Venkatakrishnan Varun, Ghode Abhijeet, Tulsian Nikhil K, Anand Ganesh S
Department of Chemistry, Pennsylvania State University, University Park, PA, United States.
Department of Biological Sciences, National University of Singapore, Singapore, Singapore.
Front Mol Biosci. 2023 Sep 21;10:1202268. doi: 10.3389/fmolb.2023.1202268. eCollection 2023.
Acrodysostosis represents a group of rare genetic disorders characterized by defective skeletal development and is often accompanied by intellectual disabilities. Mutations in the 3'5'cyclic AMP (cAMP)-dependent protein kinase (PKA) type I regulatory subunit isoform α (RIα) and phosphodiesterase (PDE) PDE4D have both been implicated in impaired PKA regulation in acrodysostosis. How mutations on PDEs and RIα interfere with the regulation of cAMP-PKA signaling is not understood. cAMP-PKA signaling can be described in two phases. In the activation phase, cAMP binding to RIα dissociates the free C-subunit (Catalytic subunit). PDEs hydrolyze cAMP bound to RIα, priming the cAMP-free RIα for reassociation with the C-subunit, thereby completing one PKA activation cycle. Signal termination is thus critical for resetting PKA to its basal state and promoting adaptation to hormonal hyperstimulation. This proceeds through formation of a transient signal termination RIα: PDE complex that facilitates cAMP channeling from the cAMP-binding domain of RIα to the catalytic site of PDE. Signal termination of cAMP-PKA proceeds in three steps: Step 1) Channeling: translocation of cAMP from the CNB of RIα to the PDE catalytic site for hydrolysis. Step 2) Processivity: binding of free cAMP from the cytosol at both CNBs of RIα. Step 3) Product (5'AMP) release from the PDE hydrolysis site through competitive displacement by a new molecule of cAMP that triggers subsequent activation cycles of PKA. We have identified the molecular basis for two acrodysostosis mutants, PDE (PDE8 T690P) and RIα (T207A), that both allosterically impair cAMP-PKA signal termination. A combination of amide hydrogen/deuterium exchange mass spectrometry (HDXMS) and fluorescence polarization (FP) reveals that PDE8 T690P and RIα T207A both blocked processive hydrolysis of cAMP by interfering with competitive displacement of product 5'AMP release from the nucleotide channel at the end of each round of cAMP hydrolysis. While T690P blocked product 5'AMP release from the PDE, T207A greatly slowed the release of the substrate from RIα. These results highlight the role of processivity in cAMP hydrolysis by RIα: PDE termination complexes for adaptation to cAMP from GPCR hyperstimulation. Impairment of the signal termination process provides an alternate molecular basis for acrodysostosis.
肢端发育不全症是一组罕见的遗传性疾病，其特征是骨骼发育缺陷，常伴有智力障碍。3',5'-环磷酸腺苷（cAMP）依赖性蛋白激酶（PKA）I型调节亚基同工型α（RIα）和磷酸二酯酶（PDE）PDE4D中的突变均与肢端发育不全症中PKA调节受损有关。PDEs和RIα上的突变如何干扰cAMP-PKA信号传导尚不清楚。cAMP-PKA信号传导可分为两个阶段。在激活阶段，cAMP与RIα结合使游离的C亚基（催化亚基）解离。PDEs水解与RIα结合的cAMP，使无cAMP的RIα重新与C亚基结合，从而完成一个PKA激活循环。因此，信号终止对于将PKA重置为基础状态并促进对激素过度刺激的适应至关重要。这通过形成瞬时信号终止RIα：PDE复合物来实现，该复合物促进cAMP从RIα的cAMP结合域向PDE的催化位点的传递。cAMP-PKA的信号终止分三步进行：步骤1）传递：cAMP从RIα的CNB转运到PDE催化位点进行水解。步骤2）持续性：游离的cAMP从细胞质与RIα的两个CNB结合。步骤3）产物（5'-AMP）通过新的cAMP分子的竞争性取代从PDE水解位点释放，从而触发PKA的后续激活循环。我们已经确定了两种肢端发育不全症突变体PDE（PDE8 T690P）和RIα（T207A）的分子基础，它们均通过变构作用损害cAMP-PKA信号终止。酰胺氢/氘交换质谱（HDXMS）和荧光偏振（FP）相结合的方法表明，PDE8 T690P和RIα T207A均通过干扰每轮cAMP水解结束时产物5'-AMP从核苷酸通道的竞争性取代来阻断cAMP的持续性水解。虽然T690P阻断了产物5'-AMP从PDE的释放，但T207A大大减缓了底物从RIα的释放。这些结果突出了持续性在RIα：PDE终止复合物对cAMP水解中的作用，以适应来自GPCR过度刺激的cAMP。信号终止过程的受损为肢端发育不全症提供了另一种分子基础。
