The objective of our research is to examine the molecular basis of photoreception and transduction in photoreceptor cells. In this five year plan, we propose experiments addressing the following two specific aims: 1. In vitro expression and mutagenesis of recombinant DNA fragments encoding the molecular photoreceptor rhodopsin will be applied to examine its structure and activity. Expression in both E. coli and mammalian cells is proposed. Rhodopsin and the corresponding mutant receptors will be examined for properties of structural folding, regulation of absorption wavelength, and functional interactions of the cytoplasmic domains with components of the cGMP cascade, e.g. G-protein, opsin kinase, and 48K-protein. 2. Techniques for isolation and characterization of two photoreceptor plasma membrane macromolecules, the Na+/Ca++ exchanger and the cGMP/light-dependent ion channel, that control cell signaling are outlined. We propose to identify and characterize the genes and mRNA encoding each of the molecules, to identify the predicted protein structure from genetic information obtained, and eventually to use in vitro expression and mutagenesis to study their structure and functional activities. Our long term research objective is to develop recombinant DNA techniques to supplement traditional biochemical isolation/characterization methods for study of proteins functional in visual transduction. As noted in aim 2, such techniques would enable us to study proteins that are important for visual function but present in only few copies per cell. In addition, we wish to initiate studies that will enable us to study the expression of genes encoding visual transduction proteins in the retina. These studies contribute basic information about macromolecules in photoreceptors that are somehow affected by genetic defects in retinitis pigmentosa or retinoblastomas.
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