Date of Award


Document Type


Degree Name

Doctor of Philosophy (PhD)


Molecular and Cellular Biology and Pathobiology


College of Graduate Studies

First Advisor

Louis M. Luttrell

Second Advisor

Lauren E. Ball

Third Advisor

Joe B. Blumer

Fourth Advisor

Michael G. Janech

Fifth Advisor

Donald C. Menick


Arrestins are cytosolic G protein-coupled receptor (GPCR) binding proteins that regulate several facets of GPCR signaling. Once bound to agonist-occupied receptors, arrestins recruit elements of the clathrin-dependent endocytic machinery, resulting in removal of GPCRs from the plasma membrane. The fate of internalized receptors is determined by the stability of the GPCR-arrestin complex, which is itself dictated by several factors, including ligand structure, receptor structure, and arrestin post-translational modifications. We hypothesized that information about ligand and receptor structure is encoded in the conformation of the intracellular domains of an activated receptor and transferred allosterically to receptor-bound arrestin to dictate which of its many cellular functions it will perform. To test this hypothesis we developed a panel of arrestin3 intramolecular FlAsH BRET biosensors that allow detection of conformational shifts between the arrestin N-terminus and six positions within the protein. Measuring the effect of receptor activation on arrestin conformation generates an arrestin3 ‘conformational signature’ in a live cell, real time, multiwell plate format. Using a panel of structurally distinct angiotensin type 1A receptor (AT1AR) ligands, we show that GPCR-arrestin complex avidity correlates directly with the ligand-induced Δ Net BRET of an arrestin3 FlAsH-BRET sensor located within the arrestin3 C-terminal globular domain. We further hypothesized that perturbation of arrestin3 post-translational modifications that influence complex stability would similarly be reflected by loss of conformational shifts of arrestin characteristic of stable complex formation. Ubiquitination of arrestin3 at Lysines 11 and 12 is necessary to stabilize complexes with the AT1AR, but not the vasopressin type 2 receptors (V2R). We found that introduction of an arrestin3 K11/12R mutation, which changes the AT1AR-arrestin interaction from stable to transient, reduced the arrestin3 C-terminal FlAsH-BRET shift produced by AT1AR, but not by the V2R, whose trafficking is unaffected by the mutation. We further tested the impact of the K11/12R mutation on two previously unstudied receptors, the bradykinin type 2 receptor (B2R) and the type 1 parathyroid hormone receptor (PTH1R). Mutation resulted in loss arrestin3 FlAsH-BRET signal induced by B2R, but not PTH1R. Examination of arrestin trafficking by confocal microscopy demonstrated that the K11/12R mutation altered B2R, but not PTH1R, trafficking. We conclude that activation-induced changes in arrestin3 conformation, observable through intramolecular FlAsHBRET, reflect the impact of ligand structure and post translational-modification on its intracellular functions. Biophysical probes such as these, which predict the function of intracellular signaling proteins upon receptor activation, may have application in drug discovery efforts to identify “biased” ligands that tailor GPCR efficacy to elicit specific downstream signaling events.


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