Extracellular Application of CRMP2 Increases Cytoplasmic Calcium through NMDA Receptors.

Collapsin Response Mediator Protein 2 (CRMP2) is an intracellular protein involved in axon and dendrite growth and specification. In this study, CRMP2 was identified in a conditioned media derived from degenerated sciatic nerves (CM). On cultured rat hippocampal neurons, acute extracellular application of CM or partially purified recombinant CRMP2 produced an increase in cytoplasmic calcium. The increase in cytoplasmic calcium was mostly mediated through NMDA receptors, with a minor contribution of N-type VDCC, and it was maintained as long as CM was present. By using live-labeling of CRMP2, Ca2+ channel binding domain 3 (CBD3) peptide derived from CRMP2, and recombinant CRMP2, we demonstrated that that this effect was mediated by an action on the extracellular side of the NMDA receptor. This is the first report of an extracellular action of CRMP2. Prolonged exposure to extracellular CRMP2, may contribute to neuronal calcium dysregulation and neuronal damage.

The gene product of a Trypanosoma equiperdum ortholog of the cAMP-dependent protein kinase regulatory subunit is a monomeric protein that is not capable of binding cyclic nucleotides.

The full gene sequence encoding for the Trypanosoma equiperdum ortholog of the cAMP-dependent protein kinase (PKA) regulatory (R) subunits was cloned. A poly-His tagged construct was generated [TeqR-like(His)8], and the protein was expressed in bacteria and purified to homogeneity. The size of the purified TeqR-like(His)8 was determined to be ∼57,000 Da by molecular exclusion chromatography indicating that the parasite protein is a monomer. Limited proteolysis with various proteases showed that the T. equiperdum R-like protein possesses a hinge region very susceptible to proteolysis. The recombinant TeqR-like(His)8 did not bind either [3H] cAMP or [3H] cGMP up to concentrations of 0.40 and 0.65 µM, respectively, and neither the parasite protein nor its proteolytically generated carboxy-terminal large fragments were capable of binding to a cAMP-Sepharose affinity column. Bioinformatics analyses predicted that the carboxy-terminal region of the trypanosomal R-like protein appears to fold similarly to the analogous region of all known PKA R subunits. However, the protein amino-terminal portion seems to be unrelated and shows homology with proteins that contained Leu-rich repeats, a folding motif that is particularly appropriate for protein-protein interactions. In addition, the three-dimensional structure of the T. equiperdum protein was modeled using the crystal structure of the bovine PKA RIα subunit as template. Molecular docking experiments predicted critical changes in the environment of the two putative nucleotide binding clefts of the parasite protein, and the resulting binding energy differences support the lack of cyclic nucleotide binding in the trypanosomal R-like protein.