Annexin A4 is a novel direct regulator of adenylyl cyclase type 5

Alexander Heinick; Xenia Husser; Kirsten Himmler; Uwe Kirchhefer; Frank Nunes; Jan S Schulte; Matthias D Seidl; Christina Rolfes; John R Dedman; Marcia A Kaetzel; Volker Gerke; Wilhelm Schmitz; Frank U Müller

doi:10.1096/fj.14-269837

Annexin A4 is a novel direct regulator of adenylyl cyclase type 5

FASEB J. 2015 Sep;29(9):3773-87. doi: 10.1096/fj.14-269837. Epub 2015 May 28.

Affiliations

¹ *Institute of Pharmacology and Toxicology, Institute of Medical Biochemistry, Center for Molecular Biology of Inflammation, and Interdisciplinary Clinical Research Center, University of Münster, Münster, Germany; and Department of Genome Science, University of Cincinnati Genome Research Institute, Cincinnati, Ohio, USA a.heinick@uni-muenster.de.
² *Institute of Pharmacology and Toxicology, Institute of Medical Biochemistry, Center for Molecular Biology of Inflammation, and Interdisciplinary Clinical Research Center, University of Münster, Münster, Germany; and Department of Genome Science, University of Cincinnati Genome Research Institute, Cincinnati, Ohio, USA.

PMID: 26023182
DOI: 10.1096/fj.14-269837

Abstract

Annexin A4 (AnxA4), a Ca(2+)- 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 ± 162 vs. 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.

Keywords: CRE-mediated transcription; CREB; cAMP; β-AR stimulation.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Adenylyl Cyclases / genetics
Adenylyl Cyclases / metabolism*
Animals
Annexin A4 / genetics
Annexin A4 / metabolism*
CREB-Binding Protein / genetics
CREB-Binding Protein / metabolism
Cell Membrane / genetics
Cell Membrane / metabolism*
HEK293 Cells
Humans
Mice
Mice, Knockout
Myocardium / metabolism*
Myocytes, Cardiac / metabolism*
Phosphorylation / physiology

Substances

Annexin A4
CREB-Binding Protein
CREBBP protein, human
Crebbp protein, mouse
Adenylyl Cyclases
adenylyl cyclase type V