FWF - GPCR - Determinants of alpha-arrestin-mediated GPCR down-regulation

G protein-coupled receptors (GPCRs) are complex proteins located in the plasma membrane that surrounds every cell. The function of a GPCR is to act as an antenna that senses a particular extracellular stimulus and initiates an appropriate intracellular response. Use of a GPCR as a mechanism to detect an extracellular cue developed very early in evolution; hence, GPCRs are found in the plasma membrane of the cells in all eukaryotic organisms - from unicellular microbes, such as yeast, to humans. GPCRs in the human body are responsible for our senses of sight, smell, certain tastes and other perception, especially response to many hormones and other signal molecules. Medically, GPCRs are primary targets of many clinically used therapeutic agents, both activators (agonists) and inhibitors (antagonists), depending on the condition being treated. However, chronic stimulation of GPCRs can cause inflammatory diseases and certain cancers. Consequently, multiple feedback mechanisms have evolved that act to attenuate GPCRinitiated responses. Recently, it has been shown that special adapter proteins located inside the cell, called alpha-arrestins, can bind to a GPCR and stimulate withdrawal of the GPCR from the plasma membrane, thereby elegantly terminating the cellular response to an excessive stimulus. Much remains to be learned about how the alpha-arrestins operate and how they, in turn, are controlled. To examine the properties and regulation of alpha-arrestins this study will use yeast cells, which are simple, inexpensive and easy-tohandle, yet exhibit all of the relevant components found in human cells (GPCRs, alpha-arrestins, and regulatory factors). A particular kind of chemical change to an alpha-arrestin, namely attachment of phosphate groups through the action of enzymes known as protein kinases, has been shown to block the ability of an alpha-arrestins to withdraw its cognate GPCR from the plasma membrane. Hence, this project will first investigate how various cellular protein kinases act on alpha-arrestins. To understand the full range of plasma membrane proteins influenced by alpha-arrestins, the second part of this project will apply novel strategies to delineate all the interaction partners of each yeast alpha-arrestin. These studies will be conducted at UC Berkeley. Finally, the knowledge gained will be applied to human alpha-arrestins and human GPCRs in the lab in Graz. Results of this work could have important impact on the development of next-generation pharmaceuticals because recent results suggest that modulation of alpha-arrestin function might allow existing drugs to achieve greater potency and suggests routes for the development of new compounds.