Formation of glycine receptor clusters and their accumulation at synapses - PubMed (original) (raw)

. 2000 Aug:113 ( Pt 15):2783-95.

doi: 10.1242/jcs.113.15.2783.

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Formation of glycine receptor clusters and their accumulation at synapses

J Meier et al. J Cell Sci. 2000 Aug.

Abstract

The glycine receptor is highly enriched in microdomains of the postsynaptic neuronal surface apposed to glycinergic afferent endings. There is substantial evidence suggesting that the selective clustering of glycine receptor at these sites is mediated by the cytoplasmic protein gephyrin. To investigate the formation of postsynaptic glycine receptor domains, we have examined the surface insertion of epitope-tagged receptor alpha subunits in cultured spinal cord neurons after gene transfer by polyethylenimine-adenofection. Expression studies were also carried out using the non-neuronal cell line COS-7. Immunofluorescence microscopy was performed using wild-type isoforms and an alpha mutant subunit bearing the gephyrin-binding motif of the beta subunit. In COS-7 cells, transfected glycine receptor alpha subunits had a diffuse surface distribution. Following cotransfection with gephyrin, only the mutant subunit formed cell surface clusters. In contrast, in neurons all subunits were able to form cell surface clusters after transfection. These clusters were not colocalized with detectable endogenous gephyrin, and the GlyR beta subunit could not be detected in transfected cells. Therefore, exogenous receptors were not assembled as heteromeric complexes. A quantitative analysis demonstrated that newly synthesized glycine receptor progressively populated endogenous gephyrin clusters, since association of both proteins increased as a function of time after the onset of receptor synthesis. This phenomenon was accelerated when glycine receptor contained the gephyrin-binding domain. Together with previous results, these data support a two-step model for glycinergic synaptogenesis whereby the gephyrin-independent formation of cell surface clusters precedes the gephyrin-mediated postsynaptic accumulation of clusters.

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