PATHWAYS OF RECEPTOR SIGNALING AND CHEMOTAXIS

趋化细胞在很浅的化学梯度中定向和移动。随着细胞的极化，不同的结构和信号成分在空间上被限制在细胞的前缘或后方。受体信号下游的反馈环整合了激活和抑制通路，以在这样的梯度内建立细胞极性。虽然许多工作都集中在定义激活途径上，但抑制性网络在很大程度上还没有被探索。 真核生物中的许多受体介导的通路适应/失活持续刺激。虽然Dictyostelius中的MAP激酶ERK2被认为适应cAMP与其7-跨膜受体CAR的持续结合，但我们已经证明，相反，ERK2在这种条件下仍然保持活性。负责ERK2激活的上游磷酸化途径对CAR刺激有短暂的反应，而ERK2去磷酸化(去激活)则被持续刺激抑制。我们认为，当细胞外cAMP浓度恒定时，最终结果是ERK2持续活跃，趋化细胞中分泌cAMP的振荡产生/破坏解释了观察到的ERK2的振荡活性。我们还表明，控制ERK2激活/失活的CAR依赖通路的功能不依赖于G蛋白和配体诱导的细胞内cAMP的产生和随后的PKA激活。这种调节使ERK2既能以一种对趋化至关重要的振荡方式发挥作用，又能以一种持久的方式发挥作用，这是基因表达所必需的，因为在发育后期分泌的cAMP增加。这项工作重新定义了CAR信号对DictyostelialERK2的调控机制，并为控制趋化过程中的信号传递奠定了新的基础。 7-TMR通过异源三聚体G蛋白依赖和非依赖途径激活多个下游信号级联通路，控制广泛的生物过程。与配体结合后，7-TMR在胞浆丝氨酸和苏氨酸残基上被特定的受体激酶磷酸化，功能是将异源三聚体G蛋白途径从受体信号中分离出来，并激活G蛋白不依赖的途径。在网柄苔藓中，CAR信号调节趋化作用，G蛋白依赖和非依赖信号级联。由于CARS在cAMP结合时被磷酸化，我们分析了只表达非磷酸化CAR变体的细胞对cAMP做出反应的能力。这些细胞在腺苷环化酶适应、细胞极化和趋化能力方面存在缺陷。虽然已知受体磷酸化可以将哺乳动物细胞中某些G蛋白介导的信号通路与7-TMR解偶联，但我们的研究首次表明，这种机制也在调节趋化作用的通路中发挥作用。

Chemotactic cells orient and move directionally in very shallow chemical gradients. As cells polarize, distinct structural and signaling components become spatially restricted to the leading edge or rear of cells. Feedback loops downstream of receptor signaling integrate both activating and inhibiting pathways to establish cell polarity within such gradients. While much effort has focused on defining activating pathways, inhibitory networks have been largely unexplored. Many receptor-mediated pathways in eukaryotes adapt/deactivate to persistent stimulation. While the MAP kinase ERK2 in Dictyostelium has been assumed to adapt to continuous engagement of cAMP with its 7-transmembrane, cell surface receptor CAR, we have shown, to the contrary, that ERK2 remains active under such conditions. The upstream phosphorylation pathway that is responsible for ERK2 activation transiently responds to CAR stimulation, whereas ERK2 de-phosphorylation (deactivation) is inhibited by continuous stimulation. We argue that the net result is persistently active ERK2 when the extracellular cAMP concentration is constant and that the oscillating production/destruction of secreted cAMP in chemotaxing cells accounts for the observed oscillatory activity of ERK2. We also showed that CAR-dependent pathways that control ERK2 activation/deactivation function independently of G proteins and of ligand-induced production of intracellular cAMP and the consequent activation of PKA. This regulation enables ERK2 to function both in an oscillatory manner, critical for chemotaxis, and in a persistent manner, necessary for gene expression, as secreted cAMP increases during later development. This work redefines mechanisms of ERK2 regulation by CAR signaling in Dictyostelium and establishes new implications for control of signal-relay during chemotaxis. 7-TMRs activate multiple downstream signaling cascades via heterotrimeric G protein-dependent and -independent pathways and control a wide range of biological processes. Upon ligand binding, 7-TMRs become phosphorylated at cytoplasmic serine and threonine residues by specific receptor kinases, functioning to uncouple heterotrimeric G protein pathways from receptor signaling and to activate G protein-independent pathways. In Dictyostelium, CAR signaling regulates chemotaxis and both G protein-dependent and -independent signaling cascades. Since the CARs become phosphorylated upon cAMP binding, we analyzed the ability of cells that only express non-phosphorylatable CAR variants to respond to cAMP. These cells were defective in adenylyl cyclase adaptation and in cell polarization and chemotaxis. While receptor phosphorylation is known to uncouple certain G protein-mediated signaling pathways from 7-TMRs in mammalian cells, our studies are the first to show that such mechanisms also function in pathways that regulate chemotaxis.