Connecdenn, A Novel DENN Domain-Containing Protein of Neuronal Clathrin-Coated Vesicles Functioning in Synaptic Vesicle Endocytosis

Abstract

Clathrin-coated vesicles (CCVs) are responsible for the endocytosis of multiple cargo, including synaptic vesicle membranes. We now describe a new CCV protein, termed connecdenn, that contains an N-terminal DENN (differentially expressed in neoplastic versus normal cells) domain, a poorly characterized protein module found in multiple proteins of unrelated function and a C-terminal peptide motif domain harboring three distinct motifs for binding the ␣-ear of the clathrin adaptor protein 2 (AP-2). Connecdenn coimmunoprecipitates and partially colocalizes with AP-2, and nuclear magnetic resonance and peptide competition studies reveal that all three ␣-earbinding motifs contribute to AP-2 interactions. In addition, connecdenn contains multiple Src homology 3 (SH3) domain-binding motifs and coimmunoprecipitates with the synaptic SH3 domain proteins intersectin and endophilin A1. Interestingly, connecdenn is enriched on neuronal CCVs and is present in the presynaptic compartment of neurons. Moreover, connecdenn has a uniquely stable association with CCV membranes because it resists extraction with Tris and high-salt buffers, unlike most other CCV proteins, but it is not detected on purified synaptic vesicles. Together, these observations suggest that connecdenn functions on the endocytic limb of the synaptic vesicle cycle. Accordingly, disruption of connecdenn interactions with its binding partners through overexpression of the C-terminal peptide motif domain or knock down of connecdenn through lentiviral delivery of small hairpin RNA both lead to defects in synaptic vesicle endocytosis in cultured hippocampal neurons. Thus, we identified connecdenn as a component of the endocytic machinery functioning in synaptic vesicle endocytosis, providing the first evidence of a role for a DENN domain-containing protein in endocytosis.

Overexpression of a DENND1A isoform produces a polycystic ovary syndrome theca phenotype

“…Therefore, the truncated form of DENND1A.V2 could potentially affect insulin or luteinizing hormone (LH)-receptor turnover and sensitivity in theca and granulosa cells, further affecting ovarian function and steroid biosynthesis in PCOS women. DENND1A has also been shown to be associated with lipids, particularly phosphoinsitol-3-phosphate, and other endocytosis/endosome proteins (31,34), and could potentially be involved in insulin and LH-receptor signaling. Alternatively, DENND1A.V2 may have a more direct role in controlling gene expression.…”

“…The DENN domains function as Rab-specific guanine nucleotide-exchange factors (31,32). The DENND1A gene consists of 22 exons extending over 500,000 bases, and encodes protein connecdenn 1, which has a clathrinbinding domain and is thought to facilitate endocytosis and receptor-mediated turnover (31,33,34). Connecdenn 1 is a guanine nucleotide-exchange factor that interacts with members of the Rab family of small GTPases, which are involved in membrane trafficking (31).…”

Interplay between Rab35 and Arf6 controls cargo recycling to coordinate cell adhesion and recycling

“…Rabbit polyclonal antibodies recognizing GFP (A6455) and pY397-FAK (44624G) were from Invitrogen (Carlsbad, CA), c-myc (C-3959) and pan-cadherin (C3678) were from Sigma, Arf6 (Ab77581) and b1-integrin (Ab52971) were from Abcam (Cambridge, MA), EGF receptor (1005) from Santa Cruz, and Akt (9272), pT308-Akt (9275), pY1068-EGF receptor (D7A5), and pY1148-EGF were from Cell Signaling. Polyclonal antibodies against CLCs, connecdenn 1 (3776), and Rab35 have been described previously (Allaire et al, 2006;Poupon et al, 2008;Allaire et al, 2010). Alexa-Fluor-647-EGF and phalloidin-TRITC were from Invitrogen.…”

Scyl1, Mutated in a Recessive Form of Spinocerebellar Neurodegeneration, Regulates COPI-mediated Retrograde Traffic

“…Thus, Scyl1 appears to have higher affinity than p24␣2 for COPI coats. To examine whether the two motifs bind to the same or different sites on COPI coats, we performed peptide competition experiments in which GSTScyl1 was incubated with brain extracts in the presence of increasing amounts of a peptide encoding the 16 C-terminal most residues of Scyl1 including RKLD-COO Ϫ , the 15 C-terminal most residues of p24␣2 including KKLV-COO Ϫ , or an epsin peptide encoding DPW motifs and previously shown to compete binding of DPW proteins to the ␣-ear of AP-2 (29). Binding of COPI coats was monitored by ␤COP Western blot.…”