Biomedical subjects
Publications and source records attributed to Wen C Xiong.
How do presynaptic inputs regulate synapse formation? In this issue of Neuron, Lin et al. show that the neurotransmitter acetylcholine decreases the stability of AChR clusters. This dispersing activity, which requires the serine/threonine kinase Cdk5, cooperates with positive signals from motoneurons to ensure high concentration of AChRs at the neuromuscular junction.
Neuregulin-induced expression of the acetylcholine receptor (AChR) contributes to high concentration of the receptor at the neuromuscular junction (NMJ). Neuregulin-1 activates ErbB tyrosine kinases and subsequently intracellular kinases including Erk that is required for induced AChR expression. Recent studies demonstrate that ligand-induced internalization may regulate signaling of various receptor tyrosine kinases. However, the role of induced ErbB endocytosis in regulating AChR expression was unclear. Here we provide evidence that ErbB tyrosine kinases became rapidly internalized in response to neuregulin. The internalization required the kinase activity of ErbB proteins and involved a clathrin-dependent endocytic pathway. Moreover, neuregulin-induced Erk activation and AChR expression were attenuated when ErbB endocytosis was blocked. These results indicate that ErbB proteins undergo endocytosis in response to neuregulin, and this process is required for neuregulin signaling and induced AChR expression.
The type I receptor-like protein tyrosine kinase MuSK is essential for the neuromuscular junction formation. MuSK expression is tightly regulated during development, but the underlying mechanisms were unclear. Here we identified a novel mechanism by which MuSK expression may be regulated. A cyclic AMP response element (CRE)-like element in the 5'-flanking region of the MuSK gene binds to CREB1 (CRE-binding protein 1). Mutation of this element increases the MuSK promoter activity, suggesting a role for CREB1 in attenuation of MuSK expression. Interestingly, CREB mutants unable to bind to DNA also inhibit MuSK promoter activity, suggesting a CRE-independent inhibitory mechanism. In agreement, CREB1 could inhibit a mutant MuSK transgene reporter whose CRE site was mutated. We provide evidence that CREB interacts directly with MyoD, a myogenic factor essential for MuSK expression in muscle cells. Suppression of CREB expression by small interfering RNA increases MuSK promoter activity. These results demonstrate an important role for CREB1 in the regulation of MuSK expression.
Neuregulins are a family of EGF domain-containing factors that play an important role in development. In the nervous system, they promote glial differentiation, induce neurotransmitter receptor expression, and regulate synaptic plasticity. Recent studies indicate that ErbB protein tyrosine kinases, neuregulin receptors, translocate to lipid raft microdomains in the plasma membrane in response to neuregulin. Localization of ErbB proteins in lipid rafts appeared to be necessary for neuregulin signaling and regulation of synaptic plasticity. We will review recent studies of lipid rafts and neuregulin function and discuss possible roles of lipid rafts in compartmentalized neuregulin signaling and translocation of ErbB proteins to synapses.
Agrin activates the transmembrane tyrosine kinase MuSK to mediate acetylcholine receptor (AChR) clustering at the neuromuscular junction (NMJ). However, the intracellular signaling mechanism downstream of MuSK is poorly characterized. This study provides evidence that geranylgeranyltransferase I (GGT) is an important signaling component in the Agrin/MuSK pathway. Agrin causes a rapid increase in tyrosine phosphorylation of the alpha(G/F) subunit of GGT and in GGT activity. Inhibition of GGT activity or expression prevents muscle cells from forming AChR clusters in response to Agrin and attenuates the formation of neuromuscular synapses in spinal neuron-muscle cocultures. Importantly, transgenic mice expressing an alpha(G/F) mutant demonstrate NMJ defects with wider endplate bands and smaller AChR plaques. These results support the notion that prenylation is necessary for AChR clustering and the NMJ formation and/or maintenance, revealing an active role of GGT in Agrin/MuSK signaling.
MuSK is a receptor tyrosine kinase essential for neuromuscular junction formation. Expression of the MuSK gene is tightly regulated during development and at the neuromuscular junction. However, little is known about molecular mechanisms regulating its gene expression. Here we report a characterization of the promoter of the mouse MuSK gene. The transcription of MuSK starts at multiple sites with a major site 51 nt upstream of the translation start site. We have identified an E-box-like cis-element that is both required and sufficient for differentiation-dependent transcription. Interestingly, the promoter activity of the MuSK gene did not respond to neuregulin, a factor believed to mediate the synapse-specific transcription of acetylcholine receptor subunit genes. Rather, MuSK expression is increased in muscle cells stimulated with Wnt or at conditions when the Wnt signaling was activated. These results suggest a novel mechanism for the MuSK synapse-specific expression.
Neuregulin (NRG) regulates synapse formation and synaptic plasticity, but little is known about the regulation of NRG signaling at synapses. Here we show that the NRG receptor ErbB4 was localized in anatomically defined postsynaptic densities in the brain. In cultured cortical neurons, ErbB4 was recruited to the neuronal lipid raft fraction after stimulation by NRG. Along with ErbB4, adaptor proteins Grb2 and Shc were translocated to lipid rafts by NRG stimulation. In transfected human embryonic kidney 293 cells, the partitioning of ErbB4 into a detergent-insoluble fraction that includes lipid rafts was increased by PSD-95 (postsynaptic density-95), through interaction of the ErbB4 C terminus with the PDZ [PSD-95/Discs large/zona occludens-1] domains of PSD-95. Disruption of lipid rafts inhibited NRG-induced activation of Erk and prevented NRG-induced blockade of induction of long-term potentiation at hippocampal CA1 synapses. Thus, our results indicate that NRG stimulation causes translocation of ErbB4 into lipid rafts and that lipid rafts are necessary for signaling by ErbB4.
Assembly of ACh receptors is under tight quality control. Only functional ACh receptors are expressed on the cell surface; unassembled subunits are retained in the endoplasmic reticulum (ER). In a recent elegant study, Wang and colleagues have identified a novel motif in the M1 domain of the ACh-receptor subunit that is responsible for ER retention and degradation of unassembled subunits. This signal appears to play an important role in regulating surface trafficking of functional ACh receptors.
We present evidence here that Erbin is a negative regulator of the Ras-Raf-Erk signaling pathway. Expression of Erbin decreases transcription of the AChR epsilon-subunit gene, an event that is mediated by Erk activation. Although it interacts with the ErbB2 C terminus through the PDZ domain, Erbin has no effect on ErbB2 tyrosine phosphorylation or binding to the adaptor proteins Shc and Grb2. In contrast, expression of Erbin greatly impairs activation of Erk, but not Akt, by ligands that activate receptor tyrosine kinases. Moreover, Erbin inhibits the Erk activation by active Ras, while it fails to do so in the presence of active Raf-1. Erbin associates with active Ras, but not inactive Ras nor Raf. Consistently, Erbin interferes with the interaction between Ras and Raf both in vivo and in vitro. Finally, overexpression of Erbin leads to inhibition of NGF-induced neuronal differentiation of PC12 cells, whereas down-regulation of endogenous Erbin by specific siRNA exhibits an opposite effect. Collectively, our study has identified Erbin as a novel suppressor of the Ras signaling by disrupting the Ras-Raf interaction.
An important aspect of synapse development is the clustering of neurotransmitter receptors in the postsynaptic membrane. Although MuSK is required for acetylcholine receptor (AChR) clustering at the neuromuscular junction (NMJ), the underlying molecular mechanisms remain unclear. We report here that in muscle cells, MuSK interacts with Dishevelled (Dvl), a signaling molecule important for planar cell polarity. Disruption of the MuSK-Dvl interaction inhibits Agrin- and neuron-induced AChR clustering. Expression of dominant-negative Dvl1 in postsynaptic muscle cells reduces the amplitude of spontaneous synaptic currents at the NMJ. Moreover, Dvl1 interacts with downstream kinase PAK1. Agrin activates PAK, and this activation requires Dvl. Inhibition of PAK1 activity attenuates AChR clustering. These results demonstrate important roles of Dvl and PAK in Agrin/MuSK-induced AChR clustering and reveal a novel function of Dvl in synapse development.
The synapse-specific synthesis of the acetylcholine receptor (AChR) is mediated by multiple mechanisms including compartmentalized signaling induced by neuregulin (NRG). This paper presents evidence that NRG receptors--ErbB receptor tyrosine kinases interact with distinct PDZ domain-containing proteins that are localized at the neuromuscular junction (NMJ). ErbB4 associates with the PSD-95 (also known as SAP90)-family members including PSD-95, SAP97, and SAP102 whereas ErbB2 interacts with Erbin and PICK1. Although, ErbB kinases are concentrated at the NMJ, they are not colocalized with the AChR in cultured muscle cells even in the presence of agrin. Co-expression of PSD-95 causes ErbB4 to form clusters in COS cells. We propose that PDZ domain-containing proteins play a role in anchoring ErbB proteins at the neuromuscular junction, and/or mediating downstream signaling pathways. Such mechanisms could be important for the maintenance and function of the synapse.
The receptor tyrosine kinase MuSK is activated by agrin, an extracellular matrix protein believed to be utilized by motoneurons to regulate the formation or maintenance of the neuromuscular junction (NMJ). Recent studies have shed light on intracellular signaling mechanisms downstream of MuSK. Agrin enhances the activity of Rho GTPases and PAK, which is required for AChR clustering. Activation of these enzymes requires not only the kinase activity of MuSK, but also its interaction with proteins such as Dishevelled. These results suggest that MuSK may function as a scaffold tyrosine kinase that forms a multi-molecule complex for AChR clustering.