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

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 minutes 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.

ErbB4 reduces synaptic GABA A currents independent of its receptor tyrosine kinase activity

“…3G). Importantly, over 88% of cultured hippocampal interneurons are also ErbB4+ in wild-type cultures (29). In these ErbB4 −/− cultured interneurons, NRG2 was without effect (Fig.…”

“…Triton X-100-solubilized synaptic plasma membranes or hippocampal culture lysates were normalized for total protein, and immunoprecipitation was performed essentially as described (8). Biotinylation reactions of hippocampal cultures were performed at 4°C, washed, and processed essentially as described (29) using NeutrAvidin UltraLink Resin (Pierce).…”

“…ErbB4 has been shown to accumulate at interneuron glutamatergic synapses, where it associates with PSD-95 at excitatory postsynaptic densities (3,22). However, coimmunofluorescence analysis in dissociated hippocampal neurons reveals a substantial fraction of ErbB4 immunoreactivity that does not overlap with PSD-95 (29) (Fig. S1A).…”

“…4E, NRG2 results in phosphoactivation of PKCγ but not PKCα/βII. To selectively block PKC activation, we included an inhibitory PKC (19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36) peptide (10 μM) in the intracellular solution. As illustrated in Fig.…”

Endocytosis and intracellular trafficking of ErbBs

“…Ligand binding typically causes down-regulation of these receptors [11,12,163], albeit to an extent that is significantly lower than that observed with EGFR down-regulation [164][165][166]. The neuregulin-induced internalization of ErbB3 and ErbB4 has been observed in various cells, including neurons [12,164,165,167,168]. However, as in the case of ErbB2, the internalization of ErbB3 and ErbB4 is slower than that of EGFR [165,166].…”