A rat homologue of CED-6 is expressed in neurons and interacts with clathrin

Abstract

We isolated from a brain library a cDNA encoding an isoform of rat CED-6 that has not been previously described. This transcript results from alternative splicing of the ced-6 gene present on chromosome 9. We expressed this isoform as his-tagged protein in E. coli and used the purified protein to raise antibodies to investigate the expression of CED-6 in rat brain. Immunoblot analysis showed the presence of CED-6 as a doublet of approximately 34 and 33 kDa in cortex, hippocampus and cerebellum, indicating that the protein was present in different regions of the brain. Subcellular fractionation experiments showed that CED-6 immunoreactivity did not concentrate in GFAP-containing glial vesicles, whereas it showed a distribution similar to the synaptotagmin in synaptosomes-enriched fractions, suggesting that CED-6 is present in neurons. CED-6 immunoreactivity was also investigated using immunohistochemistry analysis and it was found in several brain regions, being particularly strong in the cell body of some groups of neurons such as Purkinje cell layer of cerebellum, and pyramidal cells of the hippocampal formation and also in epithelial cells from the choroid plexus. Importantly, CED-6 immunoreactivity colocalized with a neuronal marker but not with a glial marker. Considering that several PTB-containing proteins bind clathrin, we investigated whether rat CED-6 would also have this property. Yeast two-hybrid and GST pull-down analysis indicated that ratCED-6 interacts with clathrin and in cultured cells we detected colocalization between CED-6 and clathrin-coated vesicles. The present findings suggest that CED-6 may have a role in endocytic trafficking or signaling in neurons.

Introduction

In multicellular organisms cells that undergo programmed cell death are rapidly recognized and removed by phagocytes prior to their lysis. Clearance of cellular corpses prevents leakage of proteases and other toxic intracellular contents that could damage neighboring tissues and helps to avoid an inflammatory response (Henson et al., 2001).

In C. elegans, two partially redundant signaling pathways function in the engulfment of apoptotic cells. One pathway involves proteins CED-2, CED-5 and CED-12 (mammalian homologues CrkII, Dock180 and ELMO, respectively). Based on studies with the mammalian homologues, it has been suggested that CED-2, CED-5 and CED-12 promote activation of CED-10 (mammalian homologue Rac), a GTPase that is involved in the actin cytoskeleton rearrangement required during cell migration and phagocytosis (Gumienny et al., 2001).

The other pathway involves the proteins CED-1, CED-6 and CED-7 (mammalian homologues of CD91/LRP/SREC; hCED-6/GULP; ABC1, respectively). CED-1 is a transmembrane receptor that is expressed at the plasma membrane of engulfing cells that seems to bind to cell corpses and initiate intracellular signaling for uptake (Zhou et al., 2001). The most probable mammalian homologue of CED-1 is the low-density lipoprotein receptor-related protein CD91/LRP, a receptor involved in the phagocytosis of apoptotic cells in mammals (Ogden et al., 2001, Su et al., 2002). Binding of CED-1 to an unknown ligand on the apoptotic cell requires the function of CED-7, which is thought to play a role in membrane dynamics. Intracellular signaling depends on the presence of two motifs, NPXY and YXXL, present at the cytoplasmic tail of CED-1 (Zhou et al., 2001).

In C. elegans, CED-6 is a signaling adapter protein that functions within engulfing cells downstream of CED-1 (Kinchen et al., 2005, Su et al., 2002). The protein contains an N-terminal phosphotyrosine-binding domain (PTB), a central leucine-zipper and a proline-rich C-terminal region (Liu and Hengartner, 1998, Su et al., 2002). The Drosophila, human and mouse homologues have been cloned and all show the same structural organization and a high degree of sequence similarity, suggesting an evolutionary conserved role (Liu and Hengartner, 1999, Smits et al., 1999, Su et al., 2002). In fact, overexpression of human CED-6 rescues the engulfment defect of ced-6 mutants in C. elegans (Liu and Hengartner, 1999). In addition, overexpression of human CED-6 in mouse J774 macrophages appears to increase phagocytosis of apoptotic cells (Smits et al., 1999). These data suggest that at least some of the CED-6 functions may be conserved in mammals.

The PTB domain of CED-6/GULP has been shown to interact directly to the FXNPXY motif in the cytoplasmic tail of CED-1 (Su et al., 2002). Moreover, microscopy analysis of transgenic worms shows that CED-1 tagged with YFP at the C-terminus(CED-1:YFP) and CED-6 tagged with CFP at the N-terminus (CFP:CED-6) colocalize in clusters around apoptotic cells during the early stages of the engulfment process while no clustering of CFP:CED-6 is observed in worms that express a mutant CED-1 that lacks the cytoplasmic tail (Kinchen et al., 2005).

NPXY motifs are believed to mediate rapid internalization of proteins from the plasma membrane (Bonifacino and Traub, 2003). As most of the proteins containing NPXY-signals are internalized via clathrin-coated vesicles, there has been an intensive search for clathrin-coat components that could work as recognition proteins. Although some studies suggested that clathrin and AP-2 bind directly to peptides containing the FXNPXY sequence (Boll et al., 2002, Kibbey et al., 1998), recent evidence indicates that this motif is actually recognized by PTB-containing proteins, such as ARH, Dab2, Dab1 and numb, which seem to collaborate with AP-2 to promote optimal clathrin recruitment and assembly (Traub, 2003). Interestingly, the CED-6 PTB domain is closely related to the PTB domains of ARH, Dab2 and Numb, suggesting that CED-6 may also have a role as a cargo-specific endocytic adaptor.

Although there is some information on the function of CED-6 in C. elegans, there is much less known on the localization, gene arrangement and expression of CED-6 in mammals. This information may help in the understanding of the role of CED-6 in mammalian physiology. In this report, we used different approaches to investigate the expression of ratCED-6. We observed that CED-6 immunoreactivity is present in some group of neurons while glial cells do not seem to express the protein. We also demonstrate that CED-6 binds to clathrin and shows some colocalization to clathrin coated-vesicles in cultured cells. The present findings suggest that CED-6 may have alternative roles in endocytic trafficking or signaling in neurons.