Development and Characterization of Pepducins as Gs-biased Allosteric Agonists*

The β 2 -adrenergic receptor (β 2 AR) is a prototypical G protein-coupled receptor that mediates many hormonal responses, including cardiovascular and pulmonary function. β-Agonists used to combat hypercontractility in airway smooth muscle stimulate β 2 AR-dependent cAMP production that ultimately promotes airway relaxation. Chronic stimulation of the β 2 AR 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. β 2 AR desensitization to β-agonists is primarily mediated by G protein-coupled receptor kinases and β-arrestins that attenuate receptor-G s coupling and promote β 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 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 β 2 AR, 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 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 β 2 AR 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 G s -biased activator of the β 2 AR and provides valuable tools for the study of β 2 AR function.A G s -biased agonist for the β 2 -adrenergic receptor (β 2 AR) has yet to be reported. Results A screen of β 2 AR pepducins identified receptor-dependent and receptor-independent pepducins that selectively activate G s . Conclusion Receptor-dependent pepducins promote a G s -biased conformation of the β 2 AR, whereas receptor-independent pepducins directly activate G s . Significance G s -biased pepducins provide a valuable tool for the continued study of β 2 AR function and may prove useful as next-generation asthma therapeutics.

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