A critical role for PI 3-kinase in cytokine-induced Fcα-receptor activation

Blood ◽  
2000 ◽  
Vol 95 (6) ◽  
pp. 2037-2043 ◽  
Author(s):  
Madelon Bracke ◽  
Evert Nijhuis ◽  
Jan-Willem J. Lammers ◽  
Paul J. Coffer ◽  
Leo Koenderman
Keyword(s):  
Ligand Binding ◽  
Signaling Pathways ◽  
Critical Role ◽  
Immunoglobulin A ◽  
Fc Receptors ◽  
Receptor Activation ◽  
Molecular Techniques ◽  
Dominant Negative ◽  
Receptor Modulation ◽  
Inside Out

Abstract Fc-receptors, such as FcR and FcγRII, play an important role in leukocyte activation, and rapid modulation of ligand binding (“activation”) is critical for receptor regulation. We have previously demonstrated that ligand binding to Fc-receptors on human eosinophils is dependent on cytokine stimulation. Utilization of pharmacological inhibitors provided evidence that the phenomenon of interleukin (IL)-5 induced immunoglobulin A (IgA) binding to human eosinophils requires activation of phosphatidylinositol 3-kinase (PI3K). However, eosinophils are refractory to manipulation by molecular techniques such as DNA transfection or viral infection. Here we utilize an IL-3 dependent pre-B cell line to investigate the molecular mechanism of cytokine-mediated ligand binding to FcR. In this system, IgA binding is dependent on IL-3, similarly to the requirement for IL-5 of eosinophils. We show that IL-3-mediated activation of FcR (CD89) requires the activation of PI3K, independent of p21ras activation. Co-expression of dominant negative (▵p85) and active (p110_K227E) forms of PI3K demonstrate that the affinity switch regulating FcR activation requires PI3K. Moreover, overexpression of PI3K is both necessary and sufficient for activation of FcR. Furthermore, we show that IL-3/IL-5/GM-CSF induced inside-out signaling pathways activating FcR require the involvement of protein kinase C downstream of PI3K. Finally, we show that these inside-out signaling pathways responsible for Fc-receptor modulation require CD89, independent of its association with the FcRγ chain.

Focal Adhesion Kinase (FAK) Is Required for CXCL12-Induced Dhemotaxis and Adhesion to VCAM-1 in B Lymphocytes and Hematopoietic Stem/Progenitor Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1325-1325
Author(s):  
Leslie E. Silberstein ◽  
Derek Dykxhoorn ◽  
Yi Le ◽  
Marek Honczarenko ◽  
Judy Lieberman ◽  
...  
Keyword(s):  
Cell Migration ◽  
B Cells ◽  
Signaling Pathways ◽  
Progenitor Cells ◽  
Critical Role ◽  
Hematopoietic Progenitor Cell ◽  
Hematopoietic Stem ◽  
Affect Cell Viability ◽  
Chemokine Cxcl12 ◽  
Inside Out

Abstract The chemokine CXCL12 (SDF-1) and its receptor CXCR4 play a critical role in hematopoietic progenitor cell migration and positioning within the bone marrow (BM). However, CXCL12/CXCR4-induced signaling pathways in blood cell migration are poorly characterized. Based on previous studies of BM progenitor B cells indicating a strong correlation between FAK signaling and CXCL12 induced adhesion to VCAM-1, we speculated that FAK might be an important signaling component in CXCL12-induced chemotaxis and integrin-mediated adhesion. Here, we used two approaches to reduce FAK expression in (human and mouse) progenitor B cells and mouse Sca-1+, Kit+, Lin− stem/progenitor cells. FAK specific siRNAs reduced FAK expression by 80% and abolished both CXCL12-induced chemotaxis and adhesion to VCAM-1 in the pro-B cell line, REH. FAK knock-down did not change expression levels of CXCR4 and VLA-4 integrin. FAK expression was rescued by transfection with wild type, chicken FAK, which also restored both CXCL12-induced chemotaxis and adhesion. Furthermore, we found that in FAK deficient cells CXCL12-induced activation of the GTP-ase Rap1 was reduced, suggesting the importance of FAK in CXCL12-mediated inside-out integrin activation. CXCL12-mediated chemotaxis was also impaired in primary progenitor B cells and hematopoietic stem/progenitor cells (HSC/P) isolated from FAK floxed mice, in which FAK was deleted by Cre-mediated excision of FAK floxed alleles. Cre-mediated FAK deletion did not affect cell viability or induce apoptosis. These studies suggest that FAK may function as a key intermediary in signaling pathways controlling hematopoietic cell migration and lineage development.

scholarly journals Expression of a dominant negative PKA mutation in the kidney elicits a diabetes insipidus phenotype

2015 ◽  
Vol 308 (6) ◽  
pp. F627-F638 ◽  
Author(s):  
Merle L. Gilbert ◽  
Linghai Yang ◽  
Thomas Su ◽  
G. Stanley McKnight
Keyword(s):  
Diabetes Insipidus ◽  
Apical Membrane ◽  
Collecting Duct ◽  
Critical Role ◽  
Physiological Role ◽  
Receptor Activation ◽  
Dominant Negative ◽  
Regulatory Subunit ◽  
Water Excretion ◽  
Protein Levels

PKA plays a critical role in water excretion through regulation of the production and action of the antidiuretic hormone arginine vasopressin (AVP). The AVP prohormone is produced in the hypothalamus, where its transcription is regulated by cAMP. Once released into the circulation, AVP stimulates antidiuresis through activation of vasopressin 2 receptors in renal principal cells. Vasopressin 2 receptor activation increases cAMP and activates PKA, which, in turn, phosphorylates aquaporin (AQP)2, triggering apical membrane accumulation, increased collecting duct permeability, and water reabsorption. We used single-minded homolog 1 ( Sim1)-Cre recombinase-mediated expression of a dominant negative PKA regulatory subunit (RIαB) to disrupt kinase activity in vivo and assess the role of PKA in fluid homeostasis. RIαB expression gave rise to marked polydipsia and polyuria; however, neither hypothalamic Avp mRNA expression nor urinary AVP levels were attenuated, indicating a primary physiological effect on the kidney. RIαB mice displayed a marked deficit in urinary concentrating ability and greatly reduced levels of AQP2 and phospho-AQP2. Dehydration induced Aqp2 mRNA in the kidney of both control and RIαB-expressing mice, but AQP2 protein levels were still reduced in RIαB-expressing mutants, and mice were unable to fully concentrate their urine and conserve water. We conclude that partial PKA inhibition in the kidney leads to posttranslational effects that reduce AQP2 protein levels and interfere with apical membrane localization. These findings demonstrate a distinct physiological role for PKA signaling in both short- and long-term regulation of AQP2 and characterize a novel mouse model of diabetes insipidus.

scholarly journals An Innovative High-Throughput Screening Approach for Discovery of Small Molecules That Inhibit TNF Receptors

2017 ◽  
Vol 22 (8) ◽  
pp. 950-961 ◽  
Author(s):  
Chih Hung Lo ◽  
Nagamani Vunnam ◽  
Andrew K. Lewis ◽  
Ting-Lan Chiu ◽  
Benjamin E. Brummel ◽  
...  
Keyword(s):  
Tumor Necrosis Factor ◽  
Ligand Binding ◽  
Small Molecules ◽  
High Throughput ◽  
High Throughput Screening ◽  
Tumor Necrosis ◽  
Critical Role ◽  
Receptor Activation ◽  
Computational Docking ◽  
Necrosis Factor

Tumor necrosis factor receptor 1 (TNFR1) is a transmembrane receptor that binds tumor necrosis factor or lymphotoxin-alpha and plays a critical role in regulating the inflammatory response. Upregulation of these ligands is associated with inflammatory and autoimmune diseases. Current treatments reduce symptoms by sequestering free ligands, but this can cause adverse side effects by unintentionally inhibiting ligand binding to off-target receptors. Hence, there is a need for new small molecules that specifically target the receptors, rather than the ligands. Here, we developed a TNFR1 FRET biosensor expressed in living cells to screen compounds from the NIH Clinical Collection. We used an innovative high-throughput fluorescence lifetime screening platform that has exquisite spatial and temporal resolution to identify two small-molecule compounds, zafirlukast and triclabendazole, that inhibit the TNFR1-induced IκBα degradation and NF-κB activation. Biochemical and computational docking methods were used to show that zafirlukast disrupts the interactions between TNFR1 pre-ligand assembly domain (PLAD), whereas triclabendazole acts allosterically. Importantly, neither compound inhibits ligand binding, proving for the first time that it is possible to inhibit receptor activation by targeting TNF receptor–receptor interactions. This strategy should be generally applicable to other members of the TNFR superfamily, as well as to oligomeric receptors in general.

An Important Role for Rac1 in Glycoprotein Ib-IX Induced Platelet Activation,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3250-3250
Author(s):  
Michael Keegan Delaney
Keyword(s):  
Shear Stress ◽  
Ligand Binding ◽  
Signaling Pathways ◽  
Platelet Activation ◽  
Platelet Adhesion ◽  
Critical Role ◽  
Mapk Signaling ◽  
Glycoprotein Ib ◽  
Platelet Receptor ◽  
Induced Aggregation

Abstract Abstract 3250 The platelet receptor for von Willebrand factor, the glycoprotein Ib-IX (GPIb-IX) complex, not only plays a critical role in mediating platelet adhesion at sites of vascular injury, but also transmits signals leading to platelet activation. GPIb-IX mediated activation of the ligand binding function of integrin αIIbβ3 is required for stable platelet adhesion under high shear rate conditions. Previous studies have suggested that ligand occupancy of GPIb-IX leads to activation of the Src family kinase Lyn, phosphoinositide 3-kinase (PI3K), Akt, cGMP-dependent protein kinase, and the MAPK and ITAM signaling pathways. However, it remains unclear how the PI3K/Akt pathway is activated during GPIb-IX dependent signal transduction. Using platelet-specific conditional Rac1−/− mice and the Rac1 specific inhibitor NSC23766 (NSC), we examined the role of Rac1 in GPIb-IX induced platelet activation. Platelet-specific conditional Rac1−/− mice were generated by crossing Pf4-Cre transgenic mice with mice containing the Rac1 conditional allele. Rac1−/− mouse platelets had a defect in VWF/botrocetin induced aggregation, TXA2 synthesis, and ATP secretion. Rac1−/− mouse platelets were also defective in stable adhesion to VWF under shear stress and in spreading on immobilized VWF, as compared to WT. The defects observed in Rac1−/− mouse platelets were recapitulated in human platelets treated with the Rac1 inhibitor NSC23766. Importantly, we show that ligand binding to GPIb-IX induced Rac1 activation, which was abolished in Lyn−/− mouse platelets. We also show that Rac1−/− (or NSC-treated) platelets have a defect in GPIb-IX induced Akt and P38 MAPK activation. Altogether, this study reveals that Rac1 plays an important role in GPIb-IX dependent platelet activation and stable adhesion to VWF under shear stress. Rac1 is activated downstream of Lyn and is important for GPIb-IX and Lyn-dependent activation of Akt and MAPK signaling pathways. Disclosures: No relevant conflicts of interest to declare.

A Mutation in the Extracellular Cysteine-Rich Repeat Region of the β3 Subunit Activates Integrins IIbβ3 and Vβ3

Blood ◽  
1999 ◽  
Vol 93 (8) ◽  
pp. 2559-2568 ◽  
Author(s):  
Hirokazu Kashiwagi ◽  
Yoshiaki Tomiyama ◽  
Seiji Tadokoro ◽  
Shigenori Honda ◽  
Masamichi Shiraga ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Structural Changes ◽  
Chinese Hamster ◽  
Receptor Activation ◽  
Single Amino Acid ◽  
Structural Basis ◽  
Relevant Variable ◽  
High Affinity ◽  
293 Cells ◽  
Inside Out

Abstract Inside-out signaling regulates the ligand-binding function of integrins through changes in receptor affinity and/or avidity. For example, IIbβ3 is in a low-affinity/avidity state in resting platelets, and activation of the receptor by platelet agonists enables fibrinogen to bind. In addition, certain mutations and truncations of the integrin cytoplasmic tails are associated with a high-affinity/avidity receptor. To further evaluate the structural basis of integrin activation, stable Chinese hamster ovary (CHO) cell transfectants were screened for high-affinity/avidity variants of IIbβ3. One clone (AM-1) expressed constitutively active IIbβ3, as evidenced by (1) binding of soluble fibrinogen and PAC1, a ligand-mimetic antiIIbβ3antibody; and (2) fibrinogen-dependent cell aggregation. Sequence analysis and mutant expression in 293 cells proved that a single amino acid substitution in the cysteine-rich, extracellular portion of β3(T562N) was responsible for receptor activation. In fact, T562N also activated Vβ3, leading to spontaneous binding of soluble fibrinogen to 293 cells. In contrast, neither T562A nor T562Q activated IIbβ3, suggesting that acquisition of asparagine at residue 562 was the relevant variable. T562N also led to aberrant glycosylation of β3, but this was not responsible for the receptor activation. The binding of soluble fibrinogen to IIbβ3(T562N) was not sufficient to trigger tyrosine phosphorylation of pp125FAK, indicating that additional post-ligand binding events are required to activate this protein tyrosine kinase during integrin signaling. These studies have uncovered a novel gain-of-function mutation in a region of β3 intermediate between the ligand-binding region and the cytoplasmic tail, and they suggest that this region is involved in integrin structural changes during inside-out signaling.

Human disorders caused by nuclear receptor gene mutations

2003 ◽  
Vol 75 (11-12) ◽  
pp. 1785-1796 ◽  
Author(s):  
J. C. Achermann ◽  
J. L. Jameson
Keyword(s):  
Nuclear Receptors ◽  
Nuclear Receptor ◽  
Receptor Gene ◽  
Critical Role ◽  
Receptor Activation ◽  
Dominant Negative ◽  
Loss Of Function ◽  
Orphan Nuclear Receptors ◽  
Naturally Occurring ◽  
Inactivating Mutations

The identification of naturally occurring nuclear receptor mutations highlights the critical role that many of these transcription factors play in human endocrine development and function. Inactivating mutations in the ligand-dependent nuclear receptors (TRβ, VDR, ERα, GR, MR, AR) are well characterized in patients with conditions such as androgen insensitivity syndrome (AIS) and vitamin D resistance. On the other hand, mutations in TRβ act in a dominant negative manner to cause hormone resistance. Inactivating mutations in orphan nuclear receptors have also been identified (PPARγ2, HNF4α, PNR, NURR1, SF1, DAX1, SHP) and reveal important developmental and metabolic functions for this group of receptors with previously elusive physiologic roles. In addition to loss of function mutations, receptor activation can result from mutations that confer constitutive activity or altered ligand responsiveness to the receptor (MR, AR), or from genetic duplication (DAX1) or the expression of fusion proteins (RARA, PPARγ1). Together, these naturally occurring mutations provide fascinating insight into key structural and functional receptor domains to reveal the diverse role nuclear receptors play in human biology.

A Mutation in the Extracellular Cysteine-Rich Repeat Region of the β3 Subunit Activates Integrins IIbβ3 and Vβ3

Blood ◽  
1999 ◽  
Vol 93 (8) ◽  
pp. 2559-2568 ◽  
Author(s):  
Hirokazu Kashiwagi ◽  
Yoshiaki Tomiyama ◽  
Seiji Tadokoro ◽  
Shigenori Honda ◽  
Masamichi Shiraga ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Structural Changes ◽  
Chinese Hamster ◽  
Receptor Activation ◽  
Single Amino Acid ◽  
Structural Basis ◽  
Relevant Variable ◽  
High Affinity ◽  
293 Cells ◽  
Inside Out

Inside-out signaling regulates the ligand-binding function of integrins through changes in receptor affinity and/or avidity. For example, IIbβ3 is in a low-affinity/avidity state in resting platelets, and activation of the receptor by platelet agonists enables fibrinogen to bind. In addition, certain mutations and truncations of the integrin cytoplasmic tails are associated with a high-affinity/avidity receptor. To further evaluate the structural basis of integrin activation, stable Chinese hamster ovary (CHO) cell transfectants were screened for high-affinity/avidity variants of IIbβ3. One clone (AM-1) expressed constitutively active IIbβ3, as evidenced by (1) binding of soluble fibrinogen and PAC1, a ligand-mimetic antiIIbβ3antibody; and (2) fibrinogen-dependent cell aggregation. Sequence analysis and mutant expression in 293 cells proved that a single amino acid substitution in the cysteine-rich, extracellular portion of β3(T562N) was responsible for receptor activation. In fact, T562N also activated Vβ3, leading to spontaneous binding of soluble fibrinogen to 293 cells. In contrast, neither T562A nor T562Q activated IIbβ3, suggesting that acquisition of asparagine at residue 562 was the relevant variable. T562N also led to aberrant glycosylation of β3, but this was not responsible for the receptor activation. The binding of soluble fibrinogen to IIbβ3(T562N) was not sufficient to trigger tyrosine phosphorylation of pp125FAK, indicating that additional post-ligand binding events are required to activate this protein tyrosine kinase during integrin signaling. These studies have uncovered a novel gain-of-function mutation in a region of β3 intermediate between the ligand-binding region and the cytoplasmic tail, and they suggest that this region is involved in integrin structural changes during inside-out signaling.

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