Molecular organization of presynaptic active zones by the ERC family of multidomain proteins

Abstract

ERC (ELKS-Rab6-interaction protein-CAST) is a family of multidomain proteins with two known members (ERC1 and ERC2). ERC is implicated in the organization of presynaptic active zones, but the underlying mechanism is poorly understood. In the first part of this study, I demonstrate that ERC directly interacts with liprin- α/SYD2, a family of multidomain proteins involved in the development of presynaptic active zones. In vitro results including yeast two hybrid, pulldown, coimmunoprecipitation and coclustering assays indicate that ERC2, an active-zone specific isoform, interacts with all of the known four isoforms of liprin-α and that liprin-α1 associates with both ERC2 and ERC1b, a splice variant of ERC1 that distributes to both cytosolic and active zone regions. ERC2 colocalizes with liprin-α1 in cultured neurons and forms a complex with liprin-α1 in rat brain. liprin-α1, when expressed alone in cultured neurons, shows a partial synaptic localization. When coexpressed with ERC2, however, liprin-α1 is redistributed to synaptic sites. Moreover, roughly the first half of ERC2, which contains the liprin-α-binding region, is sufficient for the synaptic loalization of liprin-α1 while the second half is not. In the second part, I identify that ERC directly interacts with syntenin-1, a tandem PDZ domain-containing protein. In vitro experiments indicate that syntenin-1 interacts with ERC1b and ERC2. mRNAs of syntenin-1 and ERC1b/2 show overlapping expression patterns in rat brains. Syntenin-1 and ERC proteins partially colocalize in dissociated cultured hippocampal neurons, and form a complex in rat brain. Interestingly, expression of active-zone specific ERC2 in cultured neurons induces a marked presynaptic clustering of both endogenous and exogenous syntenin-1. This clustering requires, in addition to the PDZ interaction, the multimerization of both proteins. Importantly, neuronal activity selectively increases the expression levels and synaptic localizations of ERC2...