Abstract
Purpose: :
CaBP5 is a calmodulin-like calcium-binding protein that is expressed in retinal bipolar cells and in cochlear inner hair cells. We showed previously that CaBP5 suppressed calcium-dependent inactivation of Cav1.2. However, CaBP5 knockout mice have a mild phenotype that is a ~50% reduction of sensitivity of retinal ganglion cell light responses. The purpose of this study is to identify other interacting proteins of CaBP5 in the retina that would help to understand how CaBP5 participates in the normal transmission of light signals throughout the bipolar cells.
Methods: :
Potential interacting partners for CaBP5 were identified using in vivo screening of a retina cDNA library with the yeast two-hybrid system and in vitro biochemical approaches. Mass spectrometry was used to identify CaBP5 interacting proteins eluted from a CaBP5 affinity column loaded with a bovine retinal extract. Candidates were further analyzed for colocalization in transfected cells and in the mouse retina using immunohistochemistry, subcellular fractionation and coimmunoprecipitation.
Results: :
Mass spectrometry analysis of interacting partners revealed that cytoskeleton proteins (microfilaments, intermediate filaments and microtubules), motor proteins, ATPase, HSPs and proteins involved in synaptic vesicle release binds to CaBP5. From the list of candidate proteins, myosins, syntaxin3 and munc18-1 were further investigated. Munc18-1 was also identified as an interacting partner for CaBP5 in the yeast two-hybrid screening. Those candidates partially colocalize with CaBP5 in the mouse retina and transfected cells using immunohistochemistry and western blotting.
Conclusions: :
We have discovered novel interacting partners for CaBP5 that includes proteins involved in the transport and release of synaptic vesicles. Future studies will determine if CaBP5 is involved in synaptic vesicles trafficking.
Keywords: bipolar cells • calcium • retina