Reconstitution of the Cytoplasmic Interaction between Phospholamban and Ca²⁺-ATPase of Cardiac Sarcoplasmic Reticulum

Abstract

Phospholamban (PLN) reversibly inhibits the Ca2ϩ -ATPase of cardiac sarcoplasmic reticulum (SERCA2a) through a direct protein-protein interaction, playing a pivotal role in the regulation of intracellular Ca 2ϩ 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 M. 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.The Ca 2ϩ -ATPase of cardiac sarcoplasmic reticulum (SERCA2a) plays a pivotal role in the contraction and relaxation of heart muscle. It pumps Ca 2ϩ from the cytoplasm into the lumen of the sarcoplasmic reticulum (SR), resulting in muscle relaxation. In turn, the release of Ca 2ϩ from SR induces muscle contraction (Fleischer and Inui, 1989). The activity of SERCA2a is regulated by another SR membrane protein, phospholamban (PLN) (Tada and Katz, 1982). PLN in the dephosphorylated state inhibits SERCA2a by lowering its apparent affinity for Ca 2ϩ (Inui et al., 1986). Phosphorylation of PLN by cAMP-dependent protein kinase reduces the inhibition, resulting in facilitation of the Ca 2ϩ -pumping activity of SERCA2a, which leads to the acceleration of relaxation and the following increase in contractility of heart muscle. This mechanism constitutes a principal intracellular signaling pathway involved in the positive inotropic effects of ␤-adrenergic stimulation. Thus, the SERCA2a-PLN system plays a key role in the regulation of cardiac contractility and relaxation (Kadambi and Kranias, 1997;Simmerman and Jones, 1998;Tada et al., 1988).PLN is a 52-amino acid integral membrane protein consisting of three domains: the N-terminal cytoplasmic helical domain (domain Ia), the less structured cytoplasmic region (domain Ib), and the C-terminal membrane helix (domain II) (Fujii et al., 1987). Chemical cross-linking between purified PLN an...

Close Proximity between Residue 30 of Phospholamban and Cysteine 318 of the Cardiac Ca2+ Pump Revealed by Intermolecular Thiol Cross-linking

“…However, this strategy of purification and reconstitution followed by photoaffinity labeling (9) is difficult to execute with PLB and SERCA2a and has not been duplicated in other laboratories. More recent approaches to address physical interactions between PLB and SERCA2a are cryoelectron microscopy of PLB/SERCA2a co-crystals (20) and assessment of fusion protein interactions (21).…”

In Vitro Selection and Characterization of DNA Aptamers Specific for Phospholamban

“…Conversely, mAb-A1 did not interfere with the binding of Apt-9 to PLN 1-26 -PDZ-Myc-His 6 at concentrations up to 50 g/ml (Fig. 3B), even though the antibody at this concentration almost completely inhibited the interaction between PLN 1-26 -PDZ-Myc-His 6 and the cytoplasmic domain of SERCA2a (Kimura and Inui, 2002) and maximally stimulated SERCA2a activity of cardiac SR vesicles (Kimura et al, 1991).…”

“…To obtain DNA aptamers specific for the cytoplasmic region of phospholamban, we performed the SELEX protocol with an aptamer library based on a random 40-nucleotide sequence and with a fusion protein consisting of Met1-Gln26 of human phospholamban fused to the PDZ domains of PSD-95 followed by Myc epitope and His 6 tags (PLN 1-26 -PDZ-Myc-His 6 ), which retains the ability to interact with the cytoplasmic domain of SERCA2a (Kimura and Inui, 2002). PDZ-Myc-His 6 was used to remove aptamers that might bind nonspecifically to the fusion protein.…”

“…The recombinant proteins were purified by Ni-NTA column chromatography as described previously (Kimura and Inui, 2002). The members of the DNA library for SELEX consisted of two 20-base primer regions and a 40-base random region (5Ј-ATGACCATGACCCTCCACAC-N 40 -TCAGACTGTGGCAGGGAAAC-3Ј).…”

Effects of Phospholipids on the Oligomeric State of Phospholamban of the Cardiac Sarcoplasmic Reticulum

“…11 Removal of phospholamban-mediated inhibition of SERCA2a activity is thus a potential therapeutic option for treatment of heart failure and cardiomyopathy. 12,13 Chemical cross-linking of purified proteins suggests that phospholamban binds directly to SERCA2a. 14 Site-specific mutagenesis and transient expression of the mutant proteins in heterologous cells have revealed that 3 sites of interaction in the cytoplasmic domains and membrane helices of both phospholamban and SERCA2a contribute to the inhibitory effect of the former on the latter.…”