Cupidin, an Isoform of Homer/Vesl, Interacts with the Actin Cytoskeleton and Activated Rho Family Small GTPases and Is Expressed in Developing Mouse Cerebellar Granule Cells

A developmentally regulated Homer/Vesl isoform, Cupidin (Homer 2a/Vesl-2Δ11), was isolated from postnatal mouse cerebellum using a fluorescent differential display strategy. The strongest expression of Cupidin was detected in the cerebellar granule cells at approximately postnatal day 7. Cupidin was enriched in the postsynaptic density fraction, and its immunoreactivity was concentrated at glomeruli of the inner granular layer when active synaptogenesis occurred. Cupidin protein could be divided into two functional domains: the N-terminal portion, which was highly conserved among Homer/Vesl family proteins, and the C-terminal portion, which consisted of a putative coiled-coil structure, including several leucine zipper motifs. The N-terminal fragment of Cupidin, which was able to associate with metabotropic glutamate receptor 1 (mGluR1), also interacted with F-actin in vitro. In keeping with this, F-actin immunocytochemically colocalized with Cupidin in cultured cerebellar granule cells, and a Cupidin–mGluR1–actin complex was immunoprecipitated from crude cerebellar lysates using an anti-Cupidin antibody. On the other hand, the C-terminal portion of Cupidin bound to Cdc42, a member of Rho family small GTPases, in a GTP-dependent mannerin vitro, and Cupidin functionally interacted with activated-Cdc42 in a heterologous expression system. Together, our findings indicate that Cupidin may serve as a postsynaptic scaffold protein that links mGluR signaling with actin cytoskeleton and Rho family proteins, perhaps during the dynamic phase of morphological changes that occur during synapse formation in cerebellar granule cells.

[1]  U. Francke,et al.  Wiskott–Aldrich Syndrome Protein, a Novel Effector for the GTPase CDC42Hs, Is Implicated in Actin Polymerization , 1996, Cell.

[2]  M. Kondo,et al.  Combinations of AMPA Receptor Subunit Expression in Individual Cortical Neurons Correlate with Expression of Specific Calcium-Binding Proteins , 1997, The Journal of Neuroscience.

[3]  P Siekevitz,et al.  Isolation and characterization of postsynaptic densities from various brain regions: enrichment of different types of postsynaptic densities , 1980, The Journal of cell biology.

[4]  Y. Jan,et al.  Differential effects of the Rac GTPase on Purkinje cell axons and dendritic trunks and spines , 1996, Nature.

[5]  J. Wehland,et al.  Mena, a Relative of VASP and Drosophila Enabled, Is Implicated in the Control of Microfilament Dynamics , 1996, Cell.

[6]  P. Somogyi,et al.  The metabotropic glutamate receptor (mGluRlα) is concentrated at perisynaptic membrane of neuronal subpopulations as detected by immunogold reaction , 1993, Neuron.

[7]  H. Kotani,et al.  Regulation of Cell–Cell Adhesion by Rac and Rho Small G Proteins in MDCK Cells , 1997, The Journal of cell biology.

[8]  P. Rakić,et al.  Neuron‐glia relationship during granule cell migration in developing cerebellar cortex. A Golgi and electonmicroscopic study in Macacus rhesus , 1971, The Journal of comparative neurology.

[9]  K. Inokuchi,et al.  Novel Members of the Vesl/Homer Family of PDZ Proteins That Bind Metabotropic Glutamate Receptors* , 1998, The Journal of Biological Chemistry.

[10]  J. Spudich,et al.  Purification of muscle actin. , 1982, Methods in enzymology.

[11]  K. Fujisawa,et al.  Different Regions of Rho Determine Rho-selective Binding of Different Classes of Rho Target Molecules* , 1998, The Journal of Biological Chemistry.

[12]  M E Hatten,et al.  Motility and cytoskeletal organization of migrating cerebellar granule neurons , 1995, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[13]  M. Sheng,et al.  PDZs and Receptor/Channel Clustering: Rounding Up the Latest Suspects , 1996, Neuron.

[14]  S. Narumiya,et al.  Rho effectors and reorganization of actin cytoskeleton , 1997, FEBS letters.

[15]  K. Mikoshiba,et al.  Inositol 1,4,5‐Trisphosphate Receptor‐Mediated Ca2+ Signaling in the Brain , 1995, Journal of neurochemistry.

[16]  K. Mikoshiba,et al.  Expression of the metabotropic glutamate receptor mGluR1α and the ionotropic glutamate receptor GluR1 in the brain during the postnatal development of normal mouse and in the cerebellum from mutant mice , 1993, Journal of neuroscience research.

[17]  L. Lim,et al.  The Ras-related protein Cdc42Hs and bradykinin promote formation of peripheral actin microspikes and filopodia in Swiss 3T3 fibroblasts , 1995, Molecular and cellular biology.

[18]  K. Inokuchi,et al.  vesl, a gene encoding VASP/Ena family related protein, is upregulated during seizure, long‐term potentiation and synaptogenesis 1 , 1997, FEBS letters.

[19]  N. Nomura,et al.  Identification of IQGAP as a Putative Target for the Small GTPases, Cdc42 and Rac1* , 1996, The Journal of Biological Chemistry.

[20]  S. Nakanishi,et al.  Glutamate and Quisqualate Regulate Expression of Metabotropic Glutamate Receptor mRNA in Cultured Cerebellar Granule Cells , 1993, Journal of neurochemistry.

[21]  D. Linden,et al.  Homer Binds a Novel Proline-Rich Motif and Links Group 1 Metabotropic Glutamate Receptors with IP3 Receptors , 1998, Neuron.

[22]  Takashi Ito,et al.  Fluorescent differential display: Arbitrarily primed RT‐PCR fingerprinting on an automated DNA sequencer , 1994, FEBS letters.

[23]  T. Yamamoto,et al.  Rho‐associated kinase, a novel serine/threonine kinase, as a putative target for small GTP binding protein Rho. , 1996, The EMBO journal.

[24]  A. Craig,et al.  Role of Actin in Anchoring Postsynaptic Receptors in Cultured Hippocampal Neurons: Differential Attachment of NMDA versus AMPA Receptors , 1998, The Journal of Neuroscience.

[25]  U. Walter,et al.  The 46/50 kDa phosphoprotein VASP purified from human platelets is a novel protein associated with actin filaments and focal contacts. , 1992, The EMBO journal.

[26]  K. Mikoshiba,et al.  Pharmacological and immunocytochemical characterization of metabotropic glutamate receptors in cultured Purkinje cells , 1992, The Journal of neuroscience : the official journal of the Society for Neuroscience.

[27]  J. D. Pardee,et al.  [18] Purification of muscle actin , 1982 .

[28]  John H. Lewis,et al.  Crystal Structures of a Complexed and Peptide-Free Membrane Protein–Binding Domain: Molecular Basis of Peptide Recognition by PDZ , 1996, Cell.

[29]  K. Fujisawa,et al.  Citron, a Rho-Target, Interacts with PSD-95/SAP-90 at Glutamatergic Synapses in the Thalamus , 1999, The Journal of Neuroscience.

[30]  S. Nakanishi,et al.  Distribution of the mRNA for a metabotropic glutamate receptor (mGluR1) in the central nervous system: An in situ hybridization study in adult and developing rat , 1992, The Journal of comparative neurology.

[31]  C. Barnes,et al.  Homer: a protein that selectively binds metabotropic glutamate receptors , 1997, Nature.

[32]  P. Worley,et al.  Homer Regulates the Association of Group 1 Metabotropic Glutamate Receptors with Multivalent Complexes of Homer-Related, Synaptic Proteins , 1998, Neuron.

[33]  M. Hatten,et al.  Genes involved in cerebellar cell specification and differentiation , 1997, Current Opinion in Neurobiology.

[34]  S. Nagata,et al.  pEF-BOS, a powerful mammalian expression vector. , 1990, Nucleic acids research.

[35]  Shuh Narumiya,et al.  A novel partner for the GTP‐bound forms of rho and rac , 1995, FEBS letters.

[36]  P. Greengard,et al.  Synapsin I (protein I), a nerve terminal-specific phosphoprotein. III. Its association with synaptic vesicles studied in a highly purified synaptic vesicle preparation , 1983, The Journal of cell biology.

[37]  Richard Threadgill,et al.  Regulation of Dendritic Growth and Remodeling by Rho, Rac, and Cdc42 , 1997, Neuron.