The Connecdenn Family, Rab35 Guanine Nucleotide Exchange Factors Interfacing with the Clathrin Machinery

Department of Neurology and Neurosurgery, Montreal Neurological Institute, McGill University, Montreal, Quebec H3A 2B4, Canada.
Journal of Biological Chemistry (Impact Factor: 4.57). 02/2010; 285(14):10627-37. DOI: 10.1074/jbc.M109.050930
Source: PubMed


Rabs constitute the largest family of monomeric GTPases, yet for the majority of Rabs relatively little is known about their activation and recruitment to vesicle-trafficking pathways. We recently identified connecdenn (DENND1A), which contains an N-terminal DENN (differentially expressed in neoplastic versus normal cells) domain, a common and evolutionarily ancient protein module. Through its DENN domain, connecdenn functions enzymatically as a guanine-nucleotide exchange factor (GEF) for Rab35. Here we identify two additional connecdenn family members and demonstrate that all connecdenns function as Rab35 GEFs, albeit with different levels of activity. The DENN domain of connecdenn 1 and 2 binds Rab35, whereas connecdenn 3 does not, indicating that Rab35 binding and activation are separable functions. Through their highly divergent C termini, each of the connecdenns binds to clathrin and to the clathrin adaptor AP-2. Interestingly, all three connecdenns use different mechanisms to bind AP-2. Characterization of connecdenn 2 reveals binding to the beta2-ear of AP-2 on a site that overlaps with that used by the autosomal recessive hypercholesterolemia protein and betaarrestin, although the sequence used by connecdenn 2 is unique. Loss of connecdenn 2 function through small interference RNA knockdown results in an enlargement of early endosomes, similar to what is observed upon loss of Rab35 activity. Our studies reveal connecdenn DENN domains as generalized GEFs for Rab35 and identify a new AP-2-binding motif, demonstrating a complex link between the clathrin machinery and Rab35 activation.

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    • "A recent GWAS conducted in Han Chinese women identified single nucleotide polymorphisms (SNPs) at LHCGR, THADA, and DENND1A genes to be strongly associated with PCOS (Chen et al., 2011; Cui et al., 2013). DENND1A encodes the protein DENN/MADD, a member of the connecdenn family which functions as a component of clathrinmediated endocytosis machinery (Marat and McPherson, 2010), and Rab35 activated endocytotic trafficking, whereby it controls recycling of endosomal cargo (Marat and McPherson, 2010; Allaire et al., 2010). "
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    ABSTRACT: Recent genome-wide association studies and replication analyses reported an association between variants of DENND1A gene and polycystic ovary syndrome (PCOS), mostly in Asians. We therefore examined whether the common DENND1A SNPs rs10818854, rs2479106, and rs10986105 are associated with PCOS in Bahraini Arab population. This case-control study involved 191 women with PCOS diagnosed according to the Rotterdam criteria, and 202 control women. SNP genotyping was performed by the allelic discrimination in real-time PCR. The outcome was that the minor allele frequencies of SNPs rs10818854, rs2479106, and rs10986105 were similar between women with PCOS and control women (P>0.05), even before correcting for multiple testing, and none of the tested DENND1A SNPs were associated with PCOS under co-dominant, dominant, or recessive genetic models. None of the tested DENND1A variants were associated with PCOS features (hirsutism, insulin sensitivity, menses pattern, free testosterone, and free androgen index). Taking common GTA haplotype as reference (OR=1.00), [rs10818854/rs2479106/rs10986105] 3-locus haplotype analysis demonstrated lack of association of any of the DENND1A haplotypes with PCOS, even before correcting for multiple testing. To conclude we demonstrated lack of association of DENND1A SNPs rs10818854, rs2479106, and rs10986105, previously associated with PCOS in Asians, with PCOS in Bahraini Arab women. Copyright © 2015 Elsevier B.V. All rights reserved.
    Gene 01/2015; 560(1). DOI:10.1016/j.gene.2015.01.034 · 2.14 Impact Factor
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    • "rs2479106 is located in an intron of the gene DENND1A and encodes the protein DENND1A. This protein contains a conserved protein domain DENN, which is implicated in clathrin-mediated endocytosis [17] [18]. A recent study investigated the impact of rs13405728, rs13429458 and rs2479106 variants on PCOS susceptibility in Austrian, Caucasian PCOS patients diagnosed according to the Rotterdam criteria [19]. "
    Dataset: EJOGRB

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    • "The SCD1 protein contains a DENN domain (Falbel et al., 2003), which has been shown biochemically to have specific Rab GEF activity in all 17 members of the human DENN domain protein family (Yoshimura et al., 2010; Marat et al., 2011), defining the DENN domain as protein motif characteristic of vesicle trafficking regulators . The DENN domain–containing Rab GEFs connecdenn 1, 2, and 3 have also been shown to bind clathrin and the clathrin adaptor AP2 (Marat and McPherson, 2010), and connecdenn 3 has recently been shown to bind actin (Marat et al., 2012), linking activation of Rab35 with clathrin and cytoskeletal dynamics. Rab35 mediates late secretory pathway vesicle trafficking (i.e., PM to endosome and vice versa) (Patino-Lopez et al., 2008; Sato et al., 2008) and is necessary for the late stages of animal cytokinesis, which involves both anterograde and retrograde vesicle trafficking (Kouranti et al., 2006) reminiscent of the vesicular trafficking required for CP assembly in plant cells. "
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    ABSTRACT: STOMATAL CYTOKINESIS DEFECTIVE1 (SCD1) encodes a putative Rab guanine nucleotide exchange factor that functions in membrane trafficking and is required for cytokinesis and cell expansion in Arabidopsis thaliana. Here, we show that the loss of SCD2 function disrupts cytokinesis and cell expansion and impairs fertility, phenotypes similar to those observed for scd1 mutants. Genetic and biochemical analyses showed that SCD1 function is dependent upon SCD2 and that together these proteins are required for plasma membrane internalization. Further specifying the role of these proteins in membrane trafficking, SCD1 and SCD2 proteins were found to be associated with isolated clathrin-coated vesicles and to colocalize with clathrin light chain at putative sites of endocytosis at the plasma membrane. Together, these data suggest that SCD1 and SCD2 function in clathrin-mediated membrane transport, including plasma membrane endocytosis, required for cytokinesis and cell expansion.
    The Plant Cell 10/2013; 25(10). DOI:10.1105/tpc.113.115162 · 9.34 Impact Factor
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