Synaptic adhesion molecules such as neuroligin are involved in synapse formation, whereas ionotropic transmitter receptors mediate fast synaptic transmission. In mutant mice deficient in the glutamate receptor ␦2 subunit (␦2), the number of synapses between granule neurons (GNs) and a Purkinje neuron (PN) in the cerebellum is reduced. Here, we have examined the role of ␦2 in synapse formation using culture preparations. First, we found that the size and number of GN presynaptic terminals on a PN in the primary culture prepared from knockout mice were smaller than those in control culture. Next we expressed ␦2 in nonneuronal human embryonic kidney (HEK) cells and cocultured them with GNs. Punctate structures expressing marker proteins for glutamatergic presynaptic terminals were accumulated around the HEK cells. Furthermore, HEK cells expressing both ␦2 and GluR1, a glutamate receptor subunit forming a functional glutamate-gated ion channel, showed postsynaptic current. Deletion of the extracellular leucine/isoleucine/valine binding protein (LIVBP) domain of ␦2 abolished the induction ability, and the LIVBP domain directly fused to a transmembrane sequence was sufficient to induce presynaptic differentiation. Furthermore, a mutant GluR1 whose LIVBP domain was replaced with the ␦2 LIVBP domain was sufficient by itself to establish synaptic transmission. Another member of ␦ glutamate receptor family ␦1 also induced presynaptic differentiation. Thus, the ␦ glutamate receptor subfamily can induce the differentiation of glutamatergic presynaptic terminals and contribute to the establishment of synaptic transmission.cerebellum ͉ granule neuron ͉ Purkinje neuron ͉ synapse formation S ynapse formation requires the accumulation and organization of multiple proteins on both the pre-and postsynaptic sides (1, 2). Previous studies have shown that expression of postsynaptic neuroligin in a nonneuronal cell triggers presynaptic differentiation in a contacting axon through interaction with presynaptic neurexin (3, 4). The involvement of other synaptic adhesion proteins, such as SynCAM and cadherin, in the formation or maintenance of synaptic structures has also been reported (5-7). For synaptic function, the most important postsynaptic proteins are receptors for neurotransmitters. Glutamate is the most prevalent neurotransmitter in the central nervous system, and there are ionotropic glutamate receptors (iGluRs) and metabotropic glutamate receptors on the postsynaptic membrane. iGluR opens its pore domain to allow the permeation of cations when bound by glutamate, and mediates fast excitatory synaptic transmission (8, 9).So far, iGluR has not been shown to be directly related to synapse formation. However, in mutant mice deficient in the glutamate receptor ␦2 subunit (␦2), the number of synapses between granule neurons (GNs) and a Purkinje neuron (PN) is reduced, suggesting the involvement of ␦2 in synapse formation or maintenance (10, 11). ␦2 is selectively expressed in cerebellar PNs and has been classified as a member of the...

No abstract

Glutamate receptor delta2 (GluD2) is selectively expressed on the postsynaptic spines at parallel-fiber (PF)-Purkinje neuron (PN) synapses. GluD2 knockout mice show a reduced number of PF-PN synapses, suggesting that GluD2 is involved in synapse formation. Recent studies revealed that GluD2 induces presynaptic differentiation in a manner dependent on its N-terminal domain (NTD) through binding of Cbln1 secreted from cerebellar granule neurons. However, the underlying mechanism of the specific binding of the NTD to Cbln1 remains elusive. Here, we have identified the flap loop (Arg321-Trp339) in the NTD of GluD2 (GluD2-NTD) as a crucial region for the binding to Cbln1 and the induction of presynaptic differentiation. Both induction of presynaptic differentiation and binding of Cbln1 were abolished in the HEK cells expressing not wild-type GluD2 but GluD2 with mutations in the flap loop. Especially, single amino acid substitution of either Arg321 or Trp323 to alanine was sufficient to disable the GluD2 function. Finally, a homology model of GluD2-NTD suggested that the flap loop is located at the distal end, which appears consistent with an interaction with Cbln1 and a presynaptic varicosity.

No abstract