The Arf6 GEF GEP100/BRAG2 Regulates Cell Adhesion by Controlling Endocytosis of β1 Integrins

Abstract

The small GTPase Arf6 has been shown to regulate the post-endocytic trafficking of a subset of membrane proteins, including beta1 integrins, and inhibition of Arf6 function impairs both cell adhesion and motility. The activity of Arf GTPases is regulated by a large family of guanine nucleotide exchange factors (GEFs). Arf-GEP100/BRAG2 is a GEF with reported specificity for Arf6 in vitro, but it is otherwise poorly characterized. Here we report that BRAG2 exists in two ubiquitously expressed isoforms, which we call BRAG2a and BRAG2b, both of which can activate Arf6 in vivo. Depletion of endogenous BRAG2 by siRNA leads to dramatic effects in the cell periphery; one such effect is an accumulation of beta1 integrin on the cell surface and a corresponding enhancement of cell attachment and spreading on fibronectin-coated substrates. In contrast, depletion of Arf6 leads to intracellular accumulation of beta1 integrin and reduced adhesion and spreading. These findings suggest that Arf6 regulates both endocytosis and recycling of beta1 integrins and that BRAG2 functions selectively to activate Arf6 during integrin internalization.

Brag2 differentially regulates β1- and β3-integrin-dependent adhesion in endothelial cells and is involved in developmental and pathological angiogenesis

“…In addition, silencing of Brag2 also slightly increased a3b1-and a6b1-integrin on the EC surface (data not shown). In line with our results, Dunphy et al [15] demonstrated that silencing of Brag2 in HeLa cells increased surface expression of b1-integrins and cell spreading. Moreover, another study suggested that Brag2 may promote b1-integrin endocytosis [33].…”

“…Furthermore, Brag2 was shown to be involved in trafficking processes such as the endocytosis of synaptic AMPA-receptors, E-Cadherin recycling, and phagocytosis of monocytes [21,46,47]. Silencing of Brag2 in HeLa cells increased the surface expression of b1-integrins [15] and a later study indicated that Brag2 might contribute to b1-integrin endocytosis in HeLa cells [33]. However, the role of Brag2 in the regulation of integrins in EC was not studied thus far.…”

The BRAG/IQSec family of Arf GEFs

“…Depletion of BRAG2 in Hela cells results in the accumulation of b1-integrin on the cell surface and a corresponding enhancement of attachment and spreading on fibronectin coated substrates. 18 This effect is selective for b1-integrin, as surface transferrin receptor levels were unaffected. Interestingly, Arf6-depleted cells do not accumulate b1-integrin on the cell surface and actually spread more slowly on fibronectin, in agreement with numerous reports that Arf6 regulates integrin recycling.…”

Regulation of actin cytoskeleton dynamics by Arf-family GTPases

“…The use of markers has revealed that these tails exist in live cells, but neither their function nor their regulation is currently understood. Another feature typical of Arf6 activation is the loss of actin stress fibers [27][28][29]. Again, the mechanisms through which this occurs are not well understood, but they are likely to involve crosstalk between Arf6 and Rho family GTPases.…”