Human epidermal growth factor (EGF) module-containing mucin-like hormone receptor 3 is a new member of the EGF-TM7 family that recognizes a ligand on human macrophages and activated neutrophils.

The epidermal growth factor (EGF)-TM7 subgroup of G-protein-coupled receptors is composed predominantly of leukocyte-restricted glycoproteins defined by their unique hybrid structure, in which extracellular EGF-like domains are coupled to a seven-span transmembrane moiety via a mucin-like stalk. The EGF-TM7 group comprises mouse F4/80, human EGF module-containing mucin-like hormone receptor (EMR) 1, human EMR2, and human and mouse CD97, the genes for which map to human chromosome 19p13 and the syntenic regions of the mouse genome. In this study we describe the cloning and characterization of EMR3, a novel human EGF-TM7 molecule, and show the existence of its cellular ligand. The EMR3 gene maps closely to the existing members of the EGF-TM7 family on human chromosome 19p13.1 and, in common with other EGF-TM7 genes, is capable of generating different protein isoforms through alternative splicing. Two alternative splice forms have been isolated: one encoding a 652-amino acid cell surface protein consisting of two EGF-like domains, a mucin stalk, and a putative G-protein-coupled receptor domain and the other encoding a truncated soluble form containing only two EGF-like domains. As with other members of the EGF-TM7 family, EMR3 mRNA displays a predominantly leukocyte-restricted expression pattern, with highest levels in neutrophils, monocytes, and macrophages. Through the use of soluble EMR3 multivalent probes we have shown the presence of a ligand at the surface of monocyte-derived macrophages and activated human neutrophils. These interactions suggest a potential role for EMR3 in myeloid-myeloid interactions during immune and inflammatory responses.

IL-12 selectively regulates STAT4 via phosphatidylinositol 3-kinase and Ras-independent signal transduction pathways.

IL-12 is an important immunomodulatory cytokine that induces tyrosine phosphorylation and activation of the signal transducer and activator of transcription (STAT)4. IL-12 induces sustained activation and nuclear translocation of STAT4 and this regulatory process is coupled to both tyrosine and serine phosphorylation of this molecule. IL-12-activated tyrosine kinases are the Janus kinases Jak2 and Tyk2 but little is known about IL-12 regulation of serine kinases. The object of the present study was to explore the role of mitogen-activated protein kinases (MAPK) Erk1 and Erk2 and phosphatidylinositol 3-kinase (PI3K) in STAT4 regulation. Here we show that the IL-12-induced STAT4 serine kinase is not sensitive to inhibitors of the PI3K or MAPK Erk1,2. Moreover, IL-12 activation of STAT4 in human peripheral blood-derived T cells is not accompanied by stimulation of the Ras guanine nucleotide binding cycle or stimulation of MAPK Erk1,2 or initiation of the PI3K signaling pathways. IL-12 is unable to initiate the serine phosphorylation of STAT 1 and 3. This reveals that the STAT1, 3 and 4 serine kinases are not coordinately regulated in human T cells and that IL-12 must regulate serine phosphorylation of STAT4 by a kinase network distinct to the MAPK Erk1,2 or PI3K pathways.

Copresentation of natural HIV-1 agonist and antagonist ligands fails to induce the T cell receptor signaling cascade.

It is not known how human immunodeficiency virus type 1 (HIV-1)-derived antagonist peptides interfere with intracellular activation of cytotoxic T lymphocytes (CTL). We identified Gag epitope variants in HIV-1-infected patients that act as antagonists of CTL responses to unmutated epitopes. We then investigated the effect that presentation of each variant has on the early events of T cell receptor (TCR) signal transduction. We found that altered peptide ligands (APL) failed to induce phosphorylation of pp36, a crucial adaptor protein involved in TCR signal transduction. We further investigated the effect that simultaneous presentation of APL and native antigen at low, physiological, peptide concentrations (1 nM) has on TCR signal transduction, and we found that the presence of APL can completely inhibit induction of the protein tyrosine phosphorylation events of the TCR signal transduction cascade.

Binding of soluble natural ligands to a soluble human T-cell receptor fragment produced in Escherichia coli.

An Escherichia coli expression system has been developed to produce milligram quantities of the variable domains of a human T-cell receptor from a cytotoxic T cell that recognizes the HLA-A2-influenza matrix peptide complex as a single polypeptide chain. The recombinant protein was purified by metal-chelate chromatography and then refolded in a redox buffer system. The refolded protein was shown to directly bind both Staphylococcus aureus enterotoxin B and the major histocompatibility complex protein-peptide complex using a BIAcore biosensor. Thus this preparation of a single-chain, variable-domain, T-cell receptor fragment can bind both of its natural ligands and some of it is therefore a functional fragment of the receptor molecule.