Structure-function studies of the yeast saccharomyces cerevisiae [alpha]-mating factor pheromone receptor ste2p

by 1970- Akal, Ayc?a

G protein–coupled receptors (GPCRs) are seven transmembrane domain cell surface proteins that respond to a variety of environmental cues. Response of these receptors to their cognate stimuli on the extracellular region of the cell results in a concurrent activation of a complex series of intracellular signaling pathways that prepare the cell for the required adjustments through regulation of gene expression levels. Participation of GPCRs in such intricate signal transduction pathways renders them important players in human diseases. The GPCR family of proteins therefore represents one of the largest classes of proteins to be targeted in the development of drug design for clinical applications. In light of the crucial role that GPCRs play in clinically important diseases, the focus of this dissertation has been on interactions between a GPCR and its ligand in a model eukaryotic organism, the budding yeast Saccharomyces cerevisiae. Very recently, the complete genome of the yeast S. cerevisiae has been sequenced. Detailed studies in this system along with the available sequence information have suggested a high conservation between the two eukaryotic organisms human and yeast. Therefore, the S. cerevisiae GPCR Ste2p and its associated pheromone ligand a–factor represent a good model system to study ligand–receptor interactions. The work presented in this dissertation describes results from a comprehensive mutagenesis approach on Ste2p aimed at determining residues of the receptor that are important in ligand binding and/or vii receptor activation. Regions of the receptor that have been the primary focus of the studies detailed in this dissertation are the first and third extracellular loops of Ste2p. Additional focus has been given to specific residues located in the transmembrane regions of Ste2p that have been predicted to interact with one another. Cys–scanning and Ala–scanning mutagenesis studies on the first extracellular loop, EL1, of Ste2p resulted in identification of a region of this loop harboring five functionally important residues that played an important role in the activation of the receptor but did not contribute to ligand binding. Structural studies on EL1 pointed to the possibility that this region of EL1 may attain a 310–helical structure in which the five functionally important residues may lie on one face of this helix. Collectively, all these studies underscored the important role of EL1 in Ste2p activation. Structure and function studies on the third extracellular loop, EL3, of Ste2p, using a Cys–scanning mutagenesis approach led to the identification of two additional residues that, upon mutation, resulted in a defective receptor. These results indicated the important role that EL3 played in the activation of the receptor–mediated signal transduction pathway. Scanning mutagenesis studies on EL1 and EL3 emphasized the importance of these loop residues in receptor structure and function. As a result, signal–deficient mutants from EL1 and EL3 were studied further to assess their functional properties after combining individual mutations rendering Ste2p defective with a constitutively activating mutation. These studies allowed identification of mutant receptors with intermediate signaling properties and intermediate conformations. Results from these viii studies, once again, underlined the importance of EL1 and EL3 residues in the activation of Ste2p, and further suggested that the activation mechanism for Ste2p followed multiple intermediate conformations. In addition to studies with the extracellular loops of Ste2p, certain residues in the transmembrane regions of the receptor became focus of this dissertation. De novo models, generated in collaboration with Dr. Nikiforovich of Washington University (St. Louis, MO), for the transmembrane regions of Ste2p proposed specific contact sites in the three–dimensional structure of Ste2p. These residues were targeted by a strategically designed mutagenesis approach to test the validity of the de novo models. Results from these studies partially corroborated predictions of these first de novo models, and provided a framework in which to incorporate the connecting loops to obtain the complete three–dimensional model for Ste2p. ix