Question: 

Cardiac atrial natriuretic peptide (ANP) regulates arterial blood pressure, moderates cardiomyocyte growth and stimulates angiogenesis and metabolism. To exert its functions, ANP binds to the transmembrane guanylyl cyclase-A (GC-A) receptor, which consequently induces the receptor's cyclase function. Presumably all effects of ANP/GC-A are mediated by cGMP as intracellular signaling molecule, however, little is known about additional protein factors involved in modulation of the ANP/GC-A response. Here we established a co-IP-based proteomics approach for analysis of the GC-A interactome in recombinant HEK293 and murine cardiac tissue.

Methology: 

HEK293 were stably transfected with the affinity-tagged GC-A construct FLAG-HA-TEV-GC-A (FHT-GC-A, in pcMV5 vector) and FLAG-specific co-IP was subsequently performed on native cell lysates. The eluates were analysed in comparison to adequate negative controls (i.e. non-transfected HEK293) by SDS-PAGE. Following gel silver staining, differential protein bands indicating co-precipitated putative GC-A interaction partners were extracted and finally identified by protein mass spectrometry.

Results and Conclusion: 

In preliminary experiments, four differential FLAG-IP bands were detected upon SDS-PAGE, resulting in mass spectrometric identification of 18 individual proteins. Three such hits were annotated as being involved in protein complex formation or stabilization, six were assigned to regulatory processes - including Hsp90, whose association with GC-A has also been reported elsewhere [Biol. Chem. 2001, 276 (14), 11371–11375], yet whose detailed role needs to be further specified. Next, we will validate the found GC-A interaction candidates by targeted co-IPs and furthermore expand our FLAG-specific co-IP studies to a transgenic mouse model displaying cardiac overexpression of the FHT-GC-A fusion protein.

(Supported by SFB 688)