The ultimate aim of this research proposal is to decode the molecular and biochemical definition of the recovery-adaptation process of phototransduction and of a new process probably linked with the synaptic transmission of the retina. The foundation of the project is the cloning the native rod outer segment membrane guanylate cyclase (ROS-GC), the enzyme pivotal to both the transduction processes of vision and also to the retinal neuronal synaptic activity. Recent evidence indicates that the enzyme exhibits these two activities by acting as a double-sensor of Ca/2+. It has two switches. One switch inhabits and the other switch stimulates the enzymes at micromolar concentrations of Ca/2+. The inhibitory switch is linked to phototransduction and the stimulatory switch to retinal synaptic activity. The major objective is to elucidate the most basic molecular mechanisms involved in the inhibitory and stimulatory switching components of ROS-GC. Recent evidences indicates that the intracellular segment of ROS-GC is modular in nature and the modules are specific for diverse Ca/2+ signals linked to the two processes. Signals related to both processes are mediated by the Ca/2+ binding proteins: GCAP1 and GCAP2 in phototransduction and S100beta in synaptic transmission. Domains of three distinct modules of ROS-GC involved in GCAPs and S100beta stimulation of ROS-GC have been localized and termed Ca/2+-regulated modules (CRMs): CRM1 is linked to GCAP1 signaling. CRM2 to S100beta and CRM3 to GCAP2. A second form of ROS-GC, ROS-GC2, has been cloned from the retina, and is also Ca/2+ modulated, suggesting its linkage to phototransduction. Neither its linkage to synaptic transduction nor its modular nature has been established.
Specific Aims (1-5) as stated below are to determine: (1) sequence motifs of CRM1, CRM2 and CRM3 which act as """"""""turn on"""""""" switches for GCAP1, GCAP2 and S100beta; (2) the Ca/2+-induced folding changes in the GCAPs and S100beta sequence motifs critical in ROS-GC; (3) sequence motifs of GCAP1, GCAP2 and S100beta needed to interact with CRM1, CRM2 and CRM3; (4) the mechanisms of ROS-GC2 activation by GCAPs, 1 and 2; and (5) if ROS-GC2 is also modulated by Ca/2+ in a stimulatory fashion. If it is, determine its mechanism of modulation. The combined tools of biochemistry, genetic remodeling, computer simulation-molecular modeling, and immunology will be used to accomplish these most basic goals, which have direct ramifications in understanding the molecular processes related to vision and retinal neurological disorders.
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