An Atypical Role for Collapsin Response Mediator Protein 2 (CRMP-2) in Neurotransmitter Release via Interaction with Presynaptic Voltage-gated Calcium Channels

Abstract

Collapsin response mediator proteins (CRMPs) specify axon/dendrite fate and axonal growth of neurons through protein-protein interactions. Their functions in presynaptic biology remain unknown. Here, we identify the presynaptic N-type Ca 2؉ channel (CaV2.2) as a CRMP-2-interacting protein. CRMP-2 binds directly to CaV2.2 in two regions: the channel domain I-II intracellular loop and the distal C terminus. Both proteins co-localize within presynaptic sites in hippocampal neurons. Overexpression in hippocampal neurons of a CRMP-2 protein fused to enhanced green fluorescent protein caused a significant increase in Ca 2؉ channel current density, whereas lentivirus-mediated CRMP-2 knockdown abolished this effect. Interestingly, the increase in Ca 2؉ current density was not due to a change in channel gating. Rather, cell surface biotinylation studies showed an increased number of CaV2.2 at the cell surface in CRMP-2-overexpressing neurons. These neurons also exhibited a significant increase in vesicular release in response to a depolarizing stimulus. Depolarization of CRMP-2-enhanced green fluorescent protein-overexpressing neurons elicited a significant increase in release of glutamate compared with control neurons. influx coincides with synapse formation, we sought to identify proteins that might modulate Ca 2ϩ channel behavior and transmitter release during synaptogenesis. A proteomic screen of growth cone-associated proteins (see supplemental Fig. S1) and proteins interacting with the ␣1B subunit of CaV2.2 (8) identified collapsin response mediator protein-2 (CRMP-2) as a candidate CaV2.2-interacting protein.CRMPs are intracellular phosphoproteins implicated in neuronal growth cone advance and migration (9 -13). In neurons, CRMPs are expressed in the lamellipodia and filopodia of growth cones, in the shafts of axons, and in cell bodies. They contribute to axon formation by binding to tubulin heterodimers and promoting assembly of microtubules (14). CRMP phosphorylation causes dissociation from microtubules, leading to axonal outgrowth arrest (10,15,16). Overexpression of CRMP-2 in cultured hippocampal neurons induces extra axons (12), whereas CRMP-3/4/5 have been shown to be necessary for neurite extension (10), branching (17), and growth cone formation (18). The breadth of interactions of CRMP proteins with motor proteins, kinases, enzymes, and endocytosis-exocytosis-related proteins suggests that CRMPs may serve as adaptors/scaffold molecules (10). Although neurotransmission was not affected in CRMP-1 knock-out mice (19), long term potentiation, spatial learning, and memory were affected in both CRMP-1 and CRMP-3 knock-out mice (19,20), suggesting a role for CRMPs in neurotransmission as well as synaptogenesis. As potential binding partners, CRMPs could affect neurotransmission by a physical interaction with either the synaptic machinery or CaV2.2 itself.

Cdk5‐mediated phosphorylation of CRMP‐2 enhances its interaction with CaV2.2

“…Influx of calcium via CaV2.2 is critical for transmitter release within many synapses [14][15][16]. In 2009, we reported the existence of a biochemical complex between CRMP-2 and CaV2.2 in hippocampal /cortical neurons [11]; the interaction was enhanced in an activitydependent fashion implying dynamic regulation [11]. Extensive reciprocal mapping revealed the binding to two regions within CaV2.2 and a short sequence within CRMP-2 [11,17].…”

“…Rat hippocampal and cortical neuron cultures were prepared from hippocampi or cortices dissected from postnatal day 2 (PN2) or embryonic day 19 (E19) rats, respectively, exactly as described [11].…”

“…In 2009, we reported the existence of a biochemical complex between CRMP-2 and CaV2.2 in hippocampal /cortical neurons [11]; the interaction was enhanced in an activitydependent fashion implying dynamic regulation [11]. Extensive reciprocal mapping revealed the binding to two regions within CaV2.2 and a short sequence within CRMP-2 [11,17]. The latter region, referred to as Ca 2+ channel binding domain 3 (CBD3), was sufficient to mediate the interaction as a synthetic peptide derived from this region could abrogate binding between the proteins [17].…”

“…Recent studies by our laboratory have identified CRMP-2 as a modulator of synaptic transmission through interactions with the presynaptic N-type voltage-gated Ca 2+ channel (CaV2.2) [11][12][13]. Influx of calcium via CaV2.2 is critical for transmitter release within many synapses [14][15][16].…”

Regulation of dendritic development by semaphorin 3A through novel intracellular remote signaling

“…One plausible mechanism is that Sema3A signaling may involve the interaction between CRMP2 and Cav2.2, a voltage-gated calcium channel belonging to the Cav2 family. 67,68 The Cav2 family includes channels containing a 1A , a 1B and a 1E , which mediate P/2 type, N-type, and R-type Ca 2C currents, respectively. 69 Sema3A activates Cdk5, which phosphorylates CRMP2 at Ser522.…”