A nanobody activating metabotropic glutamate receptor 4 discriminates between homo- and heterodimers

Abstract

There is growing interest in developing biologics due to their high target selectivity. The G protein–coupled homo- and heterodimeric metabotropic glutamate (mGlu) receptors regulate many synapses and are promising targets for the treatment of numerous brain diseases. Although subtype-selective allosteric small molecules have been reported, their effects on the recently discovered heterodimeric receptors are often not known. Here, we describe a nanobody that specifically and fully activates homodimeric human mGlu4 receptors. Molecular modeling and mutagenesis studies revealed that the nanobody acts by stabilizing the closed active state of the glutamate binding domain by interacting with both lobes. In contrast, this nanobody does not activate the heterodimeric mGlu2-4 but acts as a pure positive allosteric modulator. These data further reveal how an antibody can fully activate a class C receptor and bring further evidence that nanobodies represent an alternative way to specifically control mGlu receptor subtypes.

Distinct beta-arrestin coupling and intracellular trafficking of metabotropic glutamate receptor homo- and heterodimers

“…Ultimately, our study reveals great diversity between homo-and heterodimeric mGluR subtypes in their modes of β-arr coupling and intracellular trafficking that should pave the way for many future mechanistic and physiological studies with major clinical relevance for harnessing mGluRs as therapeutics. Beyond our focus on mGluR2, mGluR3, and mGluR8, future work should extend this analysis to other widely expressed group III subtypes, mGluR7 and mGluR4, which are both also capable of forming a variety of heterodimers (4,9,51,52,(61)(62)(63)88) and have unique trafficking properties (21,78,(89)(90)(91).…”

Accelerating GPCR Drug Discovery With Conformation-Stabilizing VHHs

“…In addition, large panels of VHHs that target extracellular epitopes of native class A (CXCR4, CXCR2, ACKR3, CX3C1, US28, CML1, APJ, OX2R, AGTR2, and OPRM), B (GCGR, GLP1R, PTH1R, and VPAC1), and C (mGluR2, CaSR, mGluR4, and mGluR5) GPCRs have been described ( Jähnichen et al, 2010 ; Huang et al, 2015 ; Peyrassol et al, 2016 ; Peyrassol et al, 2018 ; Van Hout et al, 2018 ; Koehl et al, 2019 ; Cheloha et al, 2020a ; Low et al, 2020 ; Pan et al, 2020 ; Ren et al, 2020 ; De Groof et al, 2021 ; Chen et al, 2021 ; Haubrich et al, 2021 and references in Heukers et al, 2019 ). While most of these VHHs that target extracellular epitopes of GPCRs block receptor signaling or are not demonstrated to interfere with receptor signal transduction, some of these extracellular binders are reported to ortho- or allosterically activate receptor signaling, thus inherently stabilizing active GPCR conformers ( Ma et al, 2020 ; Ren et al, 2020 ; Hong et al, 2021 ; Scholler et al, 2017 ).…”

Class A and C GPCR Dimers in Neurodegenerative Diseases

“…Aside from bivalent ligands, nanobodies, which are mostly derived from antibody fragments from the heavy chain-only antibodies of camelids, have emerged as promising alternatives due to their high target specificity [ 899 , 900 ]. Like bivalent ligands, nanobodies were also discovered in the 1980s but their utility was for long not recognized [ 901 ].…”

“…In another more recent study. nanobodies were used to modulate the activity of mGlu 4 R in the brain but not Glu 4 R heteromers with other GluRs, indicating that therapy of PD or pain could be improved through subtype-selective and blood-brain barrier permeable nanobodies [ 899 ]. While only biparatopic nanobody targeting different binding sites of the chemokine receptor CXCR2 entered phase 1 studies as potential new therapeutic for inflammation [ 906 , 907 ], nanobodies specifically targeting GPCR dimers (homo- and heterodimers) will be for sure a promising new therapeutic approach.…”