Impact of metabotropic glutamate receptor heteromerization on signaling and pharmacology

点击翻译按钮获取中文摘要

Glutamate, the major excitatory neurotransmitter in the brain, acts at eight metabotropic glutamate (mGlu) subtypes, expressed in a partially overlapping fashion in distinct brain circuits. Recent evidence indicates that specific mGlu receptor protomers can heterodimerize and exhibit dramatically different pharmacology when compared to their homomeric counterparts. These findings open important possibilities for more selective ligand modulation of mGlu receptor biology in the brain. To define mGlu heterodimer-specific pharmacology, we developed a technique termed CODA-RET (Complemented Donor Acceptor-Resonance Energy Transfer), in which only a defined dimeric pair of receptors induces a signal upon receptor activation. CODA-RET can be used in either homodimer or heterodimer mode, such that activating ligands with differential pharmacology at homo- vs. heterodimers can be revealed. We have used CODA-RET in the context of an mGlu2/4 heteromer to demonstrate that an mGlu2 negative allosteric modulator (NAM) can block the response to an mGlu4-specific agonist across the dimer interface. Furthermore, we showed that, while one structural class of mGlu4 positive allosteric modulators (PAMs) can potentiate both mGlu4/4 homomers and mGlu2/4 heterodimers, another class of mGlu4 PAMs potentiates only mGlu4/4 homomers. We used these pharmacological tools to identify input-specific pharmacology in specific neuronal circuits, with effects consistent with mGlu2/4 heterodimers at corticostriatal and thalamocortical synapses, versus mGlu4/4 receptors at striatopallidal synapses, providing an opportunity for more precise pharmacological intervention. In mice, evoked excitatory currents and induction of long-term potentiation (LTP) at Schaffer Collateral-CA1 (SC-CA1) synapses in the hippocampus are blocked by the selective mGlu7 negative allosteric modulator (NAM), ADX71743. In contrast, the mGlu7 NAM MMPIP, with a similar profile to ADX71743 in heterologous cells expressing mGlu7 homomers, fails to block these responses in brain slices. We hypothesized that this might result from heteromerization of mGlu7 with another mGlu receptor protomer. Since an mGlu8 pharmacology has also been reported at SC-CA1 synapses, we used CODA-RET to study mGlu7/8 heterodimers in vitro and found that ADX71743 blocks responses of both mGlu7/7 homodimers and mGlu7/8 heterodimers, whereas MMPIP only antagonizes responses at mGlu7/7 homodimers. Using these compounds, as well as genetic knockout of mGlu8, we will test the hypothesis that a receptor with a pharmacological signature consistent with an mGlu7/8 heterodimer is functionally expressed in the hippocampus. Our aims are: 1) To define the pharmacological profiles of mGlu7 allosteric ligands at glutamatergic and GABAergic synapses in area CA1 of the hippocampus. 2) To test the hypothesis that mGlu7/8 heterodimers lead to the MMPIP-insensitive phenotype in SC-CA1 responses. 3) To elucidate the activation mechanism and signaling properties of mGlu7/7 homodimers, mGlu8/8 homodimers, and mGlu7/8 heterodimers using CODA-RET.