Phospholamban (PLN) reversibly inhibits the Ca(2+)-ATPase of cardiac 
sarcoplasmic reticulum (SERCA2a) through a direct protein-protein interaction, 
playing a pivotal role in the regulation of intracellular Ca(2+) in heart muscle 
cells. The interaction between PLN and SERCA2a occurs at multiple sites within 
the cytoplasmic and membrane domains. Here, we have reconstituted the 
cytoplasmic protein-protein interaction using bacterially expressed fusion 
proteins of the cytoplasmic domain of PLN and the long cytoplasmic loop of 
SERCA2a. We have developed two methods to evaluate the binding of the fusion 
proteins, one with glutathione-Sepharose beads and the other with a 96-well 
plate. Essentially the same results were obtained by the two methods. The 
affinity of the binding (K(D)) was 0.70 microM. The association was inhibited by 
cAMP-dependent phosphorylation of the PLN fusion protein and by usage of 
anti-PLN monoclonal antibody. It was also diminished by substitution at the 
phosphorylation site of PLN of Ser(16) to Asp. These results suggest that PLN 
can bind SERCA2a in the absence of the membrane domains and that the 
modifications of the cytoplasmic domain of PLN that activate SERCA2a parallel 
the disruption of the association between the two fusion proteins. It has been 
shown that the removal of PLN inhibition of SERCA2a rescues cardiac function and 
morphology in the mouse dilated cardiomyopathy model. Our assay system can be 
applied to the screening of novel inotropic agents that remove the inhibition of 
SERCA2a by PLN, improving the relaxation as well as the contractility of the 
failing heart.