Interaction of the C-terminal domain of δ glutamate receptor with spectrin in the dendritic spines of cultured Purkinje cells

doi:10.1016/S0168-0102(99)00061-9

Neuroscience Research

Volume 34, Issue 4, September 1999, Pages 281-287

https://doi.org/10.1016/S0168-0102(99)00061-9 Get rights and content

Abstract

The interaction of neurotransmitter receptors with the underlying cytoskeleton via subsynaptic proteins is an important mechanism for the targeting of the receptors to synapses in the central nervous system. We show that δ glutamate receptors (δ receptors), expressed predominantly in the dendritic spines of cerebellar Purkinje cells, directly interact with spectrin, a member of the actin-binding family of proteins. Moreover, the interaction between spectrin and C-terminal domain of the δ receptor is 50% inhibited by 1 μM of Ca²⁺ in vitro, compared with that in the absence of Ca²⁺. These results suggest that δ receptors on the postsynaptic membrane of the dendritic spines of cerebellar Purkinje cells are anchored to the actin cytoskeleton via spectrin, and that Ca²⁺ elevation in the dendritic spines causes δ receptor declustering by dissociation of the receptors from spectrin. This mechanism for receptor anchoring at postsynaptic sites may regulate synaptogenesis and/or synaptic plasticity.

Introduction

δ Glutamate receptors (δ receptors) are expressed predominantly in cerebellar Purkinje cells (Yamazaki et al., 1992, Araki et al., 1993, Lomeli et al., 1993). Their receptor subunits neither coassemble with other glutamate receptor subunits nor bind to glutamate receptor ligands when expressed in a mammalian cell line (Lomeli et al., 1993, Mayat et al., 1995). However, knock-out of the δ2 receptor gene results in reduced parallel fiber synapses, persistence of surplus climbing fibers, impaired cerebellar long-term depression (LTD) and motor dyscoordination (Kashiwabuchi et al., 1995, Kurihara et al., 1997). The δ receptor is, therefore, considered to play important roles in the synaptogenesis and synaptic plasticity in the cerebellum.

δ Receptors are found in abundance on postsynaptic membranes at parallel fiber synapses with Purkinje cell dendritic spines from postnatal day 10 (P10) (Zhao et al., 1998). However, the mechanisms responsible for the localization of δ receptors to the postsynaptic spines are not yet well understood. Recent studies have shown that the interaction of neurotransmitter receptors with the cytoskeleton via subsynaptic proteins is important for the precise apposition of neurotransmitter receptors at postsynaptic sites. For example, postsynaptic inhibitory glycine receptors expressed on spinal cord neurons are anchored to microtubules via the peripheral membrane protein gephyrin (Kirsch and Betz, 1995). In the case of GABA_A receptors, GABA_A receptor-associating protein (GABARAP) was shown to mediate their binding to the tubulin cytoskeleton (Wang et al., 1999). Since among the cytoskeletal elements, actin is the major component in dendritic spines, it is reasonable to speculate that δ receptors expressed on the dendritic spines of Purkinje cells may associate with actin filaments via some anchoring protein in order for them to be localized on the postsynaptic spine membrane. In this paper, we examined whether spectrin, an actin-binding protein exhibiting high levels of expression in the spines of Purkinje cells, could act as a linker between the δ receptors and the actin filaments.

Section snippets

Cerebellar neuronal culture

Dissociated cerebellar neuronal cultures were prepared from the brains of 20–21-day-old Wistar rat fetuses according to a method described in a previous report (Furuya et al., 1998) with slight modification. In brief, 5.0×10⁶ cells were plated onto plastic coverslips (21 mm in diameter; Sumilon MS-80060; Sumitomo Bakelite, Tokyo) coated with poly-l-ornithine and placed in a humidified CO₂ incubator (5% CO₂ at 37°C). A serum-free culture medium was added to each well after 3 h. The medium was

Direct binding of spectrin to the C-terminal domain of the δ receptor

Previous studies have demonstrated a direct association of NMDA receptors with spectrin or α-actinin, members of the actin-binding family of proteins (Wyszynski et al., 1997, Wechsler and Teichberg, 1998). Since the dendritic spines of Purkinje cells have been shown to contain spectrin rather than α-actinin (Malchiodi-Albedi et al., 1993, Wyszynski et al., 1998), we examined whether the δ receptor–actin interaction was mediated by spectrin. Several spectrin subtypes have been reported in the

Discussion

The interaction between neurotransmitter receptors in the postsynaptic membrane and the underlying cytoskeleton has recently drawn considerable attention (Kirsch et al., 1995, Niethammer et al., 1998, Matsuda and Hirai, 1999, Wang et al., 1999, Xia et al., 1999), since the regulation of the interaction between these two components can modulate the density of a receptor expressed on the postsynaptic membrane, and may control synaptic efficacy and plasticity. The present study clarified that δ

Acknowledgements

We would like to thank Dr S. Furuya for kindly teaching us the techniques of Purkinje cell culture, S. Mikawa and T. Torashima for expert technical assistance.

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In 1994, Ranum and colleagues identified a ten-generation American kindred with a relatively mild autosomal dominant form of spinocerebellar ataxia (Ranum et al., 1994). The mutation was mapped to the centromeric region of chromosome 11, and the disorder designated SCA5 (Ranum et al., 1994). Using a multifaceted mapping approach, Ikeda et al. (2006) discovered that β-III spectrin (SPTBN2) mutations cause spinocerebellar ataxia type 5 (SCA5) in the American kindred and two additional independently reported SCA5 families. The American and French families have separate in-frame deletions of 39 and 15 bp, respectively, in the third of 17 spectrin repeat motifs. A third mutation, found in a German family, is located in the second calponin homology domain, a region known to bind actin and Arp1. Consistent with Purkinje cell degeneration in SCA5, β-III spectrin is highly expressed in cerebellar Purkinje cells. TIRF microscopy performed on cell lines transiently transfected with mutant or wild-type spectrin shows that mutant β-III spectrin fails to stabilize the glutamate transporter EAAT4 at the plasma membrane. Additionally, marked differences in EAAT4 and GluRδ2 were found by protein blot and cell fractionation in SCA5 autopsy tissue. This review summarizes data showing that β-III spectrin mutations are a novel cause of neurodegenerative disease, which may affect the stabilization or trafficking of membrane proteins.
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Thus, the changes that bestow ankyrin-binding specificity are subtle and involve primarily sequence changes in the terminal portions of h14C, the 14-15 linker, and in the 15B-C loop. Ankyrin and other ligands such as the Lutheran blood group antigens,17 the neural cell adhesion molecule NCAM,18 the delta glutamate receptor,43 and excitatory amino acid transporters6 bind with high specificity and affinity to the spectrin repeat (summarized in De Matteis and Morrow1). Other proteins with spectrin-like repeats such as BPAG122 and mAKAP21 also use their repeat regions for ligand binding.
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Interaction of the C-terminal domain of δ glutamate receptor with spectrin in the dendritic spines of cultured Purkinje cells

Abstract

Introduction

Section snippets

Cerebellar neuronal culture

Direct binding of spectrin to the C-terminal domain of the δ receptor

Discussion

Acknowledgements

Biochem. Biophys. Res. Commun.

Cell

Mol. Cell Neurosci.

FEBS Lett.

Neurosci. Res.

Neuron

Mol. Brain Res.

Neuron

Blood

Neuron

Biochem. Biophys. Res. Commun.

The spectrin-based membrane skeleton and micron-scale organization of the plasma membrane

Annu. Rev. Cell Biol.

Local calcium signaling by inositol-1,4,5-triphosphate in Purkinje cell dendrites

Nature

Ceramide and its interconvertible metabolite sphingosine function as indispensable lipid factors involved in survival and dendritic differentiation of cerebellar Purkinje cells

J. Neurochem.