Insight into the role of Ca2+-binding protein 5 in vesicle exocytosis.
OBJECTIVE
CaBP5 is a neuronal calmodulin-like Ca(2+)-binding protein that is expressed in the retina and in the cochlea. Although CaBP5 knockout mice displayed reduced sensitivity of retinal ganglion cell light responses, the function of CaBP5 in vivo is still unknown. To gain further insight into CaBP5 function, the authors screened for CaBP5-interacting partners.


METHODS
Potential retinal interacting partners for CaBP5 were identified using affinity chromatography followed by mass spectrometry and by yeast two-hybrid screening of a bovine retina cDNA library. Interacting partners were further analyzed using coimmunoprecipitation. Immunohistochemistry and subcellular fractionation were performed to determine their colocalization in the retina. The effect of CaBP5 on dopamine release and neurite outgrowth of PC12 cells was analyzed using ELISA and fluorescent labeling.


RESULTS
Using affinity chromatography, the authors identified Munc18-1 and myosin VI as interacting partners for CaBP5. Munc18-1 was also identified using the yeast two-hybrid system. Colocalization and coimmunoprecipitation of CaBP5 with these two proteins in retinal tissue further established their physiological interactions. Furthermore, CaBP5 expression in NGF-stimulated PC12 cells stimulates neurite outgrowth and dopamine exocytosis.


CONCLUSIONS
This study shows that CaBP5 interacts with Munc18-1 and myosin VI, two proteins involved in the synaptic vesicle cycle. Together with the effect of CaBP5 in stimulating neurite outgrowth and vesicle exocytosis in PC12 cells, these results suggest that CaBP5 plays a role in neurotransmitter release.
Department of Physiology and Biophysics, University of Washington, Seattle, WA 98195, USA.
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Haeseleer
D000818:Animals; D002135:Calcium-Binding Proteins / Q000502:physiology*; D002417:Cattle; D002846:Chromatography, Affinity; D004298:Dopamine / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D005089:Exocytosis / Q000502:physiology*; D019084:Fluorescent Antibody Technique, Indirect; D051379:Mice; D008810:Mice, Inbred C57BL; D051938:Munc18 Proteins / Q000378:metabolism*; D018995:Myosin Heavy Chains / Q000378:metabolism*; D016501:Neurites / Q000502:physiology; D011817:Rabbits; D012160:Retina / Q000378:metabolism*; D013347:Subcellular Fractions; D013572:Synaptic Vesicles / Q000378:metabolism*; D053719:Tandem Mass Spectrometry; D020798:Two-Hybrid System Techniques
C570016:CaBP5 protein, mouse; D002135:Calcium-Binding Proteins; D051938:Munc18 Proteins; C089785:myosin VI; D018995:Myosin Heavy Chains; D004298:Dopamine