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Principal Investigator: Sivaraj Sivaramakrishnan
Organization: UNIVERSITY OF MINNESOTA
Fiscal Year: 2024
Award: $477,751
Funding agency: National Institute of General Medical Sciences
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.
Terms: <Actin-Activated ATPase><Address><Assay><Autoregulation><Binding><Bioassay><Biochemical><Biological Agent><Biological Assay><Biological Mimetics><Biological Products><Biomimetics><Biophysics><Biosensor><Cell Body><Cell Communication and Signaling><Cell Function><Cell Physiology><Cell Process><Cell Signaling><Cell Surface Receptors><Cell membrane><Cells><Cellular Function><Cellular Matrix><Cellular Physiology><Cellular Process><Cellular biology><Chimera><Chimera organism><Collaborations><Communication><Coupling><Crowding><Cytoplasmic Membrane><Cytoskeletal System><Cytoskeleton><Diabetes Mellitus><Disease><Disorder><Engineering><Extracellular Signal-Regulated Kinase Gene><G Protein-Complex Receptor><G Protein-Coupled Receptor Genes><G Protein-Coupled Receptor Signaling><G-Protein-Coupled Receptors><G-Proteins><GP330 Antigen><GPCR><GPCR Signaling><GTP-Binding Proteins><GTP-Regulatory Proteins><Glycoprotein 330><Guanine Nucleotide Coupling Protein><Guanine Nucleotide Regulatory Proteins><Heart failure><Heyman's Nephritis Antigen GP330><Heymann Nephritis Antigen GP330><Homeostasis><Integrins><Integrins Extracellular Matrix><Intracellular Communication and Signaling><Intracellular Second Messenger><Isoforms><Kinetics><LDL-Receptor Related Protein 2><LRP-2><LRP-2 Receptor><Ligands><Locomotor Activity><Low-Density Lipoprotein Receptor-Related Protein-2><MAP Kinase Gene><MAPK><Maps><Measures><Mechanics><Mediating><Megalin><Membrane Protein Traffic><Membrane Traffic><Mitogen-Activated Protein Kinase Gene><Molecular><Molecular Configuration><Molecular Conformation><Molecular Interaction><Molecular Stereochemistry><Motility><Motor><Motor Activity><Myosin ATPase><Myosin Adenosine Triphosphatase><Myosin Adenosinetriphosphatase><Myosins><Nephritis Antigen GP 330><Pathway interactions><Peptides><Permeability><Physiological Homeostasis><Plasma Membrane><Protein Engineering><Protein Isoforms><R-Series Research Projects><R01 Mechanism><R01 Program><Receptor Activation><Receptor Protein><Regulation><Renal Glycoprotein GP330><Research><Research Grants><Research Project Grants><Research Projects><Role><Second Messenger Systems><Second Messengers><Shapes><Signal Transduction><Signal Transduction Systems><Signaling><Structure><Subcellular Process><Tail><Technology><Xenopus B2 antigen><biological sensor><biological signal transduction><biologics><biopharmaceutical><biophysical foundation><biophysical principles><biophysical sciences><biotherapeutic agent><cardiac failure><cell biology><chimeras><combat><conformation><conformational><conformational state><conformationally><conformations><cytosolic receptor><diabetes><experiment><experimental research><experimental study><experiments><genetic approach><genetic protein engineering><genetic strategy><gp 330><innovate><innovation><innovative><insight><intracellular skeleton><mechanic><mechanical><nanobodies><nanobody><neuropsychiatric disease><neuropsychiatric disorder><new drug treatments><new drugs><new pharmacological therapeutic><new technology><new therapeutic approach><new therapeutic intervention><new therapeutic strategies><new therapeutics><new therapy><new therapy approaches><new treatment approach><new treatment strategy><next generation therapeutics><novel><novel drug treatments><novel drugs><novel pharmaco-therapeutic><novel pharmacological therapeutic><novel technologies><novel therapeutic approach><novel therapeutic intervention><novel therapeutic strategies><novel therapeutics><novel therapy><novel therapy approach><optogenetics><pathway><peptide mimetic><peptide mimic><peptidomimetics><plasmalemma><plexin><programs><protein design><protein protein interaction><receptor><scaffold><scaffolding><sdAb><signal transduction second messengers><single domain antibodies><social role><stem><trafficking>