Annexin A4 is a novel direct regulator of adenylyl cyclase type 5
Annexin A4 is a novel direct regulator of adenylyl cyclase type 5
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DOI：
10.1096/fj.14-269837
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发表时间：
2015-09-01
期刊：
影响因子：
4.8
通讯作者：
Mueller, Frank U.
中科院分区：
文献类型：
--
作者：
Heinick, Alexander;Husser, Xenia;Mueller, Frank U.
膜联蛋白 A4 (AnxA4) 是一种 Ca2+- 和磷脂结合蛋白，在人类衰竭心脏中表达上调。在这项研究中，我们检查了 AnxA4 对 β 肾上腺素受体 (β-AR)/cAMP 依赖性信号转导的影响。用毛喉素 (FSK) 直接刺激腺苷酸环化酶 (AC) 后，人胚胎肾 (HEK)293 细胞中鼠 AnxA4 的表达剂量依赖性地抑制 cAMP 水平，通过 cAMP-Forster 共振能量转移 (EPAC-FRET) 传感器激活的交换蛋白和 ELISA 测定（对照与 +AnxA4：1956 +/- 162 与 1304 +/- 185） fmol/μg 蛋白质；n = 8)。 anxA4 基因的破坏导致分离的成年小鼠心肌细胞中细胞内 cAMP 水平的持续增加，EPAC-FRET 传感器的心脏定向表达，用 FSK 刺激，并通过 ELISA 测定，在用 β-AR 激动剂异丙肾上腺素 (ISO) 刺激的小鼠心肌细胞中也是如此（anxA4a(+/+) 与 anxA4a(-/-)：5.1 +/- 0.3对比 6.7 +/- 0.6 fmol/μ g 蛋白）或 FSK（anxA4a(+/+) 对比 anxA4a(-/-)：1891 +/- 238 对比 2796 +/- 343 fmol/μ g 蛋白；n = 9-10）。 HEK293 细胞中的免疫共沉淀实验揭示了鼠 AnxA4 与人膜结合 AC 5 型 (AC5) 的直接相互作用。作为 AnxA4 介导的 AC 抑制的功能性结果，AnxA4 在报告基因研究中抑制了由 cAMP 反应元件 (CRE) 介导的 FSK 诱导的转录激活（相对于对照为 10 倍；n = 4 次转染），并减少了蛋白质印迹上测量的 FSK 诱导的 CRE 结合蛋白 (CREB) 磷酸化（对照与 +AnxA4：150 +/- 17% 与 105 +/- 10%；n = 6)并通过使用磷酸化引起的CREB激活指示剂(ICAP)-FRET传感器，指示CREB磷酸化。 anxA4a(-/-)小鼠中AnxA4的失活与β-AR刺激的心脏反应增强有关。总之，这些结果表明 AnxA4 是 AC5 的新型直接负调节因子，为膜联蛋白的功能增添了新的方面。
Annexin A4 (AnxA4), a Ca2+- and phospholipid-binding protein, is up-regulated in the human failing heart. In this study, we examined the impact of AnxA4 on beta-adrenoceptor (beta-AR)/cAMP-dependent signal transduction. Expression of murine AnxA4 in human embryonic kidney (HEK)293 cells dose-dependently inhibited cAMP levels after direct stimulation of adenylyl cyclases (ACs) with forskolin (FSK), as determined with an exchange protein activated by cAMP-Forster resonance energy transfer (EPAC-FRET) sensor and an ELISA (control vs. +AnxA4: 1956 +/- 162 vs. 1304 +/- 185 fmol/mu g protein; n = 8). Disruption of the anxA4 gene led to a consistent increase in intracellular cAMP levels in isolated adult mouse cardiomyocytes, with heart-directed expression of the EPAC-FRET sensor, stimulated with FSK, and as determined by ELISA, also in mouse cardiomyocytes stimulated with the beta-AR agonist isoproterenol (ISO) (anxA4a(+/+) vs. anxA4a(-/-): 5.1 +/- 0.3 vs. 6.7 +/- 0.6 fmol/mu g protein) or FSK (anxA4a(+/+) vs. anxA4a(-/-): 1891 +/- 238 vs. 2796 +/- 343 fmol/mu g protein; n = 9-10). Coimmunoprecipitation experiments in HEK293 cells revealed a direct interaction of murine AnxA4 with human membrane-bound AC type 5 (AC5). As a functional consequence of AnxA4-mediated AC inhibition, AnxA4 inhibited the FSK-induced transcriptional activation mediated by the cAMP response element (CRE) in reporter gene studies (10-fold vs. control; n = 4 transfections) and reduced the FSK-induced phosphorylation of the CRE-binding protein (CREB) measured on Western blots (control vs. +AnxA4: 150 +/- 17% vs. 105 +/- 10%; n = 6) and by the use of the indicator of CREB activation caused by phosphorylation (ICAP)-FRET sensor, indicating CREB phosphorylation. Inactivation of AnxA4 in anxA4a(-/-) mice was associated with an increased cardiac response to beta-AR stimulation. Together, these results suggest that AnxA4 is a novel direct negative regulator of AC5, adding a new facet to the functions of annexins.