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 brainspecific 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 ERC1b͞2 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. R IM1␣ and -2␣ are multidomain adaptor proteins that were discovered as putative effectors for Rab3, a synaptic vesicle protein that binds GTP and regulates neurotransmitter release (1-4). RIMs are composed of an N-terminal Zn 2ϩ -finger domain, a central PDZ domain, and C-terminal C 2 A and C 2 B domains (3, 4). The binding of RIMs to Rab3 and the localization of RIM1␣ to presynaptic active zones suggested a function in neurotransmitter release, which was confirmed by genetic experiments in Caenorhabditis elegans and mice (5-7). Deletion of RIM1␣ in mice caused distinct phenotypes in different types of synapses. In excitatory synapses capable of N-methyl-Daspartate (NMDA)-dependent long-term potentiation (e.g., Schaffer collateral͞commissural fiber synapses in the CA1 region of the hippocampus) and in inhibitory synapses, deletion of RIM1␣ decreased the neurotransmitter release probability in response to Ca 2ϩ influx, leading to a decline in synaptic transmission and changes in short-term synaptic plasticity (6). In excitatory synapses that lack NMDA-dependent long-term potentiations but experience protein kinase A-dependent longterm potentiation (e.g., mossy fiber synapses in the CA3 region of the hippocampus), deletion of RIM1␣ had no detectable effect on acute neurotransmitter release but prevented protein kinase A-dependent potentiation (7). Although these results confirmed an important role for RIM1␣ and, by extension, RIM2␣, in presynaptic function, the spectrum of mutant phenotypes suggests that this role is incompletely understood.It is likely that RIMs regulate release by interacting with other proteins. Several binding partners were identified. The Nterminal Zn 2ϩ -finger domain of RIM1␣ directly binds to Rab3 [w...

Redundant Localization Mechanisms of RIM and ELKS inCaenorhabditis elegans

“…N-terminal dimerization of ELKS causes the ELKS-RET chimeric protein to oligomerize and act as an oncoprotein, leading to papillary thyroid carcinoma (Nakata et al, 2002). ELKS has since been identified as a Rab6A-interacting and -regulating protein (Monier et al, 2002), as a member of the IB kinase (IKK) complex involved in NF-B signaling (Ducut Sigala et al, 2004), and as a member of the active zone complex in the mammalian nervous system (Ohtsuka et al, 2002;Wang et al, 2002;Deguchi-Tawarada et al, 2004). The role of ELKS in the nervous system is unclear.…”

“…Mammalian RIM and ELKS proteins directly interact through the PDZ domain of RIM and the last three amino acids of ELKS, which contains a conserved three amino acid (Ile-Trp-Ala) PDZbinding motif (Ohtsuka et al, 2002;Wang et al, 2002). We tested whether the C. elegans homologs also physically interact.…”

Integration of ATP, cAMP, and Ca2+ Signals in Insulin Granule Exocytosis

“…CAZ (cytoskeletal matrix associated with the active zone) proteins have been suggested to organize the site of neurotransmitter release. These include Piccolo/Aczonin (6,7), Bassoon (8), Rim1 (9), Munc13-1 (10), and CAST/ERC (11,12). Piccolo/ Aczonin is a 500-kDa protein with zinc fingers, a PDZ (PSD-95, Dlg, and ZO-1) domain, and two C 2 domains.…”

Protein mutated in paroxysmal dyskinesia interacts with the active zone protein RIM and suppresses synaptic vesicle exocytosis

“…The N-terminal region forms a tripartite complex with Rab3 and Munc13 that orchestrates synaptic vesicle priming (17,18,(21)(22)(23)(24). The central PDZ-domain binds to the active zone protein ELKS (25)(26)(27), and it tethers N-and P/Qtype Ca 2+ channels to the active zone by forming a tripartite complex with α-subunits of P/Q-and N-type Ca 2+ channels and RIM-binding protein (16,28,29). The functions of the C-terminal RIM C2-domains during release are less well understood.…”