-arrestins were initially discovered to desensitize GPCR signaling in response to agonist stimulation. However, it is now appreciated that -arrestins can also transduce multiple effector pathways independent of G-protein signaling when receptors are stimulated by certain ligands, a concept known as biased signaling. The proposed mechanism for this signaling bias is based on the barcode hypothesis where unbiased and -arrestin-biased ligands impart distinct patterns of receptor phosphorylation by specific GPCR kinases (GRKs), thus converting a distinct ligand stabilized receptor conformation into selective -arrestin function and downstream signaling. In this proposal we plan to discover new mechanistic insights for the precise molecular details that underlie -arrestin biased signaling and determine how this may influence cardiac function. We will use a mass spectrometry phosphoproteomic based approach to determine whether Carvedilol induces a unique phosphorylation bar code of the 1AR compared to the balance agonist isoproterenol, and will translate these findings in vivo by using mice conditionally lacking -arrestin in differnt cell types of the heart and determine the effect of -arrestin bias on the transcriptional signatue and cardiac function under normal and injured conditions. We propose the following specific aims:
Aim 1 : Determine the specific phosphorylation bar code on the c-terminal tail of 1AR responsible for G?i dependent -arrestin-biased signaling.
Aim 2 : Determine the mechanism of 1AR-biased signaling in cardiac fibroblasts.
Aim 3 : Determine the cardiac cell type responsible for -arrestin signaling that promotes cardiac fibrosis in response to injury.
Aim 4 : Determine th cell-type specific RNA signature of wild type and conditionally deleted -arrestin1 and 2 KO mice in response to the biased ligand carvedilol and after cardiac injury.
The central hypothesis of this proposal is that ARs can be stabilized by different ligands to adopt multiple distinct and relevant conformations that will lea to the activation of distinct intracellular signaling pathways. Our preliminary data now identifiesa role of G?i in carvedilol induced -arrestin-biased signaling for the 1AR. In this proposal we ill determine how the 1AR that is occupied by carvedilol now becomes a G?i coupled -arrestin biased receptor and its role in fibroblast growth in vitro and the cardiac fibrotic response in viv. We believe these molecular and translational studies will lead to novel discoveries in GPCR signaling and potentially new therapeutic avenues in heart failure.
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