The B cell receptor-induced calcium flux involves a calcium mediated positive feedback loop.
The B cell receptor (BCR)-elicited calcium flux results in activation of mature B cells. We have recently shown that the adaptor protein Swiprosin-1/EFhd2 (EFhd2) amplifies the BCR-induced calcium flux in B cell lines. EFhd2 is a calcium binding adaptor protein with two predicted EF-hands. Here we asked whether these domains are functional and control its function. Using a blot-overlay assay with radioactive calcium we show that both EF-hands of EFhd2 have an intrinsic capacity to bind calcium. Equilibrium centrifugation confirmed that EFhd2 binds 2 calcium ions, with an apparent Kd of 110 μM. Point mutations revealed that the conserved residues E116 and E152, which reside in the canonical calcium binding loop in EF-hands 1 and 2, are essential for calcium binding by EFhd2. These mutations as well as deletion of the EF-hands, in particular EF-hand 1, abolished the ability of EFhd2 to restore BCR-induced calcium signaling in EFhd2-deficient WEHI231 cells. N-terminal deletions, but not C-terminal deletions, acted similarly. Thus, the N-terminal part of EFhd2 as well as calcium binding to its EF-hands control the intracellular calcium concentration in response to BCR stimulation in WEHI231 cells. Hence, EFhd2 regulates the BCR-elicited calcium flux through a calcium-dependent positive feedback mechanism in WEHI231 cells.
Division of Molecular Immunology, Department of Internal Medicine III, University of Erlangen-Nürnberg, Nikolaus Fiebiger Center, Glückstr. 6, 91054 Erlangen, Germany.
Sandra
S
Hagen
Sebastian
S
Brachs
Carmen
C
Kroczek
Barbara G
BG
Fürnrohr
Christiane
C
Lang
Dirk
D
Mielenz
D001402:B-Lymphocytes / Q000378:metabolism*; D002118:Calcium / Q000378:metabolism*; D002127:Calcium Isotopes; D020013:Calcium Signaling; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002460:Cell Line; D020832:EF Hand Motifs / Q000235:genetics; D004926:Escherichia coli; D025461:Feedback, Physiological; D015870:Gene Expression; D006801:Humans; D008213:Lymphocyte Activation; D009154:Mutation; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011947:Receptors, Antigen, B-Cell / Q000378:metabolism
D002127:Calcium Isotopes; D002135:Calcium-Binding Proteins; C502299:EFHD2 protein, human; D011947:Receptors, Antigen, B-Cell; D002118:Calcium