A family of RIM-binding proteins regulated by alternative splicing: Implications for the genesis of synaptic active zones

Abstract

RIMs are presynaptic active zone proteins that regulate neurotransmitter release. We describe two related genes that encode proteins with identical C-terminal sequences that bind to the conserved PDZ domain of RIMs via an unusual PDZ-binding motif. These proteins were previously reported separately as ELKS, Rab6-interacting protein 2, and CAST, leading us to refer to them by the acronym ERC. Alternative splicing of the C terminus of ERC1 generates a longer ERC1a variant that does not bind to RIMs and a shorter ERC1b variant that binds to RIMs, whereas the C terminus of ERC2 is synthesized only in a single RIM-binding variant. ERC1a is expressed ubiquitously as a cytosolic protein outside of brain; ERC1b is detectable only in brain, where it is both a cytosolic protein and an insoluble active zone component; and ERC2 is brain-specific but exclusively localized to active zones. Only brain-specific ERCs bind to RIMs, but both ubiquitous and brain-specific ERCs bind to Rab6, a GTP-binding protein involved in membrane traffic at the Golgi complex. ERC1a and ERC1b/2 likely perform similar functions at distinct localizations, indicating unexpected connections between nonneuronal membrane traffic at the Golgi complex executed via Rab6 and neuronal membrane traffic at the active zone executed via RIMs.

Fig 1.

Binding of ERC1b to RIM1 PDZ-domain. (A) Immunoblot analysis of rat brain proteins with affinity-purified ERC1b antibodies to the prey clone pPreyPDZ-16 that was isolated by yeast two-hybrid screening with the RIM1α PDZ domain. Total homogenates, supernatant, and pellet obtained after high-speed centrifugation were tested. (B) Pulldown of soluble brain ERC1b with a GST-RIM1α PDZ domain fusion protein but not with GST alone. (C) Pulldown of recombinant RIM PDZ domain expressed in transfected COS cells with an ERC1b maltose-binding protein fusion protein.

Fig 2.

Structure of ERC1a, -b, and -2. (A) Alignment of the human sequences. Identical residues are highlighted, and similar residues are boxed. Alternatively spliced regions identified as variable sequences in rat, mouse, or human cDNA or EST sequences are marked by “ = ” on the top of the sequences. (B) Structure of the 3′ end of the human ERC1 gene to illustrate mechanism of alternative splicing that creates ERC1a and -b (see supporting information).

Fig 3.

Tissue distribution and developmental expression of ERCs analyzed by immunoblotting with subtype-specific antibodies. (A) Proteins from the indicated rat tissues were probed with four ERC antibodies and a vasolin-containing protein control antibody. Antibodies used were: ERC1b (top section), raised against the conserved domain of ERC1b (Fig. 2); ERC1a (second section), raised against a synthetic peptide from the ERC1a-specific C terminus; ERC1b/2 (third section), raised against a peptide from the common C terminus of ERC1b and ERC2; and ERC2 (fourth section), raised against an internal ERC2-specific peptide. Numbers on the left indicate positions of molecular mass markers. (B) Expression of ERC1a and -1b/2 in mouse embryos at different stages of gestation examined with subtype-specific antibodies. Protein loads were normalized for samples from whole embryos (embryonic day E8.5–E12.5) or embryo heads (E14.5–E18.5) using the levels of VCP determined by immunoblotting. The low molecular mass band in the E8.5 and E9.5 samples (asterisk) is due to crossreactivity with the synaptotagmin 1 antibody (Syt 1) used as a positive control for a synaptic protein.

Fig 4.

Subcellular distribution of ERCs in brain. (A) Rat brain homogenate (Hom.; lane 1) was used to prepare a low-speed supernatant (S1; lane 2), which was separated into crude synaptosomes (P2; lane 3) and synaptosomal supernatant (S2; lane 4). Synaptosomes were lysed and subjected to sequential low- and high-speed centrifugations to yield crude synaptosomal membranes (LP1; lane 5) and synaptic vesicles (LP2). LP1 was used to isolate synaptic plasma membranes (SPM; lane 7) and mitochondria (Mito.; lane 8) by centrifugation on a sucrose step gradient (21). Fractions were analyzed by immunoblotting for the proteins indicated on the right (GDI, GDP-dissociation inhibitor). Numbers on the left indicate positions of molecular mass markers. (B) Brain homogenates from wild-type (WT RIM1) and RIM1α knockout mice (KO RIM1) were treated with buffer alone or with 1% of the indicated detergents, centrifuged at 100,000 × g, and the supernatants were analyzed by immunoblotting with the ERC1b/2 antibody. Numbers on the left indicate positions of molecular mass markers.

Fig 5.

Immunolocalization of ERC1b in cultured neurons and brain sections. (A and B) Double immunofluorescence labeling of cultured neurons with affinity-purified ERC1b antibodies and monoclonal antibodies to RIM (A) or to synapsin (B). (Bar = 30 μm.) (C) Immunolabeling of rat brain sections from hippocampus by immunoelectron microscopy using pre-embedding labeling with silver enhancement. (Bar = 300 nm.)