The nicotinic acetylcholine receptor of skeletal muscle is composed of five subunits that are assembled in a stepwise manner. Quality control mechanisms ensure that only fully assembled receptors reach the cell surface. Here, we show that Rer1, a putative Golgi-ER retrieval receptor, is involved in the biogenesis of acetylcholine receptors. Rer1 is expressed in the early secretory pathway in the myoblast line C2C12 and in mouse skeletal muscle, and up-regulated during myogenesis. Upon down-regulation of Rer1 in C2C12 cells, unassembled acetylcholine receptor ?-subunits escape from the ER and are transported to the plasma membrane and lysosomes, where they are degraded. As a result, the amount of fully assembled receptor at the cell surface is reduced. In vivo Rer1 knockdown and genetic inactivation of one Rer1 allele lead to significantly smaller neuromuscular junctions in mice. Our data show that Rer1 is a functionally important unique factor that controls surface expression of muscle acetylcholine receptors by localizing unassembled ?-subunits to the early secretory pathway.
The atypical protein kinase Czeta (PKCzeta) was recently shown to mediate epidermal growth factor (EGF)-induced activation of extracellular signal-regulated kinase (ERK) in head and neck squamous carcinoma (HNSCC) cells. Here, it is shown that EGF may induce tyrosine phosphorylation of PKCzeta in several HNSCC cells, breast carcinoma cells, as well as mouse embryonic fibroblasts. In COS-7 cells overexpressing EGF receptor (EGFR) and PKCzeta as a tumor cell model, we show that PKCzeta tyrosine phosphorylation by EGF is induced by catalytic activation. Using a loss-of-function mutant of PKCzeta, we can show that the tyrosine residue 417 in PKCzeta plays an important role in both PKCzeta activation and the ability of PKCzeta to mediate activation of ERK. The importance of PKCzeta in EGF-induced ERK activation can also be shown in several HNSCC and breast carcinoma cell lines as well as in PKCzeta-deficient mouse embryonic fibroblasts. In addition, we present several lines of evidence suggesting the physical association of PKCzeta with EGFR and the importance of the EGFR tyrosine kinase c-Src and the Src-specific phosphorylation site pY845-EGFR in the tyrosine phosphorylation as well as catalytic activation of PKCzeta. This study characterizes PKCzeta as a novel mitogenic downstream mediator of EGFR and indicates PKCzeta as a therapeutic target in some carcinomas.
gamma-Secretase is critically involved in the Notch pathway and in Alzheimers disease. The four subunits of gamma-secretase assemble in the endoplasmic reticulum (ER) and unassembled subunits are retained/retrieved to the ER by specific signals. We here describe a novel ER-retention/retrieval signal in the transmembrane domain (TMD) 4 of presenilin 1, a subunit of gamma-secretase. TMD4 also is essential for complex formation, conferring a dual role for this domain. Likewise, TMD1 of Pen2 is bifunctional as well. It carries an ER-retention/retrieval signal and is important for complex assembly by binding to TMD4. The two TMDs directly interact with each other and mask their respective ER-retention/retrieval signals, allowing surface transport of reporter proteins. Our data suggest a model how assembly of Pen2 into the nascent gamma-secretase complex could mask TMD-based ER-retention/retrieval signals to allow plasma membrane transport of fully assembled gamma-secretase.
Disrupted-in-Schizophrenia 1 (DISC1) is a prominent susceptibility gene for major psychiatric disorders. Previous work indicated that DISC1 plays an important role during neuronal proliferation and differentiation in the cerebral cortex and that it affects the positioning of radial migrating pyramidal neurons. Here we show that in mice, DISC1 is necessary for the migration of the cortical interneurons generated in the medial ganglionic eminence (MGE). RT-PCR, in situ hybridizations, and immunocytochemical data revealed expression of DISC1 transcripts and protein in MGE-derived cells. To study the possible functional role of DISC1 during tangential migration, we performed in utero and ex utero electroporation to suppress DISC1 in the MGE in vivo and in vitro. Results indicate that after DISC1 knockdown, the proportion of tangentially migrating MGE neurons that reached their cortical target was strongly reduced. In addition, there were profound alterations in the morphology of DISC1-deficient neurons, which exhibited longer and less branched leading processes than control cells. These findings provide a possible link between clinical studies reporting alterations of cortical interneurons in schizophrenic patients and the current notion of schizophrenia as a neurodevelopmental disorder.
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