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Adaptor protein complex-4 (AP-4) is expressed in the central nervous system neurons and interacts with glutamate receptor δ2

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Abstract

Ion channels and receptors are targeted and localized at specific postsynaptic sites to mediate neurotransmission. Receptors clustering at postsynaptic sites has been extensively studied; however, the molecular mechanisms underlying intracellular trafficking of receptors to their specific destinations remain unclear. In the present study, we found that glutamate receptor δ2 interacted directly with AP-4, a newly identified adaptor protein complex-4 that mediates protein sorting in mammalian cells. The interaction between μ4 subunit of AP-4 and the δ2 C-terminal involved multiple amino acid sequence motifs other than the classical tyrosine-based signals. AP-4 complex is expressed ubiquitously in many regions of brain, with localization on the Golgi-like structures in the cell bodies and dendrites of neurons. In addition, overexpression of μ4 substantially altered the distribution pattern of δ2 in heterologous cells. These results suggest a potential involvement of AP-4 in the trafficking of δ2 in the brain.

Introduction

The precise distribution of ion channels and receptors on the neuronal surface is critical for the processing and transmission of electrical signals. Accumulating evidence has revealed that secretory and endocytic pathways regulate α-amino-3-hydroxyl-5-methyl-4-isoxazolepropionate (AMPA2 ) receptor-mediated synaptic transmission and synaptic plasticity Ehlers 2000, Lin et al 2000; Lledo et al., 1998; Man et al., 2000; Matsuda et al., 2000). However, the molecular mechanisms underlying receptors trafficking between intracellular compartments are still poorly defined.

Targeting of integral membrane proteins to the plasma membrane in polarized cells is dependent upon the sorting signals contained within the cytoplasmic domains of the proteins. To date, four adaptor protein (AP) complexes, namely, AP-1, AP-2, AP-3, and AP-4, have been identified to participate in the sorting of membrane proteins. These complexes are composed of four subunits, two large chains (γ/α/δ/ε and β1–4), one medium (μ1–4), and one small chain (σ1–4), to form a heterotetrameric complex. They interact with the tyrosine- and dileucine-based signals in the cytoplasmic domain of transmembrane proteins via their μ subunits Ohno et al 1995, Owen and Evans 1998. AP-1 and AP-3 mediate protein sorting from the trans-Golgi network (TGN) to endosomes, while AP-2 is involved in clathrin-mediated endocytosis Le Borgne and Hoflack 1998, Schmid 1997. AP-4 has been isolated recently through homology screening of sequence database, and it is associated with perinuclear compartments, possibly the TGN Dell’Angelica et al 1999, Hirst et al 1999, Wang and Kilimann 1997. Although a recent study showed that AP-4 is mediating basolateral sorting in epithelial cells (Simmen et al., 2002), the exact function of AP-4 in membrane traffic remains to be elucidated.

The ionotropic glutamate receptor δ2 has a unique distribution, where it is exclusively expressed in cerebellar Purkinje cells Araki et al 1993, Landsend et al 1997, Zhao et al 1997. The δ2 receptor plays important roles in cerebellar Purkinje neurons as mutant mice lacking δ2 receptor showed impairments in motor learning, plasticity, and formation of parallel fiber-Purkinje cell synapses Kashiwabuchi et al 1995, Kurihara et al 1997. The distinct localization of δ2 at the parallel fiber synapses in adult implied that a specific sorting mechanism might be involved in the differential targeting of δ2 in Purkinje cells. Until now, PSD-93, delphilin, and protein tyrosine phosphatase PTP-MEG have been shown to interact with δ2 receptor for clustering at postsynaptic sites Hironaka et al 2000, Miyagi et al 2002, Roche et al 1999. However, the distribution of these interacting proteins in neurons suggested that they might not be responsible for the initial targeting of δ2 receptor to specific synapses.

To identify proteins that may be involved in the trafficking and synaptic targeting of δ2, we used the δ2 C-terminus to screen a rat brain cDNA library using the yeast two-hybrid system. One of the isolated proteins was the mu subunit of adaptor protein complex 4. Mutational analysis revealed that an assortment of various amino acid sequence motifs in the C-terminal of δ2 is responsible for the specific binding to μ4. Importantly, the distribution of δ2 receptor was altered and concentrated at the perinuclear region when coexpressed with μ4 in heterologous cells. These results strongly suggest that AP-4 might be functional in the trafficking of δ2 receptor in vivo.

Section snippets

Identification of δ2 receptor interacting proteins

To identify proteins that interact with δ2 receptor, we screened a rat brain cDNA library by yeast two-hybrid system using the last half of the C-terminal of δ2 (921–1007) as bait (Fig. 1A). Approximately 1.64 × 106 clones were screened and 45 positive genes were isolated. Among them, one encoded the full length of the mu subunit (μ4) of AP-4 complex (Fig. 1C) including 72 bp of the 5′ untranslated region. To examine whether μ4 interacted with δ2 specifically, we investigated the binding of μ4

Discussion

In the central nervous system, molecular mechanisms underlying clustering and anchoring of receptors have been well investigated, whereas intracellular machineries that mediate receptors trafficking are still largely unknown. In the present study, we found that AP-4 complex interacts directly with the C-terminus of δ2 glutamate receptor and localizes in soma and dendrites, especially around the Golgi apparatus of neurons. Although there is no direct evidence showing the AP-4 affects the

Yeast two-hybrid system

The rat wild-type C-terminus of δ2 (amino acids 921 to 1007) was fused to LexA in pEG202 and used as a bait to screen a rat brain cDNA library (DupLEX-A system, OriGene Technologies, Rockville, MD, USA) in the activation domain vector pJG4-5. Approximately 1.6 × 106 colonies were screened, and positive interactors were verified by restreaking for growth on leucine-deficient plates. For in vitro interaction study by yeast two-hybrid system, various cDNA constructs encoding different regions of

Acknowledgements

We would like to express our thanks to Dr. Margaret S. Robinson of the University of Cambridge for providing us the polyclonal antibodies for ε, β4, and μ4 subunits of AP-4, and thanks also to Dr. Koujiro Tohyama for excellent preparation of immunohistochemical pictures of brain sections.

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    Present address: NIEHS/NIH, 111 Alexander Dr., Research Triangle Park, NC.

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