The submembrane machinery for nicotinic acetylcholine receptor clustering.

Froehner, Stanley C.

doi:10.1083/jcb.114.1.1

Cited by 166 publications

(98 citation statements)

References 76 publications

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“…Deletion of amino acids 366-412 abolished rapsyn-induced phosphorylation, and conversely, the C-terminal half of rapsyn (aa 212-412) was sufficient to induce phosphorylation. Together, this implicates a C-terminal region encompassing the Zn ++ -binding RING-H2 domain (Froehner, 1991, Scotland et al, 1993, Bezakova and Bloch, 1998) and C-terminal 10 amino acids containing a consensus serine phosphorylation site (aa 406) (Froehner, 1991). The RING domain has been previously implicated in binding to β-dystroglycan (Bartoli et al, 2001) but no function has yet been ascribed to the extreme C-terminus (Han et al, 1999).…”

Section: Phosphorylation Of Achr β and δ Subunitsmentioning

confidence: 99%

“…Indeed, in agrin and MuSK knockout mice, AChR clusters are largely eliminated by birth and the mice die due to an inability to move and breath (DeChiara et al, 1996, Gautam et al, 1996. Downstream of MuSK activation, an important mediator of AChR clustering is the intracellular, peripheral membrane protein, rapsyn, which associates with the AChR in the postsynaptic membrane in approximately 1:1 stoichiometry (Froehner, 1991). When expressed in heterologous cells, rapsyn self-aggregates and is sufficient to cluster, anchor and stabilize the AChR (Froehner et al, 1990, Phillips et al, 1991, Phillips et al, 1993, Phillips et al, 1997, Wang et al, 1999.…”

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confidence: 99%

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Rapsyn carboxyl terminal domains mediate muscle specific kinase–induced phosphorylation of the muscle acetylcholine receptor

et al. 2008

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At the developing vertebrate neuromuscular junction, postsynaptic localization of the acetylcholine receptor (AChR) is regulated by agrin signaling via the muscle specific kinase (MuSK) and requires an intracellular scaffolding protein called rapsyn. In addition to its structural role, rapsyn is also necessary for agrin-induced tyrosine phosphorylation of the AChR, which regulates some aspects of receptor localization. Here, we have investigated the molecular mechanism by which rapsyn mediates AChR phosphorylation. In a heterologous COS cell system, we show that MuSK and rapsyn induced phosphorylation of β subunit tyrosine 390 (Y390) and δ subunit Y393, as in muscle cells. Mutation of β Y390 or δ Y393 did not inhibit MuSK/rapsyn-induced phosphorylation of the other subunit in COS cells, and mutation of β Y390 did not inhibit agrin-induced phosphorylation of the δ subunit in Sol8 muscle cells; thus, their phosphorylation occurs independently, downstream of MuSK activation. In COS cells, we further show that MuSK-induced phosphorylation of the β subunit was mediated by rapsyn, as MuSK plus rapsyn increased β Y390 phosphorylation more than rapsyn alone and MuSK alone had no effect. Intriguingly, MuSK also induced tyrosine phosphorylation of rapsyn itself. We then used deletion mutants to map the rapsyn domains responsible for activation of cytoplasmic tyrosine kinases that phosphorylate the AChR subunits. We found that rapsyn C-terminal domains (amino acids 212-412) are both necessary and sufficient for activation of tyrosine kinases and induction of cellular tyrosine phosphorylation. Moreover, deletion of the rapsyn RING domain (365-412) abolished MuSK-induced tyrosine phosphorylation of the AChR β subunit. Together, these findings suggest that rapsyn facilitates AChR phosphorylation by activating or localizing tyrosine kinases via its C-terminal domains. Keywords neuromuscular junction; synaptogenesis; agrin; postsynaptic membraneAt the developing neuromuscular junction in vertebrates, several nerve-derived signals combine to localize the acetylcholine receptor at postsynaptic sites (Sanes and Lichtman, 2001, Burden, 2002, Kummer et al., 2006. One essential factor is agrin, which signals via the MuSK receptor tyrosine kinase and induces and/or stabilizes clustering of the AChR in the postsynaptic membrane (reviewed in (Kummer et al., 2006 Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. is prepatterned and AChR clusters occur in the central region of the muscle prior to and even in the absence of neural innervation (Lin et al., 2001, Yang et al., 2001). However, upo...

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Section: Phosphorylation Of Achr β and δ Subunitsmentioning

confidence: 99%

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confidence: 99%

Rapsyn carboxyl terminal domains mediate muscle specific kinase–induced phosphorylation of the muscle acetylcholine receptor

et al. 2008

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“…There are two possible pathways. First, it is possible that the actin filament may interact with K,-channel proteins directly or indirectly through linker proteins and structural changes of actin may cause enough conformational changes in K+ channels to alter their activities (Froehner, 1991;Rosenmund and Westbrook, 1993;Berdiev et al, 1996;Prat and Cantiello, 1996). There are numerous examples of ion trans-porters that are regulated in this way.…”

Section: Dlscusslonmentioning

confidence: 99%

Actin Filaments Modulate Both Stomatal Opening and Inward K+-Channel Activities in Guard Cells of Vicia faba L

et al. 1997

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Actin antagonists have previously been shown to alter responses of Commelina communis stomata to physiological stimuli, implicating actin filaments i n the control of guard cell volume changes (M. Kim, P.K. Hepler, 5-0. Eun, K.S. Ha, Y. Lee [1995] Plant Physiol 109: 1077-1084). Since K+ channels i n the guard cell play an important role i n stomatal movements, we examined the possible regulation of K+-channel activities by the state of actin polymerization. Agents affecting actin polymerization altered light-induced stomatal opening and inward K+-channel activities measured by patch clamping in Vicia faba. Cytochalasin D, which induces depolymerization of actin filaments, promoted light-induced stomatal opening and potentiated the inward K+ current in guard cell protoplasts. Phalloidin, a stabilizer of filamentous actin, inhibited both light-induced stomatal opening and inward K+ current. lnward K+-channel activities in outside-out membrane patches showed responses to these agents that support results at the whole-cell current levei, suggesting that cytochalasin D facilitates and phalloidin inhibits K+ influx in intact guard cells, thus resulting in enhancement and inhibition of stomatal opening, respectively. To our knowledge, this is the first report that provides evidence that actin filaments may regulate an important physiological process by modulating the activities of ion channels i n plant cells.The regulation of stomatal aperture is critica1 to a plant's ability to balance the need for a C source while avoiding the deleterious effects of water loss. The size of the stomatal opening is established through volume changes of stomatal guard cells under the concerted influence of light, temperature, CO,, and phytohormones (Assmann, 1993). Recently, Kim et al. (1995) showed that cortical actin filaments are distributed radially, fanning out from the stomatal pore site in mature guard cells of Commelinu communis. Moreover, fungal toxins that interfere with the polymerization or depolymerization of actin filaments brought about altered stomatal responses to physiological stimuli. Thus, dynamic changes in the actin cytoskeleton

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“…The in vitro AChR clusters in the aneural myotube cultures resemble those of in vivo neuromuscular junctions in their morphology and in their associations with intracellular and extracellular components (Froehner, 1991;Bloch et al, 1989). In particular, both types are shown to be directly associated with cytoplasmic aggregations of 43K protein, and are speculated also to be directly associated with actin and @-spectrin (Froehner, 1991). However, the in vitro clusters appear to be more dynamic, appearing and then disappearing in a matter of days or less, with an even more rapid turnover of individual AChR within a cluster.…”

Section: Introductionmentioning

confidence: 99%

“…However, the mechanism of the clustering is not well understood on the molecular level. On rat muscle cells, previous studies have shown that the clustering not only occurs at the neuromuscular synapses (in vivo) but also can be induced in culture (in vitro) by contact with the substrate, nerves, or basic polypeptide-coated latex beads; or by supplementing the medium with embryonic hrain extract, basal lamina extract, or basic fibroblast growth factor (Froehner, 1991;Krikorian and Daniels, 1989;Olek et al, 1986). The AChR clusters in vitro have served as convenient model systems for understanding the mechanisms of clustering in vivo.…”

Section: Introductionmentioning

confidence: 99%

Time‐lapse total internal reflection fluorescence video of acetylcholine receptor cluster formation on myotubes

Wang

Axelrod

1994

Developmental Dynamics

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To study when and where acetylcholine receptor (AChR) clusters appear on developing rat myotubes in primary culture, we have made time-lapse movies of total internal reflection fluorescence (TIRF) overlaid with schlieren transmitted light images. The receptors, including the ones newly incorporated into the membrane, were labeled with rhodamine a-bungarotoxin (R-BT) continuously present in the medium. Since TIRF illuminates only cell-substrate contact regions where almost all of the AChR clusters are located, background fluorescence from fluorophores either in the bulk solution or inside the cells can be suppressed. Also, because TIRF minimizes the exposure of the cell interior to light, the healthy survival of the culture during imaging procedures is much enhanced relative to standard epi-(or trans-) illumination. During the experiment, cells were kept alive on the microscope stage at 37°C in an atmosphere of 10% C02. Two digital images were recorded by a CCD camera every 20 min: the schlieren image of the cells and the TIRF image of the clusters. After background subtraction, the cluster image was displayed in pseudocolors, overlaid onto the cell images, and recorded as 3 frames on a videotape. The final movies are thus able to summarize a week-long experiment in less than a minute. These movies and images show that clusters form often shortly after the myoblast fusion but sometimes much later, and the formation takes place very rapidly (a few hours). The clusters have an average lifetime of around a day, much shorter than the lifetime of a typical myotube. The brightest and largest clusters tend to be the longest-lived. The cluster formation seems to be associated with the contacts of myotubes at the glass substrate, but not with cell-cell contacts or myoblast fusion into myotubes. New AChR continuously appear in preexisting clusters: after photobleaching, the fluorescence of some clusters recovers within an hour. o 1994 WiIey-Liss, Inc.

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The submembrane machinery for nicotinic acetylcholine receptor clustering.

Cited by 166 publications

References 76 publications

Rapsyn carboxyl terminal domains mediate muscle specific kinase–induced phosphorylation of the muscle acetylcholine receptor

Rapsyn carboxyl terminal domains mediate muscle specific kinase–induced phosphorylation of the muscle acetylcholine receptor

Actin Filaments Modulate Both Stomatal Opening and Inward K+-Channel Activities in Guard Cells of Vicia faba L

Time‐lapse total internal reflection fluorescence video of acetylcholine receptor cluster formation on myotubes

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