Characterization of a New Class of Receptors: Identifying ligands for the membrane receptors of the class IIIa human adenylate cyclases.

哺乳动物中的九种腺苷酸环化酶的膜锚具有总共12个α-螺旋跨膜跨度（2 x 6 TM）。这些膜锚在进化上保守了5亿多年。自1989年以来，讨论了膜锚定之外的膜锚定的生理功能。三十年来，该实验室进行了研究，以分配生理作用的膜锚，我们使用的群体感应受体从哈维氏弧菌和嗜肺军团菌。这些受体在二聚化的单体蛋白中是六螺旋的，即它们对于哺乳动物腺苷酸环化酶的膜锚几乎是等排的。此外，已知这些群体感应受体的亲脂性配体。在包含各种III类腺苷酸环化酶和群体感应受体的嵌合体中，我们在2020年毫无疑问地证明了哺乳动物腺苷酸环化酶的膜锚可以作为减弱哺乳动物腺苷酸环化酶的Gsalpha激活的配体受体。目前，配体的化学身份是未知的。我们在鱼、鸡、牛和人的血清中鉴定了生物化学上的“配体活性”。通过各种纯化方法富集配体，将酸性亲脂化合物鉴定为配体。第一个脂质组学分析产生的数据是在可预见的未来识别配体的化学性质的提示。这是提案的第一个目的。然后，我们希望表征所有九种人类腺苷酸环化酶亚型的生物化学和配体-受体相互作用。我们将尝试确定潜在的激动剂和拮抗剂的基础上，我们的知识的化学身份的特殊配体。 已知腺苷酸环化酶受几种胞质次级修饰的调节。我们将研究配体受体控制的调节和二级修饰的调节是如何相互作用的。最后，我们将研究HEK 293细胞内cAMP水平的调节，以获得腺苷酸环化酶受体配体的生理作用的初步迹象。

The membrane anchors of the nine adenylate cyclases in mammals possess a total of 12 alpha-helical transmembrane spans (2 x 6TM). These membrane anchors are evolutionarily conserved for more than half a billion years. Since 1989 discussions took place speculating about a physiological function of the membrane anchors beyond membrane anchoring. For three decades this laboratory has carried out studies in order to assign a physiological role to the membrane anchors, We used the quorum-sensing receptors from Vibrio harveyi and Legionella pneumophila. These receptors are hexahelical in the monomeric proteins which dimerize, i.e. they are almost isosteric to the membrane anchors of the mammalian adenylate cyclases. In addition, lipophilic ligands are known for these quorum-sensing receptors. In chimeras comprising various class III adenylate cyclases and quorum.sensing receptors we demonstrated in 2020 beyond a reasonable doubt that the membrane anchor of mammalian adenylate cyclases can operate as ligand receptors which attenuate the Gsalpha activation of mammalian adenylate cyclases. Currently, the chemical identity of the ligands is unknown. We identified biochemically "ligandactivity" in serum from fish, chicken, cow, and humans, among others. Enrichment of ligands by various purification methods identified acidic lipohilic compounds as ligands. A first lipidomic analysis yielded data which are hints to identify the chemical nature of the ligands in the foreseeable future. This is the first aim of the proposal. Then we wish to characterize biochemically and pharmacologically ligand-receptor interactions for all nine human adenylate cyclase isoforms. We will attempt to identify potential agonists and antagonist based on our knowledge of the chemical identity of peculiar ligands. Adenylatcyclases are known to be regulated by several cytosolic secondary modifications. We will investigate how ligand-receptor controlled regulation and regulation by secondary modifications are interacting. Finally, we will examine the regulation of intracellular cAMP levels in HEK293 cells in order to gain initial indications for physiological actions of adenylate cyclase receptor ligands.