Adaptor Protein Crk Is Required for Ephrin-B1-induced Membrane Ruffling and Focal Complex Assembly of Human Aortic Endothelial Cells

Endothelial cell migration is an essential step in vasculogenesis and angiogenesis, in which receptor tyrosine kinases play a pivotal role. We investigated the mechanism by which ephrin-B1 promotes membrane ruffling in human aortic endothelial cells, because membrane ruffling heralds cell body migration. We especially focused on the role of Crk adaptor protein in EphB-mediated signaling. Using DsRed-tagged Crk and a fluorescent time-lapse microscope, we showed that Crk was recruited to the nascent focal complex after ephrin-B1 stimulation. Furthermore, we found that p130Cas, but not paxillin, recruited Crk to the nascent focal complex. The necessity of Crk in ephrin-B1–induced membrane ruffling was shown both by the overexpression of dominant negative Crk mutants and by the depletion of Crk by using RNA interference. Then, we examined the role of two major downstream molecules of Crk, Rac1 and Rap1. The dominant negative mutant of Rac1 completely inhibited ephrin-B1–induced membrane ruffling and focal complex assembly. In contrast, rap1GAPII, a negative regulator of Rap1, did not inhibit ephrin-B1–induced membrane ruffling. However, in rap1GAPII-expressing cells, ephrin-B1 did not induce membrane spreading, probably due to instability of the focal complex. These results indicated that Crk plays a critical role in Rac1-induced membrane ruffling and Rap1-mediated nascent focal complex stabilization contributing to ephrin-B1–induced human aortic endothelial cells migration.

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Functional expression of Kir2.x in human aortic endothelial cells: the dominant role of Kir2.2

AJP Cell Physiology ◽

10.1152/ajpcell.00077.2005 ◽

2005 ◽

Vol 289 (5) ◽

pp. C1134-C1144 ◽

Cited By ~ 47

Author(s):

Yun Fang ◽

Gernot Schram ◽

Victor G. Romanenko ◽

Congzhu Shi ◽

Lisa Conti ◽

...

Keyword(s):

Endothelial Cells ◽

Dominant Role ◽

Functional Expression ◽

Inward Rectifier ◽

Dominant Negative ◽

Current Sensitivity ◽

Aortic Endothelial Cells ◽

Human Aortic Endothelial Cells ◽

Aortic Endothelial

Inward rectifier K+ channels (Kir) are a significant determinant of endothelial cell (EC) membrane potential, which plays an important role in endothelium-dependent vasodilatation. In the present study, several complementary strategies were applied to determine the Kir2 subunit composition of human aortic endothelial cells (HAECs). Expression levels of Kir2.1, Kir2.2, and Kir2.4 mRNA were similar, whereas Kir2.3 mRNA expression was significantly weaker. Western blot analysis showed clear Kir2.1 and Kir2.2 protein expression, but Kir2.3 protein was undetectable. Functional analysis of endothelial inward rectifier K+ current ( IK) demonstrated that 1) IK current sensitivity to Ba2+ and pH were consistent with currents determined using Kir2.1 and Kir2.2 but not Kir2.3 and Kir2.4, and 2) unitary conductance distributions showed two prominent peaks corresponding to known unitary conductances of Kir2.1 and Kir2.2 channels with a ratio of ∼4:6. When HAECs were transfected with dominant-negative (dn)Kir2.x mutants, endogenous current was reduced ∼50% by dnKir2.1 and ∼85% by dnKir2.2, whereas no significant effect was observed with dnKir2.3 or dnKir2.4. These studies suggest that Kir2.2 and Kir2.1 are primary determinants of endogenous K+ conductance in HAECs under resting conditions and that Kir2.2 provides the dominant conductance in these cells.

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