In this report, we provide evidence that the primary function of CD300a in T and B cells is to limit antigen receptor mediated positive signaling and that the phosphatase SHP-1 is required for this function. Coligation of the BCR and CD300a with mAb reduced BCR stimulated Ca2+ mobilization and NFAT transcriptional activity. In the absence of SHP-1, but not SHP-2 or SHIP, CD300a mediated inhibition was significantly reduced. Additionally, we show that superantigen induced activation was inhibited when Jurkat T cells expressing the chimeric receptor KIR-CD300a were mixed with antigen presenting cells expressing the KIR2DL2 ligand HLA-Cw3. The interaction of KIR-CD300a with its ligand led to the tyrosine phosphorylation of CD300a ITIM motifs. This phosphorylation required the src kinase Lck, and provided docking sites for the binding of the phosphatases SHP-1 and SHP-2. These early events were followed by the inhibition of superantigen mediated up-regulation of activation markers CD25 and CD69.
The employment of two different models in attempting to understand CD300a inhibitory signal in lymphocytes was very important in our studies. Since DT40 chicken B cells do not express CD300a, the usage of these cells and their knockout counterparts allowed us to specifically express both CD300a and phosphatases, wild type and mutants, and to investigate the role of phosphatases in CD300a signaling transmission. In our hands, and with the available anti-CD300a mAb, we were unable to immunoprecipitate CD300a (data not shown). Due to this inability to immunoprecipitate CD300a and, because we also were interested in an experimental system that relies on receptor-ligand interaction, we generated the chimeric receptor KIR-CD300a. Results obtained with similar chimeric receptors have proved useful in gaining information about the role of the ITIMs. For example, by using a chimeric receptor consisting of KIR extracellular domain fused to the FcγRIIb intracellular tail, Gupta et al. demonstrated that the ITIMs in the intracellular tail, and not the extracellular portion, are responsible for the transmission of the inhibitory signal and determined which phosphatase was employed [41
For a more comprehensive understanding of CD300a mediated signaling on lymphocytes, mutational analysis of the ITIMs should prove helpful. Lankry et al. have undertaken these studies using the human YTS NK cell line [29
]. Their results indicated that all of the ITIMs, including the non-classical 4th
ITIM, were important for the inhibitory function of CD300a, with the 3rd
ITIM being the most essential. Results obtained in our laboratory in which we mutated tyrosine residues to phenlyalanine instead of to alanine, as described by Lankry et al. [29
], have confirmed that a single mutation of the 3rd
ITIM significantly decreased BCR stimulated Ca2+
release and NFAT transcriptional activity (data not shown).
In our KIR-CD300a chimera, the CD300a ITIMs were phosphorylated upon interaction with the KIR ligand without the requirement of superantigen stimulation. This is not surprising, since phosphorylation of KIR ITIMs by Lck also occurs independently of antigen stimulation [9
]. However, it is interesting that a single tyrosine kinase, such as Lck, can be utilized for both inhibitory and activating receptors. While the mechanism by which this occurs is still under investigation, findings obtained by Stefanova et al. [43
] may shed some light on this conundrum. In that report, antagonist and agonist peptides, defined by their different binding affinities to the TCR, were used to dissect the seemingly different roles of Lck in T cell homeostasis. The SHP-1 tyrosine phosphatase was a central player in their findings. When T cells were stimulated with a weak binding ligand, Lck phosphorylated SHP-1. Subsequent association of SHP-1 with Lck mediated the recruitment of SHP-1 to the TCR complex where it was proposed that SHP-1 then dephosphorylated Lck at Y394 leading to TCR desensitization. Alternatively, upon interaction with a strong TCR ligand, Erk was rapidly activated and phosphorylated Lck on serine residues (S59). This serine phosphorylation decreased the ability of Lck to bind SHP-1 and therefore the positive signaling proceeded. Here, we have shown that Lck is involved in CD300a phosphorylation. It may be possible that Lck also phosphorylates CD300a bound SHP-1, subsequently aiding in the recruitment of SHP-1 to the TCR complex, leading to the inhibition of positive signaling. Future studies should address this hypothesis.
Our results using SHP-1 and SHP-2 knocked down KIR-CD300a WT Jurkat T cells and specific phosphatase deficient DT40 chicken B cells indicated that SHP-1, but not SHP-2 or SHIP was necessary for CD300a mediated inhibition of BCR and TCR signaling. Although mAb cross-linking induced coimmunoprecipitation of SHIP with CD300a in mast cells [30
], the consensus binding sequences for SHIP are different from that of SHP-1 and SHP-2 and are not present in CD300a. SHIP has no preference for binding to residues N-terminal to the phosphorylated tyrosine (pY) but has a strong preference for Leu at the +2 position. Instead, SHP SH2 domains prefer a hydrophobic residue at the −2 position on the ITIM [44
]. All three classical ITIMs present in CD300a have hydrophobic residues at −2 and none of them have Leu at +2 position [28
]. Therefore, while the detection of SHIP in a complex with CD300a may indicate a role for SHIP in the control of signaling in mast cells, its direct binding to CD300a ITIM motifs is unlikely. On the other hand, the consensus binding motifs for SHP-1 and SHP-2 are similar [44
] and matched sequences are found in the CD300a intracellular tail [28
]. Indeed, both SHP-1 and SHP-2 were detected in immunoprecipitates from ligand stimulated Jurkat T cells expressing the KIR-CD300a chimeric receptor. However, according to previously published results that tested the binding of SHP SH2 domains to pY peptide libraries [44
], it may be that the chances of having both SH2 domains of a single phosphatase bound simultaneously to phosphorylated CD300a intracellular tail are greater with SHP-1. While binding of a single SH2 domain may potentiate phosphatase activity, binding of both domains further increases the activity by several fold [45
Further evidence that both SHP-1 and SHP-2 bind to CD300a comes from the SHP-1 and SHP-2 reconstitution experiments. As shown in Figure
, the expression of either SHP-1 CS or SHP-2 CS reduced the inhibitory function of CD300a. The mutation of the cysteine residue renders the phosphatases catalytically inactive, but they still are able to bind the target ITIM sequences and therefore become dominant negative. In that same line of thought, one could argue that since SHP-2 WT also competes for CD300a ITIM occupancy, it could also function as a dominant negative, and in fact, reconstitution of CD300a expressing DT40 chicken B cells lacking SHP-2 with human SHP-2 WT resulted in a decrease in the CD300a mediated inhibitory ability when compared with non-reconstituted DT40 cells lacking SHP-2 (see Figure
C). Additional studies designed to address the relative binding affinity of SHP-1 and SHP-2 to phosphorylated CD300a ITIMs and their differential role in signaling should prove interesting.