Final Report Summary - GPCR EVOLUTION (Directed evolution of class B G-protein coupled receptors) G protein-coupled receptors (GPCRs) are the largest group of integral membrane proteins in the human genome. They are involved in the transduction of a variety of external stimuli across the cellular membrane into the cell and are implicated in many physiological and pathophysiological processes in our body. As such, these molecules belong to the most important targets for drug development. Based on their sequence conservation GPCRs have been divided into five subgroups of which class B GPCRs constitute a small group of peptide hormone-binding receptors. Several of these receptors have been implicated in the pathogenesis of severe human diseases such as diabetes, osteoporosis and chronic pain which makes them attractive targets for drug discovery. For a better understanding of the underlying molecular mechanisms of signal transduction and to develop new compounds to specifically target these receptors a detailed understanding of the molecular structure is required. However, elucidation of protein structures by X-ray crystallography or NMR spectroscopy requires large quantities of pure protein. For GPCRs this prerequisite is often difficult to achieve as the vast majority of GPCRs exhibits low endogenous expression and is very unstable in solution. Moreover, these tasks are further complicated by the complex extracellular domains that are characteristic to class B GPCRs. Therefore, improved expression conditions are required for the efficient characterization of new GPCR structures and for developing new drugs targeting these receptors. We initiated this project to optimize class B GPCRs for improved heterologous expression and increased thermostability. For this purpose, specific mutations are introduced into the receptor gene which confer enhanced stability and better expression yields. To identify such mutations we use directed evolution - a combination of randomly introduced mutations and subsequent selection of the desired phenotype. Large libraries of randomized receptor genes are generated by mutagenesis and are expressed in E. coli using a system in which functional GPCR is targeted to the inner cell membrane. Mutants that display increased receptor expression levels and ligand binding are selected by flow cytometry using fluorescently-labeled ligands. Repetitive cycles of randomization and selection will allow to gradually increasing the level of protein expression and stability. With this evolutionary approach key residues within the receptor sequence can be rapidly identified that are responsible for improved biophysical properties without largely affecting the pharmacological properties of the receptor. Such GPCR mutants will be a valuable tool on the way to express high quantities of stable receptor protein for subsequent structural studies.In this project, we initially characterized the expression of all human class B GPCRs in E. coli and compared them to other expression hosts followed by binding studies using fluorescently- and radio-labeled ligands. As expression of class B GPCRs turned out to be challenging, several extensions to the intended directed evolution protocol were devised. A generic selection system was established which allows selections devoid of restrictions caused by unfavorable ligand properties. Such a system also extends applicability of the directed evolution approach to a wider range of integral membrane proteins for which no suitable fluorescent ligands are available. In addition, a new set of expression vectors was created which allow stringent tuning of protein expression on the single cell level. Such vectors may become valuable tools for all difficult to express proteins. Finally, we succeeded to evolve a set of receptor mutants which exhibited improved expression and thermostability. These variants were further characterized for ligand binding and signaling properties.Our efforts contribute several improvements for the expression and thermostabilization of integral membrane proteins by directed evolution. Foremost, difficult to express protein with unfavorable ligand properties should thereby become amenable to means of directed evolution. As such, these improved methods should be of high interest to the wide field of pharmaceutically relevant membrane proteins. Moreover, by applying these methods to class B GPRCs we were able to improve their stability and expression levels thereby making them applicable to subsequent biophysical studies.
