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- Title
Annexin A4 is a novel direct regulator of adenylyl cyclase type 5.
- Authors
Heinick, Alexander; Husser, Xenia; Himmler, Kirsten; Kirchhefer, Uwe; Nunes, Frank; Schulte, Jan S.; Seidl, Matthias D.; Rolfes, Christina; Dedman, John R.; Kaetzel, Marcia A.; Gerke, Volker; Schmitz, Wilhelm; Müller, Frank U.
- Abstract
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 β-adrenoceptor (β-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--Förster resonance energy transfer (EPAC-FRET) sensor and an ELISA (control vs. +AnxA4: 1956 ± 162vs. 1304 ± 185 fmol/µg 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 β-AR agonist isoproterenol (ISO) (anxA4a+/+ vs. anxA4a-/-: 5.1 ± 0.3 vs. 6.7 ± 0.6 fmol/µg protein) or FSK (anxA4a+/+ vs. anxA4a-/-: 1891 ± 238 vs. 2796 ± 343 fmol/µg 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 β-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.
- Subjects
ANNEXINS; CYCLIC adenylic acid; CALCIUM-binding proteins; INFLAMMATORY mediators; PHOSPHOLIPASE A2; ENZYME inhibitors; ADENYLATE cyclase
- Publication
FASEB Journal, 2015, Vol 29, Issue 9, p3773
- ISSN
0892-6638
- Publication type
Article
- DOI
10.1096/fj.14-269837