Role of the Guanine Nucleotide Exchange Factor Ost in Negative Regulation of Receptor Endocytosis by the Small GTPase Rac1

Abstract

The Rho family of GTPases has been implicated in the regulation of intracellular vesicle trafficking. Here, we investigated the mechanism underlying the negative regulation of clathrinmediated endocytosis of cell surface receptors mediated by the Rho family protein Rac1. Contrary to previous reports, only the activated mutant of Rac1, but not other Rho family members including RhoA and Cdc42, suppressed internalization of the transferrin receptor. On the other hand, down-regulation of Rac1 expression by RNA interference resulted in enhanced receptor internalization, suggesting that endogenous Rac1 in fact functions as a negative regulator. We identified a guanine nucleotide exchange factor splice variant designated Ost-III, which contains a unique C-terminal region including an Src homology 3 domain, as a regulator of Rac1 involved in the inhibition of receptor endocytosis. In contrast, other splice variants Ost-I and Ost-II exerted virtually no effect on receptor endocytosis. We also examined subcellular localization of synaptojanin 2, a putative Rac1 effector implicated in negative regulation of receptor endocytosis. Each Ost splice variant induced distinct subcellular localization of synaptojanin 2, depending on Rac1 activation. Furthermore, we isolated ␥-aminobutyric acid type A receptor-associated protein (GABARAP) as a protein that binds to the C-terminal region of Ost-III. When ectopically expressed, GABARAP was co-localized with Ost-III and potently suppressed the Ost-III-dependent Rac1 activation and the inhibition of receptor endocytosis. Lipid modification of GABARAP was necessary for the suppression of Ost-III. These results are discussed in terms of subcellular region-specific regulation of the Rac1-dependent signaling pathway that negatively regulates clathrin-mediated endocytosis.In eukaryotic cells the continuous uptake of essential nutrients such as iron-laden transferrin and the cholesterol-laden low density lipoprotein is carried out through clathrin-mediated endocytosis of their respective receptors. Additionally, clathrin-mediated endocytosis is the main pathway for internalization of various growth factor receptors. It mediates rapid clearance and down-regulation of activated receptors, thereby regulating the levels of cell surface receptors. The receptorligand complex is internalized into a clathrin-coated pit that is formed through the action of the adaptor protein complex AP-2 and a variety of accessory proteins, including AP180, Eps15, amphiphysin, and epsin. Subsequently, clathrin-coated pits pinch off to form clathrin-coated vesicles that are encapsulated by a polygonal clathrin coat and carry concentrated receptor-ligand complexes into the cell. Clathrin-coated vesicle fission is driven by the GTPase dynamin. The coat of clathrin-coated vesicles is removed shortly after the vesicle forms, generating an early endosome. This endosome fuses with a late endosome, where the low pH causes the dissociation of the receptor-ligand complex. A receptor-rich region buds off to form a separate ve...

Rho GTPases, phosphoinositides, and actin

“…This study again highlighted the need for Rac1, but also for RhoD in both of these endocytic pathways. In contrast to the previous cited study, 76 knocking down Rac1 was reported to not increase Tf uptake but, like RhoD, to increase clustering of Tf-positive endosomes close to the nucleus suggesting that these GTPases interfere with vesicle displacement and intervene in endosome maturation process. 87 Interestingly, this assay confirmed that different subsets of adaptor proteins are required for CME of EGF and Tf.…”

“…24 With the identification of PIP5K and synaptojanin-2 (Synj2) as potential binding partners for RhoA and Rac1, 68,73,75 it was proposed that Rho GTPases control vesicle progression through the endocytic pathway by imbalancing PI(4,5)P 2 production. Rac1-dependent recruitment of Synj2 at the plasma membrane was indeed sufficient to diminish CME of Tf by increasing PI(4,5)P 2 hydrolysis 73 and knocking down Rac1 by siRNA increased Tf uptake, 76 indicating that Rac1 may negatively regulate CME of Tf by promoting PI(4,5)P 2 hydrolysis. However, despite the fact that RhoA and Rac1 stimulate PI(4,5)P 2 production through PIP5K activation 68,75 and that sustained production of PI(4,5)P 2 by overexpression of PIP5K is sufficient to increase Tf uptake, 68,75,77 RhoA and Rac1 activation blocked, whereas RhoA and Rac1 inhibition increase CME of Tf.…”

Roles of Small GTPase Rac1 in the Regulation of Actin Cytoskeleton during Dengue Virus Infection

“…In MDCK cells, Rac1V12 expression decreased the rates of apical and basolateral endocytosis [35]. In Hela cells, overexpression of constitutively active forms of Rac1 causes inhibition of transferrin and EGF receptor internalization [18], [26]. One mechanism by which Rac1 might participate in clathrin-mediated endocytosis is the modulation of phosphatidylinositol (PI) lipid metabolism, which plays a key role in this process [36].…”

“…Rac1 has been shown to interact with a number of enzymes that regulate PI metabolism, including type I PI 3 -kinase and synaptojanin 2 [37], [38]. Recently, a guanine nucleotide exchange factor splice variant designated Ost-III is identified as a regulator of Rac1 involved in the inhibition of receptor endocytosis [26].…”

“…A novel affinity binding assay has been developed for in situ detection of active form of small GTPases in mammalian cultured cells [26]. Briefly, bound GST-CRIB was eluted off the beads with elution buffer (50 mM Tris-HCl, 10 mM reduced glutathione, pH 8.0) and stored at −80°C before use.…”