Membrane proteins that function as G-protein coupled receptors play a central role in mediating fundamental processes such as vision, olfaction, taste and touch. The etiology of a number of human diseases have ben traced to mutations that either affect the folding of these proteins into the correct three dimensional structures, or affect their ability to function in signal transduction. While a wealth of biochemical evidence has defined regions of these receptors that are important for ligand binding, G-protein activation, phosphorylation, arrestin binding, and regulation by other signaling proteins, many fundamental questions remain. What is their three-dimensional structure in the membrane? What are the events that occur during their biogenesis which ensure formation of the correct three- dimensional structure? What are the critical protein conformational changes that must occur upon activation? How do disease-causing mutations affect formation of the correct structure and/or steps in signal transduction? The experiments described in this proposal are aimed at answering these questions using rhodopsin, the G-protein coupled light receptor in vertebrates and in invertebrates as a model system. the main goals for this proposal period are to: (I) investigate structural requirements of the correct membrane insertion of newly synthesized bovine rhodopsin by analysis of the topologies of mutant polypeptides with varying lengths, (ii) probe the interaction of the nascent rhodopsin polypeptides with membrane protein components of the endoplasmic reticulum machinery using site-selective photoreactive probes incorporated into the growing polypeptide chain, (iii) initiate efforts to determine the three-dimensional structures of Drosophila Rh1 rhodopsin and metarhodopsin by electron diffraction analysis of two-dimensional structures of Drosophila Rh1 rhodopsin and metarhodopsin by electron diffraction analysis of two-dimensional crystals and (iv) study the mechanism of light-driven activation od Drosophila Rh1 rhodopsin by spectroscopic and biochemical studies of rhodopsin isolated from transgenic flies. I anticipate that together, these experiments will provide molecular """"""""snapshots"""""""" of the biogenesis, structure, and mechanisms of activation of rhodopsin.
