Requirement of SH2-containing protein tyrosine phosphatases SHP-1 and SHP-2 for paired immunoglobulin-like receptor B (PIR-B)-mediated inhibitory signal

Abstract

Paired immunoglobulin-like receptor B (PIR-B) (p91) molecule has been proposed to function as an inhibitory receptor in B cells and myeloid lineage cells. We demonstrate here that the cytoplasmic region of PIR-B is capable of inhibiting B cell activation. Mutational analysis of five cytoplasmic tyrosines indicate that tyrosine 771 in the motif VxYxxL plays the most crucial role in mediating the inhibitory signal. PIR-B-mediated inhibition was markedly reduced in the SH2-containing protein tyrosine phosphatases SHP-1 and SHP-2 double-deficient DT40 B cells, whereas this inhibition was unaffected in the inositol polyphosphate 5'-phosphatase SHIP-deficient cells. These data demonstrate that PIR-B can negatively regulate B cell receptor activation and that this PIR-B-mediated inhibition requires redundant functions of SHP-1 and SHP-2.

Figure 5

Reduction of FcγRIIB–PIR-B–mediated inhibitory effect in SHP-1/SHP-2 double-deficient cells. (A) SHIP, SHP-1, or SHP-2 protein expression on various gene-targeted DT40 cells. Each protein was immunoprecipitated and was detected by Western blotting analysis using anti-SHIP, anti–SHP-1, or anti–SHP-2 Ab. (B) Calcium mobilization was measured after BCR cross-linking (solid line) and coligation (dashed line) in various DT40 mutants expressing FcγRIIB–PIR-B. Surface expression levels of FcγRIIB–PIR-B are indicated in inset boxes. (C) Comparison of inhibitory effect in various mutant DT40 cells. The results are expressed as the mean from three independent clones. Error bars represent SD from the mean.

Figure 2

Inhibition of BCR-mediated cell activation by FcγRIIB– PIR-B. (A) Calcium mobilization stimulated by BCR cross-linking (solid line) and coligation of BCR to FcγRIIB–PIR-B (dashed line) in IIA1.6 cells expressing wild-type FcγRIIB–PIR-B. Calcium release from intracellular calcium store was measured in the presence of 1 mM EGTA, shown by right panel. Histogram in the inset box indicates the expression level of FcγRIIB–PIR-B by 2.4G2 staining. Unstained cells were used as a negative control (open histogram). Stimulation of FcγRIIB–PIR-B alone by 2.4G2 did not induce calcium mobilization. (B) NF-AT activation by BCR cross-linking and the coligation of BCR to FcγRIIB–PIR-B. The results are shown as relative NF-AT activity to BCR cross-linking alone.

Figure 1

Schematic diagram of FcγRIIB–PIR-B chimeric molecules. PIR-B cytoplasmic region contains four ITIM-like motifs (residues 688– 693, SLYASV; 717–722, ETYAQV; 769–774, VTYAQL; and 799–804, SVYATL). In addition to these tyrosines, the cytoplasmic domain of PIR-B contains one more tyrosine (residue 747). The open and shaded boxes represent portions of FcγRIIB and PIR-B, respectively.

Figure 3

Recruitment of SHP-1 and SHP-2 to FcγRIIB–PIR-B after coligation of BCR and the chimeric receptor. (A) Tyrosine phosphorylation of FcγRIIB–PIR-B by BCR cross-linking and coligation of BCR to FcγRIIB–PIR-B. IIA1.6 cells expressing wild-type FcγRIIB–PIR-B were incubated with F(ab′)₂ rabbit anti–mouse IgG, or with intact rabbit anti–mouse IgG. FcγRIIB–PIR-B was then immunoprecipitated with 2.4G2, separated by SDS-PAGE gel, transferred to membrane, and immunoblotted with antiphosphotyrosine mAb 4G10 (top). The same membrane was reprobed with anti–PIR-B Ab (bottom). (B) Immunoblotting with anti–SHP-1 and anti–SHP-2 Abs after BCR cross-linking and the coligation of BCR to FcγRIIB–PIR-B. IIA1.6 cells expressing wild-type FcγRIIB–PIR-B were stimulated with either F(ab′)₂ rabbit anti–mouse IgG or intact rabbit anti–mouse IgG and were lysed. FcγRIIB–PIR-B was then immunoprecipitated with 2.4G2, resolved by SDS-PAGE gel, transferred to membrane, and probed with anti–SHP-1 or anti–SHP-2 Ab. In the case of immunoblotting with anti-SHIP, in addition to IIA1.6 cells expressing wild-type FcγRIIB–PIR-B (lanes 1–3), wild-type A20 cells expressing FcγRIIB were stimulated similarly (lanes 4–6). FcγRIIB– PIR-B or FcγRIIB was immunoprecipitated with 2.4G2 followed by Western blotting with anti-SHIP Ab. Membranes were reprobed with anti–PIR-B Ab (bottom).

Figure 4

Requirement of cytoplasmic tyrosine residues in inhibitory response. (A) Calcium mobilization was monitored after BCR cross-linking (solid line) and coligation (dashed line) in IIA1.6 cells expressing various FcγRIIB–PIR-B mutants. Surface expression levels of FcγRIIB–PIR-B mutants are indicated in inset boxes. (B) Comparison of inhibitory effect by various FcγRIIB–PIR-B mutants. The percentage of [Ca²⁺]_i was given by the total calcium mobilization elicited by coligation of BCR to FcγRIIB–PIR-B over that by BCR stimulation alone. The results are expressed as the mean from two independent clones.