Inhibitory Interaction of the Plasma Membrane Na+/Ca2+ Exchangers with the 14-3-3 Proteins

The three Na+/Ca2+ exchanger isoforms, NCX1, NCX2, and NCX3, contain a large cytoplasmic loop that is responsible for the regulation of activity. We have used 347 residues of the loop of NCX2 as the bait in a yeast two-hybrid approach to identify proteins that could interact with the exchanger and r...

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Bibliographic Details
Published in:The Journal of biological chemistry 2006-07, Vol.281 (28), p.19645-19654
Main Authors: Pulina, Maria V., Rizzuto, Rosario, Brini, Marisa, Carafoli, Ernesto
Format: Article
Language:English
Subjects:
14-3-3 Proteins - metabolism
Amino Acid Sequence
Animals
Brain - metabolism
Cell Membrane - metabolism
HeLa Cells
Humans
Membrane Transport Proteins - metabolism
Molecular Sequence Data
Rats
Sodium-Calcium Exchanger - metabolism
Two-Hybrid System Techniques
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Summary:The three Na+/Ca2+ exchanger isoforms, NCX1, NCX2, and NCX3, contain a large cytoplasmic loop that is responsible for the regulation of activity. We have used 347 residues of the loop of NCX2 as the bait in a yeast two-hybrid approach to identify proteins that could interact with the exchanger and regulate its activity. Screening of a human brain cDNA library identified the ϵ and ζ isoforms of the 14-3-3 protein family as interacting partners of the exchanger. The interaction was confirmed by immunoprecipitation and in vitro binding experiments. The effect of the interaction on the homeostasis of Ca2+ was investigated by co-expressing NCX2 and 14-3-3ϵ in HeLa cells together with the recombinant Ca2+ probe aequorin; the ability of cells expressing both NCX2 and 14-3-3ϵ to dispose of a Ca2+ transient induced by an InsP3-producing agonist was substantially decreased, indicating a reduction of NCX2 activity. The 14-3-3ϵ protein also inhibited the NCX1 and NCX3 isoforms. In vitro binding experiments revealed that all three NCX isoforms interacted with multiple 14-3-3 isoforms. 14-3-3 was bound by both phosphorylated and nonphosphorylated NCX, but the phosphorylated form had much higher binding affinity.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M602033200