title: Regulation of β2-adrenergic receptor function by conformationally selective single-domain intrabodies.
abstract: The biologic activity induced by ligand binding to orthosteric or allosteric sites on a G protein-coupled receptor (GPCR) is mediated by stabilization of specific receptor conformations. In the case of the β2 adrenergic receptor, these ligands are generally small-molecule agonists or antagonists. However, a monomeric single-domain antibody (nanobody) from the Camelid family was recently found to allosterically bind and stabilize an active conformation of the β2-adrenergic receptor (β2AR). Here, we set out to study the functional interaction of 18 related nanobodies with the β2AR to investigate their roles as novel tools for studying GPCR biology. Our studies revealed several sequence-related nanobody families with preferences for active (agonist-occupied) or inactive (antagonist-occupied) receptors. Flow cytometry analysis indicates that all nanobodies bind to epitopes displayed on the intracellular receptor surface; therefore, we transiently expressed them intracellularly as "intrabodies" to test their effects on β2AR-dependent signaling. Conformational specificity was preserved after intrabody conversion as demonstrated by the ability for the intracellularly expressed nanobodies to selectively bind agonist- or antagonist-occupied receptors. When expressed as intrabodies, they inhibited G protein activation (cyclic AMP accumulation), G protein-coupled receptor kinase (GRK)-mediated receptor phosphorylation, β-arrestin recruitment, and receptor internalization to varying extents. These functional effects were likely due to either steric blockade of downstream effector (Gs, β-arrestin, GRK) interactions or stabilization of specific receptor conformations which do not support effector coupling. Together, these findings strongly implicate nanobody-derived intrabodies as novel tools to study GPCR biology.
authors: Department of Medicine (D.P.S., L.M.W., R.T.S., S.A., R.J.L.), Department of Biochemistry (R.J.L.), and Howard Hughes Medical Institute (R.J.L.), Duke University Medical Center, Durham, North Carolina; Department of Neuroscience and Pharmacology, The Panum Institute, University of Copenhagen, Copenhagen, Denmark (S.G.F.R.); Structural Biology Brussels and Structural Biology Research Institute, Vrije Universiteit Brussel, Brussels, Belgium (E.P., J.S.); and Department of Molecular and Cellular Physiology, Stanford University School of Medicine, Stanford, California (B.K.K.).
authors: Dean P
authors: DP
authors: Staus
authors: Laura M
authors: LM
authors: Wingler
authors: Ryan T
authors: RT
authors: Strachan
authors: Soren G F
authors: SG
authors: Rasmussen
authors: Els
authors: E
authors: Pardon
authors: Seungkirl
authors: S
authors: Ahn
authors: Jan
authors: J
authors: Steyaert
authors: Brian K
authors: BK
authors: Kobilka
authors: Robert J
authors: RJ
authors: Lefkowitz
mesh_terms: D000595:Amino Acid Sequence; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism; D054768:G-Protein-Coupled Receptor Kinases / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D008969:Molecular Sequence Data; D010766:Phosphorylation / Q000502:physiology; D011485:Protein Binding / Q000502:physiology; D018343:Receptors, Adrenergic, beta-2 / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism; D016415:Sequence Alignment; D061905:Single-Domain Antibodies / Q000378:metabolism*
chemical_list: D018343:Receptors, Adrenergic, beta-2; D043562:Receptors, G-Protein-Coupled; D061905:Single-Domain Antibodies; D000242:Cyclic AMP; D054768:G-Protein-Coupled Receptor Kinases