Recently, a second member of the protease-activated receptor (PAR) family, named PAR-2, has been identified. Similar to the thrombin receptor, PAR-2 appears to be activated by proteolytic-mediated exposure of a "tethered ligand" sequence and can also be activated by the corresponding synthetic peptides. Similarities in the amino acid sequence of the receptors' tethered ligand sequences suggest that their respective agonist peptides might not be absolutely specific for their particular receptors. To test this, the receptor specificity of each agonist has been determined by measuring the responses of Xenopus oocytes expressing the thrombin receptor or PAR-2 to agonist peptides or enzymes. Thrombin receptors responded to thrombin, the human thrombin receptor-activating peptide SFLLRNP-NH2 (TRAP) (EC50 = 0.1 microM), and Xenopus TRAP, TFRIFD-NH2 (EC50 = 1 microM), but did not show any increase in calcium efflux over control levels with trypsin (50 nM) or PAR-2 agonist peptides (100 microM). Human and murine PAR-2 receptors responded comparably to human and murine PAR-2 agonist peptides (SLIGKVD and SLIGRL, respectively) (EC50 = 0.5-2.0 microM) and trypsin, but not to thrombin. PAR-2 was also found to be responsive to TRAP (EC50 = 1 microM) but was unresponsive to Xenopus TRAP (50 microM). Responses to additional peptide agonist analogs suggest that an amino-terminal serine is critical for PAR-2 agonist activity.

The semaphorin family contains a large number of secreted and transmembrane proteins, some of which are known to act as repulsive axon guidance cues during development or to be involved in immune function. We report here on the identification of semaphorin K1 (sema K1), the first semaphorin known to be associated with cell surfaces via a glycosylphosphatidylinositol linkage. Sema K1 is highly homologous to a viral semaphorin and can interact with specific immune cells, suggesting that like its viral counterpart, sema K1 could play an important role in regulating immune function. Sema K1 does not bind to neuropilin-1 or neuropilin-2, the two receptors implicated in mediating the repulsive action of several secreted semaphorins, and thus it likely acts through a novel receptor. In contrast to most previously described semaphorins, sema K1 is only weakly expressed during development but is present at high levels in postnatal and adult tissues, particularly brain and spinal cord.The semaphorins constitute a large family of evolutionally conserved glycoproteins that are defined by a characteristic semaphorin domain of approximately 500 amino acids (1-3). The first vertebrate semaphorin, collapsin-1 in chick, was identified by its ability to induce growth cone collapse (4). Consistent with this function, its mammalian homologue, sema III, has been shown to repel specific subsets of sensory axons (5). As a result of these and other studies, Coll-1/sema III/D has been implicated in the patterning of sensory axon projections into the ventral spinal cord and cranial nerve projections into the periphery (6 -11).Several other semaphorins have also been implicated as repulsive and/or attractive cues in axon guidance, axon fasciculation, and synapse formation (1,(12)(13)(14)(15)(16)(17). In addition, members of the semaphorin family have been implicated in functions outside the nervous system, including bone skeleton and heart formation (9), immune function (18, 19), tumor suppression (20 -22), and conferring drug resistance to cells (23).Recent studies have identified the first semaphorin receptor as a member of the neuropilin family. Neuropilin-1 is a high affinity receptor for sema III, E, and IV, whereas neuropilin-2 binds differentially to the subfamily of secreted semaphorins (24 -27).The vertebrate semaphorin family can be classified into several phylogenetically distinct subfamilies (Ref. 15, Fig. 1B). Each subfamily has a unique structural arrangement of protein domains. The secreted members of the semaphorin family contain a characteristic semaphorin domain at the NH 2 terminus, followed by an immunoglobulin (Ig) domain and a stretch of basic amino acids in the carboxyl-terminal region. Between the NH 2 -terminal semaphorin domain and the transmembrane spanning region, the transmembrane semaphorins contain several alternative structural motifs, including either an Ig domain, a stretch of thrombospondin repeats, or a sequence with no obvious domain homology. Interestingly, semaphorin-like sequences have been identifi...

No abstract

Unexpected drug activities discovered during clinical testing establish the need for better characterization of compounds in human disease-relevant conditions early in the discovery process. Here, we describe an approach to characterize drug function based on statistical analysis of protein expression datasets from multiple primary human cell-based models of inflammatory disease. This approach, termed Biologically Multiplexed Activity Profiling (BioMAP), provides rapid characterization of drug function, including mechanism of action, secondary or off-target activities, and insights into clinical phenomena. Using three model systems containing primary human endothelial cells and peripheral blood mononuclear cells in different environments relevant to vascular inflammation and immune activation, we show that BioMAP profiles detect and discriminate multiple functional drug classes, including glucocorticoids; TNF-alpha antagonists; and inhibitors of HMG-CoA reductase, calcineurin, IMPDH, PDE4, PI-3 kinase, hsp90, and p38 MAPK, among others. The ability of cholesterol lowering HMG-CoA reductase inhibitors (statins) to improve outcomes in rheumatic disease patients correlates with the activities of these compounds in our BioMAP assays. In addition, the activity profiles identified for the immunosuppressants mycophenolic acid, cyclosporin A, and FK-506 provide a potential explanation for a reduced incidence of posttransplant cardiovascular disease in patients receiving mycophenolic acid. BioMAP profiling can allow integration of meaningful human biology into drug development programs.

Improper regulation of B cell responses leads to excessive production of antibodies and contributes to the development of autoimmune disease. T helper 17 (Th17) cells also drive the development of autoimmune disease, but the role of B cells in shaping Th17 cell-mediated immune responses, as well as the reciprocal regulation of B cell responses by IL-17 family cytokines, remains unclear. The aim of this study was to characterize the regulation of IL-17A and IL-17F in a model of T cell-dependent B cell activation. Stimulation of primary human B cell and peripheral blood mononuclear cell (BT) co-cultures with α-IgM and a non-mitogenic concentration of superantigens for three days promoted a Th17 cell response as evidenced by increased expression of Th17-related gene transcripts, including Il17f, Il21, Il22, and Il23r, in CD4 T cells, as well as the secretion of IL-17A and IL-17F protein. We tested the ability of 144 pharmacologic modulators representing 91 different targets or pathways to regulate IL-17A and IL-17F production in these stimulated BT co-cultures. IL-17A production was found to be preferentially sensitive to inhibition of the PI3K/mTOR pathway, while prostaglandin EP receptor agonists, including PGE2, increased IL-17A concentrations. In contrast, the production of IL-17F was inhibited by PGE2, but selectively increased by TLR2 and TLR5 agonists. These results indicate that IL-17A regulation is distinct from IL-17F in stimulated BT co-cultures and that this co-culture approach can be used to identify pathway mechanisms and novel agents that selectively inhibit production of IL-17A or IL-17F.