Structural basis and mechanism of autoregulation in 3-phosphoinositide-dependent Grp1 family Arf GTPase exchange factors.
Arf GTPases regulate membrane trafficking and actin dynamics. Grp1, ARNO, and Cytohesin-1 comprise a family of phosphoinositide-dependent Arf GTPase exchange factors with a Sec7-pleckstrin homology (PH) domain tandem. Here, we report that the exchange activity of the Sec7 domain is potently autoinhibited by conserved elements proximal to the PH domain. The crystal structure of the Grp1 Sec7-PH tandem reveals a pseudosubstrate mechanism of autoinhibition in which the linker region between domains and a C-terminal amphipathic helix physically block the docking sites for the switch regions of Arf GTPases. Mutations within either element result in partial or complete activation. Critical determinants of autoinhibition also contribute to insulin-stimulated plasma membrane recruitment. Autoinhibition can be largely reversed by binding of active Arf6 to Grp1 and by phosphorylation of tandem PKC sites in Cytohesin-1. These observations suggest that Grp1 family GEFs are autoregulated by mechanisms that depend on plasma membrane recruitment for activation.
Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, MA 01605, USA.
Jonathan P
JP
DiNitto
Anna
A
Delprato
Meng-Tse
MT
Gabe Lee
Thomas C
TC
Cronin
Shaohui
S
Huang
Adilson
A
Guilherme
Michael P
MP
Czech
David G
DG
Lambright
D041721:3T3-L1 Cells; D020727:ADP-Ribosylation Factors / Q000378:metabolism*; D017667:Adipocytes / Q000166:cytology / Q000201:enzymology; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000201:enzymology; D018360:Crystallography, X-Ray; D004252:DNA Mutational Analysis; D004789:Enzyme Activation; D020662:Guanine Nucleotide Exchange Factors / Q000378:metabolism*; D051379:Mice; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010716:Phosphatidylinositols / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D018160:Receptors, Cytoplasmic and Nuclear / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
D020662:Guanine Nucleotide Exchange Factors; D010716:Phosphatidylinositols; D018160:Receptors, Cytoplasmic and Nuclear; C040985:phosphatidylinositol receptors; C483312:phosphoinositide 3-phosphate; D020727:ADP-Ribosylation Factors