title: Annexin A4 is a novel direct regulator of adenylyl cyclase type 5.
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.
authors.affiliation: *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.
authors.forename: Alexander
authors.initials: A
authors.lastname: Heinick
authors.affiliation: *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.
authors.forename: Xenia
authors.initials: X
authors.lastname: Husser
authors.affiliation: *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.
authors.forename: Kirsten
authors.initials: K
authors.lastname: Himmler
authors.affiliation: *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.
authors.forename: Uwe
authors.initials: U
authors.lastname: Kirchhefer
authors.affiliation: *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.
authors.forename: Frank
authors.initials: F
authors.lastname: Nunes
authors.affiliation: *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.
authors.forename: Jan S
authors.initials: JS
authors.lastname: Schulte
authors.affiliation: *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.
authors.forename: Matthias D
authors.initials: MD
authors.lastname: Seidl
authors.affiliation: *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.
authors.forename: Christina
authors.initials: C
authors.lastname: Rolfes
authors.affiliation: *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.
authors.forename: John R
authors.initials: JR
authors.lastname: Dedman
authors.affiliation: *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.
authors.forename: Marcia A
authors.initials: MA
authors.lastname: Kaetzel
authors.affiliation: *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.
authors.forename: Volker
authors.initials: V
authors.lastname: Gerke
authors.affiliation: *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.
authors.forename: Wilhelm
authors.initials: W
authors.lastname: Schmitz
authors.affiliation: *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.
authors.forename: Frank U
authors.initials: FU
authors.lastname: Müller
mesh_terms: D000262:Adenylyl Cyclases / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D017314:Annexin A4 / Q000235:genetics / Q000378:metabolism*; D050882:CREB-Binding Protein / Q000235:genetics / Q000378:metabolism; D002462:Cell Membrane / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D018345:Mice, Knockout; D009206:Myocardium / Q000378:metabolism*; D032383:Myocytes, Cardiac / Q000378:metabolism*; D010766:Phosphorylation / Q000502:physiology
chemical_list: D017314:Annexin A4; D050882:CREB-Binding Protein; C494363:CREBBP protein, human; C494364:Crebbp protein, mouse; D000262:Adenylyl Cyclases; C419510:adenylyl cyclase type V
keywords: CRE-mediated transcription; CREB; cAMP; β-AR stimulation