Regulation of ErbB-4 endocytosis by neuregulin in GABAergic hippocampal interneurons

doi:10.1016/j.brainresbull.2007.02.014

Brain Research Bulletin

Volume 73, Issues 4–6, 12 July 2007, Pages 210-219

https://doi.org/10.1016/j.brainresbull.2007.02.014 Get rights and content

Abstract

Neuregulin (NRG)/ErbB receptor signaling pathways have recently been implicated in the reversal of long-term potentiation at hippocampal glutamatergic synapses. Moreover, polymorphisms in NRG-1 and ErbB-4 genes have been linked to an increased risk for developing schizophrenia. ErbB-4 is highly expressed at glutamatergic synapses where it binds to PSD-95 via its carboxyl terminal T–V–V sequence. Here we investigated the expression, localization and trafficking of ErbB-4 in cultured hippocampal neurons by immunocytochemistry, surface protein biotinylation, and live labeling of native receptors. We show that neuronal ErbB-4 is detected at its highest levels in GABAergic interneurons, as observed in vivo. ErbB-4 immunoreactivity precedes PSD-95 expression, with ErbB-4 cluster initially forming in the absence of, but later associating with, PSD-95-positive puncta. By surface protein biotinylation, the fraction of ErbB-4 receptors on the plasma membrane increases from 30% to 65% between 6 and 16 days in vitro (DIV). Interestingly, 30 min of NRG stimulation triggers measurable ErbB-4 receptor internalization at DIV 16, despite increased colocalization with PSD-95. We also investigated the role of TNFα-converting enzyme (TACE)-mediated receptor processing in regulating ErbB-4 surface expression. We found that the cleavage-resistant JM-b isoform accounts for 80% of all ErbB-4 transcripts in cultured hippocampal neurons. Receptor stimulation or treatment with phorbol esters does not induce detectable ErbB-4 processing, indicating that neurons mostly rely on endocytosis of the intact receptor to regulate ErbB-4 surface expression. These results enhance our understanding of the regulation of ErbB-4—mediated signaling at glutamatergic synapses.

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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¹: Present address: Unidad de Neurobiologia Molecular, Centro de Biociencias y Medicina Molecular, Instituto de Estudios Avanzados-IDEA, Caracas, Venezuela. Tel.: +58 212 9035191.

²: Both authors contributed equally to this work.

³: Present address: 2755 Ordway Street NW, Washington, DC 20008, United States. Tel.: +1 202 462 1303.

⁴: Tel.: +1 301 594 4665.

⁵: Tel.: +1 301 451 0942.

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Research reportRegulation of ErbB-4 endocytosis by neuregulin in GABAergic hippocampal interneurons

Abstract

Introduction

Section snippets

Materials

ErbB-4 is expressed in cultured hippocampal GABAergic interneurons

Discussion

Conflict of interest

Acknowledgements

Exp. Cell. Res.

J. Biol. Chem.

Neuron

Curr. Opin. Neurobiol.

Exp. Cell Res.

J. Biol. Chem.

J. Biol. Chem.

Exp. Cell Res.

Neuroscience

Trends Cell Biol.

Neuron

Biochem. Biophys. Res. Commun.

Neuron

Neurosci. Lett.

Mol. Cell Neurosci.

J. Biol. Chem.

J. Biol. Chem.

Mol. Cell Neurosci.

J. Biol. Chem.

Role of neuregulins in glial cell development

Glia

Research report
Regulation of ErbB-4 endocytosis by neuregulin in GABAergic hippocampal interneurons