Identification of regions in the Ca(2+)-ATPase of sarcoplasmic reticulum that affect functional association with phospholamban.

When the SERCA 2 isoform of the Ca(2+)-ATPase of cardiac and slow-twitch muscle sarcoplasmic reticulum was coexpressed with phospholamban in COS-1 cells, a reduction in Ca2+ affinity (measured as Ca2+ dependence of Ca2+ transport) of 0.2-0.3 pCa units was observed. This inhibitory effect was reversed by phosphorylation of phospholamban with cAMP-dependent protein kinase A. SERCA 1 and SERCA 3, were also expressed in COS-1 cells, alone and together with phospholamban. SERCA 1 had high Ca2+ affinity which was reduced upon coexpression with phospholamban, but SERCA 3 had lower Ca2+ affinity, which was unaltered by coexpression with phospholamban. To identify which regions of the Ca2+ ATPase sequence determine its functional interaction with phospholamban, chimeric Ca(2+)-ATPases between SERCA 2 and SERCA 3 were constructed and coexpressed with phospholamban. Measurement of Ca2+ affinities for a series of chimeras showed that two separate regions of the cytoplasmic domain of SERCA 2 were required for manifestation of a functional interaction between phospholamban and the Ca(2+)-ATPase. The first is a region between amino acids 336 and 412 in the phosphorylation domain, which corresponds to a phospholamban interaction site identified earlier (James, P., Inui, M., Tada, M., Chiesi, M., and Carafoli, E. (1989) Nature 342, 90-92). The second region is the nucleotide binding/hinge domain (amino acids 467-762) which determines high Ca2+ affinity for SERCA type pumps (Toyofuku, T., Kurzydlowski, K., Lytton, J., and MacLennan, D. H. (1992) J. Biol. Chem. 267, 14490-14496).