The R1 open reading frame, the first open reading frame of RRV, encodes a transmembrane protein predicted to contain an extracellular amino-terminal domain and a cytoplasmic carboxy-terminal domain. The extracellular domain of R1 is similar in length and sequence to that of KSHV K1 and shows homology to the Ig receptor superfamily. These properties suggest that R1, like other receptor proteins, may be capable of binding a stimulating ligand, which can result in the transduction of signals inside the cell.
Our results demonstrate that upon cross-linking with an anti-CD8 antibody, B cells expressing a CD8Δ-R1C chimera protein containing the full-length cytoplasmic domain of R1 become fully activated, as indicated by the release of free calcium from intracellular stores. This event differs from that induced by BCR cross-linking by an anti-IgM antibody in that the release of free calcium is prolonged, lasting significantly longer than the BCR-induced response. Importantly, none of the R1 deletion mutants could elicit calcium mobilization to a significant degree. Mutants CD8Δ-D1 and CD8Δ-D4, which contain putative SH2 binding domains, as well as CD8Δ-D3, which contains putative ITAM-like sequences, showed only a marginal increase in calcium mobilization, suggesting that the release of calcium from intracellular stores is dependent on the full-length cytoplasmic tail. The fact that the CD8Δ-D3 mutant could not significantly mobilize calcium was surprising since it contains sequences that match well with the ITAM consensus. One possibility that we considered was that the CD8Δ-D3 mutant requires a linker sequence between the CD8 extracellular and transmembrane domains and the carboxy-terminal SH2 binding motifs in R1 in order to induce calcium mobilization. However, CD8Δ-D5, which contains such a linker sequence, was also incapable of eliciting a significant calcium response. Thus these data are supportive of the need for the full-length R1 cytoplasmic domain for calcium mobilization. The differences in the lengths of the R1 versus K1 cytoplasmic domains required to achieve calcium mobilization could possibly result from additional functions for R1 that remain to be determined or could reflect the fact that the K1 protein is more highly evolved. It seems likely that the amino acids that are required for calcium mobilization reside in two or more distinct domains in the R1 cytoplasmic tail and that proper folding of the cytoplasmic tail is needed to bring together disparate parts of the molecule.
In agreement with our calcium mobilization data, we have also observed that the R1 protein is capable of inducing NFAT activity eightfold in B lymphocytes. Both these events are indicative of B-cell activation. We determined that none of the CD8Δ-R1 chimera proteins could activate NFAT activity to a significant level in B cells by themselves, but upon addition of anti-CD8 antibody we observed a dramatic increase in the activation of NFAT by the CD8Δ-R1C chimera protein. This is suggestive of a role for the extracellular and transmembrane regions of the R1 protein for lymphocyte activation, in addition to that for a full-length cytoplasmic tail. The extracellular domain of the R1 protein may be binding a ligand present at least to some extent in the media or on the surfaces of B cells. Another possibility is that the extracellular and transmembrane domains of R1 may undergo self-oligomerization leading to the phosphorylation of the R1 cytoplasmic domain followed by B-cell activation events.
In agreement with the calcium mobilization assays, we observed that only the chimera with the full-length R1 cytoplasmic domain (CD8Δ-R1C) could induce a state of cellular phosphorylation in B cells. Cross-linking of the CD8Δ-R1C chimeric protein by an anti-CD8 antibody induced a pattern of tyrosine phosphorylation similar to that seen with BCR.
We detected complex formation between R1 and the major B-cell kinase, Syk. Although both Src and Syk kinase were capable of phosphorylating R1, only Syk kinase specifically interacted detectably with R1. The ability of Src kinase to phosphorylate R1 may be indirect. Syk induced the phosphorylation of the CD8Δ-R1C chimera. Syk also induced the phosphorylation of the deletion mutants CD8Δ-D1, CD8Δ-D3, and CD8Δ-D4, and these same mutants showed marginal responses in the calcium mobilization assay. Taken together, these data suggest that these mutants may exhibit partial function to a slight extent. In addition, stimulation of the CD8Δ-R1C cell line with an anti-CD8 antibody resulted in increased phosphorylation of Syk kinase indicative of the need for a full-length R1 cytoplasmic protein to elicit tyrosine phosphorylation in B cells.
The cytoplasmic tail of R1 is 170 amino acids in length and contains 13 tyrosine residues, 5 of which are part of YXXL motifs at the carboxyl-terminal end of the protein and match consensus sequences for ITAMs. Five of the other eight tyrosine residues could also potentially function as SH2 binding motifs since the associated sequences, YXXA, YXXP, YXXT, and YXXV, match sequences previously shown to bind SH2 domains. YXXA can bind Src, Lyn, Fyn, Shc, phosphatidylinositol 3 kinase, and phospholipase Cγ (28
). YXXP has been shown to interact with Abl, Crk, and Nck SH2 binding domains (29
). Thus the R1 protein has rich potential for interacting with a number of different signal-transducing molecules. Nonetheless, our data have shown that one contiguous stretch containing seven tyrosines and a separate overlapping stretch containing seven different contiguous tyrosines are insufficient for a significant mobilization of calcium, induction of tyrosine phosphorylation, and NFAT activity.
Both RRV R1 and KSHV K1 have been shown to transform rodent fibroblasts, to substitute for STP in HVS-induced immortalization of T cells to interleukin-2-independent growth, and to induce cellular phosphorylation and signal transduction (20
). Although these proteins have sequence and structural homology in their external domains, the length of the R1 cytoplasmic domain and the number of potential SH2 binding motifs contained in this domain differ markedly from those of K1, which has a short 38-amino-acid cytoplasmic tail and only two SH2 binding motifs, which constitute a single ITAM (18
). One striking difference in the activities of these proteins is the sustained calcium mobilization induced in B cells by R1 versus K1. This prolonged activation event may be a result of the phosphorylation of multiple tyrosine residues in the R1 cytoplasmic tail as these residues are part of SH2 binding motifs that could possibly interact with a variety of cellular kinases.
Cellular receptors, upon ligand stimulation, are capable of undergoing oligomerization and of inducing cellular proliferation. LMP1 protein of EBV mimics an activated CD40 receptor by undergoing multimerization of its transmembrane domains in a ligand-independent manner, which results in the activation of the NF-κB pathway leading to cellular proliferation (11
). It remains to be determined whether signal induction via R1 and K1 results from stimulation by an exogenous ligand or self-multimerization. If there is no self-multimerization, a ligand appears to be present, at least to some extent, on the surfaces of BJAB cells, in the culture supernatant, or in the serum. Regardless of the mechanism, both R1 and K1 appear to be able to activate B cells independent of the traditional BCR engagement.