The calmodulin-binding site of the plasma membrane Ca2+pump interacts with the transduction domain of the enzyme

Plasma membrane Ca2+-ATPase (PMCA) transports Ca2+ by a reaction cycle including phosphorylated intermediates. Calmodulin binding to the C-terminal tail disrupts autoinhibitory interactions, activating the pump. To assess the conformational changes during the reaction cycle, we studied the structure of different PMCA states using a fluorescent probe, hydrophobic photolabeling, controlled proteolysis and Ca2+-ATPase activity.  Our results show that calmodulin binds to E2P-like states, and during dephosphorylation, the hydrophobicity in the nucleotide-binding pocket decreases and the Ca2+ binding site becomes inaccessible to the extracellular medium. Autoinhibitory interactions are disrupted in E1Ca and in the E2P ground state whereas they are stabilized in the E2∙Pi product state. Finally, we propose a model that describes the conformational changes during the Ca2+ transport of PMCA.

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Dual mechanism of activation of plant plasma membrane Ca2+-ATPase by acidic phospholipids: Evidence for a phospholipid binding site which overlaps the calmodulin-binding site

Molecular Membrane Biology ◽

10.1080/09687680802508747 ◽

2008 ◽

Vol 25 (6-7) ◽

pp. 539-546 ◽

Cited By ~ 13

Author(s):

Silvia Meneghelli ◽

Tiziana Fusca ◽

Laura Luoni ◽

Maria Ida De Michelis

Keyword(s):

Plasma Membrane ◽

Binding Site ◽

Phospholipid Binding ◽

Calmodulin Binding ◽

Acidic Phospholipids ◽

Dual Mechanism ◽

Plant Plasma Membrane

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Antibodies against erythrocyte Ca2+-transport ATPase specifically inhibit the calmodulin-dependent fraction of the enzyme's activity

Biochemical Journal ◽

10.1042/bj2280479 ◽

1985 ◽

Vol 228 (2) ◽

pp. 479-485 ◽

Cited By ~ 1

Author(s):

K Gietzen ◽

J Kolandt

Keyword(s):

Smooth Muscle ◽

Plasma Membrane ◽

Oleic Acid ◽

Atpase Activity ◽

Binding Site ◽

Rat Brain ◽

Limited Proteolysis ◽

Transport Atpase ◽

Calmodulin Binding ◽

Muscle Plasma Membrane

Antibodies against purified Ca2+-transport ATPase from human erythrocytes were raised in rabbits. Immunodiffusion experiments revealed that precipitating antibodies had been developed. The immunoglobulin fraction inhibited solely the calmodulin-dependent fraction of erythrocyte Ca2+-transport ATPase activity, whereas the basal (in the absence of added calmodulin) activity of the enzyme was not significantly affected by the antibodies. The antibodies produced similar doseresponse curves for the calmodulin- and the oleic acid-stimulated enzyme. However, the immunoglobulin fraction was considerably less effective in inhibiting Ca2+-transport ATPase activated by limited proteolysis. The results obtained with our antibodies are compatible with the interpretation that at least one subpopulation of the antibodies attacks the enzyme at or close to the calmodulin-binding site of the ATPase. The antibodies also inhibited the calmodulin-regulated Ca2+-transport ATPase from pig smooth-muscle plasma membrane, though with lower potency. However, the immunoglobulin fraction failed to suppress pig cardiac sarcoplasmicreticulum Ca2+-transport ATPase activity in the concentration range investigated. In addition, the activity of phosphodiesterase from rat brain, another enzyme modulated by calmodulin, was not at all affected by the immunoglobulin fraction.

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The Calmodulin Binding Domain of the Plasma Membrane Ca2+ Pump Interacts Both with Calmodulin and with Another Part of the Pump

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)63860-8 ◽

1989 ◽

Vol 264 (21) ◽

pp. 12313-12321 ◽

Cited By ~ 4

Author(s):

A Enyedi ◽

T Vorherr ◽

P James ◽

D J McCormick ◽

A G Filoteo ◽

...

Keyword(s):

Plasma Membrane ◽

Binding Domain ◽

Calmodulin Binding

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Characterization of the Calmodulin-binding Site of Nonerythroid α-Spectrin

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)83624-9 ◽

1989 ◽

Vol 264 (10) ◽

pp. 5826-5830 ◽

Cited By ~ 4

Author(s):

T L Leto ◽

S Pleasic ◽

B G Forget ◽

E J Benz ◽

V T Marchesi

Keyword(s):

Binding Site ◽

Calmodulin Binding

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Phosphorylation of the calmodulin binding domain of the plasma membrane Ca2+ pump by protein kinase C reduces its interaction with calmodulin and with its pump receptor site.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(19)51081-x ◽

1994 ◽

Vol 269 (39) ◽

pp. 24298-24303

Author(s):

F. Hofmann ◽

J. Anagli ◽

E. Carafoli ◽

T. Vorherr

Keyword(s):

Plasma Membrane ◽

Protein Kinase C ◽

Protein Kinase ◽

Receptor Site ◽

Binding Domain ◽

Calmodulin Binding ◽

Kinase C

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Inhibition of clathrin-coated pit assembly by an Eps15 mutant

Journal of Cell Science ◽

10.1242/jcs.112.9.1303 ◽

1999 ◽

Vol 112 (9) ◽

pp. 1303-1311 ◽

Cited By ~ 16

Author(s):

A. Benmerah ◽

M. Bayrou ◽

N. Cerf-Bensussan ◽

A. Dautry-Varsat

Keyword(s):

Plasma Membrane ◽

Fusion Protein ◽

Binding Site ◽

Specific Marker ◽

Coated Pits ◽

Receptor Mediated Endocytosis ◽

Gfp Fusion Protein ◽

Protein Encoding ◽

Homology Domains

Recent data have shown that Eps15, a newly identified component of clathrin-coated pits constitutively associated with the AP-2 complex, is required for receptor-mediated endocytosis. However, its precise function remains unknown. Interestingly, Eps15 contains three EH (Eps15-Homology) domains also found in proteins required for the internalization step of endocytosis in yeast. Results presented here show that EH domains are required for correct coated pit targeting of Eps15. Furthermore, when cells expressed an Eps15 mutant lacking EH domains, the plasma membrane punctate distribution of both AP-2 and clathrin was lost, implying the absence of coated pits. This was further confirmed by the fact that dynamin, a GTPase found in coated pits, was homogeneously redistributed on the plasma membrane and that endocytosis of transferrin, a specific marker of clathrin-dependent endocytosis, was strongly inhibited. Altogether, these results strongly suggest a role for Eps15 in coated pit assembly and more precisely a role for Eps15 in the docking of AP-2 onto the plasma membrane. This hypothesis is supported by the fact that a GFP fusion protein encoding the ear domain of (alpha)-adaptin, the AP-2 binding site for Eps15, was efficiently targeted to plasma membrane coated pits.

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