title: Identification of regions in the Ca(2+)-ATPase of sarcoplasmic reticulum that affect functional association with phospholamban.
abstract: 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).
authors.affiliation: Banting and Best Department of Medical Research, University of Toronto, C. H. Best Institute, Ontario, Canada.
authors.forename: T
authors.initials: T
authors.lastname: Toyofuku
authors.forename: K
authors.initials: K
authors.lastname: Kurzydlowski
authors.forename: M
authors.initials: M
authors.lastname: Tada
authors.forename: D H
authors.initials: DH
authors.lastname: MacLennan
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001693:Biological Transport, Active; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D066298:In Vitro Techniques; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009206:Myocardium / Q000201:enzymology; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D011817:Rabbits; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D016415:Sequence Alignment; D013329:Structure-Activity Relationship; D000099211:Phospholamban
chemical_list: D002118:Calcium; D002135:Calcium-Binding Proteins; D000252:Calcium-Transporting ATPases; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D011494:Protein Kinases; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D000099211:Phospholamban