EFA6 controls Arf1 and Arf6 activation through a negative feedback loop

Guanine nucleotide exchange factors (GEFs) of the exchange factor for Arf6 (EFA6), brefeldin A-resistant Arf guanine nucleotide exchange factor (BRAG), and cytohesin subfamilies activate small GTPases of the Arf family in endocytic events. These ArfGEFs carry a pleckstrin homology (PH) domain in tandem with their catalytic Sec7 domain, which is autoinhibitory and supports a positive feedback loop in cytohesins but not in BRAGs, and has an as-yet unknown role in EFA6 regulation. In this study, we analyzed how EFA6A is regulated by its PH and C terminus (Ct) domains by reconstituting its GDP/GTP exchange activity on membranes. We found that EFA6 has a previously unappreciated high efficiency toward Arf1 on membranes and that, similar to BRAGs, its PH domain is not autoinhibitory and strongly potentiates nucleotide exchange on anionic liposomes. However, in striking contrast to both cytohesins and BRAGs, EFA6 is regulated by a negative feedback loop, which is mediated by an allosteric interaction of Arf6-GTP with the PH-Ct domain of EFA6 and monitors the activation of Arf1 and Arf6 differentially. These observations reveal that EFA6, BRAG, and cytohesins have unanticipated commonalities associated with divergent regulatory regimes. An important implication is that EFA6 and cytohesins may combine in a mixed negative-positive feedback loop. By allowing EFA6 to sustain a pool of dormant Arf6-GTP, such a circuit would fulfill the absolute requirement of cytohesins for activation by Arf-GTP before amplification of their GEF activity by their positive feedback loop.Guanine nucleotide exchange factors (GEFs), which activate small GTPases by stimulating their intrinsically very slow GDP/GTP exchange, are key players in the extraordinary diversity of small GTPases pathways (reviewed in ref. 1). Small GTPases carry little specificity determinants on their own to determine when and where they should be turned on and which pathway they should activate (2), which are instead largely monitored by their GEFs. Thus, understanding how different members of a GEF family activate an individual small GTPase in distinct patterns is a major issue in small GTPase biology in normal cells and in diseases.An important contribution to the functional specificity of GEFs is how they themselves are regulated. Crystallographic studies combined with biochemical studies that reconstituted GEF-stimulated GDP/GTP nucleotide exchange have been instrumental in uncovering a growing complexity of regulatory mechanisms (reviewed in ref. 1). These include autoinhibitory elements outside the catalytic GEF domain that block access to the active site (3–7), large conformational changes that release autoinhibition in response to various stimuli (8–11), positive feedback loops in which freshly produced GTP-bound GTPases stimulate GDP/GTP exchange (10, 12–15), and potentiation of nucleotide exchange by colocalization on membranes (11, 13, 16, 17).These previous studies demonstrated that a wide range of regulatory regimes can be achieved even at the scale of a single GEF family by regulatory mechanisms that combine in multiple ways. GEFs that activate small GTPases of the Arf family (ArfGEFs), which are major regulators of many aspects of membrane traffic and organelle structure in eukaryotic cells (reviewed in refs. 18 and 19), form one of the best-characterized GEF families to date (reviewed in ref. 1), making a comprehensive view of their regulatory repertoire within reach. ArfGEFs comprise two major groups: the BIG/GBF1 group, which functions at the Golgi, and a group composed of the exchange factor for Arf6 (EFA6), brefeldin A-resistant Arf guanine nucleotide exchange factor (BRAG), and cytohesin subfamilies, which activate Arf GTPases at the cell periphery and function in various aspects of endocytosis (reviewed in ref. 20). The actual substrates of these ArfGEFs have been difficult to establish, notably because the most abundant Arf isoform, Arf1, was long believed to be excluded from the plasma membrane where the Arf6 isoform is located. Accordingly, cytohesins and BRAGs have been described as Arf6-specific GEFs in cells but are now recognized as active Arf1-GEFs (16, 21, 22), whereas EFA6 remains the sole ArfGEF considered to be strictly Arf6-specific (23, 24).Members of the EFA6, BRAG, and cytohesin subfamilies have divergent N-terminal domains but a related domain organization in their C terminus comprising a Sec7 domain, which stimulates GDP/GTP exchange, followed by a pleckstrin homology (PH) domain, which has multiple regulatory functions. In cytohesins, the PH domain recognizes signaling phosphoinositides by its canonical lipid-binding site (25), autoinhibits the Sec7 domain by obstructing its Arf-binding site (4), and amplifies nucleotide exchange by a positive feedback loop involving its direct interaction with Arf1-GTP or Arf6-GTP (10, 13, 21). In contrast, the PH domain of BRAG is not autoinhibitory and is not involved in a feedback loop, but instead strongly potentiates nucleotide exchange by binding to polyanionic membranes without marked phosphoinositides preference (16).How members of the EFA6 subfamily are regulated is currently unknown. These ArfGEFs are found predominantly (although not exclusively) in the brain and function in the coordination of endocytosis and actin dynamics (23, 26, 27), in the maintenance of tight junctions (28), in microtubule dynamics in Caenorhabditis elegans embryos (29), and in the formation and maintenance of dendrites (30), although the molecular details of these functions remain largely unknown. Consistent with an important role in the brain, defects in EFA6 functions have been found in neurologic disorders (31) and in human gliomas (32). The PH domain of EFA6 subfamily members drives the localization of EFA6 members to the plasma membrane (26) and it binds to PIP₂ lipids (33). It is followed by a 150-residue C-terminal (Ct) domain predicted to form a coiled coil, which massively induces actin-rich membrane protrusions when expressed with the PH domain (26). The divergence of regulatory mechanisms between Sec7-PH–containing cytohesins and BRAGs prompted us to undertake a quantitative biochemical investigation of EFA6 nucleotide exchange regulation. Our findings reveal an overlooked dual specificity of EFA6 for Arf1 and Arf6 and an unprecedented regulation by a negative feedback loop, with important potential implications for the activation of Arf GTPases in endocytic events.