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Functional monoclonal antibody acts as a biased agonist by inducing internalization of metabotropic glutamate receptor 7.

ABSTRACT: BACKGROUND AND PURPOSE: The mGlu(7) receptors are strategically located at the site of vesicle fusion where they modulate the release of the main excitatory and inhibitory neurotransmitters. Consequently, they are implicated in the underlying pathophysiology of CNS diseases such as epilepsy and stress-related psychiatric disorders. Here, we characterized a selective, potent and functional anti-mGlu(7) monoclonal antibody, MAB1/28, that triggers receptor internalization. EXPERIMENTAL APPROACH: MAB1/28's activity was investigated using Western blot and direct immunofluorescence on live cells, in vitro pharmacology by functional cAMP and [(35) S]-GTP? binding assays, the kinetics of IgG-induced internalization by image analysis, and the activation of the ERK1/2 by elisa. KEY RESULTS: mGlu(7) /mGlu(6) chimeric studies located the MAB1/28 binding site at the extracellular amino-terminus of mGlu(7) . MAB1/28 potently antagonized both orthosteric and allosteric agonist-induced inhibition of cAMP accumulation. The potency of the antagonistic actions was similar to the potency in triggering receptor internalization. The internalization mechanism occurred via a pertussis toxin-insensitive pathway and did not require G?(i) protein activation. MAB1/28 activated ERK1/2 with potency similar to that for receptor internalization. The requirement of a bivalent receptor binding mode for receptor internalizations suggests that MAB1/28 modulates mGlu(7) dimers. CONCLUSIONS AND IMPLICATIONS: We obtained evidence for an allosteric-biased agonist activity triggered by MAB1/28, which activates a novel IgG-mediated GPCR internalization pathway that is not utilized by small molecule, orthosteric or allosteric agonists. Thus, MAB1/28 provides an invaluable biological tool for probing mGlu(7) function and selective activation of its intracellular trafficking.

SUBMITTER: Ullmer C

PROVIDER: S-EPMC3514759 | biostudies-literature | 2012 Dec

REPOSITORIES: biostudies-literature

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Functional monoclonal antibody acts as a biased agonist by inducing internalization of metabotropic glutamate receptor 7.

Ullmer C C Zoffmann S S Bohrmann B B Matile H H Lindemann L L Flor Pj P Malherbe P P

British journal of pharmacology 20121201 7

<h4>Background and purpose</h4>The mGlu(7) receptors are strategically located at the site of vesicle fusion where they modulate the release of the main excitatory and inhibitory neurotransmitters. Consequently, they are implicated in the underlying pathophysiology of CNS diseases such as epilepsy and stress-related psychiatric disorders. Here, we characterized a selective, potent and functional anti-mGlu(7) monoclonal antibody, MAB1/28, that triggers receptor internalization.<h4>Experimental ap ...[more]

PMID: 22747985

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Project description:Allosteric modulators, that exhibit no intrinsic agonist activity, offer the advantage of spatial and temporal fine-tuning of endogenous agonist activity, allowing the potential for increased selectivity, reduced adverse effects and improved clinical outcomes. Some allosteric ligands can differentially activate and/or modulate distinct signaling pathways arising from the same receptor, phenomena referred to as 'biased agonism' and 'biased modulation'. Emerging evidence for CNS disorders with glutamatergic dysfunction suggests the metabotropic glutamate receptor subtype 5 (mGlu5) is a promising target. Current mGlu5 allosteric modulators have largely been classified based on modulation of intracellular calcium (iCa2+) responses to orthosteric agonists alone. We assessed eight mGlu5 allosteric modulators previously classified as mGlu5 PAMs or PAM-agonists representing four distinct chemotypes across multiple measures of receptor activity, to explore their potential for engendering biased agonism and/or modulation. Relative to the reference orthosteric agonist, DHPG, the eight allosteric ligands exhibited distinct biased agonism fingerprints for iCa2+ mobilization, IP1 accumulation and ERK1/2 phosphorylation in HEK293A cells stably expressing mGlu5 and in cortical neuron cultures. VU0424465, DPFE and VU0409551 displayed the most disparate biased signaling fingerprints in both HEK293A cells and cortical neurons that may account for the marked differences observed previously for these ligands in vivo. Select mGlu5 allosteric ligands also showed 'probe dependence' with respect to their cooperativity with different orthosteric agonists, as well as biased modulation for the magnitude of positive cooperativity observed. Unappreciated biased agonism and modulation may contribute to unanticipated effects (both therapeutic and adverse) when translating from recombinant systems to preclinical models. This article is part of the Special Issue entitled 'Metabotropic Glutamate Receptors, 5 years on'.

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Functional monoclonal antibody acts as a biased agonist by inducing internalization of metabotropic glutamate receptor 7.

Publications

Functional monoclonal antibody acts as a biased agonist by inducing internalization of metabotropic glutamate receptor 7.

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