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Annexin A4 is a novel direct regulator of adenylyl cyclase type 5
Author(s) -
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.
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fj.14-269837
Subject(s) - adenylyl cyclase , creb , annexin , forskolin , hek 293 cells , signal transduction , reporter gene , leucine zipper , biology , phosphorylation , microbiology and biotechnology , chemistry , medicine , endocrinology , gene expression , receptor , transcription factor , stimulation , gene , biochemistry , flow cytometry
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.—Heinick, A., Husser, X., Himmler, K., Kirchhefer, U., Nunes, F., Schulte,J. S., Seidl, M. D., Rolfes, C., Dedman, J. R., Kaetzel, M. A., Gerke, V., Schmitz, W., Müller, F. U. Annexin A4 is a novel direct regulator of adenylyl cyclase type 5. FASEB J. 29, 3773‐3787 (2015). www.fasebj.org