitization of the receptors. Our data suggest that both, N-terminal and C-terminal region of SNX1 play critical role in the normal trafficking of the receptor. In addition, we also show here that SNX1 regulates the trafficking of mGluR1 through the interaction with Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate), a protein that has been implicated in both signaling and vesicular trafficking. Thus, these studies reveal a mechanistic role of SNX1 in the trafficking of group I mGluRs and its physiological implications.SIGNIFICANCE STATEMENT Group I mGluRs are activated by the neurotransmitter glutamate in the CNS, and play various important roles in the brain. Similar to many other receptors, trafficking plays crucial roles in controlling the precise localization as well as activity of these receptors. Despite this obvious significance very little is known about the cellular machineries that control the trafficking of these receptors. We demonstrate here, that SNX1 plays a critical role in the trafficking of mGluR1, a member of the group I mGluR family. SNX1-mediated trafficking is critical for the resensitization of the receptor. SNX1 controls the trafficking of the receptor through the interaction with another protein, Hrs. The results suggest a role for SNX1 in the regulation of group I mGluRs.

Group I Metabotropic Glutamate Receptors (mGluRs): Ins and Outs.

Glutamate is a nonessential amino acid, known to act as a major excitatory neurotransmitter in the central nervous system. Glutamate transduces its signal by activating two types of receptors, viz., ionotropic glutamate receptors and metabotropic glutamate receptors (mGluRs). mGluR1 and mGluR5 are members of the group I mGluR family, and they belong to the G-protein-coupled receptor (GPCR) family. These receptors are involved in various forms of synaptic plasticity including learning and memory. Similar to many other GPCRs, trafficking plays a critical role in controlling the spatiotemporal localization of these receptors on the cell surface, which is critical for the normal ligand/receptor interaction. Improper targeting of GPCRs results in aberrant signaling, which often leads to various diseases. Trafficking also regulates the activity of these receptors. Thus, inappropriate trafficking of these receptors might have pathological consequences. Group I mGluRs have been implicated in various neuropsychiatric disorders like Fragile X syndrome, autism, etc. In this review, we discuss the current understanding of group I mGluR trafficking in the central nervous system and its physiological importance.

A Critical Role for Ubiquitination in the Endocytosis of Glutamate Receptors.

Group I metabotropic glutamate receptors (mGluRs) play important roles in various neuronal processes and elicit changes in synaptic efficacy through AMPA receptor (AMPAR) endocytosis. Trafficking of mGluRs plays an important role in controlling the precise localization of these receptors at specific region of the cell; it also regulates the activity of these receptors. Despite this obvious significance, we know very little about the cellular mechanisms that control the trafficking of group I mGluRs. We show here that ligand-mediated internalization of group I mGluRs is ubiquitination-dependent. A lysine residue (Lys1112) at the C-terminal tail of mGluR1 (a member of the group I mGluR family) plays crucial role in this process. Our data suggest that Lys63-linked polyubiquitination is involved in the ligand-mediated endocytosis of mGluR1. We also show here that the mGluR1 internalization is dependent on a specific E3 ubiquitin ligase, Siah-1A. Furthermore, acute knockdown of Siah-1A enhances the mGluR-mediated AMPAR endocytosis. These studies reveal a novel function of ubiquitination in the regulation of group I mGluRs, as well as its role in mGluR-dependent AMPAR endocytosis.

Inside story of Group I Metabotropic Glutamate Receptors (mGluRs).

Metabotropic glutamate receptors (mGluRs) are G-protein coupled receptors (GPCRs) that are activated by the neurotransmitter glutamate in the central nervous system. Among the eight subtypes, mGluR1 and mGluR5 belong to the group I family. These receptors play important roles in the brain and are believed to be involved in multiple forms of experience dependent synaptic plasticity including learning and memory. In addition, group I mGluRs also have been implicated in various neuropsychiatric disorders like Fragile X syndrome, autism etc. The normal signaling depends on the precise location of these receptors in specific region of the neuron and the process of receptor trafficking plays a crucial role in controlling this localization. Intracellular trafficking could also regulate the desensitization, resensitization, down-regulation and intracellular signaling of these receptors. In this review I focus on the current understanding of group I mGluR regulation in the central nervous system and also their role in neuropsychiatric disorders.

Metabotropic glutamate receptor 1 recycles to the cell surface in protein phosphatase 2A-dependent manner in non-neuronal and neuronal cell lines.

Trafficking of G protein-coupled receptors plays a crucial role in controlling the precise signalling of the receptor as well as its proper regulation. Metabotropic glutamate receptor 1 (mGluR1), a G protein-coupled receptor, is a member of the group I mGluR family. mGluR1 plays a critical role in neuronal circuit formation and also in multiple types of synaptic plasticity. This receptor has also been reported to be involved in various neuropsychiatric diseases. Other than the central nervous system, mGluR1 plays crucial roles in various non-neuronal cells like hepatocytes, skin cells, etc. Although it has been reported that mGluR1 gets endocytosed on ligand application, the events after the internalization of the receptor has not been studied. We show here that mGluR1 internalizes on ligand application. Subsequent to endocytosis, majority of the receptors localize at the recycling compartment and no significant presence of the receptor was noticed in the lysosome. Furthermore, mGluR1 returned to the cell membrane subsequent to ligand-mediated internalization. We also show here that the recycling of mGluR1 is dependent on the activity of protein phosphatase 2A. Thus, our data suggest that the ligand-mediated internalized receptors recycle back to the cell surface in protein phosphatase 2A-dependent manner.

Functional selectivity in serotonin receptor 2A (5-HT2A) endocytosis, recycling, and phosphorylation.

G protein-coupled receptor (GPCR) signaling is modulated by endocytosis and endosomal sorting of receptors between degradation and recycling. Differential regulation of these processes by endogenous ligands and synthetic drugs is a poorly understood area of GPCR signaling. Here, we describe remarkable diversity in the regulation of trafficking of GPCR induced by multiple ligands. We show that the serotonin receptor 2A (5-HT(2A)), a prototypical GPCR in the study of functional selectivity at a signaling receptor, is functionally selective in endocytosis and recycling in response to five ligands tested: endogenous agonists serotonin (5-HT) and dopamine (DA), synthetic agonist 1-(2,5-dimethoxy-4-iodophenyl)-aminopropane (DOI), antagonist ketanserin, and inverse agonist and antipsychotic drug clozapine. Only four ligands (5-HT, DA, DOI, and clozapine) bring about receptor endocytosis. As we have earlier described with 5-HT and DA, there is ligand-specific requirement for protein kinase C (PKC) in endocytosis. We now show 5-HT(2A) phosphorylation by PKC is necessary for 5-HT-mediated and DOI-mediated receptor endocytosis, but DA-mediated and clozapine-mediated internalization is not affected if PKC is inhibited. Internalized receptors are recycled to the cell surface, but there is variability in the time course of recycling. 5-HT- and DA-internalized receptors are recycled in 2.5 hours while agonist DOI and antagonist clozapine bring about recycling in 7.5 hours. Recycling in response to those ligands that require PKC activation to effect receptor endocytosis is dependent on receptor dephosphorylation by protein phosphatase 2A (PP2A). Thus, internalization and phosphorylation/dephosphorylation cycles may play a significant role in the regulation of 5-HT(2A) by functionally and therapeutically important ligands.

Constitutive internalization and recycling of metabotropic glutamate receptor 5 (mGluR5).

Ligand-dependent and ligand-independent endocytic trafficking of G-protein coupled receptors (GPCRs) is critical for accurate receptor-mediated signaling and its regulation. Metabotropic glutamate receptor 5 (mGluR5) is a GPCR that plays a crucial role in circuit formation in the brain and also in various forms of synaptic plasticity including learning and memory. Outside the central nervous system this receptor also plays very important role in various other non-neuronal cells like heart cells, skin cells, hepatocytes, etc. Although the ligand-mediated endocytosis of mGluR5 has been studied in some detail, ligand-independent/constitutive endocytosis of the receptor has not been properly studied. Here, we have investigated the constitutive endocytosis of mGluR5 and also the sub-cellular fate of the receptor subsequent to internalization. We show here that mGluR5 undergoes constitutive internalization in HEK293 cells. Following endocytosis, the receptor enters the recycling compartment and no localization of the receptor was observed in the lysosome. In addition, we also report here that most of the receptors recycle to the cell surface subsequent to constitutive internalization. Thus, our data demonstrate that mGluR5 receptors internalize without the application of ligand and the internalized receptors recycle back to the cell surface following constitutive endocytosis.

Calcium binding to PICK1 is essential for the intracellular retention of AMPA receptors underlying long-term depression.

NMDA receptor (NMDAR)-dependent long-term depression (LTD) in the hippocampus is mediated primarily by the calcium-dependent removal of AMPA receptors (AMPARs) from the postsynaptic density. The AMPAR-binding, PDZ (PSD-95/Dlg/ZO1) and BAR (Bin/amphiphysin/Rvs) domain-containing protein PICK1 has been implicated in the regulation of AMPAR trafficking underlying several forms of synaptic plasticity. Using a strategy involving small hairpin RNA-mediated knockdown of PICK1 and its replacement with recombinant PICK1, we performed a detailed structure-function analysis of the role of PICK1 in hippocampal synaptic plasticity and the underlying NMDAR-induced AMPAR trafficking. We found that PICK1 is not necessary for maintenance of the basal synaptic complement of AMPARs or expression of either metabotropic glutamate receptor-dependent LTD or NMDAR-dependent LTP. Rather, PICK1 function is specific to NMDAR-dependent LTD and the underlying AMPAR trafficking. Furthermore, although PICK1 does not regulate the initial phase of NMDAR-induced AMPAR endocytosis, it is required for intracellular retention of internalized AMPARs. Detailed biophysical analysis of an N-terminal acidic motif indicated that it is involved in intramolecular electrostatic interactions that are disrupted by calcium. Mutations that interfered with the calcium-induced structural changes in PICK1 precluded LTD and the underlying NMDAR-induced intracellular retention of AMPARs. These findings support a model whereby calcium-induced modification of PICK1 structure is critical for its function in the retention of internalized AMPARs that underlies the expression of hippocampal NMDAR-dependent LTD.

N-methyl-D-aspartate receptor- and metabotropic glutamate receptor-dependent long-term depression are differentially regulated by the ubiquitin-proteasome system.

Long-term depression (LTD) in CA1 pyramidal neurons can be induced by activation of either N-methyl-D-aspartate receptors (NMDARs) or metabotropic glutamate receptors (mGluRs), both of which elicit changes in synaptic efficacy through alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptor (AMPAR) endocytosis. To address the role of the ubiquitin-proteasome system in regulating AMPAR endocytosis during these forms of LTD, we examined the effects of pharmacological inhibitors of proteasomal degradation and protein ubiquitination on endocytosis of glutamate receptor 1 (GluR1) -containing AMPARs in dissociated rat hippocampal cultures as well as LTD of excitatory synaptic responses in acute rat hippocampal slices. Our findings suggest that the contribution of the ubiquitin-proteasome system to NMDAR-induced vs. mGluR-induced AMPAR endocytosis and the consequent LTD differs significantly. NMDAR-induced AMPAR endocytosis and LTD occur independently of proteasome function but appear to depend, at least in part, on ubiquitination. In contrast, mGluR-induced AMPAR endocytosis and LTD are enhanced by inhibition of proteasomal degradation, as well as by the inhibitor of protein ubiquitination. Furthermore, the decay of mGluR-induced membrane depolarization and Erk activation is delayed following inhibition of either ubiquitination or proteasomal degradation. These results suggest that, although NMDAR-dependent LTD may utilize ubiquitin as a signal for AMPAR endocytosis, mGluR-induced signaling and LTD are limited by a feedback mechanism that involves the ubiquitin-proteasome system.

A critical role for PSD-95/AKAP interactions in endocytosis of synaptic AMPA receptors.

The endocytosis of AMPA receptors (AMPARs) underlies several forms of synaptic plasticity, including NMDA receptor (NMDAR)-dependent long-term depression (LTD), but the molecular mechanisms responsible for this trafficking remain unknown. We found that PSD-95, a major postsynaptic density protein, is important for NMDAR-triggered endocytosis of synaptic AMPARs in rat neuron cultures because of its binding to A kinase-anchoring protein 150 (AKAP150), a scaffold for specific protein kinases and phosphatases. Knockdown of PSD-95 with shRNA blocked NMDAR-triggered, but not constitutive or mGluR-triggered, endocytosis of AMPARs. Deletion of PSD-95's Src homology 3 and guanylate kinase-like domains, as well as a point mutation (L460P), both of which inhibit binding of PSD-95 to AKAP150, also blocked NMDAR-triggered AMPAR endocytosis. Furthermore, expression of a mutant AKAP150 that does not bind calcineurin inhibited this NMDAR-triggered trafficking event. Our results suggest that PSD-95's interaction with AKAP150 is critical for NMDAR-triggered AMPAR endocytosis and LTD, possibly because these scaffolds position calcineurin in the appropriate subsynaptic domain.

Endocytosis and recycling of AMPA receptors lacking GluR2/3.

Excitatory synapses in the mammalian brain contain two types of ligand-gated ion channels: AMPA receptors (AMPARs) and NMDA receptors (NMDARs). AMPARs are responsible for generating excitatory synaptic responses, whereas NMDAR activation triggers long-lasting changes in these responses by modulating the trafficking of AMPARs toward and away from synapses. AMPARs are tetramers composed of four subunits (GluR1-GluR4), which current models suggest govern distinct AMPAR trafficking behavior during synaptic plasticity. Here, we address the roles of GluR2 and GluR3 in controlling the recycling- and activity-dependent endocytosis of AMPARs by using cultured hippocampal neurons prepared from knockout (KO) mice lacking these subunits. We find that synapses and dendritic spines form normally in cells lacking GluR2/3 and that upon NMDAR activation, GluR2/3-lacking AMPARs are endocytosed in a manner indistinguishable from GluR2-containing AMPARs in wild-type (WT) neurons. AMPARs lacking GluR2/3 also recycle to the plasma membrane identically to WT AMPARs. However, because of their permeability to calcium, GluR2-lacking but not WT AMPARs exhibited robust internalization throughout the dendritic tree in response to AMPA application. Dendritic endocytosis of AMPARs also was observed in GABAergic neurons, which express a high proportion of GluR2-lacking AMPARs. These results demonstrate that GluR2 and GluR3 are not required for activity-dependent endocytosis of AMPARs and suggest that the most important property of GluR2 in the context of AMPAR trafficking may be its influence on calcium permeability.

Activation, internalization, and recycling of the serotonin 2A receptor by dopamine.

Serotonergic and dopaminergic systems, and their functional interactions, have been implicated in the pathophysiology of various CNS disorders. Here, we use recombinant serotonin (5-HT) 2A (5-HT2A) receptors to further investigate direct interactions between dopamine and 5-HT receptors. Previous studies in Xenopus oocytes showed that dopamine, although not the cognate ligand for the 5-HT2A receptor, acts as a partial-efficacy agonist. At micromolar concentrations, dopamine also acts as a partial-efficacy agonist on 5-HT2A receptors in HEK293 cells. Like 5-HT, dopamine also induces receptor-internalization in these cells, although at significantly higher concentrations than 5-HT. Interestingly, if the receptors are first sensitized or "primed" by subthreshold concentrations of 5-HT, then dopamine-induced internalization occurs at concentrations approximately 10-fold lower than when dopamine is used alone. Furthermore, unlike 5-HT-mediated internalization, dopamine-mediated receptor internalization, alone, or after sensitization by 5-HT, does not depend on PKC. Dopamine-internalized receptors recycle to the surface at rates similar to those of 5-HT-internalized receptors. Our results suggest a previously uncharacterized role for dopamine in the direct activation and internalization of 5-HT2A receptors that may have clinical relevance to the function of serotonergic systems in anxiety, depression, and schizophrenia and also to the treatment of these disorders.

Internalization and recycling of 5-HT2A receptors activated by serotonin and protein kinase C-mediated mechanisms.

Serotonin (5-HT), a major neurotransmitter, has a large number of G protein-coupled receptors in mammals. On activation by exposure to their ligand, 5-HT(2) receptor subtypes increase IP(3) levels and undergo desensitization and internalization. To visualize the receptor in cells during these processes, we have constructed a 5-HT(2A)-enhanced GFP (SR2-GFP) fusion receptor. We show that this fusion receptor undergoes internalization on exposure to its natural ligand, 5-HT. Because 5-HT(2A) receptors activate the phospholipase C pathway, we studied the effect of protein kinase C (PKC) on the internalization process and found that activation of PKC by its specific activator phorbol 12-myristate 13-acetate, in the absence of 5-HT, leads to internalization of the receptor. Moreover, inhibition of PKC by its inhibitor sphingosine in the presence of 5-HT prevents the internalization process, suggesting that activation of PKC is sufficient and necessary for the internalization of 5-HT(2A) receptors. We also show that SR2-GFP recycles back to the plasma membrane after 5-HT-dependent internalization, suggesting a mechanism for resensitization. In addition, receptors that have been internalized on addition of phorbol 12-myristate 13-acetate in the absence of 5-HT also recycle to the surface, with a time course similar to that seen after activation of the receptors by 5-HT. Our study suggests that 5-HT(2A) receptors internalize and return to the surface after both serotonin- and PKC-mediated processes. This study reveals a role for PKC in receptor internalization and also shows that 5-HT(2A) receptors are recycled.