Sadler, Fredrik2024-06-052024-06-052023-04https://hdl.handle.net/11299/263704University of Minnesota Ph.D. dissertation. April 2023. Major: Biochemistry, Molecular Bio, and Biophysics. Advisor: Sivaraj Sivaramakrishnan. 1 computer file (PDF); ix, 138 pages.The third intracellular loop (ICL3) of the G protein-coupled receptor (GPCR) fold is important for the signal transduction process downstream of receptor activation. Despite this, ICL3’s lack of defined structure, combined with its high sequence divergence among GPCRs, obfuscates characterization of its involvement in receptor signaling. Previous studies focusing on the β2 adrenergic receptor (β2AR) suggest that ICL3 is involved in the structural process of receptor activation and signaling. We derive mechanistic insights into ICL3s role in β2AR signaling, finding that ICL3 autoregulates receptor activity through a dynamic conformational equilibrium between states that block or expose the receptor’s G protein binding site. We demonstrate the importance of this equilibrium for receptor pharmacology, finding that G protein-mimetic effectors bias ICL3’s exposed states to allosterically activate the receptor. Our findings additionally reveal that ICL3 tunes signaling specificity by inhibiting receptor coupling to G protein subtypes that weakly couple to the receptor. Despite the sequence diversity of ICL3, we demonstrate that this negative G protein selection mechanism through ICL3 extends to GPCRs across the superfamily, expanding upon the framework for how receptors mediate G protein subtype selective signaling. Furthermore, our collective findings motivate ICL3 as an allosteric site for receptor and signaling pathway specific ligands.enallosteric modulatorFRETintrinsic disorderprotein engineeringreceptorThesis or Dissertation