Project Summary Cell signaling and membrane traffic emerge from an ensemble of dynamic, transient protein-protein interactions (PPIs) in a crowded milieu. Traditional structural and biochemical approaches are mostly limited to dissecting the function of stable, structured PPIs. To address emergent function stemming from transient PPIs, my research program develops innovative protein engineering and biophysical technologies. We investigate outstanding questions in GPCR-G protein selectivity and cell surface receptor activation of myosins. Studies will advance the fundamental cell biology of GPCRs and myosins, while delivering new therapeutic strategies to combat disease. Building on new technologies and conceptual advances from my lab, we propose five parallel research projects. (1) We discovered and characterized the temporal coupling of sequential GPCR-G protein interactions, leading to allokairic modulation of GPCR signaling. We will dissect the structural basis of allokairic modulation through the GPCR’s sequence-divergent third intracellular loop (ICL3). Using novel biosensors and receptor chimeras, we will define roles for ICL3 in autoregulation and G protein selection in closely related receptor isoforms. (2) We engineered a simple, accessible cell-free biosensor assay to measure the molecular efficacy of GPCR ligands. We will use this assay to identify and characterize receptor isoform-selective biologics, including peptides, peptide-mimetics, and nanobodies/affibodies. These biologics will serve as probes to advance the structural basis of GPCR-G protein selectivity and yield cell-permeable strategies to selectively target GPCRs. (3) We successfully integrated a computation-experiment collaboration to reveal the dynamic reshaping of GPCR cytosolic cavities underlying G protein selection. Using this strategy, we will map temporally persistent receptor- G protein interaction hot-spots across GPCRs, that encode G protein selectivity. We will dissect the structural basis of allosteric modulators through the dispersal of inter-residue communication networks within GPCRs. (4) We identified motor-cargo interaction kinetics and mechanical stiffness as two novel cellular regulatory mechanisms of cytoskeletal motors. We will use programmable biomimetic scaffolds to dissect myosin regulation through both receptor-adaptor and adaptor-motor ensembles. We focus on the impact of motor conformation and clustering triggered by diverse cell surface receptors including β1-integrin, plexin D1, and LRP2/megalin. (5) We will investigate a novel temporal bias mechanism in GPCR signaling, through receptor-mediated engagement of myosins during membrane traffic. We will characterize the differential regulation of motor activity through PDZ-binding motifs in the GPCR C-tail. We will use optogenetic/chemogenetic strategies to steer GPCR trafficking and map the temporal signaling profile through second messenger and Akt/MAPK pathways.