Development and Characterization of Pepducins as Gs-biased Allosteric Agonists

• Published in: The Journal of biological chemistry Vol. 289; no. 52; pp. 35668 - 35684
• Main Authors: Carr, Richard, Du, Yang, Quoyer, Julie, Panettieri, Reynold A., Janz, Jay M., Bouvier, Michel, Kobilka, Brian K., Benovic, Jeffrey L.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 26.12.2014; American Society for Biochemistry and Molecular Biology
• Subjects: Adrenergic beta-Agonists - pharmacology; Adrenergic Receptor; Allosteric Regulation; Amino Acid Sequence; Asthma; Biased Agonist; Cyclic AMP - biosynthesis; Drug Discovery; Functional Selectivity; G Protein-coupled Receptor (GPCR); GTP-Binding Protein alpha Subunits, Gs - metabolism; HEK293 Cells; Humans; Molecular Sequence Data; Pepducin; Peptide Fragments - chemistry; Peptide Fragments - pharmacology; Peptide Library; Peptides; Phosphorylation; Protein Binding; Protein Processing, Post-Translational; Protein Structure, Tertiary; Protein Transport; Receptors, Adrenergic, beta-2 - chemistry; Receptors, Adrenergic, beta-2 - metabolism; Second Messenger Systems; Signal Transduction; β2-Adrenergic Receptor; Functional Selectivity; Peptides; Signal Transduction; Adrenergic Receptor; Pepducin; β2-Adrenergic Receptor; Biased Agonist; Drug Discovery; G Protein-coupled Receptor (GPCR); Asthma
• ISSN: 0021-9258; 1083-351X; 1083-351X
• DOI: 10.1074/jbc.M114.618819

The β2-adrenergic receptor (β2AR) 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 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 Gs 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 Gs-biased pepducins, receptor-independent and receptor-dependent, as well as several β-arrestin-biased pepducins. The receptor-independent Gs-biased pepducins operate by directly stimulating G protein activation. In contrast, receptor-dependent Gs-biased pepducins appear to stabilize a Gs-biased conformation of the β2AR that couples to Gs 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 Gs-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.A Gs-biased agonist for the β2-adrenergic receptor (β2AR) has yet to be reported. A screen of β2AR pepducins identified receptor-dependent and receptor-independent pepducins that selectively activate Gs. Receptor-dependent pepducins promote a Gs-biased conformation of the β2AR, whereas receptor-independent pepducins directly activate Gs. Gs-biased pepducins provide a valuable tool for the continued study of β2AR function and may prove useful as next-generation asthma therapeutics.