The impact of CRMP4 SUMOylation on the Cav1.2 interaction, neurite outgrowth and thermal pain sensitivity

Abstract

Collapsin response mediator protein 4 (CRMP4) is critical for neuronal development. However, whether CRMP4 could be SUMOylated and how the SUMOylation regulates the interaction with the L-type voltage-gated calcium channel (Cav1.2), neurite outgrowth, and thermal pain sensitivity remain to be elucidated. To determine the SUMOylation of CRMP4, Glutathione S-transferase (GST) -Small Ubiquitin-like Modifier 1 (-SUMO1), -SUMO2, and -SUMO3 proteins were purified for GST-pulldown. Immunofluorescence staining was performed to observe colocalization of CRMP4 and SUMOs. Coimmunoprecipitation (co-IP) was performed to assess the interaction between CRMP4 and SUMO2. GST-pulldown and co-IP were performed to verify the interaction between CRMP4 and Cav1.2. The impact of SUMOylation of CRMP4 on its interaction with Cav1.2 was determined. Then, the effect of CRMP4 SUMOylation on neurite outgrowth was observed. Whole-cell patch clamping revealed the effect of CRMP4 SUMOylation on Cav1.2 mediated calcium influx. Paw withdrawal latency was measured to assess the impact of CRMP4 SUMOylation on thermal pain sensitivity in rats. The data revealed that CRMP4 K374 is a potential site for SUMO modification. SUMO1, SUMO2, and SUMO3 can all interact with CRMP4. SUMO2 interacts with CRMP4, but not a variant of CRMP4 harboring a mutation of K374. CRMP4 and SUMO proteins colocalized in neurites, and CRMP4 deSUMOylation promoted neurite outgrowth. CRMP4 interacted with Cav1.2, and deSUMOylation of CRMP4 strengthened this interaction. CRMP4 promoted calcium influx via Cav1.2, and overexpression of CRMP4 significantly increased thermal pain sensitivity in rats, which CRMP4 deSUMOylation strengthened. In conclusion, these data demonstrate the SUMOylation of CRMP4, elucidate the impacts of SUMOylation on the interaction with Cav1.2 on neurite outgrowth and thermal pain sensitivity.

ZNF354C Mediated by DNMT1 Ameliorates Lung Ischemia-Reperfusion Oxidative Stress Injury by Reducing TFPI Promoter Methylation to Upregulate TFPI

“…Following a previous study [ 30 ], the processed HUVECs were seeded onto polylysine-coated slides in 24-well plate. After 48 h, cells were fixed with 4% paraformaldehyde and permeabilized by 0.1% Triton X100.…”

Peptide and Peptidomimetic Inhibitors Targeting the Interaction of Collapsin Response Mediator Protein 2 with the N-Type Calcium Channel for Pain Relief

“…CRMP2 is known to be sensitive to divalent cations, which affect the stability of the CRMP2 tetramer and the propensity to form heterotetramers with other CRMP isoforms which share high sequence identity (∼75% for CRMPs 1–4) and very high structural similarity. , Further, CRMP4 has also been reported to be a member of synaptic vesicle recycling complexes . In our hands, expression of eGFP-CRMP4 in neurons resulted in an increase in calcium currents that was even higher than that of eGFP-CRMP2, implicating CRMP4 as an additional regulator of calcium channels, as also suggested in another study . In fact, our earliest study actually lists the CRMP1 isoform as being found in the presynaptic Ca V 2.2 complex from rat brain synaptosomes .…”

“…188 In our hands, expression of eGFP-CRMP4 in neurons resulted in an increase in calcium currents that was even higher than that of eGFP-CRMP2, implicating CRMP4 as an additional regulator of calcium channels, 189 as also suggested in another study. 190 In fact, our earliest study actually lists the CRMP1 isoform as being found in the presynaptic Ca V 2.2 complex from rat brain synaptosomes. 85 Potentially, a heteromeric form with CRMP1 could have been identified in that analysis as later work showed that CRMP1 is not involved in regulation of Ca V 2.2 7 but can form heterotetramers with CRMP2.…”

“…195,196 This conservation suggests that CRMP2 could be involved in regulating other HVA Ca V s, and since the CBD3 region is partly conserved, 100 other CRMP isoforms may be involved in calcium channel regulation as well, as noted above for CRMP4. 189,190 The L1 and C-terminal interaction sites for CRMP2 also raise the potential for two other areas of cross-talk through Ca V 2.2, namely, GPCR modulation and splice variants. Mechanistically, GPCRs regulate Ca V 2.2 through the Gproteins.…”

“…The Ca V 2.2 Loop I–II region containing the AID helix is well-conserved in high-voltage activated (L-, N-, P/Q-, and R-type) Ca V isoforms, but it differs significantly in the low-voltage activated (T-type) Ca V s, which do not appear to associate with auxiliary subunits. , This conservation suggests that CRMP2 could be involved in regulating other HVA Ca V s, and since the CBD3 region is partly conserved, other CRMP isoforms may be involved in calcium channel regulation as well, as noted above for CRMP4. , …”