Kinetic studies of the Arf activator Arno on model membranes in the presence of Arf effectors suggest control by a positive feedback loop.
Proteins of the cytohesin/Arno/Grp1 family of Arf activators are positive regulators of the insulin-signaling pathway and control various remodeling events at the plasma membrane. Arno has a catalytic Sec7 domain, which promotes GDP to GTP exchange on Arf, followed by a pleckstrin homology (PH) domain. Previous studies have revealed two functions of the PH domain: inhibition of the Sec7 domain and membrane targeting. Interestingly, the Arno PH domain interacts not only with a phosphoinositide (phosphatidylinositol 4,5-bisphosphate or phosphatidylinositol 3,4,5-trisphosphate) but also with an activating Arf family member, such as Arf6 or Arl4. Using the full-length membrane-bound forms of Arf1 and Arf6 instead of soluble forms, we show here that the membrane environment dramatically affects the mechanism of Arno activation. First, Arf6-GTP stimulates Arno at nanomolar concentrations on liposomes compared with micromolar concentrations in solution. Second, mutations in the PH domain that abolish interaction with Arf6-GTP render Arno completely inactive when exchange reactions are reconstituted on liposomes but have no effect on Arno activity in solution. Third, Arno is activated by its own product Arf1-GTP in addition to a distinct activating Arf isoform. Consequently, Arno activity is strongly modulated by competition with Arf effectors. These results show that Arno behaves as a bistable switch, having an absolute requirement for activation by an Arf protein but, once triggered, becoming highly active through the positive feedback effect of Arf1-GTP. This property of Arno might provide an explanation for its function in signaling pathways that, once triggered, must move forward decisively.
Institut de Pharmacologie Moléculaire et Cellulaire, Université de Nice Sophia Antipolis et CNRS, 06560 Valbonne, France.
Danièle
D
Stalder
Hélène
H
Barelli
Romain
R
Gautier
Eric
E
Macia
Catherine L
CL
Jackson
Bruno
B
Antonny
D020823:ADP-Ribosylation Factor 1 / Q000378:metabolism*; D000089982:ADP-Ribosylation Factor 6; D020727:ADP-Ribosylation Factors / Q000378:metabolism; D002460:Cell Line; D025461:Feedback, Physiological; D020690:GTPase-Activating Proteins / Q000378:metabolism*; D020662:Guanine Nucleotide Exchange Factors; D006160:Guanosine Triphosphate; D006801:Humans; D007700:Kinetics; D008081:Liposomes; D017434:Protein Structure, Tertiary; D055213:Retinal Pigment Epithelium / Q000166:cytology; D014162:Transfection
D000089982:ADP-Ribosylation Factor 6; D020690:GTPase-Activating Proteins; D020662:Guanine Nucleotide Exchange Factors; D008081:Liposomes; C102474:cytohesin-2; D006160:Guanosine Triphosphate; D020823:ADP-Ribosylation Factor 1; D020727:ADP-Ribosylation Factors; C000715348:ARF6 protein, human