Recognition of basic sorting motifs within synaptic membrane cargo proteins by the clathrin-adaptor complex AP-2

Abstract

Communication in the central nervous system (CNS) occurs via chemical synapses, intercellular asymmetric junctions between an axon and the dendrite of a neighbouring neuron. Synapses comprised of a presynaptic release site and a postsynaptic receptive site. Internalization of synaptic proteins by means of clathrin-coated vesicles (CCVs) plays a major role in the regulation of processes at both sides of the synapse. At the presynaptic side synaptic vesicle proteins (SVP) and excess membrane are retrieved by clathrin-mediated endocytosis (CME). At the postsynaptic active zone the strength of signal transmission can be modulated by clathrin-mediated internalization of neurotransmitter receptors. A pre-requisite for CME is the recognition of cargo proteins via sorting signals within their cytoplasmic portion by the heterotetrameric clathrin-adaptor AP-2 or other monomeric adaptors.We provide evidence here that the medium subunit µ2 of the AP-2 complex interacts directly with the cytoplasmic tail of the excitatory AMPA receptor subunit GluR2 via a dibasic recognition motif. At the presynaptic site binding of µ2-adaptin to a dibasic cluster within the C2B-domain of the synaptic vesicle protein (SVP) synaptotagmin 1 provides a link to the endocytic machinery and is implicated in SV recycling. We have used a chimeric approach to analyze the role of the AMPA receptor dibasic recognition motif in CME. The basic cluster was sufficient to facilitate clathrin-mediated internalization of a synaptotagmin 1 chimera carrying the AMPA receptor sorting motif from the plasma membrane of transfected cells. Inhibition of CME in cells transfected with siRNA directed against µ2-adaptin prevented internalization of the chimera. Thus, recognition of the dibasic sorting motif by µ2-adaptin provides a direct link to the endocytic machinery, and is thereby involved in CME of excitatory AMPA type glutamate receptors similar to the recognition of the SVP synaptotagmin 1 by AP-2. This work shows that dibasic motifs function as a common recognition signal in the regulated internalization of membrane cargo proteins from both sides of the synapse. Establishment of axonal polarity during neuronal differentiation requires the maturation of an intracellular sorting machinery that achieves directed transport of SVPs to the nascent synapse. In the second part of this work we have addressed the question if clathrin-mediated internalization from the plasma membrane of neuronal precursor cells plays a role in the formation of a SV precursor organelle.Retinoic acid induced differentiation of unpolarized embryonic precursor cells into postmitotic neurons was used as a model system to study expression patterns of SVPs. The expression of SVPs is switched on at very early stages of neuronal differentiation that precede neurite outgrowth and synaptogenesis. SVPs reside within a perinuclear compartment in neuronal precursor cells. Co-localization of internalized transferrin with endogenous SVPs in the perinuclear compartment of neuronal precursor cells hints at an endosomal origin of the compartment, although no co-localization with markers for early or late endosomes was observed. An antibody feeding assay, in which a monoclonal antibody directed against the luminal domain of the SVP synaptotagmin 1 was internalized from the plasma membrane of postmitotic neurons and sorted into the perinuclear compartment, supports a putative recycling function of the compartment.Our data support a model whereby clathrin-mediated cycling of SVPs between the plasma membrane and a perinuclear recycling compartment in neuronal precursor cells might be involved in the biogenesis of a SV precursor organelle that mediates transport of SVPs along the axon to the nascent synapse.