Development and Characterization of Pepducins as Gs-biased Allosteric Agonists

The beta(2)-adrenergic receptor (beta(2)AR) is a prototypical G protein-coupled receptor that mediates many hormonal responses, including cardiovascular and pulmonary function. beta-Agonists used to combat hypercontractility in airway smooth muscle stimulate beta(2)AR-dependent cAMP production that ultimately promotes airway relaxation. Chronic stimulation of the beta(2)ARby long acting beta-agonists used in the treatment of asthma can promote attenuated responsiveness to agonists and an increased frequency of fatal asthmatic attacks. beta(2)AR desensitization to beta-agonists is primarily mediated by G protein-coupled receptor kinases and beta-arrestins that attenuate receptor-G(s) coupling and promote beta(2)AR internalization and degradation. A biased agonist that can selectively stimulate G(s) signaling without promoting receptor interaction with G protein-coupled receptor kinases and beta-arrestins should serve as an advantageous asthma therapeutic. To identify such molecules, we screened similar to 50 lipidated peptides derived from the intracellular loops of the beta(2)AR, known as pepducins. This screen revealed two classes of G(s)-biased pepducins, receptor-independent and receptor-dependent, as well as several beta-arrestin-biased pepducins. The receptor-independent G(s)-biased pepducins operate by directly stimulating G protein activation. In contrast, receptor-dependent G(s)-biased pepducins appear to stabilize a G(s)-biased conformation of the beta(2)AR that couples to G(s) but does not undergo G protein-coupled receptor kinase-mediated phosphorylation or beta-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 G(s)-biased activator of the beta(2)AR and provides valuable tools for the study of beta(2)AR function.