SNAP-tagged nanobodies enable reversible optical control of a G protein-coupled receptor via a remotely tethered photoswitchable ligand

Abstract

G protein-coupled receptors (GPCRs) mediate the transduction of extracellular signals into complex intracellular responses. Despite their ubiquitous roles in physiological processes and as drug targets for a wide range of disorders, the precise mechanisms of GPCR function at the molecular, cellular, and systems levels remain partially understood. In order to dissect the function of individual receptors subtypes with high spatiotemporal precision, various optogenetic and photopharmacological approaches have been reported that use the power of light for receptor activation and deactivation. Here, we introduce a novel and, to date, most remote way of applying photoswitchable orthogonally remotely-tethered ligands (PORTLs) by using a SNAP-tag fused nanobody. Our nanobody-photoswitch conjugates (NPCs) can be used to target a GFP-fused metabotropic glutamate receptor by either gene-free application of purified complexes or co-expression of genetically encoded nanobodies to yield robust, reversible control of agonist binding and subsequent downstream activation. By harboring and combining the selectivity and flexibility of both nanobodies and self-labelling enzymes, we set the stage for targeting endogenous receptors in vivo.

Branched Photoswitchable Tethered Ligands Enable Ultra-efficient Optical Control and Detection of G Protein-Coupled Receptors In Vivo

“…Along these lines, we recently reported nanobody-photoswitch conjugates (NPCs) consisting of a SNAP-tagged nanobody labeled with a PORTL. NPCs containing an anti-GFP nanobody are able to photoactivate GFP-tagged mGluR2, but with limited efficiency (Farrants et al, 2018). Similar to other systems tested, 2x BGAG 12 doubled the photoswitch efficiency of NPC-mediated photoswitching of mGluR2 (Figures S5E and S5F).…”

“…Whole cell patch clamp recordings from HEK293T cells were performed 24-48 hours after transfection as previously described (Farrants et al, 2018). Briefly, voltage clamp recordings at À60 mV were performed in a high potassium (120 mM) solution to enable large inward currents upon receptor activation.…”

International Union of Basic and Clinical Pharmacology. CXI. Pharmacology, Signaling, and Physiology of Metabotropic Glutamate Receptors

“…9D). Proof of concept has been established using a nanobody recognizing a green fluorescent protein fused to the N terminus of mGlu 2 receptors, allowing photocontrol of the receptor (Farrants et al, 2018).…”

Improved GPCR Ligands Based on Genetically Impossible Nanobody-Peptide Fusions

“…In one precedent for targeting GPCRs with antibody-ligand fusions, VHHGFP equipped with a SNAP-tag was linked to a photoactivatable ligand for the GPCR mGluR2 37 . This fusion was then used to activate a GFP-tagged receptor upon photoactivation of the ligand.…”

“…This fusion was then used to activate a GFP-tagged receptor upon photoactivation of the ligand. The response induced by a saturating solution of the photoactivatable VHH-ligand conjugate was ~40% as strong as that induced by a saturating solution of natural ligand and moreover required the use of a receptor-GFP fusion 37 . This precluded the use of genetically unmodified cells or animals.…”