G protein coupled receptors (GPCRs) are a universally conserved signalling mechanism by which extracellular signals can be transduced across the plasma membrane. They make up ~3% of the human genome and ~50% of all non-antimicrobial therapeutics act upon GPCRs or their pathways. Upon binding of agonist, structural changes activate the GPCR, and allow the GPCR to bind and induce nucleotide exchange upon the alpha subunit of the heterotrimeric G protein complex. Rhodopsin represents perhaps the best understood GPCR for both GPCR activation by ligand and for the consequent binding and induction of nucleotide exchange upon the G alpha subunit of its cognate G protein, transducin. Furthermore, rhodopsin and the rhodopsin: transducin complex has been used as a prototypical GPCR to model the process of activation in general among all GPCRs. While structures of both the ground state and several photo intermediate states of rhodopsin have been solved through a variety of structural techniques, a significant gap exists in our knowledge. In order to fully understand the process of activation in rhodopsin as well we need to determine the precise molecular interactions that are responsible for (1) the process of rhodopsin going from the ground (dark) state to the fully active (Meta II) state and (2) the structural determinants for transducin binding and nucleotide exchange and activation. A high resolution structure of the Meta II activated state is needed to fully understand the sequence of events that comprises rhodopsin activation. Similarly, a large volume of biochemical and biophysical studies on transducin and G protein activation have been conducted, but without a structure of this complex, it is difficult to understand the underlying molecular basis for binding and nucleotide exchange. This proposal seeks to determine the fundamental changes in rhodopsin structure that accompany its transition to the activated Meta II state. Furthermore, we will determine the structure of the complex between rhodopsin and transducin, which will elucidate the molecular mechanisms of G protein binding and activation.
Structural studies of the molecular interactions that allow the sensing of light by the protein, rhodopsin, will greatly increase our knowledge of how this system functions as well as the underlying pathologies of some blinding diseases. Furthermore, as rhodopsin is a member of the G protein coupled receptor superfamily, it will serve to explain how important physiological processes, such as sight, smell, taste;heart rate and metabolism are governed.
