Jeanne Hirsch - US grants

Signal transduction is an important biological process by which information from extracellular stimuli is communicated to cells, resulting in changes in cellular physiology. The long-term objective of this proposal is a description at the molecular level of the mechanisms involved in transmission of the signal generated by pheromone in the yeast mating response. Yeast genetics will be used to elucidate the roles of genes thought to be involved in this process and to identify new genes through suppressor analysis. Haploid yeast cells respond to mating pheromones via two cell type- specific receptors linked to a heterotrimeric G protein. In yeast, unlike most other systems, the betagamma complex of the G protein appears to propagate the signal after release from the alpha subunit due to interaction with occupied receptor. The molecular events that take place downstream of the G protein are not well understood and constitute an important area for study. A specific aim of this proposal is to investigate the function of a gene that was isolated as a multicopy suppressor of a temperature sensitive Gbeta subunit, as well as the function of a closely-related gene. These genes form an essential redundant set because one or the other of them is required for cell viability. Analysis of the expression of these genes and of the defect that occurs when they are not transcribed will be undertaken. Constructs containing an inducible version of each gene will be created for this purpose and will also be used to isolate suppressors that overcome the lethality caused by the lack of both gene products. Alleles of the suppressor gene that are specifically defective in mating will be isolated as well. A second aim is to identify proteins that interact with the Gbeta subunits, a genetic technique for the isolation of interacting gene products. A final aim is the characterization of a receptor-mediated desensitization pathway that blocks transmission of the signal downstream of the Galpha subunit. Mutations will be isolated both in regions of the receptor gene that are involved in this function and in other genes that act downstream in the desensitization pathway. Information gained from these studies will provide insights into mammalian G protein-mediated signalling pathways which control the response to a number of diverse stimuli, including hormones, neurotransmitters, catecholamines, and light.

Cell growth is regulated by signal transduction pathways that respond to extracellular stimuli. In yeast, the signal that promotes growth is thought to be the availability of nutrients. Nutritional signaling involves the activation of Ras proteins and adenylyl cyclase through a process that is not well understood. Results presented in this proposal demonstrate that nutritional signaling in yeast also involves the activation of a G protein alpha-subunit by a G protein-coupled receptor. Several observations suggest that this pathway is activated in response to nitrogen starvation. For example, strains lacking the receptor or the G protein are defective for pseudohyphal growth, a developmental switch that is induced by nitrogen starvation. The long-term objective of this project is a description of the mechanisms employed by the G protein-mediated pathway to signal in response to nutritional conditions. Classical genetics and protein biochemistry will be used to elucidate the roles of genes and their encoded proteins in this process. The aims of this project are to characterize and identify the ligand for a newly described G protein-coupled receptor and to determine when the receptor is physiologically active. The first specific aim will investigate the conditions under which the Gpr1p receptor is bound to its ligand. These studies will be performed by coupling the Gpr1p receptor to the pheromone response pathway and using a well-established reporter construct that detects activation of the pathway. The second specific aim will employ the Gpr1p reporter system to characterize and identify the Gpr1p ligand. Identification of such a ligand would be one of the first examples of a receptor-binding molecule that activates a nutritional signaling pathway in a eukaryotic organism. The final specific aim will characterize the regulation of Gpr1p receptor expression and stability. Investigation of this novel signaling system will contribute to an understanding of nutritional signaling in eukaryotes, an area in which many important questions have not yet been addressed. Significant progress in elucidating signal transduction mechanisms has been made in organisms in which sophisticated genetic manipulations can easily be performed. A complete understanding of growth control in yeast is likely to provide insight into growth control in mammals, which is also regulated by signaling through G proteins and Ras. Such information is essential for understanding the process of carcinogenesis, in which cells escape this regulation and exhibit uncontrolled growth.

Signal transduction pathways that act through heterotrimeric G proteins mediate the response to a wide variety of extracellular signals in many different organisms. In yeast, the pheromone response pathway is activated by the binding of extracellular pheromones to G protein- coupled receptors that are specific to cells of either the MATa or MATalpha mating type. Activation of the G protein alpha-subunit results in release of the beta-gamma-subunits, which transmit the signal to downstream kinases. Studies described in this proposal will investigate a novel function of Ste3p, the a-factor receptor. This novel function, called receptor inhibition, causes a block in signal transmission by inhibiting the activity of the beta-subunit in a manner that is independent of the alpha-subunit. Receptor inhibition only occurs when cells contain both a- specific and alpha-specific proteins, and probably functions immediately after the fusion of two mating cells to ensure that the signaling pathway is rapidly turned off. This process requires the product of a newly identified a-specific gene called ASG7. Thus, when the same cell expresses both Asg7p, an a-specific protein, and Ste3p, an alpha-specific protein, a process is initiated that acts directly on the beta-subunit and inhibits its activity. This project will investigate how beta-subunit signaling is inhibited when the a-factor receptor and the novel regulator Asg7p are present in the same cell. One specific aim is to determine how the activity of the beta- subunit is blocked by this process. The effect of changes in Asg7p abundance, localization, and binding to the beta-subunit will be tested for their effects on beta-subunit activity. A screen for other genes required for receptor inhibition will also be carried out. A second specific aim is to determine how Asg7p is activated by the presence of the Ste3p receptor. The potential roles of membrane localization, posttranslational modification, and Ste3p binding in the activation of As97p will be determined. A final specific aim is to investigate the structure/function relationships of Ste3p with respect to its receptor inhibition function. By isolating specific mutations in STE3 and by constructing chimeric receptors, the region of Ste3p involved in receptor inhibition will be identified. Significant progress in elucidating signal transduction mechanisms has been made in organisms in which sophisticated genetic manipulations can easily be performed. Elucidation of the process of receptor inhibition in yeast is likely to provide information about the regulation of G protein beta-subunits in other systems in which the beta-subunit plays an active role in signal transmission.

Pseudohyphal and invasive growth in the yeast Saccharomyces cerevisiae require a signaling pathway that is mediated by the G protein beta-subunit Gpa2p and its coupled receptor Gpr1. Gpr1p is a low affinity glucose receptor that responds to high concentrations of glucose in the environment. Transmission of a signal from Gpr1p to Gpa2p results in phenotypes associated with high levels of intracellular cAMP. We have recently identified KRH1 and KRH2 as genes that encode components of the Gpa2p signaling pathway. We have shown that Krh1p and Krh2p act downstream of adenylyl cyclase to inhibit protein kinase A (PKA) by a process that does not involve production of intracellular cAMP. Activation of Gpa2p is thought to relieve the inhibition of PKA by Krh1p and Krh2p, resulting in high levels of PKA activity. The long-term objectives of this project are: 1) To obtain a complete description of the molecular processes that comprise the Gpa2p signal transduction pathway; 2) To understand the biological function of the Gpa2p pathway in terms of an entire cell population undergoing pseudohyphal growth. The first specific aim of this project is to determine how Krh1p and Krh2p regulate PKA. These studies will investigate whether binding of Krh1p and Krh2p to PKA is direct and whether binding inhibits PKA kinase activity. The second specific aim is to determine whether Krh1p and Krh2p control signaling by affecting the localization of PKA. The third specific aim is to investigate whether the GTP-bound form of Gpa2p blocks the inhibitory function of Krh1p and Krh2p by determining the effects of non-activatable and constitutive alleles of GPA2 on Krh1p and Krh2p function. The fourth specific aim is to test whether Gpa2p is specifically activated in cells at the edge of a growing colony in order to suppress stress and starvation responses and to promote growth and pseudohyphal development. The goal of this aim is to develop a reporter system to allow detection of activated Gpa2p at the level of a whole colony undergoing pseudohyphal growth. These studies will provide new information about G protein-mediated signaling pathways, which are essential for the proper functioning of many physiological processes in humans.