On the mechanism of autoinhibition of the RhoA-specific nucleotide exchange factor PDZRhoGEF.
BACKGROUND
The Dbl-family of guanine nucleotide exchange factors (GEFs) activate the cytosolic GTPases of the Rho family by enhancing the rate of exchange of GTP for GDP on the cognate GTPase. This catalytic activity resides in the DH (Dbl-homology) domain, but typically GEFs are multidomain proteins containing other modules. It is believed that GEFs are autoinhibited in the cytosol due to supramodular architecture, and become activated in diverse signaling pathways through conformational change and exposure of the DH domain, as the protein is translocated to the membrane. A small family of RhoA-specific GEFs, containing the RGSL (regulators of G-protein signaling-like) domain, act as effectors of select GPCRs via Galpha12/13, although the molecular mechanism by which this pathway operates is not known. These GEFs include p115, LARG and PDZRhoGEF (PRG).


RESULTS
Here we show that the autoinhibition of PRG is caused largely by an interaction of a short negatively charged sequence motif, immediately upstream of the DH-domain and including residues Asp706, Glu708, Glu710 and Asp712, with a patch on the catalytic surface of the DH-domain including Arg867 and Arg868. In the absence of both PDZ and RGSL domains, the DH-PH tandem with additional 21 residues upstream, is 50% autoinhibited. However, within the full-length protein, the PDZ and/or RGSL domains significantly restore autoinhibition.


CONCLUSIONS
Our results suggest a mechanism for autoinhibition of RGSL family of GEFs, in which the RGSL domain and a unique sequence motif upstream of the DH domain, act cooperatively to reduce the ability of the DH domain to bind the nucleotide free RhoA. The activation mechanism is likely to involve two independent steps, i.e. displacement of the RGSL domain and conformational change involving the autoinhibitory sequence motif containing several negatively charged residues.
Zheng
Meiying
M
Department of Molecular Physiology and Biological Physics, University of Virginia, PO Box 800736, Charlottesville, Virginia, 22908-0736, USA. mz2k@Virginia.edu
Cierpicki
Tomasz
T
Momotani
Ko
K
Artamonov
Mykhaylo V
MV
Derewenda
Urszula
U
Bushweller
John H
JH
Somlyo
Avril V
AV
Derewenda
Zygmunt S
ZS
D000818:Animals; D020134:Catalytic Domain; D020662:Guanine Nucleotide Exchange Factors / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D006801:Humans; D051379:Mice; D008956:Models, Chemical; D009154:Mutation; D041681:NIH 3T3 Cells; D054731:PDZ Domains; D064067:Rho Guanine Nucleotide Exchange Factors
C577234:ARHGEF11 protein, human; D020662:Guanine Nucleotide Exchange Factors; D064067:Rho Guanine Nucleotide Exchange Factors