Interferon-gamma receptor 2 expression as the deciding factor in human T, B, and myeloid cell proliferation or death.

The heterodimeric interferon (IFN)-gamma receptor (IFN-gammaR) is formed of two chains. Here we show that the binding chain (IFN-gammaR1) was highly expressed on the membranes of T, B, and myeloid cells. Conversely, the transducing chain (IFN-gammaR2) was highly expressed on the surfaces of myeloid cells, moderately expressed on B cells, and poorly expressed on the surfaces of T cells. Differential cell membrane expression of IFN-gammaR2 determined the number of receptor complexes that transduced the IFN-gamma signal and resulted in a different response to IFN-gamma. After IFN-gamma stimulation, high IFN-gammaR2 membrane expression induced rapid activation of signal transducer and activator of transcription-1 (STAT-1) and high levels of interferon regulatory factor-1 (IRF-1), which then triggered the apoptotic program. By contrast, low cell membrane expression resulted in slow activation of STAT-1, lower levels of IRF-1, and induction of proliferation. Because the forced expression of IFN-gammaR2 on T cells switched their response to IFN-gamma from proliferative to apoptotic, we concluded that the surface expression of IFN-gammaR2 determines whether a cell stimulated by IFN-gamma undergoes proliferation or apoptosis.

Characterization of interfacial catalysis by Aeromonas hydrophila lipase/acyltransferase in the highly processive scooting mode.

A glycerophospholipid:cholesterol acyltransferase (GCAT) that also has lipase activity is secreted by the bacterium Aeromonas hydrophila. Hydrolysis of the sn-2-ester bond of 1,2-dimyristoyl-sn-glycero-3-phosphomethanol (DMPM) vesicles by this enzyme is shown to occur in a highly processive scooting mode in which the enzyme, substrate, and the products of hydrolysis remain bound to the vesicle interface. This conclusion is based on the following observations. (a) When there is an excess of vesicles over enzyme, the hydrolysis of the sn-2-acyl group ceases after only a fraction of the total available substrate is hydrolyzed. Addition of more enzyme, but not of more substrate, leads to a new round of hydrolysis. (b) The extent of hydrolysis of vesicles per enzyme increases with the size of the vesicles, and it corresponds to the total hydrolysis of the outer monolayer of one vesicle by one enzyme. (c) The enzyme bound to vesicles composed of reaction products or of the non-hydrolyzable phospholipid 1,2-ditetradecyl-sn-glycero-3-phosphomethanol (DTPM) is not able to undergo intervesicle exchange. Instead, intervesicle transfer of the substrate or the bound enzyme due to vesicle fusion promotes hydrolysis of all of the vesicles present in the reaction mixture. (d) Addition of DTPM vesicles to a reaction mixture containing DMPM substrate vesicles and the enzyme has no noticeable effect on the course of hydrolysis. Substrate specificity studies in the scooting mode on DMPM vesicles reveal that GCAT displays essentially no selectivity in the hydrolysis of phospholipids with different polar head groups. Treatment of GCAT with trypsin, which removes a small peptide, results in an enzyme that displays comparable catalytic activity but increased affinity for the interface. Alkyltrifluoromethyl ketones are shown to be tight-binding competitive inhibitors of GCAT. The scooting mode analysis, which has previously been shown to provide a simplified approach for analyzing the steady-state kinetics of interfacial catalysis by secreted phospholipase A2, is also useful for analyzing the interfacial kinetic behavior of lipases.

Identification and functional characterization of a second chain of the interleukin-10 receptor complex.

Interleukin-10 (IL-10) is a pleiotropic cytokine which signals through a specific cell surface receptor complex. Only one chain, that for ligand binding (IL-10Ralpha or IL-10R1), was identified previously. We report here that, although human IL-10 binds to the human IL-10R1 chain expressed in hamster cells, it does not induce signal transduction. However, the co-expression of CRFB4, a transmembrane protein of previously unknown function belonging to the class II cytokine receptor family, together with the IL-10R1 chain renders hamster cells sensitive to IL-10. The IL-10:CRFB4 complex was detected by cross-linking to labeled IL-10. In addition, the IL-10R1 chain was able to be co-immunoprecipitated with anti-CRF antibody when peripheral blood mononuclear cells were treated with IL-10. These results demonstrate that the CRFB4 chain is part of the IL-10 receptor signaling complex. Thus, the CRFB4 chain, which we designate as the IL-10R2 or IL-10Rbeta chain, serves as an accessory chain essential for the active IL-10 receptor complex and to initiate IL-10-induced signal transduction events.

Signaling by covalent heterodimers of interferon-gamma. Evidence for one-sided signaling in the active tetrameric receptor complex.

Interferon-gamma (IFN-gamma) and its receptor complex are dimeric and bilaterally symmetric. We created mutants of IFN-gamma that bind only one IFN-gammaR1 chain per dimer molecule (called a monovalent IFN-gamma) to see if the interaction of IFN-gamma with one-half of the receptor complex is sufficient for bioactivity. Mutating a receptor-binding sequence in either AB loop of a covalent dimer of IFN-gamma yielded two monovalent IFN-gammas, gamma(m)-gamma and gamma-gamma(m), which cross-link to only a single soluble IFN-gammaR1 molecule in solution and on the cell surface. Monovalent IFN-gamma competes fully with wild type IFN-gamma for binding to U937 cells but only at a greater than 100-fold higher concentration than wild type IFN-gamma. Monovalent IFN-gamma had anti-vesicular stomatitis virus activity and antiproliferative activity, and it induced major histocompatibility complex class I and class II (HLA-DR) expression. In contrast, the maximal levels of activated Stat1alpha produced by monovalent IFN-gammas after 15 min were never more than half of those produced by either wild type or covalent IFN-gammas in human cell lines. These data indicate that while monovalent IFN-gamma activates only one-half of a four-chain receptor complex, this is sufficient for Stat1alpha activation, major histocompatibility complex class I surface antigen induction, and antiviral and antiproliferative activities. Thus, while interaction with both halves of the receptor complex is required for high affinity binding of IFN-gamma and efficient signal transduction, interaction with only one-half of the receptor complex is sufficient to initiate signal transduction.

An antagonist peptide-EPO receptor complex suggests that receptor dimerization is not sufficient for activation.

Dimerization of the erythropoietin (EPO) receptor (EPOR), in the presence of either natural (EPO) or synthetic (EPO-mimetic peptides, EMPs) ligands is the principal extracellular event that leads to receptor activation. The crystal structure of the extracellular domain of EPOR bound to an inactive (antagonist) peptide at 2.7 A resolution has unexpectedly revealed that dimerization still occurs, but the orientation between receptor molecules is altered relative to active (agonist) peptide complexes. Comparison of the biological properties of agonist and antagonist EMPs with EPO suggests that the extracellular domain orientation is tightly coupled to the cytoplasmic signaling events and, hence, provides valuable new insights into the design of synthetic ligands for EPOR and other cytokine receptors.