The neuregulin receptor ErbB-4 interacts with PDZ-containing proteins at neuronal synapses

Abstract

Neuregulins regulate the expression of ligand- and voltage-gated channels in neurons and skeletal muscle by the activation of their cognate tyrosine kinase receptors, ErbB 1-4. The subcellular distribution and mechanisms that regulate the localization of ErbB receptors are unknown. We have found that ErbB receptors are present in brain subcellular fractions enriched for postsynaptic densities (PSD). The ErbB-4 receptor is unique among the ErbB proteins because its C-terminal tail (T-V-V) conforms to a sequence that binds to a protein motif known as the PDZ domain. Using the yeast two-hybrid system, we found that the C-terminal region of ErbB-4 interacts with the three related membrane-associated guanylate kinases (MAGUKs) PSD-95/SAP90, PSD-93/chapsyn-110, and SAP 102, which harbor three PDZ domains, as well as with beta(2)-syntrophin, which has a single PDZ domain. As with N-methyl-D-aspartate (NMDA) receptors, ErbB4 interacts with the first two PDZ domains of PSD-95. Using coimmunoprecipitation assays, we confirmed the direct interactions between ErbB-4 and PSD-95 in transfected heterologous cells, as well as in vivo, where both proteins are coimmunoprecipitated from brain lysates. Moreover, evidence for colocalization of these proteins was also observed by immunofluorescence in cultured hippocampal neurons. ErbB-4 colocalizes with PSD-95 and NMDA receptors at a subset of excitatory synapses apposed to synaptophysin-positive presynaptic terminals. The capacity of ErbB receptors to interact with PDZ-domain proteins at cell junctions is conserved from invertebrates to mammals. As discussed, the interactions found between receptor tyrosine kinases and MAGUKs at neuronal synapses may have important implications for activity-dependent plasticity.

Figure 1

ErbB receptors are present in the post synaptic density fraction. The subcellular distribution of ErbB receptors in rat forebrain fractions enriched in synaptic membranes (SM) and postsynaptic densities (PSD) was analyzed by Western blot. The blots were probed with specific antisera raised against ErbB-2, ErbB-3, and ErbB-4, and the postsynaptic proteins PSD-95 and NR1. Antisera against synaptophysin (SynP) and glutamate transporter-1 (GLT-1) were used to monitor the presence of presynaptic and glial markers, respectively, in the SM and PSD fractions. All lanes contain 37 μg of protein.

Figure 2

Coimmunoprecipitation of ErbB-4 receptor and PSD-95 from forebrain lysates. (A) Total proteins from rat forebrain deoxycholate homogenates (Western, Left), or proteins immunoprecipitated from these lysates (IP, Right), were resolved by SDS/PAGE and probed on Western blots with ErbB-4 (E) and PSD-95 (P) antisera. The antibodies specifically recognize proteins of approximately 180 kDa (small arrowhead) and 95 kDa (large arrowhead), respectively. Migration of the prestained molecular weight markers (myosin, β-galactosidase, and BSA) are indicated. (B) Coimmunoprecipitations were performed from forebrain deoxycholate lysates using PSD-95 (Left) or ErbB-4 (Right) antibodies, and probed on Western blots using anti-ErbB4 or anti-PSD-95 antisera. As negative controls, the immunoprecipitations were performed either in the absence of antibodies (−), normal rabbit immunoglobulins (IgG), or monoclonal antibodies raised against the unrelated transcription factor E2F1 (an IgG2a that is the same isotype as the PSD-95 monoclonal).

Figure 3

ErbB-4 directly associates with PSD-95 in heterologous cells by interactions requiring its C terminus. QT-6 fibroblasts were transiently transfected with ErbB-4 and PSD-95 expression vectors to analyze for interactions between these proteins. (A) Coimmunoprecipitation assays were performed from lysates of cells transfected with constructs encoding PSD-95 (P), ErbB-4 (E), and both proteins (E/P) using ErbB-4 or PSD-95 antisera for immunoprecipitation (IP Ab), and probing the Western blots with anti-PSD-95 and anti-ErbB-4 antisera. A mock transfection (M) using an empty expression vector was used as negative control. (B) A truncated form of ErbB-4, missing the C-terminal 48 amino acids, fails to interact with PSD-95. Coimmunoprecipitation experiments and Western blots were performed as above, using lysates from cells transfected with constructs encoding the truncated ErbB-4 receptor alone (tE) or in combination with PSD-95 (tE/P).

Figure 4

Immunocytochemical localization of ErbB-4 at synaptic sites of cultured hippocampal cells. Double immunofluorescence was performed on dissociated hippocampal neurons cultured for 3 wk. (A–C) ErbB-4 receptor puncta (red) are located at synaptic sites apposed to a subset of the synaptophysin-positive presynaptic terminals (green). The yellow color in the enlarged view (C) indicates the extent of immunofluorescence overlap. (D–F) ErbB-4 receptors (red) and PSD-95 (green) colocalize in a subset of synaptic puncta (yellow). (G–I) ErbB-4 receptors (red) and the NR1 subunit of the NMDA receptor (green) also colocalize at puncta (yellow). Areas that are boxed are enlarged (Bottom), and the arrows indicate examples of colocalization.