Intracellular interaction of EBV/C3d receptor (CR2) with p68, a calcium-binding protein present in normal but not in transformed B lymphocytes.
To analyze direct intracellular interactions of CR2 in normal human B lymphocytes, we used polyclonal anti-Id anti-CR2 antibodies (Ab2) prepared against the highly purified CR2 molecule (gp140) as original immunogen. We previously demonstrated that this Ab2 contained specificities that mimicked extracellular and intracellular domains of CR2 and was helpful for identifying CR2-specific ligands. Indeed, some Ab2 specificities recognized human C3d and EBV, two extracellular CR2 ligands. In addition, other Ab2 specificities interacted directly, as CR2, with the intracellular p53 antioncoprotein that is expressed in transformed cells and not in normal cells. We demonstrate herein that Ab2 detected in normal B lymphocytes a 68-kDa protein, p68, that was not expressed in transformed B cells. p68 was localized in purified plasma membranes and cytosol fractions. Direct interaction of purified CR2 with purified p68 was demonstrated. Competitive studies supported that CR2 and Ab2 interacted with identical sites on p68. These interactions were calcium dependent. p68 was identified as a calcium-binding protein by its ability to be solubilized from B lymphocyte membranes by EGTA, a calcium-chelating agent, to bind specifically on phenothiazine-Sepharose in a calcium-dependent interaction, and to be recognized by specific antibodies directed against human p68, a calcium-binding protein of the annexin VI family. Thus, demonstration of different intracellular interactions of CR2 with distinct regulatory proteins, such as p53, the antioncoprotein, and p68, a calcium-binding protein, supports involvement of two regulatory pathways of signal transduction through CR2, depending on the normal or transformed state of human B lymphocytes.
Barel
M
M
Immunochimie des Antigènes de Membrane et des Interactions Cellulaires, Centre INSERM, Hôpital Saint-Antoine, Paris, France.
Gauffre
A
A
Lyamani
F
F
Fiandino
A
A
Hermann
J
J
Frade
R
R
D017317:Annexin A6; D000888:Antibodies, Anti-Idiotypic / Q000276:immunology; D000944:Antigens, Differentiation, B-Lymphocyte / Q000276:immunology / Q000378:metabolism; D001402:B-Lymphocytes / Q000737:chemistry*; D002135:Calcium-Binding Proteins / Q000032:analysis / Q000378:metabolism*; D015933:Complement C3d / Q000378:metabolism*; D006801:Humans; D008223:Lymphoma / Q000378:metabolism*; D008565:Membrane Proteins / Q000032:analysis; D009363:Neoplasm Proteins / Q000032:analysis / Q000378:metabolism*; D011951:Receptors, Complement / Q000276:immunology / Q000378:metabolism*; D017464:Receptors, Complement 3d; D016159:Tumor Suppressor Protein p53 / Q000032:analysis
D017317:Annexin A6; D000888:Antibodies, Anti-Idiotypic; D000944:Antigens, Differentiation, B-Lymphocyte; D002135:Calcium-Binding Proteins; D008565:Membrane Proteins; D009363:Neoplasm Proteins; D011951:Receptors, Complement; D017464:Receptors, Complement 3d; D016159:Tumor Suppressor Protein p53; D015933:Complement C3d