SIGNIFICANCE: G protein-coupled receptors (GPCRs) are the largest class of membrane proteins in the human genome and are implicated in a wide array of cardiovascular and respiratory diseases. Notably, GPCRs are the targets of one third of FDA approved therapeutics, including drugs prescribed for coronary heart disease, hypertension, and asthma. However, of the ~800 known GPCRs, ~120 are orphan receptors (oGPCRs), which are considered druggable targets but remain understudied in basic research efforts as they lack a known ligand. Over the past two decades, extensive efforts to deorphanize oGPCRs have resulted in little turnout. It is evident there is a need to increase the rate of discovery of ligands for oGPCRs. This proposal outlines a yeast directed evolution platform for systematic, high-throughput discovery of antibody-based ligands for oGPCRs. Deorphanizing a single oGPCR alone would open the door to understanding new biology and development of a potential therapeutic agent. BACKGROUND: oGPCR deorphanization efforts have been historically slow and low-throughput. Conventional screens for oGPCR ligands can handle many targets, but due to the sheer amount of possibilities, including protons, photons, lipids, hormones, peptides, and larger proteins, success has been limited. Nanobodies, or small antibody fragments, offer distinct advantages for ligand screening, namely their suitability to directed evolution. Directed evolution allows for billions of nanobodies to be screened for binding and modulation of oGPCRs with a simple growth selection. Using a yeast-based hypermutation directed evolution platform, and a precise, scalable, high-throughput continuous culture device, directed evolution campaigns can be scaled up to ensure a deep search of nanobody sequence space. SYNOPSIS OF AIMS: In this proposal are two related, but not interdependent aims. AIM1 establishes a systematic workflow for the functional reconstitution of human GPCRs in yeast, which will (1) create a library of yeast strains with orphan and non-orphan GPCRs, and (2) elucidate general guidelines to increase success rates of GPCR expression in yeast. AIM2 takes oGPCRs found to be functional in yeast and uses them to run directed evolution campaigns to discover nanobody ligands and validate these ligands in human cells. Both aims can be developed in parallel, with results from AIM1 being progressively integrated into AIM2. Together, these innovative platforms will lead to the deorphanization of many oGPCRs, thereby enabling the exploration of new GPCR biology and pharmacology.