Regulation ofβ₂-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 b 2 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 b 2 -adrenergic receptor (b 2 AR). Here, we set out to study the functional interaction of 18 related nanobodies with the b 2 AR 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 b 2 AR-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, b-arrestin recruitment, and receptor internalization to varying extents. These functional effects were likely due to either steric blockade of downstream effector (G s , b-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.

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

“…Although ICL3-9 appears to be the first reported G s -biased activator of the ␤ 2 AR, Staus et al (55) previously reported stimulation of biased signaling from the ␤ 2 AR using intracellularly expressed nanobodies (intrabodies). In this report, intrabodies against agonist-activated or inactive ␤ 2 AR were selective for inhibiting G protein activation or GRK and ␤-arrestin engagement.…”

Outward-facing conformers of LacY stabilized by nanobodies

“…Nbs represent a unique type of single-domain antibodies with flexible antigen-binding loops containing CDR3, which is able to insert into clefts and cavities of membrane proteins and stabilize specific conformers (23)(24)(25)(26)(27). Therefore, Nbs were prepared against LacY mutant G46W/G262W, which is in an outward-open conformation, anticipating that such Nbs would interact with epitopes within the open periplasmic cavity to stabilize outward-facing conformers of WT LacY.…”

“…In this regard, remarkable progress has been made with G protein-coupled receptors through the use of camelid single-domain nanobodies (Nbs), which stabilize specific conformers (23)(24)(25)(26)(27). Advantages of Nbs include small size and a unique structure that allows flexible antigen-binding loops to insert into clefts and cavities.…”

Crystal structure of a LacY–nanobody complex in a periplasmic-open conformation

“…To characterize intermediates in the LacY transport cycle, stable conformers are essential, particularly with respect to crystallization, and remarkable progress has been made in this direction with G protein-coupled receptors through the use of camelid single-domain nanobodies (Nbs) (25)(26)(27)(28)(29). Nbs are small in size and have a unique structure that allows flexible loops with complementarity-determining regions (CDRs) to insert into nooks and crannies.…”