Development and characterization of pepducins as Gs-biased allosteric agonists.
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.
From the Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107.
Richard
R
Carr
the Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, California 94305.
Yang
Y
Du
the Department of Biochemistry and Institute for Research in Immunology and Cancer, Université de Montréal, Montreal, Quebec H3C 3J7, Canada.
Julie
J
Quoyer
the Department of Medicine, Pulmonary, Allergy and Critical Care Division, Airways Biology Initiative, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania 19104, and.
Reynold A
RA
Panettieri
Anchor Therapeutics, Cambridge, Massachusetts 02139.
Jay M
JM
Janz
the Department of Biochemistry and Institute for Research in Immunology and Cancer, Université de Montréal, Montreal, Quebec H3C 3J7, Canada.
Michel
M
Bouvier
the Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, California 94305.
Brian K
BK
Kobilka
From the Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, jeffrey.benovic@jefferson.edu.
Jeffrey L
JL
Benovic
D000318:Adrenergic beta-Agonists / Q000494:pharmacology*; D000494:Allosteric Regulation; D000595:Amino Acid Sequence; D000242:Cyclic AMP / Q000096:biosynthesis; D019205:GTP-Binding Protein alpha Subunits, Gs / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000494:pharmacology*; D019151:Peptide Library; D010766:Phosphorylation; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D018343:Receptors, Adrenergic, beta-2 / Q000737:chemistry / Q000378:metabolism; D015290:Second Messenger Systems
D000318:Adrenergic beta-Agonists; D010446:Peptide Fragments; D019151:Peptide Library; D018343:Receptors, Adrenergic, beta-2; D000242:Cyclic AMP; D019205:GTP-Binding Protein alpha Subunits, Gs
Adrenergic Receptor; Asthma; Biased Agonist; Drug Discovery; Functional Selectivity; G Protein-coupled Receptor (GPCR); Pepducin; Peptides; Signal Transduction; β2-Adrenergic Receptor