Insights into the oligomerization process of the C-terminal domain of human plasma membrane Ca²+-ATPase.
Plasma membrane calcium pumps (PMCAs) sustain a primary transport system for the specific removal of cytosolic calcium ions from eukaryotic cells. PMCAs are characterized by the presence of a C-terminal domain referred to as a regulatory domain. This domain is target of several regulatory mechanisms: activation by Ca²+-calmodulin complex and acidic phospholipids, phosphorylation by kinase A and C, proteolysis by calpain and oligomerization. As far as oligomerization is concerned, the C-terminal domain seems to be crucial for this process. We have cloned the C-terminal domain of the human PMCA isoform 1b, and characterized its properties in solution. The expressed protein maintains its tendency to oligomerize in aqueous solutions, but it is dissociated by amphipathic molecules such as diacylglycerol and sodium dodecyl sulphate. The presence of sodium dodecyl sulphate stabilizes the domain as a compact structure in monomeric form retaining the secondary structure elements, as shown by small angle neutron scattering and circular dichroism measurements. The importance of oligomerization for the regulation of PMCA activity and intracellular calcium concentration is discussed.
Department of Biology, University of Padova, Viale G. Colombo 3, I-35131 Padova, Italy.
Federico
F
Benetti
Ivan
I
Mičetić
Flavio
F
Carsughi
Francesco
F
Spinozzi
Luigi
L
Bubacco
Mariano
M
Beltramini
D001483:Base Sequence; D002462:Cell Membrane / Q000201:enzymology; D018076:DNA, Complementary / Q000235:genetics; D004795:Enzyme Stability; D006801:Humans; D066298:In Vitro Techniques; D046529:Microscopy, Electron, Transmission; D008958:Models, Molecular; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000648:ultrastructure; D053499:Plasma Membrane Calcium-Transporting ATPases / Q000737:chemistry* / Q000235:genetics / Q000648:ultrastructure; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000648:ultrastructure; D012967:Sodium Dodecyl Sulfate
C506952:ATP2B1 protein, human; D018076:DNA, Complementary; D010446:Peptide Fragments; D011994:Recombinant Proteins; D012967:Sodium Dodecyl Sulfate; D053499:Plasma Membrane Calcium-Transporting ATPases