Intercellular signaling during terminal differentiation

描述（申请人提供）：盘基网柄藻中前孢子和前柄细胞之间的通讯协调终末分化。信号包括34个氨基酸的肽SDF-2，其具有与神经肽DBI（地西泮结合抑制剂）相关的序列。神经递质GABA也通过与哺乳动物的GABAB受体类似的G蛋白偶联受体起作用而在网骨藻中用作信号。另一方面，SDF-2受体DhkA是与DBI受体GABAA和“外周型”BZ受体完全无关的“双组分”组氨酸激酶。似乎细胞间信号在长期的进化过程中一直保持不变，而受体在某些情况下发生了变化。我们将进一步阐明在这个遗传上易处理的系统中细胞间信号传导的详细机制，因为这些结果似乎与哺乳动物中枢神经系统中的信号传导有关，并可能揭示神经精神疾病的潜在机制。在前孢子和前柄细胞分别准备包囊和液泡化时，它们之间有复杂的信息交换。SDF-2是由前孢子细胞释放的蛋白质AcbA的蛋白水解加工产生的。加工发生在由低水平的SDF-2或GABA引发后的前柄细胞的表面上。前孢子细胞通过从囊泡中快速释放AcbA来响应引发。AcbA是一种酰基辅酶A结合蛋白，也是DBI的前体。使用定点诱变，我们将确定这种活性是否是必需的积累AcbA的内涵体和随后的释放。我们将确定是否胞吐在低水平的SDF-2或GABA的响应是依赖于通过cAMP依赖性蛋白激酶PKA的信号转导。胞外AcbA被膜包埋的前茎特异性蛋白酶TagC蛋白水解切割，其仅在低水平的SDF-2或GABA引发后暴露。我们将确定这一过程是否也依赖于PKA活性。我们的研究结果表明，哺乳动物神经肽DBI，ACBP的前体，可以在一种细胞类型中合成，储存在囊泡中，并由另一种细胞类型加工成活性肽。这种相互作用可以调节这种天然配体的水平，这种天然配体与广泛使用的药物安定（安定）的靶点结合。我们观察到GABA似乎诱导快速胞吐作用，这不仅解释了其在盘基网柄菌中的启动活性，而且为更好地理解控制胞吐作用的基本过程提供了一个测试系统。这些研究将为改善神经系统疾病的诊断和治疗奠定基础。

DESCRIPTION (provided by applicant): Communication between prespore and prestalk cells coordinates terminal differentiation in Dictyostelium discoideum. The signals include a 34 amino acid peptide, SDF-2, that has a sequence related to that of the neuropeptide DBI (Diazepam Binding Inhibitor). The neurotransmitter GABA is also used as a signal in Dictyostelium by acting through a G-protein coupled receptor similar to the GABAB receptor of mammals. The SDF-2 receptor, DhkA, on the other hand, is a "two-component" histidine kinase completely unrelated to the DBI receptors, GABAA and "peripheral type" BZ receptors. It seems that intercellular signals have been conserved over long periods of evolution while the receptors have changed in some cases. We will further elucidate the detailed mechanisms of intercellular signaling in this genetically tractable system since the results appear to have bearing on signaling in the mammalian central nervous system and may shed light on mechanisms underlying neuropsychiatric disorders. There is a complicated interchange of information between prespore and prestalk cells as they prepare to encapsulate and vacuolize, respectively. SDF-2 is generated by proteolytic processing of a protein, AcbA, released from prespore cells. Processing occurs on the surface of prestalk cells after priming by low levels of SDF-2 or by GABA. Prespore cells respond to priming by rapid release of AcbA from vesicles. AcbA is an acyl-CoA binding protein as is the precursor of DBI. Using site-directed mutagenesis we will determine whether this activity is essential for accumulation of AcbA in endosomes and subsequent release. We will determine whether exocytosis in response to either low levels of SDF-2 or GABA is dependent on signaling through the cAMP dependent protein kinase PKA. Extracellular AcbA is proteolytically cleaved by the membrane embedded prestalk specific protease TagC, which is only exposed following priming by either low levels of SDF-2 or GABA. We will determine whether this process is also dependent on PKA activity. Our results suggest that the precursor of the mammalian neuropeptide DBI, ACBP, may be synthesized in one cell type, stored in vesicles, and processed to the active peptides by another cell type. Such interactions could modulate the levels of this natural ligand that binds at the target of the widely used drug diazepam (Valium). Our observations that GABA appears to induces rapid exocytosis not only accounts for its priming activity in Dictyostelium but provides a test system for better understanding of the basic processes controlling exocytosis. Together these studies will add to the foundation for improvements in the diagnosis and treatment of neurological diseases.