Reconstitution of the cytoplasmic interaction between phospholamban and Ca(2+)-ATPase of cardiac sarcoplasmic reticulum.
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.
Kimura
Yoshihiro
Y
Department of Pharmacology, Yamaguchi University School of Medicine, Ube, Yamaguchi, Japan.
Inui
Makoto
M
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000737:chemistry / Q000378:metabolism*; D003593:Cytoplasm / Q000201:enzymology / Q000378:metabolism; D017353:Gene Deletion; D006801:Humans; D008274:Magnesium / Q000378:metabolism; D009206:Myocardium / Q000201:enzymology*; D010446:Peptide Fragments / Q000378:metabolism; D010766:Phosphorylation; D017354:Point Mutation; D017434:Protein Structure, Tertiary; D011817:Rabbits; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000099211:Phospholamban
D000255:Adenosine Triphosphate; D000911:Antibodies, Monoclonal; D002118:Calcium; D002135:Calcium-Binding Proteins; D000252:Calcium-Transporting ATPases; D008274:Magnesium; D010446:Peptide Fragments; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000099211:Phospholamban