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

Heinick A., Husser X., Himmler K., Kirchhefer U., Nunes F., Schulte J., Seidl M., Rolfes C., Dedman J., Kaetzel M., Gerke V., Schmitz W., Müller F.

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 b-adrenoceptor (b-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/mg 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 b-AR agonist isoproterenol (ISO) (anxA4a+/+ vs. anxA4a-/-: 5.160.3 vs. 6.760.6 fmol/mg protein) or FSK (anxA4a+/+ vs. anxA4a-/-: 18916238 vs. 2796 ± 343 fmol/mg protein; n = 9-10). Coimmunoprecipitation experiments in HEK293 cells revealed a direct interaction of murine AnxA4 with human membranebound AC type 5 (AC5). As a functional consequence of AnxA4-mediated AC inhibition, AnxA4 inhibited the FSKinduced transcriptional activation mediated by the cAMP response element (CRE) in reporter gene studies (10-fold vs. control; n = 4 transfections) and reduced the FSKinduced 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 b-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.