Epstein-Barr virus/C3d receptor (CR2, CD21) interacts with three intracellular proteins: the p53 anti-oncoprotein expressed in human B lymphoma cells, the p68 calcium binding protein expressed in normal B lymphocytes and the nuclear p120 ribonucleoprotein (RNP). We previously demonstrated that p53 and p68 interacted with the intracytoplasmic carboxy-terminal domain of CR2. To analyse the amino acid sequence of CR2 binding sites for p53 and p68, we synthesized different peptides whose sequences were derived from this carboxy-terminal domain. Thus, CR2 bound to p53 and p68 through two distinct binding sites localized on the N-terminal and on the central part of its carboxy-terminal domain, characterized by the amino acid sequences of KHRERNYYTD and KEAFHLEARE, respectively. CR2 site reacting with the nuclear p120RNP was determined using either anti-CR2 mAb directed against its extracellular domain or pep34, pep14/SCR3 and pep14/SCR4, synthetic peptides whose sequences corresponded to the intracellular 34 amino acid domain or to sites of the extracellular domain of CR2, respectively. Data support that CR2 interacts with p120RNP through the DEGYRLQGPPSSRC amino acid sequence of its extracellular SCR4 domain. Furthermore, phosphorylation of CR2 inhibits its interaction with the nuclear p120RNP. Binding of CR2, through its intracellular and extracellular domains, with the p53 oncoprotein and p120RNP, respectively, and the co-localization of these three proteins on nuclear interchromatin fibrils, suggest that CR2 could act as a crosslinker between these two nuclear proteins to regulate their functions.

EBV/C3d receptor (CR2) interacts with the p53 anti-oncoprotein expressed in the human B lymphoma cells, Raji but not in normal B cells, and with the p68 calcium-binding protein, expressed in normal B lymphocytes but not in transformed B lymphocytes. To characterize the CR2 domain interacting with these two intracellular proteins, we synthesized a 34-amino acid peptide, pep34, corresponding to its intracytoplasmic carboxy-terminal domain and analyzed its binding and antigenic properties. Binding of 125I-labeled p53 or 125I-labeled p68 on immobilized pep34 was specific, additive, and totally inhibited by unlabeled p53 or p68, respectively, but not by unlabeled p68 or p53, respectively. Antigenic properties of pep34 were analyzed by immunizing rabbits with particle-bound pep34. Polyclonal anti-pep34 Ab carried anti-CR2 specificities that recognized only the intracellular domain of CR2. In addition, anti-pep34 Ab also carried anti-p53 or anti-p68 specificities. Anti-p53 or anti-p68 specificities were not due to putative common structural or conformational antigenic determinants between the pep34 synthetic peptide and the p68 or p53 proteins. These anti-p53 and anti-p68 specificities were identified as anti-idiotypic anti-CR2 Ab mimicking either p53 or p68 binding sites of CR2. These data clearly establish that despite its short length, the intracytoplasmic C-terminal tail of CR2 is involved in direct protein-protein interactions with the two intracellular regulatory proteins, p53 and p68. An additional feature of these data is the demonstration that particle-bound pep34 triggered "in vivo" anti-Id Ab restricted to either p53 or p68 specificities.

A monoclonal antibody was raised against p57, a serine proteinase, characterized by an apparent molecular weight of 57 kDa, and purified from human erythrocyte membranes. P57 proteinase cleaves the human third component of complement, C3. The antibody selected, MP1, of IgG2a isotype, precipitated specifically the p57 antigen which carried the C3/C3b-cleaving activity present in membrane crude extract of human erythrocytes. P57 proteinase eluted from MP1-sepharose was inhibited by 5 x 10(-4) M PMSF, enhanced by 0.5% SDS and generated C3 fragments identical to those generated by membrane crude extract of human erythrocytes. All these properties were identical to those of the p57 previously purified by biochemical procedures. In addition, 5000 binding sites were detected on cell surface. This MP1 monoclonal antibody will be helpful to analyse the role of p57 in human erythrocytes.

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.

We present herein the first evidence that human C3 and, with a higher efficiency, trypsin-cleaved C3 enhanced in vitro phosphorylation of a cellular component, characterized by an apparent molecular weight of 105 kDa, pp105, present in the human B lymphoma cells, Raji. This regulatory activity was associated with C3d fragment generated in trypsin-cleaved C3. A 16 amino-acid peptide, carrying the LYNVEA sequence of C3d reacting with the C3d receptor (CR2), was synthetized. P16 enhanced, in a dose-dependent curve between 0.3 to 10 μM, in vitro phosphorylation of pp105, as well as C3d fragments present in trypsin-cleaved C3. A fibrinogen-related synthetic peptide of 15 amino acids, used as control, had no effect on pp105 phosphorylation. P16 and trypsin-cleaved C3 regulate pp105 phosphorylation through identical pathways. Thus, p16 represents the 16 amino-acid sequence of C3 which regulated in vitro phosphorylation of pp105.