Mechanisms Regulating B2AR Sorting to Signaling Microdomains

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PROJECT SUMMARY The beta 2 adrenergic receptor (B2AR) is a G Protein Coupled Receptor (GPCR) that plays a significant role in catecholamine signaling in the heart, especially during periods of heart failure. B2AR signaling in heart failure is incompletely understood, with contradictory data indicating both cardioprotective and deleterious impacts. It has become clear in recent years that changes in B2AR localization within the cell are major regulators of signaling and contribute to variation in response to differential stimulation at the same receptor. Once activated, B2AR can signal via the cognate G protein Gs at the cell surface and in intracellular endosomal compartments. The endosomal signaling promotes transcription of particular genes, most of which are not stimulated by B2AR activation at the plasma membrane. Gs activation by B2AR at endosomes is tightly controlled by posttranslational modifications of the B2AR C-terminal tail. Phosphorylation of serines 345 and 346 (SS345/6) on the receptor tail by protein kinase A (PKA) following agonist stimulation is required for B2AR sorting to specific tubular domains from which it can signal via Gs. PKA inhibition or mutating SS345/6 to alanine residues that cannot be phosphorylated prevents B2AR signaling from endosomes. These manipulations also increase the rate at which B2AR recycles to the plasma membrane by allowing the receptor to enter additional bulk recycling tubules which are unavailable to wild type B2AR and from which it cannot signal. The protein interactions governing this regulation are unknown. Phosphorylation of SS345/6 is also regulated by the presence of palmitoylation at B2AR cysteine 341. Abolition of palmitoylation at this site results in a significant increase in basal SS345/6 phosphorylation in the absence of agonist stimulation. This proposal tests the role of specific protein complexes in localizing B2AR to specific intracellular membrane domain, the role of phosphorylation and palmitoylation in this localization, and the functional relevance of this localization to signaling in the heart. I will use fluorescence microscopy and quantitative real-time PCR to determine the impact of candidate proteins on B2AR sorting to endosomal microdomains and signaling from these compartments in both HEK293 cells and primary cardiomyocytes. I will also use novel unbiased proximity labeling approaches to identify and quantify transient interactions of regulatory proteins with wild type, phosphorylation-deficient, and palmitoylation-deficient B2AR. The role of palmitoylation in regulating this process will be examined using functional genetics and microscopy with conformational biosensors that can identify both B2AR localization and signaling. The proposed research, by characterizing pathways with significant impact on the function of cardiomyocytes, will potentially identify new druggable targets that improve the function of the failing heart and alleviate heart failure.