Janet M. Allen

Loss of function of a lupus-associated FcγRIIb polymorphism through exclusion from lipid rafts

Dysfunction of receptors for IgG (FcγRs) has been thought to be involved in the pathogenesis of systemic lupus erythematosus (SLE). We show that a recently described SLE-associated polymorphism of FcγRIIb (FcγRIIbT232), encoding a single transmembrane amino acid substitution, is functionally impaired. FcγRIIbT232 is unable to inhibit activatory receptors because it is excluded from sphingolipid rafts, resulting in the unopposed proinflammatory signaling thought to promote SLE.

Pituitary Adenylyl Cyclase-activating Peptide Stimulates Extracellular Signal-regulated Kinase 1 or 2 (ERK1/2) Activity in a Ras-independent, Mitogen-activated Protein Kinase/ERK Kinase 1 or 2-dependent Manner in PC12 Cells

Sustained activation of extracellular signal-regulated kinase 1/2 (ERK1/2) is critical for initiating differentiation of the PC12 cell to a sympathetic-like neurone. The neuropeptide, pituitary adenylyl cyclase-activating peptide (PACAP), has been demonstrated to cause cells to adopt a neuronal phenotype, although the mechanism of this activity is unclear. PACAP through its type I receptor stimulates a biphasic activation of ERK1/2; a >10-fold increase within 5 min, followed by a >5-fold increase that is sustained for ≥60 min. An equivalent stimulation is seen in PC12 cells expressing a dominant negative Ras mutant. However, the mitogen-activated kinase/ERK kinase 1/2 (MEK1/2) inhibitor PD98059 blocked both PACAP-induced stimulation of ERK1/2 activity and neurite outgrowth. Thus, the activation signal from the PACAP type I receptor on the ERK1/2 cascade pathway is received downstream of Ras, either at Raf or MEK. Down-regulation of protein kinase C or its inhibition by calphostin C blocked the ability of PACAP to stimulate ERK1/2. We conclude that activation of PACAP type I receptor activates protein kinase C, which then activates the ERK1/2 cascade in a Ras-independent manner at either Raf or MEK1/2.

Two distinct regions of FC gamma RI initiate separate signalling pathways involved in endocytosis and phagocytosis.

Cross‐linking of the high affinity receptor for IgG, Fc gamma RI, can result in both endocytosis of immune complexes and phagocytosis of opsonized particles in myeloid cells, although the cytoplasmic domain of the receptor lacks the tyrosine activation motif which has been implicated in signal transduction triggered by cross‐linking of other Fc receptors. To identify the structural determinants of Fc gamma RI‐mediated ligand internalization, we have expressed Fc gamma RI or truncated versions of Fc gamma RI in COS cells, either alone or in the presence of the Fc epsilon RI gamma subunit (which contains a classical tyrosine activation motif and associates with Fc gamma RI in myeloid cells), and assessed their ability to mediate endocytosis and phagocytosis. We have found that Fc gamma RI alone (in the absence of the gamma subunit) is capable of mediating endocytosis in COS cells and that the process occurs via a novel, tyrosine kinase‐independent signalling pathway. Activation of this pathway following cross‐linking appears to require only the receptor extracellular domain. In contrast, Fc gamma RI phagocytic function in COS cells is dependent on an interaction between the receptor transmembrane domain and the gamma subunit and is mediated by recruitment of tyrosine kinase activity. Our data therefore indicate that distinct domains of the receptor regulate ligand internalization following receptor cross‐linking by either immune complexes (endocytosis) or opsonized particles (phagocytosis) and that these functions are mediated by different intracellular signalling pathways.

A molecular switch changes the signalling pathway used by the FcγRI antibody receptor to mobilise calcium

BACKGROUND Leukocytes express Fc gamma receptors, which are specific for the constant region of immunoglobulin G. Aggregation of these receptors activates a repertoire of responses that can lead to targeted cell killing by antibody-directed cellular cytotoxicity. The nature of the myeloid response to Fc gamma receptor aggregation is highly variable and depends on the maturation state of the cell, but little is known about the signalling mechanisms underlying this variability. RESULTS We show here that differentiation of a monocytic cell line, U937, to a more macrophage phenotype resulted in an absolute and fundamental switch in the nature of the phospholipid signalling pathway recruited following Fc gamma receptor aggregation. In cytokine-primed monocytes, aggregation of the high-affinity receptor Fc gamma RI resulted in the activation of phospholipase D and sphingosine kinase, which in turn led to the transient release of stored calcium; these effects were mediated by the gamma chain, an Fc gamma RI accessory protein. In contrast, in cells differentiated to a more macrophage type, aggregation of Fc gamma RI resulted in the Fc gamma RIIa-mediated activation of phospholipase C, and the resulting calcium response was prolonged as calcium entry was stimulated. CONCLUSIONS The switch in Fc gamma RI signalling pathways upon monocyte differentiation is mediated by a switch in the accessory molecule recruited by Fc gamma RI, which lacks its own intrinsic signal transduction motif. As many immune receptors have separate polypeptide chains for ligand binding and signal transduction (allowing a similar switch in signalling pathways), the mechanism described here is likely to be widely used.

C‐reactive protein‐mediated phagocytosis and phospholipase D signalling through the high‐affinity receptor for immunoglobulin G (FcγRI)

C‐reactive protein (CRP) is the prototypic acute‐phase protein in man which performs innate immune functions. CRP‐mediated phagocytosis may be indirect, through activation of complement and complement receptors, or direct, through receptors for the Fc portion of immunoglobulin G (IgG; FcγRs) or even a putative CRP‐specific receptor. No strong evidence has been shown to indicate which receptors may be responsible for phagocytosis or signalling responses. Using BIAcore technology, we confirm that CRP binds directly to the extracellular portion of FcγRI with a threefold higher affinity than IgG (KD = 0·81 × 10−9 m). Binding is Ca2+ dependent and is inhibited by IgG1 but not by phosphorylcholine (PC). CRP opsonization (using CRP concentrations within the normal human serum range) of PC‐conjugated sheep erythrocytes increased phagocytosis of these particles by COS‐7 cells transfected with FcγRI‐II chimaera or FcγRI/γ‐chain. Interferon‐γ‐treated U937 cells, which signal through FcγRI to activate phospholipase D (PLD) in response to cross‐linked IgG, were also activated by CRP without any requirement for further cross‐linking. These studies indicate that CRP is capable of binding to and cross‐linking FcγRI thereby resulting in PLD activation and increased phagocytosis. Uptake by FcγRI has been reported to promote various acquired immune responses suggesting that CRP could act in a similar way.

Differentiation‐dependent switch in protein kinase C isoenzyme activation by FcγRI, the human high‐affinity receptor for immunoglobulin G

Aggregation of receptors for the constant region (Fc) of immunoglobulin G on myeloid cells results in endocytosis or phagocytosis and cellular activation. Previous work has shown, using the cell line U937, that the high‐affinity immunoglobulin G receptor, FcγRI, activates alternate intracellular signalling pathways depending on the cell differentiation state, which results in a marked change in the nature of calcium transients within the cell. Here, we show that protein kinase C (PKC) is activated in both interferon‐γ (IFN‐γ) ‐primed and dibutyryl cyclic AMP (dbcAMP) ‐differentiated cells but that the nature of the particular isoenzymes recruited differs. Thus, in IFN‐γ‐primed U937 cells, FcγRI aggregation results in an increase of PKC activity which is essentially calcium independent resulting from the translocation to the membrane of the novel PKCs, δ and ε, together with the atypical PKC ζ. However, in cells differentiated to a more macrophage phenotype, all PKC enzyme activity after receptor aggregation is calcium dependent. Consistent with this finding, the isoenzymes translocated to the nuclear‐free membrane fraction are the conventional PKCs α, β and γ; results consistent with our previous finding that FcγRI couples to phospholipase C in such dbcAMP‐differentiated cells. Thus, the nature of PKC isoenzyme activated following FcγRI aggregation is defined by differentiation. The calcium dependence of the PKC isoenzyme is consistent with the duration of calcium transients previously reported in the two differentiation states.

FcγRI activation of phospholipase Cγ1 and protein kinase C in dibutyryl cAMP‐differentiated U937 cells is dependent solely on the tyrosine‐kinase activated form of phosphatidylinositol‐3‐kinase

The human high affinity receptor for immunoglobulin G, FcγRI, in dibutyryl cyclic AMP (dbcAMP)‐differentiated U937 cells, is coupled to the activation of phospholipase C (PLC) and the conventional protein kinase C (PKC) isoforms, α, β, and γ. Here we demonstrate that aggregation of FcγRI activates the tyrosine‐kinase regulated form of phosphatidylinositol‐3‐kinase (PI‐3‐kinase) and that an increase of phosphatidylinositol trisphosphate (PIP3) is essential for the activation and translocation of PLCγ1 in these cells. In addition, activation of the PKC isoforms was ablated by specific inhibitors of PI3‐kinase or by overexpression of a dominant negative p85 subunit of PI3‐kinase. The findings reported here demonstrate that PLCγ1 and PKC activation by FcγRI are downstream of PI3‐kinase, and that in contrast to cytokine primed cells, only the tyrosine‐kinase activated isoform of PI3‐kinase is coupled to FcγRI in dbcAMP‐differentiated cells.

Regulation of the Y1 neuropeptide Y receptor gene expression in PC12 cells

The Y1 receptor for neuropeptide Y (NPY-Y1) is constitutively expressed in PC12 cells. In this study, we examined the role of nerve growth factor (NGF), pituitary adenylyl cyclase activating polypeptide (PACAP) and dexamethasone on the expression of the gene encoding the rat NPY-Y1 receptor in PC12 cells. A fusion gene (pY1-Luc) was constructed where the reporter enzyme firefly luciferase was placed under the control of 700 bp of the promoter region of the rat NPY-Y1 receptor gene. This promoter region contains recognition consensus sequences for various transcription factors, including one activation protein-1 (AP-1) site, two cyclic AMP responsive element sites, one estrogen receptor element site and four glucocorticoid receptor element sites. NGF increased luciferase activity in a concentration dependent manner. This increase was inhibited by K-252a, a trk A receptor inhibitor, and calphostin C, a PKC inhibitor. PACAP-38 increased luciferase activity in a concentration dependent manner. This activation was inhibited by H-89. Dexamethasone increased transcription of NPY-Y1 gene in PC12 cells. These results indicate that differentiation of PC12 cells into endocrine-like phenotype by dexamethasone and into a neuronal-like phenotype by either NGF or PACAP-38 increases the transcriptional activity of the NPY-Y1 receptor gene in PC12 cells.

The interaction between human FCγRI and the γ-chain is mediated solely via the 21 amino acid transmembrane domain of FCγRI

We have established a biological assay to investigate the nature of the non-covalent interaction between two integral type I membrane proteins, Fc gamma RI and gamma-chain. Fc gamma RI, the human high affinity receptor for immunoglobulin G (IgG), is expressed on the surface of macrophages and monocytes and mediates a broad range of important immunological functions. Fc gamma RI relies on a functional interaction with a second integral type I membrane protein, gamma-chain, to mediate many of these functions. For example, Fc gamma RI can only mediate phagocytosis of IgG-coated particles in COS cells when co-expressed with gamma-chain. We have previously shown that the cytoplasmic domain of Fc gamma RI is not necessary for this functional interaction. In this study we have used the phagocytosis assay to investigate the role of the transmembrane region of Fc gamma RI in mediating this functional interaction with gamma-chain by using mutant and chimeric forms of the receptor. Three mutants, which introduce or remove charged residues from a conserved 10 amino acid stretch of amino acids in the proximal transmembrane region of Fc gamma RI, were able to mediate phagocytosis of IgG-coated particles. In contrast, two chimeric receptors, In which 21 of the amino acids in the distal transmembrane region of Fc gamma RI were replaced with the transmembrane region of the related receptors CD2 or LFA3, were expressed but failed to interact functionally with gamma-chain to mediate phagocytosis. Thus, these mutants demonstrate that the interaction between human Fc gamma RI and gamma-chain is mediated solely via these 21 amino acids in the transmembrane domain of Fc gamma RI.

The human high‐affinity immunoglobulin G receptor activates SH2‐containing inositol phosphatase (SHIP)

On cytokine‐primed U937 cells, aggregation of the human high‐affinity immunoglobulin receptor, FcγRI, initiates signal transduction cascades which lead to the release of calcium from intracellular stores and no significant calcium influx. In these cells, aggregation of FcγRI results in no significant increase in inositol trisphosphate production, but rather phospholipase D is activated. Here we show that, in interferon‐γ (IFN‐γ)‐primed cells, the SH2 containing inositol 5′ phosphatase, SHIP, is constitutively associated with the membrane fraction. Following aggregation of FcγRI, SHIP is rapidly and transiently tyrosine phosphorylated and becomes associated with the adapter molecule Shc. Shc also becomes tyrosine phosphorylated and translocates from the cytoplasm to the membrane fraction concomitant with the association between Shc and SHIP. Further, SHIP and Shc appear to be recruited to membrane‐associated immune complexes following FcγRI aggregation. As no immunoreceptor inhibitory motif has been demonstrated to associate with FcγRI, these data suggest that SHIP may be recruited to the receptor through an SH2 domain interaction with Shc.