Research reportRegulation of ErbB-4 endocytosis by neuregulin in GABAergic hippocampal interneurons
Introduction
ErbB receptors, including the EGF receptor (ErbB-1), ErbB-2, ErbB-3 and ErbB-4, comprise a family of receptor tyrosine kinases involved in numerous signaling processes including the control of cell growth and differentiation. In particular, ErbB2-4 and their cognate neuregulin (NRG) ligands have long been recognized as critical mediators of cell fate, proliferation, migration and differentiation processes in the developing peripheral and central nervous system (for review, see [1], [6], [10], [12], [16]. Although NRGs and ErbB receptors continue to be expressed at high levels, until recently their functions in the adult brain were unknown. Work from our laboratory, as well as others, has implicated NRG/ErbB signaling in regulating plasticity of glutamatergic synapses. At CA3-to-CA1 synapses in the hippocampus, activation of ErbB receptor signaling can prevent or revert long-term potentiation (LTP) [21], [23]. Depotentiation of LTP in hippocampal neurons is mediated by the internalization of GluR1-containing AMPA receptors without affecting NMDA receptor-evoked postsynaptic currents. Conversely, stimulation of NRG/ErbB signaling in the prefrontal cortex leads to altered surface expression of NMDA receptors at glutamatergic synapses on pyramidal neurons [18]. Consistent with its ability to regulate synaptic plasticity, there is a rapidly growing body of evidence supporting the involvement of the NRG/ErbB pathway, and interactions between ErbB-4 and PSD-95, in the pathogenesis underlying schizophrenia [9], [19], [20].
By in situ hybridization, ErbB2-4 receptor genes are expressed in the CA1-CA3 regions of the hippocampus, with highest ErbB-4 mRNA levels found in interneurons [17], [24], [25]. The distribution of ErbB-4 protein in different neuronal populations is less clear; nevertheless, there is general agreement that the highest receptor levels are in GABAergic neurons (see Section 4). ErbB-4 and NMDA receptors colocalize in postsynaptic densities (PSDs) at glutamatergic synapses. ErbB-4 physically interacts via its c-terminal T–V–V sequence with membrane-associated guanylate kinases (MAGUKs) such as PSD-95, SAP-102 and PSD-93 [14], [21]. PSD-95 is a major scaffolding component of PSDs, and together with other MAGUKs, plays an important role in organizing the intricate network of receptors, signaling molecules and cytoskeletal adaptor proteins that together mediate synaptic transmission and plasticity (see, for example: [4], [15], [22]). The association with the PSD positions ErbB-4 as a potentially important modulator of synaptic plasticity, and further supports the notion that NRG/ErbB signaling regulates synaptic plasticity in vivo.
Many growth factor receptors are internalized upon ligand binding to gradually attenuate receptor signaling and to desensitize the cell to excess ligand availability, but also to target activated receptors to other intracellular substrates [37]. Unlike the EGF receptor (ErbB-1), rapid ligand-dependent receptor internalization is notably low for all other ErbB receptors in cancer cell lines expressing ErbB-2 or ErbB-3, or transfected NIH 3T3 cells overexpressing ErbB-4 [3]. Rather, ligand-mediated proteolytic processing has been proposed as a densitization mechanism for ErbB-4 [7], [40]. Specifically, it was found that ligand-induced cleavage of the 120 kDa ectodomain by TNF-alpha converting enzyme (TACE; syn. Adam 17) serves to shed the receptor from the surface [31], [35], [36], [40]. Sequence determinants for susceptibility to TACE-dependent ErbB-4 processing reside in a juxtamembrane region that is included in JM-a but that is missing from JM-b transcripts [11]. Interestingly, a recent study reported that JM-a isoforms were upregulated in postmortem brains from schizophrenic individuals [33].
Trafficking of ErbB-4 in neurons has been studied mainly with regard to its NRG-dependent recruitment to lipid rafts [27], a process which is believed to help targeting the ErbB-4 signaling complex to synapses [38]. However, to better understand the emerging role of ErbB receptors, in particular of ErbB-4, as modulators of synaptic plasticity in the adult brain, it is critical to know how receptor availability is regulated in neurons. As our present knowledge about endocytosis of ErbB receptors is largely based on cell lines lacking the functional specializations of mature neurons such as the postsynaptic density, it is unclear if, and how, ErbB-4 receptor processing and endocytosis are regulated in neurons in response to NRG binding. To address this question, we have investigated the surface expression and NRG-stimulated endocytosis of ErbB-4 in dissociated hippocampal neurons by surface protein biotinylation and live cell labeling of endogenous receptors, as well as the role of TACE-mediated ectodomain shedding in regulating surface ErbB-4.
Section snippets
Materials
Human NRG1β1 EGF domain peptide (amino acids 176–246; R&D systems (Minneapolis, MN)) was reconstituted at 5 μM in 0.1% bovine serum albumin (BSA) in phosphate-buffered saline (PBS). 4-[(3-bromophenyl)amino]-6-(methylamino)-pyrido[3,4-d]pyrimidine (PD158780), PMA (4α-Phorbol 12-myristate 13-acetate) and the TACE inhibitor TAPI-2 were from Calbiochem (La Jolla, CA). Mouse monoclonal antibodies ab77 and ab72 against the extracellular domain of ErbB-4 [8] and Ab-4 against tubulin were from Lab
ErbB-4 is expressed in cultured hippocampal GABAergic interneurons
Previous evidence from in situ hybridization and immunohistochemistry analyses in the hippocampus [17], [25] suggested highest levels of ErbB-4 expression in GABAergic interneurons, based on cell position and morphology. These observations were later corroborated by double-immunofluorescence of the receptor [39]. We double-labeled dissociated hippocampal neurons (DIV 14) with antibodies against GABA and ErbB-4 to test if this expression pattern is maintained in culture. By immunofluorescence,
Discussion
In the past few years, increasing attention has been directed to the functions of the NRG/ErbB signaling pathway in the adult nervous system. This interest was fueled by two separate but potentially converging lines of research. On the one hand, NRG signaling has been recognized as a potent modulator of synaptic plasticity at glutamatergic synapses of the hippocampus and the prefrontal cortex [18], [21], [23]. Although the mechanisms by which it regulates synaptic plasticity appear to be
Conflict of interest
None.
Acknowledgements
This work was supported by NICHD, and by a fellowship from NINDS to M.C.-H.
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AAK1 identified as an inhibitor of neuregulin-1/ErbB4-dependent neurotrophic factor signaling using integrative chemical genomics and proteomics
2011, Chemistry and BiologyCitation Excerpt :Given our isolation of AAK1 and AP2 adaptor complex-associated proteins with our K252a-based affinity probes, and the observation of the elevated total levels of ErbB4 and its redistribution toward cellular membranes upon treatment with K252a (1) and RNAi-mediated gene silencing of AAK1, it is tempting to speculate that the loss of function of AAK1 leads to an ErbB4 internalization defect due to altered clathrin-mediated endocytosis (Conner and Schmid, 2002; Ricotta et al., 2002; Smythe and Ayscough, 2003). Although the mechanism of internalization of ErbB4 is poorly understood (Baulida et al., 1996; Sorkin and Goh, 2008), several recent studies have demonstrated that endocytosis of ErbB4 does occur in various cellular systems, and regulation of this process may be a critical component of the dynamic regulation of Nrg1-ErbB4 signaling, particularly in neurons (Liu et al., 2007; Longart et al., 2007; Sundvall et al., 2008). However, besides playing a key role in regulating AP2 recruitment to endocytic cargo through phosphorylation of μ2 (Ricotta et al., 2002), AAK1 has been shown to function at multiple steps in receptor transport including receptor recycling from the early endosome and pathways involved in protein degradation (Henderson and Conner, 2007).
The neuregulin signaling pathway and schizophrenia: From genes to synapses and neural circuits
2010, Brain Research BulletinCitation Excerpt :Ultrastructural analysis in CA1 interneurons using immunoelectron microscopy revealed abundant ErbB4 expression in the somatodendritic compartment where it accumulates at, and adjacent to, glutamatergic postsynaptic sites [150]. By contrast, we found no evidence for presynaptic expression in cultured GAD67-positive hippocampal interneurons and in CA1 basket cell terminals [98,150,113]. The localization of ErbB4 at excitatory synapses on GABAergic neurons, but not excitatory neurons, identifies these synapses as a primary target of NRG signaling in the hippocampus and indicates that ErbB4 serves as a selective marker for PSDs on GABAergic neurons [150].
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