Abstract
The biological activity induced by ligand binding to orthosteric or allosteric sites on a 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, recently a monomeric single domain antibody (nanobody) from the Camelid family was 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 β2 adrenergic receptor 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 (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 inhibited G-protein activation (cyclic AMP accumulation), 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.
- The American Society for Pharmacology and Experimental Therapeutics