Identification of ligands that bind to the orphan delta2 receptor

描述(申请人提供)：谷氨酸受体家族由18个基因组成，这些基因可以结合形成数十个不同的异构体受体复合体。这类受体介导哺乳动物大脑中大多数兴奋性突触传递，并在学习和记忆的细胞模型以及广泛的神经病理中发挥核心作用。在这些基因产物中，有两个孤儿受体亚基(Delta1和Delta2)在序列同源性的基础上被指定为谷氨酸受体家族的成员，尽管还没有发现激活配体。Delta2亚单位在小脑突触中表达，多种电生理学研究表明，Delta2在小脑发育和突触可塑性中起重要作用。此外，缺乏delta2亚单位或表达该受体的构成活性突变形式(delta2-lurcher)的小鼠，表现出不同的表型和运动障碍。因此，存在一种悖论，即Delta2谷氨酸受体亚单位被认为在大脑功能中发挥重要作用，但我们对该受体的了解却存在真空。我们的合作者最近使用结晶学方法确定了一个小的氨基酸(D-丝氨酸)可以与delta2配体结合域结合，形成了一组与其他谷氨酸受体亚基上的谷氨酸结合一致的原子接触。有趣的是，尽管D-丝氨酸与Delta2亚基结合，但它不会在野生型受体中引起任何电流反应。然而，Delta2亚基中自然发生的点突变(Lurcher突变)会产生结构性活性的同源通道，对这些通道施加D-丝氨酸会抑制流经这些结构性活性通道的紧张性电流。这些数据表明，D-丝氨酸的结合可以导致门控的变化，并增加了将Delta2亚基整合到异构体受体组装中可能赋予谷氨酸受体一个D-丝氨酸的调节位点的可能性。这些新数据为探索Delta2受体的结构、功能和药理学提供了前所未有的机会。我们建议在两个实验中利用这一最新发现：目标1：识别有效激活或抑制delta2受体的新配体。目的2：评价Delta2与谷氨酸受体其他亚基的共表达是否增加了受体对D-丝氨酸的敏感性。这些研究的完成将为这一受体类别提供第一个全面的药理学评估，以及研究其在大脑中的功能和调节的新工具。与公共卫生相关：我们最近的发现D-丝氨酸可以与孤儿谷氨酸受体delta2结合，这为我们提供了一个极大地提高我们对该受体功能的理解的机会，尽管体内发现在小脑发育和突触可塑性方面具有重要作用，但该受体的功能十多年来一直是个谜。这些研究的目的是确定新的和选择性的药理制剂(激活剂和拮抗剂)，这些药剂可能是了解组织中delta2功能的有用研究工具。此外，我们还将测试Delta2亚基是否改变D-丝氨酸对异源多聚谷氨酸受体的影响。这些研究的完成将促进我们对Delta2功能的理解，Delta2功能对神经元信号传递、突触可塑性和小脑神经元发育具有重要意义。这些研究还可能为靶向delta2受体的新治疗药物提供洞察力。

DESCRIPTION (provided by applicant): The glutamate receptor family is comprised of 18 genes that can combine to form dozens of different heteromeric receptor complexes. This receptor class mediates most excitatory synaptic transmission in the mammalian brain, and plays a central role in cellular models of learning and memory as well as a wide range of neuropathologies. Of these gene products, two orphan receptor subunits (delta1 and delta2) have been designated to be in the glutamate receptor family on the basis of sequence homology even though no activating ligand has been identified. The delta2 subunit is expressed at cerebellar synapses, and a variety of electrophysiological studies suggest that delta2 plays an important role in cerebellar development and synaptic plasticity. In addition, mice that either lack the delta2 subunit or express a constitutively active mutant form of this receptor (delta2-lurcher), show distinct phenotypes with motor disturbances. Thus, a paradox exists whereby the delta2 glutamate receptor subunit is thought to play an important role in brain function yet there is a vacuum regarding what we know about the receptor. Our collaborators have recently used crystallography to determine that a small amino acid (D-serine) can bind to the delta2 ligand binding domain, making a set of atomic contacts that are consistent with glutamate binding at other glutamate receptor subunits. Interestingly, although D-serine binds to the delta2 subunit, it does not induce any current response in wild- type receptors. However, a naturally occurring point mutation within the delta2 subunit (the lurcher mutation) produces constitutively active homomeric channels, and application of D- serine to these channels inhibits the tonic current flowing through these constitutively active channels. These data suggest that binding of D-serine can lead to changes in gating, and raises the possibility that incorporation of the delta2 subunit into heteromeric receptor assemblies may endow glutamate receptors with a regulatory site for D-serine. These new data provide an unprecedented opportunity to explore delta2 receptor structure, function, and pharmacology. We propose to exploit this recent finding in 2 lines of experimentation: Aim 1: Identify new ligands that potently activate or inhibit delta2 receptors. Aim 2: Evaluate whether co-expression of delta2 with other glutamate receptor subunits adds D-serine sensitivity to the receptors. Completion of these studies will provide the first comprehensive pharmacological evaluation of this receptor class, as well as new tools with which to study its function and regulation in brain. PUBLIC HEALTH RELEVANCE: Our recent findings that D-serine can bind to the orphan glutamate receptor delta2 has provided an opportunity to significantly advance our understanding of the function of this receptor, which has been enigmatic for over a decade despite in vivo findings that suggest an important role in cerebellar development and synaptic plasticity. The goal of these studies is to identify novel and selective pharmacological agents (activators and antagonists) that could be useful research tools for understanding delta2 function in tissue. In addition, we will also test whether delta2 subunit alters the effects of D-serine on hetero-mulitmeric glutamate receptors. Completion of these studies will advance our understanding of delta2 function, which holds implications for neuronal signaling, synaptic plasticity, and neuronal development in the cerebellum. These studies may also provide insight into new therapeutic agents that target the delta2 receptor.