Rab GEFs and GAPs: The Enigma Variations - Ras Superfamily Small G Proteins: Biology and Mechanisms 2 - page 87

a divergent group of DENN-related proteins with four subgroups FAM116A/B,
KIAA1147, AVL9 and FAM45A (Sato et al. 2008 ; Allaire et al. 2010 ; Yoshimura
et al. 2010 ; Linford et al. 2012 ). DENN domain proteins were initially implicated as
Rab GEFs when the protein MADD was purified as a Rab3 GEF from brain and
subsequently the C. elegans orthologue AEX-3 found to act as a regulator of Rab3
in vivo (Wada et al. 1997 ; Brown and Howe 1998 ; Iwasaki et al. 1997 ; Figueiredo
et al. 2008 ). Wider appreciation that DENN domain proteins were Rab GEFs came
once it was shown that the C. elegans receptor-mediated endocytosis defective
mutant rme-4 encoded a DENN domain protein homologous to mammalian
DENND1A and regulated the Rab35 family GTPase RME-5 (Sato et al. 2008 ).
Both RME-4 and DENND1A have specific Rab35 GEF activity (Sato et al. 2008 ;
Allaire et al. 2010 ; Yoshimura et al. 2010 ). A family wide study of the human
DENN domain proteins then defined the substrate specificity of this entire family of
Rab GEFs. This defines the following Rab-GEF pairs: Rab35-DENND1A/B,
Rab13-DENND1C, Rab2-DENND2A-D, Rab12-DENND3, Rab10-DENND4A/B,
Rab39-DENND5A/B, Rab28-MTMR5/13 and Rab3/27A/B-MADD, respectively
(Yoshimura et al. 2010 ). The best evidence confirming these biochemical assign-
ments has been obtained for Rab3-MADD, Rab35-DENND1 and Rab10-DENND4.
As already mentioned Rab35 and DENND1 are required for endocytic uptake in
C. elegans and other work in human cells suggests they act at an early stage in the
endocytic trafficking pathway and are needed for transport from the cell surface to
the Golgi (Sato et al. 2008 ; Allaire et al. 2006 , 2010 ; Yoshimura et al. 2010 ).
Studies in Drosophila melanogaster provide compelling evidence that Rab10 and
the DENND4 orthologue CRAG are required for polarised traffic of collagen and
other cargo to the basolateral surface in epithelial cells (Denef et al. 2008 ; Lerner
et al. 2013 ). These defects are reminiscent of those seen when Rab10 is inactivated
in human cells (Denef et al. 2008 ; Schuck et al. 2007 ). Less is known about the
function of the DENN-related GEFs and a defined Rab target and function has been
found for only one subfamily member. FAM116A/B have been shown to be specific
GEFs for Rab14, and to act in endocytic recycling by activation of Rab14. This
pathway is important for the recycling of cell surface ADAM proteases and the
control of cadherin family cell-cell adherens junctions in migrating cells (Linford
et al. 2012 ).
A high-resolution X-ray crystal structure has been obtained for only one DENN
domain protein, human DENND1B with Rab35 (Wu et al. 2011 ). This structure was
a major advance, since it provided crucial new information about general themes in
Rab GEF architecture and mechanism. Despite sharing no sequence similarity this
structure revealed that the DENND1 has a longin fold similar to the Bet3 and Bet5
subunits of TRAPP, and a unique C-terminal domain (Levine et al. 2013 ; Cai
et al. 2008 ; Wu et al. 2011 ). Both these domains make contacts with the Rab35
TIGID RabF1 motif, and the conserved tryptophan and polypeptide backbone of the
WDTAGQE switch II sequence. The specificity of DENND1 for the GDP form of
Rab35 may be explained by the availability of the TIGID RabF1 motif, since the
threonine residue in this motif makes interactions with the magnesium ion in the
GTP form of Rabs. Unlike TRAPP and Vps9 there are no insertions from the GEF