G protein-coupled receptor (GPCR) drug development has traditionally focused on the concepts of orthosteric agonism and antagonism, in which receptor structure determines the nature of the downstream signal and ligand efficacy determines its intensity. Over the past decade, the newer paradigms of 'pluridimensional efficacy'and 'functional selectivity'have established that GPCRs mediate biological effects by engaging both classical G protein-dependent and novel G protein-independent signaling networks, and that ligand structure can 'bias'signaling by stabilizing active receptor states in different proportions than the native ligand. Such 'biased agonists'are novel pharmacological entities with the ability to qualitatively change GPCR signaling, in effect creating 'new receptors with distinct efficacy profiles driven by ligand structure. The promise of biased agonism resides with this ability to engender 'mixed'effects, and work during the current term of this project has demonstrated that arrestin-pathway selective agonism can produce potentially beneficial effects in vivo that are not attainable with conventional agonists or antagonists. At the same time, our data suggest that activating GPCRs in 'unnatural'ways can lead to unpredictable downstream biological consequences, especially when using ligands that selectively activate as-yet poorly characterized arrestin-dependent signaling networks. Efforts to develop biased therapeutics are thus hampered by an inability to predict the in vivo actions of arrestin-biased ligands based on their in vitro efficacy. The central hypothesis of this proposal is that the in vivo consequences arrestin-dependent signaling arises from activation of a relatively discrete and conserved set of biological responses that can be profiled in vitro using cell-based assays that are amenable to drug discovery research. During the next term, we plan to test this hypothesis using state-of-the-art biophysical and biochemical approaches combined with in vivo characterization of biased ligand action. Using intramolecular FlAsH BRET sensors that monitor conformational changes in arrestins following receptor activation, Aim 1 will examine the factors that determine the arrestin 'conformational signature'and determine the relationship between arrestin conformation and receptor/ligand efficacy in vitro. Using multi-dimensional efficacy profiling and SILAC-based whole cell phosphoproteomic analysis of signaling networks, Aim 2 will determine the range of biased ligand effects attainable in vitro and test whether the arrestin-dependent signaling network is conserved between different GPCRs.
Aim 3 will then 'close the loop'by determining the relationship between arrestin conformation, in vitro ligand efficacy, and the biological phenotype associated with in vivo exposure to arrestin-selective biased agonists. These experiments will employ biased PTH1 parathyroid hormone receptor ligands in a murine model of bone formation. Completion of this project will help establish a rational framework for relating in vitro ligand efficacy to the in vivo actions of arrestin pathway-selective biased GPCR ligands, providing information and tools that are critical to the development of novel biased therapeutics.

Public Health Relevance

The capacity of arrestin pathway-selective biased agonists to qualitatively change G protein-coupled receptor (GPCR) signaling holds the promise of new drugs that exploit ligand 'bias'to improve clinical effectiveness. This project will employ state-f-the-art state biophysical and biochemical approaches combined with in vivo characterization of biased ligand action to test the hypothesis that the in vivo consequences arrestin- dependent signaling arise from activation of a relatively discrete and conserved set of biological responses that can be captured in vitro using cell-based assays that are amenable to drug discovery research.

National Institute of Health (NIH)
Research Project (R01)
Project #
Application #
Study Section
Molecular and Integrative Signal Transduction Study Section (MIST)
Program Officer
Abraham, Kristin M
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Medical University of South Carolina
Internal Medicine/Medicine
Schools of Medicine
United States
Zip Code
Kendall, Ryan T; Lee, Mi-Hye; Pleasant, Dorea L et al. (2014) Arrestin-dependent angiotensin AT1 receptor signaling regulates Akt and mTor-mediated protein synthesis. J Biol Chem 289:26155-66
Leonard, Anthony P; Appleton, Kathryn M; Luttrell, Louis M et al. (2013) A high-content, live-cell, and real-time approach to the quantitation of ligand-induced *-Arrestin2 and Class A/Class B GPCR mobilization. Microsc Microanal 19:150-70
Appleton, Kathryn M; Lee, Mi-Hye; Alele, Christian et al. (2013) Biasing the parathyroid hormone receptor: relating in vitro ligand efficacy to in vivo biological activity. Methods Enzymol 522:229-62
Kendall, Ryan T; Strungs, Erik G; Rachidi, Saleh M et al. (2011) The beta-arrestin pathway-selective type 1A angiotensin receptor (AT1A) agonist [Sar1,Ile4,Ile8]angiotensin II regulates a robust G protein-independent signaling network. J Biol Chem 286:19880-91
Dey, Mamon; Baldys, Aleksander; Sumter, Dezmond B et al. (2010) Bradykinin decreases podocyte permeability through ADAM17-dependent epidermal growth factor receptor activation and zonula occludens-1 rearrangement. J Pharmacol Exp Ther 334:775-83
Wang, Liyun; Martin, Bronwen; Brenneman, Randall et al. (2009) Allosteric modulators of g protein-coupled receptors: future therapeutics for complex physiological disorders. J Pharmacol Exp Ther 331:340-8
Lee, Mi-Hye; Klein, Richard L; El-Shewy, Hesham M et al. (2008) The adiponectin receptors AdipoR1 and AdipoR2 activate ERK1/2 through a Src/Ras-dependent pathway and stimulate cell growth. Biochemistry 47:11682-92
Fan, Hongkuan; Luttrell, Louis M; Tempel, George E et al. (2007) Beta-arrestins 1 and 2 differentially regulate LPS-induced signaling and pro-inflammatory gene expression. Mol Immunol 44:3092-9
Jafri, Farahdiba; El-Shewy, Hesham M; Lee, Mi-Hye et al. (2006) Constitutive ERK1/2 activation by a chimeric neurokinin 1 receptor-beta-arrestin1 fusion protein. Probing the composition and function of the G protein-coupled receptor "signalsome". J Biol Chem 281:19346-57
Gooz, Monika; Gooz, Pal; Luttrell, Louis M et al. (2006) 5-HT2A receptor induces ERK phosphorylation and proliferation through ADAM-17 tumor necrosis factor-alpha-converting enzyme (TACE) activation and heparin-bound epidermal growth factor-like growth factor (HB-EGF) shedding in mesangial cells. J Biol Chem 281:21004-12

Showing the most recent 10 out of 31 publications