Barth, Patrick DanielKéri, Dániel2021-04-082021-04-082021-04-08202110.5075/epfl-thesis-10631https://infoscience.epfl.ch/handle/20.500.14299/177044G protein-coupled receptors (GPCRs) are 7-transmembrane alpha-helical integral membrane proteins on which cells heavily rely to receive information regarding their external environment. These receptors are able to transfer information to intracellular downstream effectors upon stimulation from their cognate ligands, which can be considerably diverse. They can include light, small molecules, peptides, protons; Due to its evolutionary success, over millions of years of evolution, this protein family has expanded to include over 800 members. Despite the large size of the family and the diverse inputs they accept, the structure and the mechanism for relaying signals to their downstream partners, G proteins and B-arrestins, remains largely the same. As these receptors are involved in a great deal of biological processes such as sight (rhodopsin), cognition (dopamine, serotonin, opioid receptors), immunity (chemokine receptors), heart function (adrenergic, angiotensin receptors), etc.; a great deal of effort has gone into understanding their structure and function. Our efforts have gone into understanding their signaling properties and rationally modifying them. Most proteins are fairly flexible entities and their structures tend to oscillate and move around, sampling a number of conformations. Nature has taken advantage of these natural motions to regulate protein function from distant binding sites, in a phenomenon called allostery. GPCRs are unique in the sense that their primary function relies on allostery. By tweaking the allosteric signaling of GPCRs, we hope to have a better understand allostery, use the principles we learn to create designer GPCR biosensors with the desired sensitivity, and to eventually create de novo allosteric proteins. In this work, I present our progress in this effort, which has been significant. We have created a highly accurate in silico method to allosterically alter relative stabilities of the active and inactive states of the GPCR dopamine D2 receptor, but also to independently control its allosteric signaling strength. With our methodology we have been able to create receptors which have high basal activities by selectively stabilizing the active state of the receptor. This has already facilitated the structure determination of the active, G protein-bound dopamine D2 receptor; a first for this GPCR. Additionally, we have created a number of dopamine D2 variants with roughly 100 times higher sensitivity to dopamine than the WT receptor. These efforts continue to characterize the functional effects of these allosteric mutations. Furthermore, we are also working on a collaboration to rewire the signaling pathway of the adenosine A2A receptor. Adenosine is a common immunosuppresant in solid tumor micro-environments (TMEs). By rewiring the downstream signaling of the A2A receptor, we hope to reverse the response of T-cells in these TMEs to activate and attack the solid tumor.enG protein-coupled receptorGPCRG proteinallosteryprotein designbiosensorCAR T-cellimmunotherapyRosettabiased signalingthesis::doctoral thesis