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Regulation of β2-adrenergic receptor function by conformationally selective single-domain intrabodies.

Publication ,  Journal Article
Staus, DP; Wingler, LM; Strachan, RT; Rasmussen, SGF; Pardon, E; Ahn, S; Steyaert, J; Kobilka, BK; Lefkowitz, RJ
Published in: Mol Pharmacol
March 2014

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.

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Published In

Mol Pharmacol

DOI

EISSN

1521-0111

Publication Date

March 2014

Volume

85

Issue

3

Start / End Page

472 / 481

Location

United States

Related Subject Headings

  • Single-Domain Antibodies
  • Sequence Alignment
  • Receptors, G-Protein-Coupled
  • Receptors, Adrenergic, beta-2
  • Protein Binding
  • Phosphorylation
  • Pharmacology & Pharmacy
  • Molecular Sequence Data
  • Humans
  • HEK293 Cells
 

Citation

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Staus, D. P., Wingler, L. M., Strachan, R. T., Rasmussen, S. G. F., Pardon, E., Ahn, S., … Lefkowitz, R. J. (2014). Regulation of β2-adrenergic receptor function by conformationally selective single-domain intrabodies. Mol Pharmacol, 85(3), 472–481. https://doi.org/10.1124/mol.113.089516
Staus, Dean P., Laura M. Wingler, Ryan T. Strachan, Soren G. F. Rasmussen, Els Pardon, Seungkirl Ahn, Jan Steyaert, Brian K. Kobilka, and Robert J. Lefkowitz. “Regulation of β2-adrenergic receptor function by conformationally selective single-domain intrabodies.Mol Pharmacol 85, no. 3 (March 2014): 472–81. https://doi.org/10.1124/mol.113.089516.
Staus DP, Wingler LM, Strachan RT, Rasmussen SGF, Pardon E, Ahn S, et al. Regulation of β2-adrenergic receptor function by conformationally selective single-domain intrabodies. Mol Pharmacol. 2014 Mar;85(3):472–81.
Staus, Dean P., et al. “Regulation of β2-adrenergic receptor function by conformationally selective single-domain intrabodies.Mol Pharmacol, vol. 85, no. 3, Mar. 2014, pp. 472–81. Pubmed, doi:10.1124/mol.113.089516.
Staus DP, Wingler LM, Strachan RT, Rasmussen SGF, Pardon E, Ahn S, Steyaert J, Kobilka BK, Lefkowitz RJ. Regulation of β2-adrenergic receptor function by conformationally selective single-domain intrabodies. Mol Pharmacol. 2014 Mar;85(3):472–481.
Journal cover image

Published In

Mol Pharmacol

DOI

EISSN

1521-0111

Publication Date

March 2014

Volume

85

Issue

3

Start / End Page

472 / 481

Location

United States

Related Subject Headings

  • Single-Domain Antibodies
  • Sequence Alignment
  • Receptors, G-Protein-Coupled
  • Receptors, Adrenergic, beta-2
  • Protein Binding
  • Phosphorylation
  • Pharmacology & Pharmacy
  • Molecular Sequence Data
  • Humans
  • HEK293 Cells