Regular articleCoincidence in dendritic clustering and synaptic targeting of homer proteins and NMDA receptor complex proteins NR2B and PSD95 during development of cultured hippocampal neurons
Introduction
Homer family proteins represent a prominent component of glutamatergic postsynaptic density (PSD) protein complexes Brakeman et al 1997, Xiao et al 1998, Kato et al 1998, Shiraishi et al 1999. The N-terminal Enabled/Vasp homology 1 (EVH1)-like region is highly conserved in the family and binds to various postsynaptic proteins containing a proline-rich motif (PPxxF), including group I metabotropic glutamate receptor types 1α and 5 (mGluR1α/5), inositol 1,4,5-trisphosphate receptor (IP3R), ryanodine receptor (RyR) Tu et al 1998, Xiao et al 1998, and Shank/ProSAP/ CortBP1, a scaffold protein for the N-methyld-aspartate (NMDA) receptor (NMDAR) complex Tu et al 1999, Naisbitt et al 1999, Tu et al 1999. The C-terminal region consists of a coiled-coil structure followed by leucine zipper motifs and is likely involved in multimerization Kato et al 1998, Sun et al 1998. We found that an isoform Cupidin/Homer 2 binds to filamentous actin (F-actin) (Shiraishi et al., 1999) and Drebrin (Mizutani et al., 1999, in preparation), an actinbinding and remodeling protein in dendritic spines Hayashi et al 1996, Hayashi and Shirao 1999, at the N-terminal region and to GTP-bound Cdc42, a Rhofamily small GTPase, at the C-terminal region. In postsynapses, Homer would therefore be a good candidate for forming a PSD protein cluster in which two glutamate receptor complexes, glutamate-mediated calcium release complex (mGluR1α/5-Homer-IP3R) and glutamate-mediated calcium influx complex (Homer-Shank-GKAP-PSD95-NMDAR), and the actin cytoskeletal reorganizing complex (F-actin-Drebrin-Homer-activated Cdc42) are physically and/or functionally linked (for reviews, see Scannevin and Huganir 2000, Xiao et al 2000, Sheng 2001.
Understanding dendritic clustering and synaptic targeting of Homer proteins in relation to other synaptic proteins including the binding partners will provide clues to how postsynaptic complexes are organized during synaptic differentiation. Several studies utilizing cultured neurons have introduced exogenous Homer genes to express cDNA-derived Homer proteins. In hippocampal cell cultures, Sala et al. (2001) reported that overexpression of Homer 1b together with Shank by transfection induced accumulation of IP3R in dendritic spines and changes of spine morphology and synaptic function. Okabe et al. (2001) reported that green fluorescent protein (GFP)-fused PSD-Zip45/ Homer 1c expressed by adenovirus vector gene transfer demonstrated activity-dependent redistribution of synaptic clustering. In cerebellar granule cell cultures, Ango et al. (2000) reported that cotransfected Homer 1b/c and mGluR5 were synaptically targeted. We have previously demonstrated that clustering of both endogenous and transfected Cupidin/Homer 2 in cerebellar in preparation/granule cells is both developmentally (Shiraishi et al., 1999) and activity-regulated (Shiraishi et al., in preparation). At present, little is known about developmental changes in the cellular localization of endogenous proteins for each Homer isoform during dendrogenesis and synaptogenesis.
In this study, we generated isoform-specific anti-Homer antibodies to determine whether endogenous Homer family proteins undergo changes in cellular localization during dendritic and synaptic differentiation in cultured hippocampal neurons. We describe how developmental clustering and synaptic targeting of endogenous Homer protein isoforms coincides with those of the NMDA receptor-PSD95 complex, rather than the actin-cytoskeletal binding partners F-actin and Drebrin.
Section snippets
Isoform-specific anti-Homer antibodies
To characterize developmental changes in cellular localizations of Homer family proteins, we produced polyclonal rabbit antibodies specific to each Homer isoform and rat antibody to Cupidin/Homer 2 isoform (see Experimental Methods). Specificity of antibodies was verified using Western blot analysis of protein extracts prepared from HeLa cells that were specifically expressing each Homer isoform following cDNA transfection (Fig. 1A). Isoform-specific rabbit anti-Homer 1b/c, anti-Cupidin/Homer
Discussion
We report herein the spatio-temporal changes in cellular localizations of Homer family proteins in cultured hippocampal neurons at 7, 14, and 21 DIV, which are basically consistent with the early, middle, and late stages of dendritic and synaptic development described by Rao et al., (1998) and Zhang and Benson (2001). Our study revealed that: (1) Homer proteins form clusters around the base to proximal region of dendrites during the early stage and thereafter behave as clusters throughout
Cell culture
Hippocampal dissociated primary cultures were prepared from embryonic day 18 Wistar rat (Nippon SLC, Shizuoka, Japan). Excised hippocampi were treated with 45 units of papain (Worthington, PAPL, Lakewood, NJ), 0.01% DNase I (Boehringer-Mannheim, Indianapolis, IN), 0.02% dl-cystein, 0.02% BSA, and 0.5% glucose in PBS for 20 min at 37°C. After adding 20% bovine serum, cells were dissociated by repeated passage through a 1-mL plastic pipette tip. Dispersed cells were plated at a density of 1.1 × 10
Acknowledgements
We thank Dr. David R. Coleman (Mount Sinai School of Medicine, NY) for anti-N-cadherin antibody and Dr. Shelley Halpain and Dr. S. Graber (The Scripps Research Institute, CA) for critical comments and helpful suggestions. This research was supported by grants-inaid for Scientific Research from the Japanese Ministry of Education, Culture, Sports, Science and Technology and the Japan Society for the Promotion of Science (to T.F.), a Research Fellowship grant (12780573) from the Japan Society for
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Present address: Department of Cell Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037.