Abstract
Abstract: :
Purpose: Biochemical, structural and functional characterization of a novel Ca2+–modulated cyclic GMP signaling pathway in the inner retinal neurons. Methods:Protein purification, MALDI–MS, Western blotting, Immunohistochemical analyses, surface plasmon resonance spectroscopy and in vitro reconstitution experiments in conjunction with enzyme assays were employed. Results:Neurocalcin delta was purified from the inner plexiform layer of the retina. MALDI–MS analyses showed that the native neurocalcin d is myristoylated. The myristoylated form of neurocalcin delta migrates from the cytoplasm to the membranes in the presence of submicromolar levels of Ca2+. At the IPL membrane, it modulates ROS–GC1 – the cyclase previously characterized from the photoreceptor outer segments. ROS–GC1 and neurocalcin delta reside in close proximity in IPL membranes as demonstrated by cross–linking. The myr–neurocalcin delta binds to its specific ROS–GC1 domain and stimulates it in a Ca2+–dependent fashion (EC50= 0.8 µM). Binding of myr–neurocalcin delta to ROS–GC1 occurs with KD of 426 nM. Neurocalcin delta is not present in outer segments. It is co–localized with ROS–GC1 in defined regions of the IPL and a sub–population of ganglion cells. Conclusions: The neurocalcin delta:ROS–GC1 signaling pathway is absent in the outer segments, is dissociated from phototransduction and is linked specifically with the visual transduction machinery in the IPL and ganglion cells of the retina.
Keywords: calcium • signal transduction • synapse