The proposed work seeks to define the structural requirements of a Ca2+-dependent GCAP that are essential for activation of the photoreceptor-specific GC during the recovery process that follows visual excitation. Mutations will be introduced into GCAP and the mutant protein expressed using a baculovirus expression system. Targets for mutagenesis include: (1) a point mutation at the site of myristoylation; (2) point mutations for each of the three Ca2+ binding sites; (3) N-terminal deletion mutants which retain the myristoylation site but lack regions thought to be involved in GC activation, and; (4) C-terminal deletions. The mutants will be analyzed for their ability to activate GC in a Ca2+-dependent manner. Relevant mutants will be further characterized to determine changes in protein conformation, ability to bind Ca2+, ability to interact with membranes, kinetics of activation of GC, and their physiological effect on the recovery of the photoresponse in intact photoreceptors.
| Howes, K; Bronson, J D; Dang, Y L et al. (1998) Gene array and expression of mouse retina guanylate cyclase activating proteins 1 and 2. Invest Ophthalmol Vis Sci 39:867-75 |
