Cell surface receptors coupled to the heterotrimeric GTP-binding proteins are universally responsible for the transmembrane transmission of extracellular messengers such as hormones, neurotransmitters and a variety of sensory stimuli. Because of this direct involvement in the regulation of the most crucial cellular functions, G-protein coupled receptors (GPCRs) are among the most important targets of therapeutic intervention. It's estimated that about 50% of drugs in use act on GPCRs. Thus, understanding of the receptor and the G- protein functions at the molecular level is among the highest priorities of public health research. Several competing models aim at describing the universal mechanism of G- protein activation by GPCRs, but none has presented compelling and conclusive experimental evidence so far. HYPOTHESIS: G-protein 23-subunit complex is a key molecular switch at the center of the gear-shift model of G-protein activation. We will test this hypothesis using the prototypical GPCR rhodopsin (R) and the G-protein transducin (Gt) responsible for phototransduction in retinal rod cells as a model system. Three interconnected Specific Aims will test various aspects of the hypothesis, such as questions of the molecular organization of the receptor- G-protein complex, the high-resolution picture of the receptor-G-protein interface, the mechanism of signal transfer from the receptor, and the roles of individual G- protein subunits, especially the G23 subunit complex, in this dynamic process. This project aims at understanding the universal principles underlying cell- to-cell communications and cellular responses to a variety of sensory stimuli. Several competing theories describing these basic molecular mechanisms will be tested to gain insights into the inner workings of cell surface receptor proteins and specific protein-protein interactions. Because almost half of all therapeutics on the market today target these signaling pathways, knowledge obtained as a result of these studies will be essential in new drug design and fighting a wide range of diseases such as heart problems, asthma and vision disorders. ? ? ?
