Project Grant R35GM154813

MOLECULAR MECHANISMS OF EPH RECEPTOR SIGNALING - PROJECT SUMMARY THE SURVIVAL OF EVERY MULTICELLULAR ORGANISM RELIES ON EFFECTIVE CELL-CELL COMMUNICATION. DESPITE THE DIVERSITY OF SIGNALS THAT CELLS NEED TO COMMUNICATE TO THEIR NEIGHBORS, EVOLUTION APPEARS TO HAVE SETTLED ON ONLY A FEW PHYSICAL MECHANISMS FOR TRANSFERRING INFORMATION ACROSS MEMBRANES. ONE SUCH MECHANISM IS THE OLIGOMERIZATION, OR CLUSTERING, OF MEMBRANE-SPANNING RECEPTOR PROTEINS, WHEREIN THE RECEIPT OF A SIGNAL ON ONE SIDE OF THE MEMBRANE IS CONVERTED INTO A BIOCHEMICAL RESPONSE ON THE OTHER SIDE. THE MECHANISMS AND REGULATION OF OLIGOMERIC TRANSITIONS REMAIN POORLY UNDERSTOOD FOR MOST FAMILIES OF RECEPTOR PROTEINS DUE TO THE INHERENT EXPERIMENTAL DIFFICULTY OF DETECTING AND PROBING THESE SUBTLE TRANSITIONS. MY LAB IS INTERESTED IN HARNESSING THE LATEST DEVELOPMENTS IN OPTOGENETICS, SINGLE-MOLECULE MICROSCOPY, GENOME EDITING, AND HIGH- THROUGHPUT SEQUENCING TO DISSECT THE RECEPTOR CLUSTERING EVENTS THAT ALLOW CELLS TO "TALK" TO EACH OTHER IN A SYSTEMATIC AND QUANTITATIVE WAY. THE PROPOSED RESEARCH PROGRAM WILL TACKLE THE ACTIVATION AND REGULATION OF EPH RECEPTORS IN HUMAN CELLS. EPH RECEPTORS CONSTITUTE THE LARGEST KNOWN FAMILY OF RECEPTOR TYROSINE KINASES (RTK'S) AND ARE LINKED TO A BROAD RANGE OF BIOLOGICAL PROCESSES IN BOTH HEALTH AND DISEASE, RANGING FROM TISSUE PATTERNING AND EMBRYONIC DEVELOPMENT TO NEURODEGENERATION AND CANCER. THE MECHANISTIC DETAILS BEHIND EPH RECEPTOR ACTIVATION REMAIN OBSCURE, IN LARGE PART BECAUSE OF THE LARGE NUMBER OF RECEPTOR TYPES THAT ARE OFTEN EXPRESSED IN A SINGLE CELL AND THEIR APPARENT ABILITY TO FROM BOTH HOMO- AND HETERO-OLIGOMERS OF VARYING STOICHIOMETRIES. OVER THE NEXT FIVE YEARS, WE WILL DEVELOP A COMBINED APPROACH FOR PRECISELY MANIPULATING AND DETECTING HOMO- AND HETERO-OLIGOMERIZATION OF EPH RECEPTORS, ALLOWING US TO DIRECTLY DETERMINE THE EFFECTS OF CLUSTER SIZE AND COMPOSITION ON THE DOWNSTREAM SIGNALING OUTCOMES. THIS APPROACH WILL ALSO ALLOW US TO REVEAL THE CRYPTIC ROLES OF HETERODIMERS BETWEEN EPH RECEPTORS OF DIFFERENT TYPES, INCLUDING THE TWO CATALYTICALLY INACTIVE MEMBERS OF THE FAMILY. LIGHT-INDUCIBLE OPTOGENETIC CLUSTERING IN LIVING CELLS WILL BE CROSS-VALIDATED WITH IN VITRO ENZYMOLOGY AND DIRECT OBSERVATION OF CLUSTERING IN SINGLE-MOLECULE TRACKING EXPERIMENTS, ENSURING THAT WE ARE FAITHFULLY RECAPITULATING THE BIOLOGICALLY RELEVANT OLIGOMERIC TRANSITIONS. IN ADDITION TO ITS IMMEDIATE SIGNIFICANCE TO THE EPH RECEPTOR FIELD, THE PROPOSED WORK WILL DEVELOP A BIOPHYSICAL FRAMEWORK FOR ANALYZING THE SIGNALING OF OTHER RECEPTOR FAMILIES AT THE CELL-CELL INTERFACE. FINALLY, THE SPATIALLY DEFINED AND NON-INVASIVE NATURE OF LIGHT MEANS THAT DEVELOPMENT OF OPTICALLY CONTROLLED RECEPTORS WILL SERVE AS A POWERFUL TOOL FOR THE STUDY OF CELL-CELL SIGNALING AT THE ORGANOID OR ORGANISMAL LEVEL.