The connecdenn family, Rab35 guanine nucleotide exchange factors interfacing with the clathrin machinery.
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.
Department of Neurology and Neurosurgery, Montreal Neurological Institute, McGill University, Montreal, Quebec H3A 2B4, Canada.
Andrea L
AL
Marat
Peter S
PS
McPherson
D033962:Adaptor Protein Complex 2 / Q000378:metabolism*; D000595:Amino Acid Sequence; D015153:Blotting, Western; D002966:Clathrin / Q000378:metabolism*; D022163:Clathrin-Coated Vesicles / Q000378:metabolism*; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D020662:Guanine Nucleotide Exchange Factors / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D008969:Molecular Sequence Data; D034741:RNA, Small Interfering / Q000494:pharmacology; D017386:Sequence Homology, Amino Acid; D020691:rab GTP-Binding Proteins / Q000378:metabolism*
D033962:Adaptor Protein Complex 2; D002966:Clathrin; D020662:Guanine Nucleotide Exchange Factors; D034741:RNA, Small Interfering; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; C511183:RAB35 protein, human; D020691:rab GTP-Binding Proteins