CD6 regulates T-cell responses through activation-dependent recruitment of the positive regulator SLP-76.

Deciphering the role of lymphocyte membrane proteins depends on dissecting the role of a protein in the steady state and on engagement with its ligand. We show that expression of CD6 in T cells limits their responsiveness but that engagement by the physiological ligand CD166 gives costimulation. This costimulatory effect of CD6 is mediated through phosphorylation-dependent binding of a specific tyrosine residue, 662Y, in its cytoplasmic region to the adaptor SLP-76. A direct interaction between SLP-76 and CD6 was shown by binding both to a phosphorylated peptide (equilibrium dissociation constant [K(D)] = 0.5 muM at 37 degrees C) and, using a novel approach, to native phosphorylated CD6. Evidence that CD6 and SLP-76 interact in cells was obtained in coprecipitation experiments with normal human T cells. Analysis of human CD6 mutants in a murine T-cell hybridoma model showed that both costimulation by CD6 and the interaction between CD6 and SLP-76 were dependent on 662Y. The results have implications for regulation by CD6 and the related T-cell surface protein, CD5.

A novel form of the membrane protein CD147 that contains an extra Ig-like domain and interacts homophilically.

BACKGROUND: CD147 is a broadly distributed integral membrane glycoprotein with two Ig-like domains implicated in a wide range of functions. It is associated at the cell surface with the monocarboxylate transporters MCT1 and 4 but interactions of the extracellular region have not been characterised. RESULTS: We report the characterisation of a form of CD147 with an additional membrane-distal Ig-like domain. In contrast to the two domain form, this three domain form of CD147 interacts homophilically. Surface plasmon resonance analysis using recombinant proteins showed that the interaction was of low affinity (KD approximately 40 microM) and this is typical of many interactions between membrane proteins. cDNA for the 3 domain form are rare but have been identified in human and mouse retina. CONCLUSION: The finding that the three domain form of CD147 has an extracellular ligand, that is it interacts homophilically, suggests this interaction may be important in aligning lactate transporters in the retina where lactate is an important metabolite.

Direct and indirect interactions of the cytoplasmic region of CD244 (2B4) in mice and humans with FYN kinase.

Engagement of the receptor CD244 (2B4) by its ligand CD48 has inhibitory and activating potential, and this differs depending on experimental systems in mouse and human. We show that, in both mouse and human upon engagement of its ligand CD48, CD244 can give a negative signal to natural killer cells, implying conservation of function between the two species. The signaling mechanisms used by CD244 in both human and mouse are conserved as shown by quantitative analyses of the direct molecular interactions of the SH2 domains of the adaptors SLAM-associated protein (SAP) and EAT-2 and of FYN kinase with CD244 together with the indirect interactions of the FYN SH2 domain with EAT-2. Functional experiments support the biochemical hierarchy of interactions and show that EAT-2 is not inhibitory per se. The data are consistent with a model in which the mechanism of signal transduction by CD244 is to regulate FYN kinase recruitment and/or activity and the outcome of CD48/CD244 interactions is determined by which other receptors are engaged.

Human herpesvirus 8 K14 protein mimics CD200 in down-regulating macrophage activation through CD200 receptor.

Many viral proteins limit host immune defenses, and their genes often originate from their hosts. CD200 (OX2) is a broadly distributed cell surface glycoprotein that interacts with a receptor on myeloid cells (CD200R) that is implicated in locally preventing macrophage activation. Distant, but recognizable, homologues of CD200 have been identified in many herpesviruses and poxviruses. Here, we show that the product of the K14 open reading frame from human herpesvirus 8 (Kaposi's sarcoma-associated herpesvirus) interacts with human CD200R and is expressed at the surfaces of infected cells solely during the lytic cycle. Despite sharing only 40% primary sequence identity, K14 and CD200 interacted with CD200R with an almost identical and low affinity (K(D) = 0.5 microM), in contrast to other characterized viral homologue interactions. Cells expressing CD200 or K14 on the cell surface were able to inhibit secretion by activated macrophages of proinflammatory cytokines such as tumor necrosis factor alpha, an effect that could be specifically relieved by addition of monoclonal antibodies and soluble monomeric CD200 protein. We conclude that CD200 delivers local down-modulatory signals to myeloid cells through direct cell-cell contact and that the K14 viral homologue closely mimics this.

Characterization of the CD200 receptor family in mice and humans and their interactions with CD200.

CD200 (OX2) is a broadly distributed cell surface glycoprotein that interacts with a structurally related receptor (CD200R) expressed on rodent myeloid cells and is involved in regulation of macrophage function. We report the first characterization of human CD200R (hCD200R) and define its binding characteristics to hCD200. We also report the identification of a closely related gene to hCD200R, designated hCD200RLa, and four mouse CD200R-related genes (termed mCD200RLa-d). CD200, CD200R, and CD200R-related genes were closely linked in humans and mice, suggesting that these genes arose by gene duplication. The distributions of the receptor genes were determined by quantitative RT-PCR, and protein expression was confirmed by a set of novel mAbs. The distribution of mouse and human CD200R was similar, with strongest labeling of macrophages and neutrophils, but also other leukocytes, including monocytes, mast cells, and T lymphocytes. Two mCD200 receptor-like family members, designated mCD200RLa and mCD200RLb, were shown to pair with the activatory adaptor protein, DAP12, suggesting that these receptors would transmit strong activating signals in contrast to the apparent inhibitory signal delivered by triggering the CD200R. Despite substantial sequence homology with mCD200R, mCD200RLa and mCD200RLb did not bind mCD200, and presently have unknown ligands. The CD200 receptor gene family resembles the signal regulatory proteins and killer Ig-related receptors in having receptor family members with potential activatory and inhibitory functions that may play important roles in immune regulation and balance. Because manipulation of the CD200-CD200R interaction affects the outcome of rodent disease models, targeting of this pathway may have therapeutic utility.