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1تقرير
المؤلفون: Rotin, D., Barsagi, D., Obrodovich, H., Merilainen, J., Lehto, V. P., Canessa, C. M., Rossier, B. C., Downey, G. P.
مصطلحات موضوعية: ion channel
الوصف: The amiloride-sensitive Na+ channel constitutes the rate-limiting step for Na+ transport in epithelia. Immunolocalization and electrophysiological studies have demonstrated that this channel is localized at the apical membrane of polarized epithelial cells. This localization is essential for proper channel function in Na+ transporting epithelia. In addition, the channel has been shown to associate with the cytoskeletal proteins ankyrin and alpha-spectrin in renal epithelia. However, the molecular mechanisms underlying the cytoskeletal interactions and apical membrane localization of this channel are largely unknown. In this study we show that the putative pore forming subunit of the rat epithelial (amiloride-sensitive) Na+ channel (alpha rENaC) binds to alpha-spectrin in vivo, as determined by co-immunoprecipitation. This binding is mediated by the SH3 domain of alpha-spectrin which binds to a unique proline-rich sequence within the C-terminal region of alpha rENaC. Accordingly, the C-terminal region is sufficient to mediate binding to intact alpha-spectrin from alveolar epithelial cell lysate. When microinjected into the cytoplasm of polarized primary rat alveolar epithelial cells, a recombinant fusion protein containing the C-terminal proline-rich region of alpha rENaC localized exclusively to the apical area of the plasma membrane, as determined by confocal microscopy. This localization paralleled that of alpha-spectrin. In contrast, microinjected fusion protein containing the N-terminal (control) protein of alpha rENaC remained diffuse within the cytoplasm. These results suggest that an SH3 binding region in alpha rENaC mediates the apical localization of the Na+ channel. Thus, cytoskeletal interactions via SH3 domains may provide a novel mechanism for retaining proteins in specific membranes of polarized epithelial cells.
العلاقة: Rotin, D., Barsagi, D., Obrodovich, H., Merilainen, J., Lehto, V. P., Canessa, C. M., Rossier, B. C., Downey, G. P. (October 1994) An Sh3 Binding Region in the Epithelial Na+ Channel (Alpha-Renac) Mediates Its Localization at the Apical Membrane. Embo Journal, 13 (19). pp. 4440-4450. ISSN 0261-4189
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2تقرير
المؤلفون: Bar-Sagi, D., Rotin, D., Batzer, A., Mandiyan, V., Schlessinger, J.
مصطلحات موضوعية: phospholipase C, signal transduction
الوصف: In this study we describe the cellular distribution of the SH2 and SH3 domains of phospholipase C-gamma (PLC-gamma) and of the adaptor protein GRB2 following their microinjection into living rat embryo fibroblasts. Using immunofluorescence microscopy, we show that a truncated protein composed of the SH2 and SH3 domains of PLC-gamma was localized to the actin cytoskeleton. A similar localization pattern was observed when only the SH3 domain of PLC-gamma was microinjected. In contrast, a truncated protein composed of only the SH2 domains of PLC-gamma exhibited diffuse cytoplasmic distribution. Microinjected GRB2 protein was localized primarily to membrane ruffles, as was GRB2 protein containing SH2 loss-of-function point mutations. Hence, the localization of GRB2 to membrane ruffles does not require interaction with tyrosine-phosphorylated moieties. However, GRB2 proteins with SH3 loss-of-function point mutations exhibited diffuse cytoplasmic distribution. These results indicate that SH3 domains are responsible for the targeting of signaling molecules to specific subcellular locations.
العلاقة: Bar-Sagi, D., Rotin, D., Batzer, A., Mandiyan, V., Schlessinger, J. (July 1993) SH3 domains direct cellular localization of signaling molecules. Cell, 74 (1). pp. 83-91. ISSN 0092-8674 (Print)
الإتاحة: https://doi.org/10.1016/0092-8674Test(93)90296-3
http://repository.cshl.edu/id/eprint/32490Test/
http://www.ncbi.nlm.nih.gov/pubmed/8334708Test
