Allosteric nanobodies uncover a role of hippocampal mGlu2 receptor homodimers in contextual fear consolidation

Abstract

Antibodies have enormous therapeutic and biotechnology potential. G protein-coupled receptors (GPCRs), the main targets in drug development, are of major interest in antibody development programs. Metabotropic glutamate receptors are dimeric GPCRs that can control synaptic activity in a multitude of ways. Here we identify llama nanobodies that specifically recognize mGlu2 receptors, among the eight subtypes of mGluR subunits. Among these nanobodies, DN10 and 13 are positive allosteric modulators (PAM) on homodimeric mGlu2, while DN10 displays also a significant partial agonist activity. DN10 and DN13 have no effect on mGlu2-3 and mGlu2-4 heterodimers. These PAMs enhance the inhibitory action of the orthosteric mGlu2/mGlu3 agonist, DCG-IV, at mossy fiber terminals in the CA3 region of hippocampal slices. DN13 also impairs contextual fear memory when injected in the CA3 region of hippocampal region. These data highlight the potential of developing antibodies with allosteric actions on GPCRs to better define their roles in vivo.

Cinacalcet corrects biased allosteric modulation of CaSR by AHH autoantibody

“…Aromatic L-amino acids have been reported to enhance calcium-stimulated signals by binding to sites associated with calcium-binding sites (60). Interestingly, Pin's group has recently reported that a nanobody interacting with the VFT domain of the mGlu2 receptor acts as its positive allosteric modulator (61). It is thus possible that our patient's autoantibodies modulate CaSR signaling by reacting with a site near to one of the residues that is important for calcium binding.…”

“…It is thus possible that our patient's autoantibodies modulate CaSR signaling by reacting with a site near to one of the residues that is important for calcium binding. It is likely that our patient's autoantibodies affect the relative movement of the VFT domains, an essential step in the activation process of this class c GPCR (61).…”

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

“…In contrast to a recent study using an antibody to deliver an irreversible photosensitizer to inactivate AMPA receptors[23] this work establishes the suitability of immunochemistry for targeting a membrane protein for reversible optical control, opening the door to the manipulation of native proteins with high spatiotemporal precision. Similarly, Scholler et al[24] recently reported allosteric nanobodies to manipulate mGluR2, but these tools also lack reversibility and were not shown to permit genetic encoding for cell-type targeting, limiting their utility for precise, dynamic manipulation of receptors. In addition, the ability of NPCs to manipulate signaling via the orthosteric binding site via PORTLs permits a more physiological receptor perturbation through a defined, native mechanism.…”

A toolbox of nanobodies developed and validated for diverse neuroscience research applications

“…Their ability to precisely target specific proteins in living cells, and/or label them in post vivo samples with a high degree of efficacy and spatial resolution make nAbs attractive for numerous biomedical research applications, including in neuroscience research (Sudhof, 2018). However, their uses in neuroscience have largely been limited to samples exogenously expressing GFP-tagged proteins [e.g., (Chamma et al, 2016;Ekstrand et al, 2014;Fang et al, 2018;Joensuu et al, 2016;Modi et al, 2018;Tang et al, 2013)], or in studies targeting proteins expressed in non-neuronal brain cells (Fang et al, 2018), although a set of recent studies have employed nAbs against neuronal targets [e.g., (Maidorn et al, 2019;Schenck et al, 2017;Scholler et al, 2017;Schoonaert et al, 2017). Mammalian brain neurons are distinguished from other cells by extreme molecular and structural complexity that is intimately linked to the array of intra-and inter-cellular signaling events that underlie brain function.…”