PATHWAYS OF RECEPTOR SIGNALING AND CHEMOTAXIS

趋化细胞在非常浅的化学梯度中定向和移动。当细胞伸展时，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部。受体信号传导下游的反馈环整合激活和抑制途径以在这种梯度内建立细胞极性。虽然许多努力都集中在定义激活途径，抑制网络在很大程度上尚未探索。 真核生物中许多受体介导的通路适应/失活持续刺激。虽然已经假设网骨藻中的MAP激酶ERK 2适应cAMP与其7-跨膜细胞表面受体CAR的连续接合，但我们已经证明，相反，ERK 2在这种条件下保持活性。负责ERK 2激活的上游磷酸化途径对CAR刺激短暂响应，而ERK 2去磷酸化（失活）被连续刺激抑制。我们认为，净结果是持续活跃的ERK 2时，细胞外cAMP浓度是恒定的，振荡生产/破坏分泌的cAMP在趋化细胞占观察到的振荡活性的ERK 2。我们还表明，控制ERK 2激活/失活的CAR依赖性途径的功能独立于G蛋白和配体诱导的细胞内cAMP的产生以及随后的PKA激活。这种调节使ERK 2能够以振荡的方式发挥作用，这对趋化性至关重要，并且以持续的方式发挥作用，这是基因表达所必需的，因为分泌的cAMP在以后的发育过程中增加。这项工作重新定义了网骨藻中CAR信号转导对ERK 2调节的机制，并为趋化过程中信号中继的控制建立了新的意义。 7-TMR通过异源三聚体G蛋白依赖性和非依赖性途径激活多个下游信号级联，并控制广泛的生物过程。在配体结合后，7-TMR在细胞质丝氨酸和苏氨酸残基处被特异性受体激酶磷酸化，起到将异源三聚体G蛋白途径与受体信号传导解偶联并激活G蛋白非依赖性途径的作用。在网骨藻中，CAR信号调节趋化性以及G蛋白依赖性和非依赖性信号级联。由于汽车在cAMP结合后变得磷酸化，我们分析了仅表达不可磷酸化的CAR变体的细胞响应cAMP的能力。这些细胞在腺苷酸环化酶适应和细胞极化和趋化性方面有缺陷。虽然已知受体磷酸化将某些G蛋白介导的信号通路与哺乳动物细胞中的7-TMR解偶联，但我们的研究首次表明此类机制也在调节趋化性的通路中起作用。

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.