Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge, CB2 1PD, UK.
J Cell Sci. 2012 Feb 15;125(Pt 4):869-86. doi: 10.1242/jcs.091090. Epub 2012 Mar 7.
The central and pervasive influence of cAMP on cellular functions underscores the value of stringent control of the organization of adenylyl cyclases (ACs) in the plasma membrane. Biochemical data suggest that ACs reside in membrane rafts and could compartmentalize intermediary scaffolding proteins and associated regulatory elements. However, little is known about the organization or regulation of the dynamic behaviour of ACs in a cellular context. The present study examines these issues, using confocal image analysis of various AC8 constructs, combined with fluorescence recovery after photobleaching and fluorescence correlation spectroscopy. These studies reveal that AC8, through its N-terminus, enhances the cortical actin signal at the plasma membrane; an interaction that was confirmed by GST pull-down and immunoprecipitation experiments. AC8 also associates dynamically with lipid rafts; the direct association of AC8 with sterols was confirmed in Förster resonance energy transfer experiments. Disruption of the actin cytoskeleton and lipid rafts indicates that AC8 tracks along the cytoskeleton in a cholesterol-enriched domain, and the cAMP that it produces contributes to sculpting the actin cytoskeleton. Thus, an adenylyl cyclase is shown not just to act as a scaffold, but also to actively orchestrate its own micro-environment, by associating with the cytoskeleton and controlling the association by producing cAMP, to yield a highly organized signalling hub.
环磷酸腺苷（cAMP）对细胞功能的核心和普遍影响突显了严格控制质膜中环腺苷酸（AC）组成的重要性。生化数据表明，AC 存在于膜筏中，并且可以分隔中间支架蛋白和相关调节元件。然而，对于细胞环境中 AC 的动态行为的组织或调节知之甚少。本研究使用各种 AC8 构建体的共焦图像分析，结合光漂白后荧光恢复和荧光相关光谱学，研究了这些问题。这些研究表明，AC8 通过其 N 端增强质膜处的皮质肌动蛋白信号；通过 GST 下拉和免疫沉淀实验证实了这种相互作用。AC8 还与脂筏动态相关；在Förster 共振能量转移实验中证实了 AC8 与固醇的直接结合。肌动球蛋白细胞骨架和脂筏的破坏表明，AC8 在富含胆固醇的区域中沿着肌动球蛋白细胞骨架追踪，并且它产生的 cAMP 有助于塑造肌动球蛋白细胞骨架。因此，腺嘌呤核苷酸环化酶不仅可以作为支架发挥作用，而且还可以通过与细胞骨架结合并通过产生 cAMP 来控制其自身的微环境的关联，从而产生高度组织化的信号枢纽。
