The β2 adrenergic receptor cross-linked interactome identifies 14-3-3 proteins as regulating the availability of...

The emerging picture of G protein-coupled receptor function suggests that the global signaling response is an integrated sum of a multitude of individual receptor responses, each regulated by their local protein environment. The β2 adrenergic receptor (B2AR) has long served as an example receptor in the development of this model. However, the mechanism and the identity of the protein-protein interactions that govern the availability of receptors competent for signaling remain incompletely characterized. To address this question, we characterized the interactome of agonist-stimulated B2AR in human embryonic kidney 293 cells using FLAG coimmunoprecipitation coupled to stable isotope labeling by amino acids in cell culture and mass spectrometry. Our B2AR cross-linked interactome identified 190 high-confidence proteins, including almost all known interacting proteins and 6 out of 7 isoforms of the 14-3-3 family of scaffolding proteins. Inhibiting 14-3-3 proteins with the peptide difopein enhanced isoproterenol-stimulated adrenergic signaling via cAMP approximately 3-fold and increased both miniGs and arrestin recruitment to B2AR more than 2-fold each, without noticeably changing EC₅₀ with respect to cAMP signaling or effector recruitment upon stimulation. Our results show that 14-3-3 proteins negatively regulate downstream signaling by inhibiting access of B2AR to effector proteins. We propose that 14-3-3 proteins maintain a dynamic pool of B2AR that has reduced signaling efficacy in response to acute agonist stimulation, limiting the number of signaling-competent receptors at the plasma membrane. SIGNIFICANCE STATEMENT: This study presents a new interactome of the agonist-stimulated β2 adrenergic receptor, a paradigmatic G protein-coupled receptor that is both a model system for members of this class and an important signaling protein in respiratory, cardiovascular, and metabolic regulation. We identify 14-3-3 proteins as responsible for restricting β2 adrenergic receptor access to signaling effectors and maintaining a receptor population that is insensitive to acute stimulation by agonists.