Regulation of Adrenergic and Proton-Sensing Receptor Function by Protein-Protein Interactions

Abstract

G protein-coupled receptors (GPCRs) form the largest class of signaling proteins through which mammalian cells derive the ability to sense and respond to the external environment. This family includes both paradigmatic receptors like the beta 2 adrenergic receptor (B2AR) and members with comparatively unknown regulatory mechanisms, such as the proton-sensing receptor GPR65. Investigation of these receptors aids our understanding of both the mechanisms of canonical GPCR function and the applicability of these findings to less conventional receptors. In addition, both are significant regulatory proteins in physiological and pathophysiological systems, with B2AR playing major roles in cardiovascular and respiratory health and emerging interest in GPR65 for the modulation of the immune system and treatment of certain types of cancer. Here we probe the interactome of B2AR in search of new and understudied regulators of receptor function, in particular for proteins that preferentially associate with B2AR following phosphorylation by protein kinase A (PKA). Using both co- immunoprecipitation and proximity labeling approaches, we identify the scaffolding protein endofin and the CORVET and COMMANDER endosomal sorting complexes as interactors of B2AR, with the former showing preferential association with PKA- phosphorylated receptor. Our findings provide a comprehensive picture of the B2AR interactome and a useful dataset for further investigation of this important receptor. Focusing on one class of understudied interactors identified by our proteomic research, we determine that 14-3-3 proteins bind to unstimulated B2AR, contrary to their canonical function as phosphorylation-specific scaffolds. 14-3-3 proteins act to impair B2AR signaling via the second messenger cAMP as well as recruitment of β-arrestin, with xv no effect on signaling via the MAP kinase cascade nor bulk receptor trafficking. We propose that 14-3-3 proteins are responsible for maintaining a population of B2AR in a sequestered pool at the plasma membrane, limiting access to effectors and inhibiting acute signaling. We also clarify the landscape of B2AR interaction with proteins containing PDZ domains that bind to the receptor’s distal C-terminus. Using microscopy, luciferase- complementation assays, and biochemical approaches, we show that B2AR switches binding from the protein NHERF1 to SNX27 upon stimulation, but that neither of these scaffolding proteins mediate the effects on ERK phosphorylation regulated by the B2AR PDZ ligand. In addition our results indicate that, despite previous reports, the PDZ protein MAGI3 does not bind to B2AR, nor does it significantly regulate signaling via the MAP kinase pathway. Turning to our other receptor of study, we determine that canonical mechanisms of GPCR trafficking are not sufficient to explain the constitutive internalization of GPR65. GPR65 continues to internalize even in the absence of G protein-coupled receptor kinases or β-arrestins, although we find that internalization is enhanced by acidic extracellular pH. Despite not requiring canonical GRKs, the GPR65 C-terminus does undergo phosphorylation that promotes internalization, with the kinase or kinases and protein-protein interactions responsible remaining unknown. Our findings advance the field of GPCR biology from two directions: expanding our understanding of the protein-protein interactions governing function of the model receptor B2AR and applying our knowledge of canonical GPCR function to reveal non- canonical workings of the understudied receptor GPR65. These investigations identify novel regulatory mechanisms for both receptors, providing new points for modulation of these important signaling proteins.