Stimulation of ERK Phosphorylation by Isoproterenol and Select β-Blockers at the β-2 Adrenergic Receptor is Dependent on Receptor Internalization

Introduction: Recently, it has been found that different ligands can have distinct efficacy profiles at a single receptor; a concept known as functional selectivity. The β1 and β2 adrenergic receptors are among several that demonstrate this characteristic. This has been revealed through the study of various β-receptor ligands, specifically receptor antagonists commonly known as β-blockers, which have an inhibitory effect on the traditional G-protein mediated signaling pathway but stimulate a separate pathway that leads to activation of ERK independently of G-proteins. While the mechanism and identification of all key elements of this pathway have yet to be fully elucidated, previous studies have shown that ERK activation through ligand stimulation of the β-1 adrenergic receptor is dependent on a number of different components including Src, EGFR co-stimulation, β-arrestin, and receptor internalization. Objective: The objective of our current study is to determine and compare the signaling pathways of drug-stimulated ERK phosphorylation among various β-blockers at the β-2 adrenergic receptor by inhibiting specific signaling components, specifically focusing on diverse mechanisms of receptor internalization. Methods: HEK293 cell lines that stably expressed the wildtype β-2 adrenergic receptor (HEK-β2) were created using clonal selection with G418. The activation of both cAMP accumulation and ERK phosphorylation due to stimulation of different β-2 ligands, including isoproterenol and various β-blockers, were measured using a radioactive binding assay and Western blotting technique, respectively. PP2, a Src inhibitor, and AG1478, an EGFR inhibitor, were introduced to determine if these components were required for drug-stimulated ERK phosphorylation with the agents being studied. Several inhibitors of receptor internalization with unique mechanisms were also introduced to further define the role of internalization in these two pathways. Conclusions: Preliminary results have shown that drug-stimulated ERK phosphorylation through the β-2 adrenergic receptor for isoproterenol and several β-blockersis at least partially dependent on Src, activation of EGFR, and receptor internalization