Direct Ligand Binding Research Articles

Ganglioside GM3 Blocks the Activation of Epidermal Growth Factor Receptor Induced by Integrin at Specific Tyrosine Sites

The epidermal growth factor receptor (EGFR) can be activated by both direct ligand binding and cross-talk with other molecules, such as integrins. This integrin-mediated cross-talk with growth factor receptors participates in regulating cell proliferation, survival, migration, and invasion. Previous studies have shown that ligand-dependent EGFR activation is inhibited by GM3, the predominant ganglioside of epithelial cells, but the effect of GM3 on ligand-independent, integrin-EGFR cross-talk is unknown. Using a squamous carcinoma cell line we show that endogenous accumulation of GM3 disrupts the ligand-independent association of the integrin beta1 subunit with EGFR and results in inhibition of cell proliferation. Consistently, endogenous depletion of GM3 markedly increases the association of EGFR with tyrosine-phosphorylated integrin beta1 and promotes cell proliferation. The ligand-independent stimulation of EGFR does not require focal adhesion kinase phosphorylation or cytoskeletal rearrangement. Stimulation of EGFR and mitogen-activated protein kinase signaling by GM3 depletion involves the phosphorylation of EGFR at tyrosine residues 845, 1068, and 1148 but not 1086 or 1173. The specific blockade of phosphorylation at Tyr-845 with Src family kinase inhibition and at Tyr-1148 with phosphatidylinositol 3-kinase inhibition suggests that GM3 inhibits integrin-induced, ligand-independent EGFR phosphorylation (cross-talk) through suppression of Src family kinase and phosphatidylinositol 3-kinase signaling.

Glycosylation profile of integrin α 3β 1 changes with melanoma progression

Glycosylation of integrins has been implicated in the modulation of their function. Characterisation of carbohydrate moieties of α 3 and β 1 subunits from non-metastatic (WM35) and metastatic (A375) human melanoma cell lines was carried out on peptide- N-glycosidase F-released glycans using matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). β 1 integrin subunit from both cell lines displayed tri- and tetraantennary oligosaccharides complex type glycans, but only in A375 cell line was the sialylated tetraantennary complex type glycan (Hex 7HexNAc 6FucSia 4) present. In contrast, only α 3 subunit from metastatic cells possessed β1–6 branched structures. Our data indicate that the β 1 and α 3 subunits expressed by the metastatic A375 cell line carry β1–6 branched structures, suggesting that these cancer-associated glycan chains may modulate tumor cell adhesion by affecting the ligand binding properties of α 3β 1 integrin. In direct ligand binding assays, α 3β 1 integrin from both cell lines binds strongly to fibronectin and to much lesser degree to placental laminin. No binding to collagen IV was observed. Enzymatic removal of sialic acid residues from purified α 3β 1 integrin stimulates its adhesion to all examined ECM proteins. Our data suggest that the glycosylation profile of α 3β 1 integrin in human melanoma cells correlates with the acquisition of invasive capacity during melanoma progression.

Role of Toll-like receptors in pathogen recognition.

The innate immune system relies on a vast array of non-clonally expressed pattern recognition receptors for the detection of pathogens. Pattern recognition receptors bind conserved molecular structures shared by large groups of pathogens, termed pathogen-associated molecular patterns. The Toll-like receptors (TLRs) are a recently discovered family of pattern recognition receptors which show homology with the Drosophila Toll protein and the human interleukin-1 receptor family. Engagement of different TLRs can induce overlapping yet distinct patterns of gene expression that contribute to an inflammatory response. The TLR family is characterized by the presence of leucine-rich repeats and a Toll/interleukin-1 receptor-like domain, which mediate ligand binding and interaction with intracellular signaling proteins, respectively. Most TLR ligands identified so far are conserved microbial products which signal the presence of an infection, but evidence for some endogenous ligands that might signal other danger conditions has also been obtained. Molecular mechanisms for pathogen-associated molecular pattern recognition still remain elusive but seem to be more complicated than initially anticipated. In most cases, direct binding of microbial ligands to TLRs still has to be demonstrated. Moreover, Drosophila TLRs bind endogenous ligands, generated through a proteolytic cascade in response to an infection. In the case of endotoxin, recognition involves a complex of TLR4 and a number of other proteins. Moreover, TLR heterodimerization further extends the spectrum of ligands and modulates the response towards specific ligands. The fact that TLR expression is regulated in both a cell type- and stimulus-dependent fashion further contributes to the complexity.

Glucose Regulates Receptor Glycosylation

Vascular smooth muscle cells (VSMCs) respond to changes in extracellular glucose concentration, and exposure to high glucose promotes VSMC responsiveness to angiotensin II (AngII), which may contribute to the vascular complications associated with diabetes. Konishi and Berk show that only in the presence of high glucose do cultured VSMCs exhibit phosphorylation of the kinases Akt and extracellular signal-regulated kinases (ERK1 and ERK2) in response to AngII. AngII, which binds a heterotrimeric GTP-binding protein (G protein)-coupled receptor, may transactivate epidermal growth factor receptors (EGFRs) to mediate stimulation of the Akt and mitogen-activated protein kinase (MAPK) cascades. The authors found that the glycosylation state of EGFR was altered in response to changes in extracellular glucose concentration or in response to the presence of other metabolizable sugars, but not those that were not metabolizable. A time-dependent increase in the presence of a lower-molecular-weight form of EGFR (145 kD rather than 170 kD) appeared when the cells were cultured in low-glucose medium, consistent with glucose consumption. Furthermore, the 145-kD EGFR was not phosphorylated in response to AngII, which is consistent with finding the cells refractory to AngII when cultured in low-glucose medium. Although the 145-kD EGFR was phosphorylated in response to direct ligand binding by either heparin-binding EGF-like growth factor (HB-EGF) or by transforming growth factor-α (TGF-α), this form of the EGFR was not transactivated by G protein-coupled receptor ligands thrombin or sphingosine 1-phosphate. Thus, low glucose appears to promote a change in the glycosylation of the EGFR that alters the receptor's responsiveness to transactivation. The mechanism for this altered transactivation sensitivity may result from a redistribution of the 145-kD form from membrane microdomains, because cells grown in low-glucose medium showed a loss of EGFR from more dense fractions on sucrose density gradients. Thus, altered glycosylation of the EGFR leading to redistribution of the receptors within the plasma membrane may be a mechanism by which metabolic activity of VSMCs controls EGFR transactivation by physiological ligands, such as AngII. A. Konishi, B. C. Berk, Epidermal growth factor receptor transactivation is regulated by glucose in vascular smooth muscle cells. J. Biol. Chem. 278 , 35049-35056 (2003). [Abstract] [Full Text]

Induction of CYP1A by the N‐imidazole derivative, 1‐benzylimidazole

AbstractXenobiotics can induce cytochrome P4501A (CYP1A) by ligand binding to the aryl hydrocarbon receptor (AhR). Typical AhR ligands are polycyclic aromatic compounds with planar molecular conformation. The present work investigated the ability of the N‐imidazole derivative, 1‐benzylimidazole (BIM), to induce CYP1A in rainbow trout hepatocytes. Benzylimidazole increased hepatocellular CYP1A catalytic activity (determined as 7‐ethoxyresorufin‐O‐deethylase [EROD] activity) and CYP1A mRNA in a concentration‐dependent way. Computational studies on the molecular structure of BIM indicated that the energetically most stable BIM conformer has the imidazole ring and the phenyl ring in different planes, i.e., does not take a planar conformation. This property of BIM does not agree with the structural requirements of a typical AhR ligand. In line with this observation, we found that the AhR antagonist, α‐naphthoflavone (αNF), was not able to inhibit BIM induction of EROD activity and CYP1A mRNA, although it inhibited the induction of CYP1A by the prototypic AhR ligand, β‐naphthoflavone (βNF). The results suggest that transcriptional activation of CYP1A by the N‐imidazole derivative, BIM, is not mediated through direct ligand binding to the AhR.

Structure of the extracellular region of HER2 alone and in complex with the Herceptin Fab.

HER2 (also known as Neu, ErbB2) is a member of the epidermal growth factor receptor (EGFR; also known as ErbB) family of receptor tyrosine kinases, which in humans includes HER1 (EGFR, ERBB1), HER2, HER3 (ERBB3) and HER4 (ERBB4). ErbB receptors are essential mediators of cell proliferation and differentiation in the developing embryo and in adult tissues, and their inappropriate activation is associated with the development and severity of many cancers. Overexpression of HER2 is found in 20-30% of human breast cancers, and correlates with more aggressive tumours and a poorer prognosis. Anticancer therapies targeting ErbB receptors have shown promise, and a monoclonal antibody against HER2, Herceptin (also known as trastuzumab), is currently in use as a treatment for breast cancer. Here we report crystal structures of the entire extracellular regions of rat HER2 at 2.4 A and human HER2 complexed with the Herceptin antigen-binding fragment (Fab) at 2.5 A. These structures reveal a fixed conformation for HER2 that resembles a ligand-activated state, and show HER2 poised to interact with other ErbB receptors in the absence of direct ligand binding. Herceptin binds to the juxtamembrane region of HER2, identifying this site as a target for anticancer therapies.

INDUCTION OF CYP1A BY THE N-IMIDAZOLE DERIVATIVE, 1-BENZYLIMIDAZOLE

Xenobiotics can induce cytochrome P4501A (CYP1A) by ligand binding to the aryl hydrocarbon receptor (AhR). Typical AhR ligands are polycyclic aromatic compounds with planar molecular conformation. The present work investigated the ability of the N-imidazole derivative, 1-benzylimidazole (BIM), to induce CYP1A in rainbow trout hepatocytes. Benzylimidazole increased hepatocellular CYP1A catalytic activity (determined as 7-ethoxyresorufin-O-deethylase [EROD] activity) and CYP1A mRNA in a concentration-dependent way. Computational studies on the molecular structure of BIM indicated that the energetically most stable BIM conformer has the imidazole ring and the phenyl ring in different planes, i.e., does not take a planar conformation. This property of BIM does not agree with the structural requirements of a typical AhR ligand. In line with this observation, we found that the AhR antagonist, alpha-naphthoflavone (alphaNF), was not able to inhibit BIM induction of EROD activity and CYP1A mRNA, although it inhibited the induction of CYP1A by the prototypic AhR ligand, beta-naphthoflavone (betaNF). The results suggest that transcriptional activation of CYP1A by the N-imidazole derivative, BIM, is not mediated through direct ligand binding to the AhR.

Protein Kinase Cα Is Required for Vanilloid Receptor 1 Activation: EVIDENCE FOR MULTIPLE SIGNALING PATHWAYS

Activation of vanilloid receptor (VR1) by protein kinase C (PKC) was investigated in cells ectopically expressing VR1 and primary cultures of dorsal root ganglion neurons. Submicromolar phorbol 12,13-dibutyrate (PDBu), which stimulates PKC, acutely activated Ca(2+) uptake in VR1-expressing cells at pH 5.5, but not at mildly acidic or neutral pH. PDBu was antagonized by bisindolylmaleimide, a PKC inhibitor, and ruthenium red, a VR1 ionophore blocker, but not capsazepine, a vanilloid antagonist indicating that catalytic activity of PKC is required for PDBu activation of VR1 ion conductance, and is independent of the vanilloid site. Chronic PDBu dramatically down-regulated PKC(alpha) in dorsal root ganglion neurons or the VR1 cell lines, whereas only partially influencing PKCbeta, -delta, -epsilon, and -zeta. Loss of PKC(alpha) correlated with loss of response to acute re-challenge with PDBu. Anandamide, a VR1 agonist in acidic conditions, acts additively with PDBu and remains effective after chronic PKC down-regulation. Thus, two independent VR1 activation pathways can be discriminated: (i) direct ligand binding (anandamide, vanilloids) or (ii) extracellular ligands coupled to PKC by intracellular signaling. Experiments in cell lines co-expressing VR1 with different sets of PKC isozymes showed that acute PDBu-induced activation requires PKC(alpha), but not PKC(epsilon). These studies suggest that PKC(alpha) in sensory neurons may elicit or enhance pain during inflammation or ischemia.

Preface: All you need is love.

Retinoid X receptors (RXRs) control a wide variety of functions by virtue of their dimerization with other nuclear hormone receptors (NRs), contributing thereby to activities of different signaling pathways. We review known RXR ligands as transcriptional modulators of specific RXR-dimers and the associated biological processes. We also discuss the physiological relevance of such ligands, which remains frequently a matter of debate and which at present is best met by member(s) of a novel family of retinoids, postulated as Vitamin A5. Through comparison with other natural, but also with synthetic ligands, we discuss high diversity in the modes of ligand binding to RXRs resulting in agonistic or antagonistic profiles and selectivity towards specific subtypes of permissive heterodimers. Despite such diversity, direct ligand binding to the ligand binding pocket resulting in agonistic activity was preferentially preserved in the course of animal evolution pointing to its functional relevance, and potential for existence of other, species-specific endogenous RXR ligands sharing the same mode of function.

The Binding of Oxidized Low Density Lipoprotein to Mouse CD36 Is Mediated in Part by Oxidized Phospholipids That Are Associated with Both the Lipid and Protein Moieties of the Lipoprotein

There is growing evidence that CD36 has an important physiological function in the uptake of oxidized low density lipoprotein (OxLDL) by macrophages. However, the ligand specificity and the nature of the ligands on OxLDL that mediate the binding to CD36 remain ill defined. Results from recent studies suggested that some of the macrophage scavenger receptors involved in the uptake of OxLDL recognized both the lipid and the protein moieties of OxLDL, but there was no conclusive direct evidence for this. The present studies were undertaken to test whether a single, well characterized OxLDL receptor, CD36, could bind both the lipid and protein moieties of OxLDL. COS-7 cells transiently transfected with mouse CD36 cDNA bound intact OxLDL with high affinity. This binding was very effectively inhibited ( approximately 50%) both by the reconstituted apoB from OxLDL and by microemulsions prepared from OxLDL lipids. The specific binding of both moieties to CD36 was further confirmed by direct ligand binding analysis and by demonstrating reciprocal inhibition, i.e. apoB from OxLDL inhibited the binding of the OxLDL lipids and vice versa. Furthermore, a monoclonal mouse antibody that recognizes oxidation-specific epitopes in OxLDL inhibited the binding of intact OxLDL and also that of its purified protein and lipid moieties to CD36. This antibody recognizes the phospholipid 1-palmitoyl 2-(5'-oxovaleroyl) phosphatidylcholine. This model of an oxidized phospholipid was also an effective competitor for the CD36 binding of both the resolubilized apoB and the lipid microemulsions from OxLDL. Our results demonstrate that oxidized phospholipids in the lipid phase or covalently attached to apoB serve as ligands for recognition by CD36 and, at least in part, mediate the high affinity binding of OxLDL to macrophages.

Lipoxins, aspirin-triggered 15-epi-lipoxin stable analogs and their receptors in anti-inflammation: a window for therapeutic opportunity.

LXs and 15-epimer LXs are generated during cell-cell interactions that occur during multicellular host response to inflammation, tissue injury or host defense. Results indicate that they are present in vivo during human illness and carry predominantly counter-regulatory biological actions opposing the action of well-characterized mediators of inflammation that appear to lead to resolution of the inflammatory response or promotion of repair and wound healing. The first selective receptor of LXA4 was identified by direct ligand binding and was cloned and characterized. Its signaling involves a novel polyisoprenyl-phosphate pathway that directly regulates PLD (Levy et al. 1999a). LX- and 15-epimer-LX-stable analogs that resist metabolic inactivation were designed, synthesized and shown to be potent LX mimetics and novel topically active anti-inflammatory agents in animal models. These new investigational tools enable structure-function studies of LX signal transduction, further elucidation of the role of LX and 15-epimer LX in host responses and exploitation of their potent bioactions in the design of novel pharmacologic agents.

Identification of calcium binding sites in the trypanosome flagellar calcium-acyl switch protein

The 24 kDa flagellar calcium binding protein (FCaBP) of the protozoan Trypanosoma cruzi is a calcium-acyl switch protein. FCaBP is modified by the addition of myristate and palmitate at its amino terminal segment and both modifications are required for calcium-modulated flagellar membrane association. FCaBP has four sequence motifs for potential calcium binding, and comparison to other calcium-acyl switch proteins, such as recoverin, suggested that only two of these sites are functional. Because it is not possible to predict with certainty the calcium binding affinity or selectivity based on motif analysis alone, we determined the quantitative calcium binding activity of FCaBP by direct ligand binding using the flow dialysis method. The results demonstrated the presence of two calcium binding sites in the full length FCaBP and in a mutant (FCaBPΔ12) lacking the amino terminal pair of sites. FCaBPΔ12 retains its ability to localize to the flagellum. A mutant FCaBP lacking the two carboxyl-terminal sites (FCaBPΔ34), did not bind calcium with high affinity and selectivity under the conditions used. The calcium binding properties of FCaBP are therefore distinct from other myristoyl switch proteins such as recoverin. The results add to a growing body of knowledge about the correlation of sequence motifs with calcium binding activity. Moreover, they demonstrate the need to determine the apparently novel mechanism by which FCaBP undergoes calcium modulated flagellar membrane association and its relation to calcium signal transduction.

Recombinant soluble human alpha 3 beta 1 integrin: purification, processing, regulation, and specific binding to laminin-5 and invasin in a mutually exclusive manner.

Using insect cells, we expressed large quantities of soluble human integrin alpha 3 beta 1 ectodomain heterodimers, in which cytoplasmic and transmembrane domains were replaced by Fos and Jun dimerization motifs. In direct ligand binding assays, soluble alpha 3 beta 1 specifically bound to laminin-5 and laminin-10, but not to laminin-1, laminin-2, fibronectin, various collagens, nidogen, thrombospondin, or complement factors C3 and C3b. Soluble alpha 3 beta1 integrin also bound to invasin, a bacterial surface protein, that mediates entry of Yersinia species into the eukaryotic host cell. Invasin completely displaced laminin-5 from the alpha 3 beta 1 integrin, suggesting sterically overlapping or identical binding sites. In the presence of 2 mM Mg2+, alpha 3 beta 1's binding affinity for invasin (Kd = 3.1 nM) was substantially greater than its affinity for laminin-5 (Kd > 600 nM). Upon addition of 1 mM Mn2+, or activating antibody 9EG7, binding affinity for both laminin-5 and invasin increased by about 10-fold, whereas the affinity decreased upon addition of 2 mM Ca2+. Thus, functional regulation of the purified soluble integrin alpha 3 beta 1 ectodomain heterodimer resembles that of wild-type membrane-anchored beta 1 integrins. The integrin alpha 3 subunit was entirely cleaved into disulfide-linked heavy and light chains, at a newly defined cleavage site located C-terminal of a tetrabasic RRRR motif. Within the alpha 3 light chain, all potential N-glycosylation sites bear N-linked mannose-rich carbohydrate chains, suggesting an important structural role of these sugar residues in the stalk-like region of the integrin heterodimer. In conclusion, studies of our recombinant alpha 3 beta 1 integrin have provided new insights into alpha 3 beta1 structure, ligand binding function, specificity, and regulation.

Deficiency of β1 Integrins in Teratoma Interferes with Basement Membrane Assembly and Laminin-1 Expression

Subcutaneous injection of β1 integrin-deficient embryonic stem cells in mice causes the formation of teratomas although they occur with a lower frequency and are smaller than wild-type cells. Immunofluorescence analysis of these deficient tumors indicates a disorganized deposition of several basement membrane proteins. This was confirmed by electron microscopy which demonstrated frequent gaps in cell-associated basement membranes or loss of close contacts to the cells. Further aberrant features were multilaminar structures and amorphous deposits, indicating a strong impairment of correct basement membrane assembly. Quantitative radioimmunoassays were used to determine the levels of specific proteins in successive tissue extracts with neutral buffer in the absence and presence of EDTA and with 6 M guanidine. This demonstrated a more than 90% decrease in the content of laminin-1 (α1β1γ1) and a 70% decrease in nidogen in the β1 integrin-deficient teratomas. No significant changes were detected for other matrix proteins (perlecan, fibronectin, fibulins). This selective change impaired the formation of laminin-nidogen complex and enhanced nidogen degradation. Northern blots also demonstrated a distinctly reduced expression of laminin α1, β1, and γ1 chains. Similar reductions were also observed in cultured embryonic stem cells prior to any differentiation. No or only smaller changes were observed for laminin α2 and β2 chain, nidogen, and perlecan mRNA. These data emphasize a distinct role of β1 integrins in the correct assembly of basement membranes which may occur through direct ligand binding and/or regulatory events at the transcriptional level.

Bivalence of EGF-like ligands drives the ErbB signaling network.

Signaling by epidermal growth factor (EGF)-like ligands is mediated by an interactive network of four ErbB receptor tyrosine kinases, whose mechanism of ligand-induced dimerization is unknown. We contrasted two existing models: a conformation-driven activation of a receptor-intrinsic dimerization site and a ligand bivalence model. Analysis of a Neu differentiation factor (NDF)-induced heterodimer between ErbB-3 and ErbB-2 favors a bivalence model; the ligand simultaneously binds both ErbB-3 and ErbB-2, but, due to low-affinity of the second binding event, ligand bivalence drives dimerization only when the receptors are membrane anchored. Results obtained with a chimera and isoforms of NDF/neuregulin predict that each terminus of the ligand molecule contains a distinct binding site. The C-terminal low-affinity site has broad specificity, but it prefers interaction with ErbB-2, an oncogenic protein acting as a promiscuous low-affinity subunit of the three primary receptors. Thus, ligand bivalence enables signal diversification through selective recruitment of homo- and heterodimers of ErbB receptors, and it may explain oncogenicity of erbB-2/HER2.

Folding-dependent binding of thyrotropin (TSH) and TSH receptor autoantibodies to the murine TSH receptor ectodomain.

The mouse TSH receptor ectodomain (mTSHR-ecd) was amplified from murine thyroid complementary DNA and ligated into the pAcGP67B insect cell vector, and the nucleotide sequence was confirmed. Employing a baculovirus-insect cell system, the mTSHR-ecd (amino acids 22-415) was expressed as a fusion protein with the gp67 insect cell signal sequence at the NH2-terminus and a C-terminal six-histidine tag. Protein expression was assessed by Western blot using a murine monoclonal antibody (recognizing amino acids 22-35) and a rabbit antipeptide antibody (recognizing amino acids 397-415). These antibodies detected two principal species of mTSHR-ecd, one glycosylated (66 kDa) and one nonglycosylated (52 kDa), in cell lysates of infected insect cells. More than 10% of these species were present in a water-soluble (cytosolic) fraction. This fraction was then used to purify, under native conditions, 100-microg amounts of mTSHR-ecd using nickel-nitrilo-triacetic (Ni-NTA) resin chromatography. The purified cytosolic mTSHR-ecd migrated as a homogeneous 66-kDa band visible on Coomassie blue-stained gels and was confirmed by Western blotting. We also purified the mTSHR-ecd from total cell lysates under denaturing conditions, followed by "in vitro" refolding on the Ni-NTA column. Under these conditions, milligram amounts of soluble mTSHR-ecd were obtained. This material consisted primarily of the 66-kDa glycosylated form, but in addition contained four or five lower molecular mass, partially glycosylated intermediates and the 52-kDa nonglycosylated form. Deglycosylation with either endoglycosidase F or H, reduced all mTSHR-ecd glycosylated species to a 52-kDa nonglycosylated form. Both the cytosolic and refolded mTSHR-ecd preparations inhibited the binding of [125I]TSH to the full-length human TSHR expressed in Chinese hamster ovary cells in a dose-dependent manner, with similar affinities. The affinity of such interactions was 3 orders of magnitude less than observed with native porcine TSHR and was further reduced by unfolding the mTSHR-ecd preparations. The cytosolic and refolded mTSHR-ecd were also recognized by hTSHR autoantibodies in the serum of patients with hyperthyroid Graves' disease. Such autoantibody binding to mTSHR-ecd was also markedly reduced by unfolding the antigen. These results demonstrated the successful production of large quantities of well characterized, biologically active, mTSHR-ecd antigen. In addition, the data showed that although the ectodomain of the mTSHR bound TSH, intact holoreceptor may be required for high affinity ligand binding. Whether the transmembrane region is required for direct ligand binding, as seen for other G protein-linked receptors, or whether it is needed to stabilize the ligand binding to the ectodomain and maintain a correctly folded state, remains unclear.

The Urokinase-receptor (CD87) Is Expressed in Cells of the Megakaryoblastic Lineage

Megakaryocytopoiesis is governed in the bone marrow microenvironment by cellular interactions that include various adhesion receptor systems and pericellular proteolysis for proper regulation of cell motility and differentiation. In order to define the role of cell surface molecules required for these processes, we searched for protease receptors on these cells. In an in vitro system utilizing different cell lines of the megakaryoblastic lineage (MEG-01, Dami), low level surface expression of the urokinase (uPA) receptor was noted. Following stimulation with phorbolester (PMA), a 3-6 fold higher expression of uPA receptor over a period of up to 5 days could be observed by fluorescent activated cell-sorting as well as by direct ligand-binding of amino-terminal fragment of uPA or vitronectin. Together with elevated expression of alpha IIb beta 3-integrin (glycoprotein IIb/IIIa complex), double immuno-fluorescence staining of stimulated cells confirmed the increased cell surface localization of uPA receptor. Semi-quantitative RT-PCR, ligand blot analysis and measurement of cell-bound proteolytic activity revealed a differentiation-dependent upregulation of the uPA receptor expression in megakaryoblastic cell lines as in monocytic cells. Due to its glycolipid anchorage, incubation with phosphatidylinositol-specific phospholipase C reduced uPA receptor-mediated ligand binding by about 60%, uPA receptor mRNA was expressed in cultured megakaryocytes derived from bone marrow, whereas no uPA receptor mRNA was detectable in platelets. These results indicate a differentiation-dependent increase in the expression of uPA receptor in megakaryoblastic cells. The characteristics of surface expression and functionality of the receptor on megakaryocytic cells may influence their maturation by regulating cellular communication in the bone marrow micro-environment.

Identification of Transmembrane Domain Residues Determinant in the Structure-Function Relationship of the Human Platelet-activating Factor Receptor by Site-directed Mutagenesis

Platelet-activating factor (PAF) is a potent phospholipid mediator that produces a wide range of biological responses. The PAF receptor is a member of the seven-transmembrane GTP-binding regulatory protein-coupled receptor superfamily. This receptor binds PAF with high affinity and couples to multiple signaling pathways, leading to physiological responses that can be inhibited by various structurally distinct PAF antagonists. We have used site-directed mutagenesis and functional expression studies to examine the role of the Phe97 and Phe98 residues located in the third transmembrane helix and Asn285 and Asp289 of the seventh transmembrane helix in ligand binding and activation of the human PAF receptor in transiently transfected COS-7 cells. The double mutant FFGG (Phe97 and Phe98 mutated into Gly residues) showed a 3-4-fold decrease in affinity for PAF, but not for the specific antagonist WEB2086, when compared with the wild-type (WT) receptor. The FFGG mutant receptor, however, displayed normal agonist activation, suggesting that these two adjacent Phe residues maintain the native PAF receptor conformation rather than interacting with the ligand. On the other hand, substitution of Ala for Asp289 increased the receptor affinity for PAF but abolished PAF-dependent inositol phosphate accumulation; it did not affect WEB2086 binding. Substitution of Asn for Asp289, however, resulted in a mutant receptor with normal binding and activation characteristics. When Asn285 was mutated to Ala, the resulting receptor was undistinguishable from the WT receptor. Surprisingly, substitution of Ile for Asn285 led to a loss of ligand binding despite normal cell surface expression levels of this mutant, as verified by flow cytometric analysis. Our data suggest that residues 285 and 289 are determinant in the structure and activation of the PAF receptor but not in direct ligand binding, as had been recently proposed in a PAF receptor molecular model.

Evidence for glycosphingolipid modification of the type 1 IFN receptor.

P1-determinant glycolipids that include two membrane glycosphingolipids, globotriaosylceramide (Gal alpha 1-4Gal beta 1-4Glc-ceramide, Gb3) and galabiosylceramide (Gal alpha 1-4Gal-ceramide, Gb2) are receptors for an Escherichia coli-derived subunit toxin, verotoxin (VT-1). Studies with Daudi cells and glycosphingolipid-deficient Daudi mutants and U937 cells identified that the presence of Gb2/Gb3 correlates with IFN-alpha sensitivity. Comparison of amino acid sequences between VT-1 and the extracellular region of the 63-kDa IFN-alpha beta receptor (IFNAR) peptide reveals regions of identity, specifically in those domains in the VT-1 B subunit, that have been implicated as Gb2/Gb3 binding sites. In direct ligand binding studies, we show that membrane Gb2/Gb3 content affects the binding capacity of cells for IFN-alpha, although IFNAR cell surface expression is unaffected. Binding of IFN-alpha to the receptor leads to kinase-associated phosphorylation of the latent transcription factor, ISGF3, which activates transcription by binding to IFN-stimulated regulatory elements in IFN-sensitive genes. Electrophoretic mobility band shift assays indicated that U-937 and Daudi mutant cells, deficient in Gb2 and Gb3, exhibited reduced nuclear factor binding to the human 2-5A synthetase IFN-stimulated regulatory element when compared with wild-type Daudi cells, after exposure to IFN-alpha. Moreover, when Daudi cells were treated with a ceramide analogue, 1-phenyl-2-(decanoylamino)-3-morpholino-1-propanol, Gb2 and Gb3 synthesis was inhibited and a concomitant reduction in IFN-induced ISGF3 activation was noted. IFNAR cell surface expression was unaffected by 1-phenyl-2-(decanoylamino)-3-morpholino-1-propanol treatment. By using a fusion protein of the extracellular domain of IFNAR linked at the carboxyl terminus to the Fc portion of IgG1, we demonstrate that IFNAR is able to bind preferentially to Gb2. These results suggest that an association of IFNAR with membrane Gal alpha 1-4Gal containing glycolipids facilitates receptor-mediated signaling.

Immunological identification and functional quantitation of retinoic acid and retinoid X receptor proteins in human skin.

We have determined protein levels of total and individual nuclear retinoic acid (RAR-alpha, -beta, -gamma) and retinoid X (RXR-alpha, -beta, -gamma) receptors by ligand binding, Western analysis, and gel shift assays, in adult human skin, a major retinoid-responsive tissue. Total RARs and RXRs, measured by direct binding of specific ligands, were 0.24 +/- 0.01 fmol/micrograms (n = 13) and 1.26 +/- 0.08 fmol/micrograms (n = 7), respectively. These values calculated on an average per cell basis were 1790 RARs/cell and 9400 RXRs/cell. Similar results were obtained with competitive ligand binding assays. RAR-alpha, -beta, and -gamma were each specifically immunoprecipitated, and their levels determined by ligand binding assays of supernatants and Western analysis of precipitates. RAR-gamma was the most abundant, representing 87% of RAR protein. The remaining 12-14% of RAR protein was RAR-alpha. No RAR-beta was detected. Similar immunoprecipitation studies revealed that RXR-alpha represented 90% of RXR protein expressed in human skin. No RXR-beta or RXR-gamma proteins were detected by Western blot. Supershift gel retardation with antibodies to RARs detected probe-RAR-alpha and probe-RAR-gamma complexes in a 1 to 4 ratio. No probe-RAR-beta complex was detected. With antibodies to both RAR-gamma and RXR, a double supershifted complex was formed, indicating that RAR-gamma/RXR heterodimers bound to the probe. These data demonstrate 1) protein levels of RXRs are five times greater than RARs, 2) relative protein levels of RAR and RXR family members are compatible with their previously described relative mRNA levels, and 3) RXR-alpha/RAR-gamma heterodimers are the major retinoid receptors that have the potential to regulate transcription of target genes, in adult human skin.