Development and characterization of pepducins as Gs-biased allosteric agonists

The β₂-adrenergic receptor (β₂AR) is a prototypical G pro-tein- coupled receptor that mediates many hormonal responses, including cardiovascular and pulmonary function. β-Agonists used to combat hypercontractility in airway smooth muscle stimulate β₂AR-dependent cAMP production that ultimately promotes airway relaxation. Chronic stimulation of the β₂AR by long acting β-agonists used in the treatment of asthma can promote attenuated responsiveness to agonists and an increased frequency of fatal asthmatic attacks. β₂AR desensitization to β-agonists is primarily mediated by G protein-coupled receptor kinases and β-arrestins that attenuate receptor-Gs coupling and promote β₂AR internalization and degradation. A biased agonist that can selectively stimulate G_s signaling without promoting receptor interaction with G protein-coupled receptor kinases and β-arrestins should serve as an advantageous asthma therapeutic. To identify such molecules, we screened ∼50 lipidated peptides derived from the intracellular loops of the β₂AR, known as pepducins. This screen revealed two classes of G_s- biased pepducins, receptor-independent and receptor-dependent, as well as several β-arrestin-biased pepducins. The recep-tor- independent G_s-biased pepducins operate by directly stimulating G protein activation. In contrast, receptor-dependentG _s-biased pepducins appear to stabilize aGs-biased conformation of the β₂AR that couples to G_s but does not undergo G protein-coupled receptor kinase-mediated phosphorylation or β-arrestin-mediated internalization. Functional studies in primary human airway smooth muscle cells demonstrate that G_s- biased pepducins are not subject to conventional desensitization and thus may be good candidates for the development of next generation asthma therapeutics. Our study reports the first Gs-biased activator of the β₂AR and provides valuable tools for the study of β₂AR function.