2004 Fiscal Year Final Research Report Summary
Intracellular and intercellular signaling mediated by Eph tyrosine kinase receptor in vascular endothelial cells
| Project/Area Number |
14380342
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Cell biology
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| Research Institution | National Cardiovascular Center Research Institute |
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Principal Investigator |
MOCHIZUKI Naoki National Cardiovascular Center Research Institute, Structural Analysis, Director, 循環器形態部, 部長 (30311426)
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| Co-Investigator(Kenkyū-buntansha) |
ONISHI Hirofumi National Cardiovascular Center Research Institute, Structural Analysis, Section chief, 循環器形態部, 室長 (80092542)
MASUDA Michitaka National Cardiovascular Center Research Institute, Structural Analysis, Section chief, 循環器形態部, 室長 (00190364)
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| Project Period (FY) |
2002 – 2004
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| Keywords | signal transduction / tyrosine kinase / receptor / cell-cell adhesion / angiogenesis |
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| Research Abstract |
In this research project, we have identified a Rho guanine nucleotide exchange factor(GEF)espetially expressed in vascular smooth muscle cells. We named this exchange factor Vsm-RhoGEF(Vascular smooth muscle specific GEF) after its expression and characterized the exchange specificity and localization. Vsm-RhoGEF functions downstream of EphA4 receptor and is expressed exclusively in the arterial smooth muscle cells (VSMCs), where EphA4 receptor is localized/. Furtheremore, Vsm-RhoGEF exhibits GEF activity for Rac as well as Rho. Consistently, when VSMCs were stimulated with ephrin-A1,they showed remarkable membrane ruffling, which is a hallmark of Rac activation. Vsm-RhoGEF is localized on actin stress fiber in unstimulated VSMCs, whereas it is dissociated from stress fiber to peripheral ruffled membrane upon ephrin-A1 simulation. Thus, while the Vsm-RhoGEF on stress fiber may function as a RhoGEF for Rho-RhoKinase signaling to regulate acttin-myosin coupling, the dissocicated Vsm-RhoG
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EF activates Rac for membrane extension.
We further investigated the Ras family GTPases, especially R-Ras family functioning downstream of Eph tyrosine kinase. R-Ras has been suggested to function for extracellular matrix-cell adhesion when simulated by ephrin. We hypothesized that actomyosin is regulated by R-Ras, because MysoinIX contains Ras binding domain in its ajnino-terminus of the myosin head. R-Ras family consists of R-Ras, M-Ras, and TC21. We found that among them R-Ras and TC21 preferentially bind to myosinIX. Other Ras family members, H-Ras, Rap1 and Ral do not bind to MysoinIX. Thus Eph-activated R-Ras and TC21 may promote myosinIX-based motor function such as vesicular frafficking or muscle contraction.
Eph tyrosine kinase receptor is endocytosed similarly to other tyrosine kinase receptor family members. We first explored the endocytosis of carboxy-terminally EGFP-tagged EphB receptors upon ephrin-B1 stimulation. PECAM-1-EGFP was used as a negative control. The cells expressing either EphB1-EGFP or PECAM-1-EGFP stimulated with ephrin-B1 were time-lapse imaged. We found that EphB1-EGFP exhibited the oligomerization upon stimulation while PECAM-1 EGFP did not. In addition to oligomerization, we found that oligomerized EphB1-EGFP was endocytosed into the cell body f from the plasma membrane. These results suggest that Eph-ephrin-mediated intracellular signaling is triggerd upon cell-cell contact and modulated by endocytosis. We will prceed to examine the molecular mechanism how Vsm-RhoGEF functions as GEF switch for Rho and/or Rac. Less
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Research Products
(28 results)
