Howard Hughes Medical Institute
Duke University Medical Center
Durham, North Carolina
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Research description - Structural Biology Division

A major focus of our research program is to understand the structural and mechanistic basis of biased signaling through 7TMRs. How a given receptor is able to couple to and signal selectively through one or another signaling pathway. A range of biophysical studies have provided unequivocal evidence that conformational flexibility of the 7TMRs and effectors such as beta arrestins lies at the heart of biased signaling. Using a chemical labeling approach coupled with mass spectrometry, we have recently demonstrated the existence of multiple ligand specific conformations in the beta 2 AR (Kahsai et al., 2011) which are likely to be responsible for distinct signaling outcomes. More importantly, we also found that Carvedilol, a beta arrestin biased ligand, was able to induce a unique conformation of the receptor.

Using a Bioluminescence Resonance Energy Transfer (BRET) based biosensor, we have shown that receptors occupied with biased ligands or biased receptor mutants induce distinct conformations in beta arrestin compared to unbiased ligand occupied receptor (Shukla et al., 2008). This finding in fact suggests that the relay of conformational information from the receptor to the intracellular effector beta arrestin governs the subsequent functional outcome.

We use a range of accessory proteins such as antibody fragments, nanobodies and RNA aptamers to selectively capture specific receptor and beta arrestin conformations. One of the goals is to reveal the structural differences among multiple active conformations of the receptor and beta arrestins by X-ray crystallography. Moreover, we are taking a combinatorial approach of rational design coupled with directed “in vitro” evolution to generate receptor variants with biased signaling properties. We hope that these studies will help us map the structural basis of biased signaling which in turn will help design even better biased ligands with unique therapeutic potential.

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