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Nanobodies to Study G Protein-Coupled Receptor Structure and Function.

ABSTRACT: Ligand-induced activation of G protein-coupled receptors (GPCRs) is a key mechanism permitting communication between cells and organs. Enormous progress has recently elucidated the structural and dynamic features of GPCR transmembrane signaling. Nanobodies, the recombinant antigen-binding fragments of camelid heavy-chain-only antibodies, have emerged as important research tools to lock GPCRs in particular conformational states. Active-state stabilizing nanobodies have elucidated several agonist-bound structures of hormone-activated GPCRs and have provided insight into the dynamic character of receptors. Nanobodies have also been used to stabilize transient GPCR transmembrane signaling complexes, yielding the first structural insights into GPCR signal transduction across the cellular membrane. Beyond their in vitro uses, nanobodies have served as conformational biosensors in living systems and have provided novel ways to modulate GPCR function. Here, we highlight several examples of how nanobodies have enabled the study of GPCR function and give insights into potential future uses of these important tools.

SUBMITTER: Manglik A

PROVIDER: S-EPMC5500200 | biostudies-literature | 2017 Jan

REPOSITORIES: biostudies-literature

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Nanobodies to Study G Protein-Coupled Receptor Structure and Function.

Manglik Aashish A Kobilka Brian K BK Steyaert Jan J

Annual review of pharmacology and toxicology 20161207

Ligand-induced activation of G protein-coupled receptors (GPCRs) is a key mechanism permitting communication between cells and organs. Enormous progress has recently elucidated the structural and dynamic features of GPCR transmembrane signaling. Nanobodies, the recombinant antigen-binding fragments of camelid heavy-chain-only antibodies, have emerged as important research tools to lock GPCRs in particular conformational states. Active-state stabilizing nanobodies have elucidated several agonist- ...[more]

PMID: 27959623

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Project description:G-protein-coupled receptors (GPCRs) modulate many physiological processes by transducing a variety of extracellular cues into intracellular responses. Ligand binding to an extracellular orthosteric pocket propagates conformational change to the receptor cytosolic region to promote binding and activation of downstream signalling effectors such as G proteins and ?-arrestins. It is well known that different agonists can share the same binding pocket but evoke unique receptor conformations leading to a wide range of downstream responses (‘efficacy’). Furthermore, increasing biophysical evidence, primarily using the ?2-adrenergic receptor (?2AR) as a model system, supports the existence of multiple active and inactive conformational states. However, how agonists with varying efficacy modulate these receptor states to initiate cellular responses is not well understood. Here we report stabilization of two distinct ?2AR conformations using single domain camelid antibodies (nanobodies)—a previously described positive allosteric nanobody (Nb80) and a newly identified negative allosteric nanobody (Nb60). We show that Nb60 stabilizes a previously unappreciated low-affinity receptor state which corresponds to one of two inactive receptor conformations as delineated by X-ray crystallography and NMR spectroscopy. We find that the agonist isoprenaline has a 15,000-fold higher affinity for ?2AR in the presence of Nb80 compared to the affinity of isoprenaline for ?2AR in the presence of Nb60, highlighting the full allosteric range of a GPCR. Assessing the binding of 17 ligands of varying efficacy to the ?2AR in the absence and presence of Nb60 or Nb80 reveals large ligand-specific effects that can only be explained using an allosteric model which assumes equilibrium amongst at least three receptor states. Agonists generally exert efficacy by stabilizing the active Nb80-stabilized receptor state (R80). In contrast, for a number of partial agonists, both stabilization of R80 and destabilization of the inactive, Nb60-bound state (R60) contribute to their ability to modulate receptor activation. These data demonstrate that ligands can initiate a wide range of cellular responses by differentially stabilizing multiple receptor states.

Dataset Information

Nanobodies to Study G Protein-Coupled Receptor Structure and Function.

Publications

Nanobodies to Study G Protein-Coupled Receptor Structure and Function.

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