Impact of metabotropic glutamate receptor heteromerization on signaling and pharmacology

谷氨酸是大脑中主要的兴奋性神经递质，作用于八个代谢型谷氨酸（mGlu） 亚型，在不同的脑回路中以部分重叠的方式表达。最近的证据表明， 特异性mGlu受体原聚体可以异源二聚化，并表现出显著不同的药理学， 与同体的同类相比。这些发现为更有选择性的配体提供了重要的可能性 调节脑中的mGlu受体生物学。 为了定义mGlu异二聚体特异性药理学，我们开发了一种称为CODA-RET的技术， （互补供体受体共振能量转移），其中仅限定的受体二聚体对 在受体激活时诱导信号。CODA-RET可以同二聚体或异二聚体模式使用， 从而可以揭示在同源二聚体与异源二聚体上具有不同药理学的活化配体。我们有 在mGlu 2/4异聚体的背景下使用CODA-RET来证明mGlu 2负变构 调节剂（NAM）可以阻断对mGlu 4特异性激动剂的反应穿过二聚体界面。此外，委员会认为， 我们发现，虽然一类mGlu 4正变构调节剂（PAM）可以增强这两种作用， mGlu 4/4同聚体和mGlu 2/4异二聚体，另一类mGlu 4 PAM仅增强mGlu 4/4同聚体。 我们使用这些药理学工具来识别特定神经元回路中的输入特异性药理学， 与mGlu 4/4相比，mGlu 2/4异二聚体在皮质纹状体和丘脑皮质突触上的作用一致 受体的纹状体苍白球突触，提供了一个机会，更精确的药物干预。 在小鼠中，在Schaffer Collateral-CA 1诱发兴奋性电流和长时程增强（LTP）诱导 海马中的SC-CA 1突触被选择性mGlu 7负变构调节剂（NAM）阻断， ADX71743。相比之下，mGlu 7 NAM MMPIP在异源细胞中具有与ADX 71743相似的特征， 表达mGlu 7同聚体，未能阻断脑切片中的这些反应。我们假设这可能 mGlu 7与另一mGlu受体原聚体的异聚化。由于mGlu 8药理学 在SC-CA 1突触中也有报道，我们使用CODA-RET在体外研究mGlu 7/8异二聚体，发现 ADX 71743阻断mGlu 7/7同源二聚体和mGlu 7/8异源二聚体的反应，而MMPIP 仅拮抗mGlu 7/7同源二聚体的应答。使用这些化合物，以及基因敲除 我们将检验一种假设，即具有与mGlu 7/8一致的药理学特征的受体 异二聚体在海马中功能性表达。我们的目的是：1）确定药理学 在海马CA 1区谷氨酸能和GABA能突触的mGlu 7变构配体分布图 海马体。2)为了检验mGlu 7/8异二聚体导致MMPIP不敏感性的假设， SC-CA 1反应的表型。3)为了阐明的激活机制和信号特性， 使用CODA-RET的mGlu 7/7同源二聚体、mGlu 8/8同源二聚体和mGlu 7/8异源二聚体。

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.