UCLA

In this dissertation, I describe the development and application of a multiplexed method for high-throughput screening of receptor-ligand interactions. Such interactions underpin our cells’ ability to sense and respond to their environment and represent a primary venue for therapeutic intervention. By leveraging advancements in DNA synthesis, genome editing, and next-generation sequencing, we have built a platform to measure the activity of a mixed population of receptors through RNA-seq of barcoded genetic reporters. We demonstrate the utility of the method for large-scale identification of chemical-receptor interactions and biochemical characterization of receptor function.

First, small molecules can interact with many biological targets in an organism, and uncovering these relationships is critical for modulating their function. Mammalian olfactory receptors (ORs), a large family of G protein-coupled receptors (GPCRs), mediate the sense of smell through activation by odorant small molecules. Each OR can respond to many odorants, and vice versa, making exploring this space one interaction at a time difficult. We used the platform to screen chemicals against a multiplexed library of ORs. We screened three concentrations of 181 odorants, where in each well we record the activity of 39 ORs simultaneously, and identified 79 novel associations, including ligands for 15 orphan receptors.

Second, GPCRs are ubiquitous throughout mammalian biology. They are conformationally dynamic which is essential to their function, but makes them recalcitrant to many techniques of structural determination. Here, we mutagenize and characterize all 7,828 possible missense variants of the beta-2-adrenergic receptor. On a broad scale, we find positions that respond similarly to mutation share certain properties of their environment and functional role within the protein. We recapitulate the importance of known critical residues and motifs and identify new residues important for function. Additionally, we describe an unreported, conserved extracellular motif maintained in both the inactive and active conformation of the protein that is essential for function.

As a whole, multiplexed screening enables the investigation of many outstanding questions in receptor biology. It is applicable to the disparate biological niches and systems that receptors occupy. As demonstrated in this dissertation, it has the potential to be a powerful tool for mapping receptor-ligand interactions and understanding receptor biochemistry.