A high-affinity receptor for urokinase plasminogen activator on human keratinocytes: characterization and potential modulation during migration.

Low passage cultures of normal human keratinocytes produce several components of the plasminogen activator/plasmin proteolytic cascade, including urokinase plasminogen activator (uPA), tissue plasminogen activator (tPA), and two specific inhibitors. Studies here presented demonstrate that these cells also contain a high-affinity (Kd = 3 x 10(-10) M) plasma membrane-binding site for uPA. High molecular weight uPA, either as the single-chain precursor or two-chain activated form, bound to the receptor; however, low molecular weight (33 kD) uPA, tPA, or epidermal growth factor did not compete for binding, demonstrating specificity. Acid treatment, which removed endogenous uPA from the receptor, was required to detect maximal binding (45,000 sites per cell). To investigate the possibility that the uPA receptor on keratinocytes may be involved in epithelial migration during wound repair, cultures were wounded and allowed to migrate into the wounded site. Binding sites for uPA were localized by autoradiographic analysis of 125I-uPA binding as well as by immunocytochemical studies using anti-uPA IgG. With both techniques uPA binding sites were detected selectively on the plasma membrane of cells at the leading edge of the migrating epithelial sheet. This localization pattern suggests that uPA receptor expression on keratinocytes may be coupled to cell migration during cutaneous wounding.     

The binding cascade of SecB to SecA to SecY/E mediates preprotein targeting to the E. coli plasma membrane

The export of many E. coli proteins such as proOmpA requires the cytosolic chaperone SecB and the membrane-bound preprotein translocase. Translocase is a multisubunit enzyme with the SecA protein as its peripheral membrane domain and the SecY/E protein as its integral domain. SecB, by binding to proOmpA in the cytosol, prevents its aggregation or association with membranes at nonproductive sites. The SecA receptor binds the proOmpA-SecB complex (Kd approximately 6 x 10(-8) M) through direct recognition of both the SecB (Kd approximately 2 x 10(-7) M) as well as the leader and mature domains of the precursor protein. SecB has a dual function in stabilizing the precursor and in passing it on to membrane-bound SecA, the next step in the pathway. SecA itself is bound to the membrane by its affinity (Kd approximately 4 x 10(-8) M) for SecY/E and for acidic lipids. The functions of SecB and SecA as a two-stage receptor system are linked by their affinity for each other.     

Binding of epidermal growth factor by human colon carcinoma cell (Caco-2) monolayers

The objective of this study was to investigate whether Caco-2 cells bind and internalize epidermal growth factor (EGF). [125I]EGF was presented to the apical (AP) or basolateral (BL) side of Caco-2 monolayers, grown on microporous membranes, at different times in culture. At day 10, [125I]EGF binding (at 37 degrees C) to the BL membrane was 2-3 times greater than binding to the AP membrane. Of that [125I]EGF bound to the AP membrane 76% was internalized within 3 h while internalization from the BL membrane was 90%. At lower temperatures membrane-bound [125I]EGF increased while internalization decreased. At day 16, AP and BL binding decreased and then remained constant through day 25. [125I]EGF was bound to the BL membrane of 10 days old monolayers with a Kd of 0.67 nM. There was a single binding site whose numbers in the BL membrane was about 5500/cell.     

Modulation of the expression of the VIP receptor by serum factors on the human melanoma cell line IGR39.

IGR39 cells, isolated from a human superficial melanoma, display at their surface high and low affinity receptors for the vasoactive intestinal peptide (VIP). When grown in DME medium supplemented with 10% fetal calf serum, cells display 1.6 x 10(5) high affinity (Kd 0.74 nM) and 5.6 x 10(5) low affinity (Kd 55 nM) VIP binding sites per cell. When cultured in a chemically defined medium containing EGF, transferrin, and selenium, IGR39 cells display many neurite-like extensions. Following these morphological changes, the specific [125I]VIP binding is increased four- to fivefold after 6 days in culture. This phenomenon is reversible and is the result of an increased number of VIP binding sites available at the cell surface, without modification of their affinities. The molecular mass of the binding sites is also unchanged whatever cell culture conditions. Increase in [125I]VIP binding is inversely correlated to the serum concentration in the culture medium. When added to the chemically defined medium, sera from various origins as well as some serum substitutes reduce [125I]VIP binding to the same extent as that of the serum. The total cAMP production by VIP-stimulated IGR39 cells is enhanced by a factor of six to seven when cells are cultured in serum-free medium, in good correlation with the increase of VIP binding capacity. These data suggest that factor(s) present in fetal calf serum inhibit(s) the expression of VIP receptor, thus demonstrating the importance of a strict control of cell culture conditions for in vitro studies.     

Specific binding of [3H]heparin to human carcinoma SW-13 and other mammalian cells

This study reports on the specific binding of [3H]heparin to human adrenocortical carcinoma cell line SW-13. Heparin binding to SW-13 cells is specific, saturable, and time- and temperature-dependent with maximum binding occurring between 90 and 120 min at 22 degrees C. Scatchard analysis revealed two classes of binding sites. The apparent Kd for high-affinity receptors is 2.14 x 10(-8) M with 1.48 x 10(6) sites per cells. Six other tested mammalian cell lines also have specific binding sites for heparin.     

Binding of lys-plasminogen to E-fragment of fibrinogen

The interaction of Lys-plasminogen and its fragments with fibrinogen fragment E was studied by equilibrium affinity binding. A quantitative analysis of binding parameters revealed two types of binding sites responsible for Lys-plasminogen interaction with the immobilized fragment E, i.e., with a high (Kd = 1.5 x 10(-6) M) and low (Kd = 82 x 10(-6) M) affinity ones. Among plasminogen fragments, only miniplasminogen and KI-3 bound immobilized fragment E and were eluted by epsilon-aminocaproic acid. Hence, two lysine binding sites may be involved in the binding of Lys-plasminogen to fragment E; they are localized in the KI-3 and K5 kringle structures.     

Characterization of an intracellular hyaluronic acid binding site in isolated rat hepatocytes.

125I-HA, prepared by chemical modification at the reducing sugar, specifically binds to rat hepatocytes in suspension or culture. Intact hepatocytes have relatively few surface 125I-HA binding sites and show low specific binding. However, permeabilization of hepatocytes with the nonionic detergent digitonin results in increased specific 125I-HA binding (45-65%) and a very large increase in the number of specific 125I-HA binding sites. Scatchard analysis of equilibrium 125I-HA binding to permeabilized hepatocytes in suspension at 4 degrees C indicates a Kd = 1.8 x 10(-7) M and 1.3 x 10(6) molecules of HA (Mr approximately 30,000) bound per cell at saturation. Hepatocytes in primary culture for 24 h show the same affinity but the total number of HA molecules bound per cell at saturation decreases to approximately 6.2 x 10(5). Increasing the ionic strength above physiologic concentrations decreases 125I-HA binding to permeable cells, whereas decreasing the ionic strength above causes an approximately 4-fold increase. The divalent cation chelator EGTA does not prevent binding nor does it release 125I-HA bound in the presence of 2 mM CaCl2, although higher divalent cation concentrations stimulate 125I-HA binding. Ten millimolar CaCl2 or MnCl2 increases HA binding 3-6-fold compared to EGTA-treated cells. Ten millimolar MgCl2, SrCl2, or BaCl2 increased HA binding by 2-fold. The specific binding of 125I-HA to digitonin-treated hepatocytes at 4 degrees C increased greater than 10-fold at pH 5.0 as compared to pH 7.(ABSTRACT TRUNCATED AT 250 WORDS)     

Desmoglein endocytosis and desmosome disassembly are coordinated responses to pemphigus autoantibodies

Desmosomes are adhesive intercellular junctions prominent in the skin and heart. Loss of desmosome function is associated with severe congenital and acquired disorders characterized by tissue fragility. Pemphigus vulgaris (PV) is an autoimmune disorder in which antibodies are directed against the desmosomal adhesion molecule Dsg3, resulting in severe mucosal erosions and epidermal blistering. To define the mechanisms by which Dsg3 autoantibodies disrupt keratinocyte adhesion, the fate of PV IgG and various desmosomal components was monitored in primary human keratinocytes exposed to PV patient IgG. PV IgG initially bound to keratinocyte cell surfaces and colocalized with desmosomal markers. Within 6 h after PV IgG binding to Dsg3, electron microscopy revealed that desmosomes were dramatically disrupted and keratinocyte adhesion was severely compromised. Immunofluorescence analysis indicated that PV IgG and Dsg3 were rapidly internalized from the cell surface in a complex with plakoglobin but not desmoplakin. Dsg3 internalization was associated with retraction of keratin filaments from cell-cell borders. Furthermore, the internalized PV IgG-Dsg3 complex colocalized with markers for both endosomes and lysosomes, suggesting that Dsg3 was targeted for degradation. Consistent with this possibility, biotinylation experiments demonstrated that soluble Dsg3 cell surface pools were rapidly depleted followed by loss of detergent-insoluble Dsg3. These findings demonstrate that Dsg3 endocytosis, keratin filament retraction, and the loss of keratinocyte cell-cell adhesion are coordinated responses to PV IgG.     

A B-type PDGF receptor lacking most of the intracellular domain escapes degradation after ligand binding

The characteristics of the human B-type platelet-derived-growth-factor (PDGF) receptor expressed in Chinese hamster ovary (CHO) cells, were compared with those of a mutant receptor lacking all but 19 amino acids of the intracellular domain. The transfected wild-type receptor was synthesized as a 160-kDa precursor that was processed to 190 kDa. Each CHO cell expressed 30,000-100,000 receptors which bound PDGF-BB with a Kd of about 0.5 nM. Analysis of PDGF-AB binding yielded non-linear Scatchard plots; the major part of the binding sites had a Kd of 6 nM. PDGF-AA was not bound. The receptors expressed in CHO cells were down-regulated after binding of PDGF-BB, and mediated degradation of 125I-PDGF-BB with similar efficiency as PDGF-B-type receptors in human fibroblasts. The transfected receptor also transduced a mitogenic signal. The mutant receptor was synthesized as a 90-kDa precursor and was processed to 120 kDa with a slightly faster rate than the wild-type receptor. Cells expressing the mutant receptor generally had around 10(6) ligand-binding sites/cell, with a Kd for binding of PDGF-BB of 3 nM. The mutant receptor, which did not transduce a mitogenic response, mediated degradation of 125I-PDGF-BB, albeit less efficiently compared to the wild-type receptor. In contrast to the wild-type receptor, it was down-regulated only to a limited extent and not degraded in response to ligand binding. These findings indicate a role for the intracellular part of the receptor, not only in mitogenic signaling, but also in receptor internalization and intracellular routing.     

Modulation of interleukin 2 high-affinity binding by lymphocyte-derived tetrahydrobiopterin: pterins as potential participants in the control of interleukin 2 receptor assembly

In this report, we have examined whether (6R)-tetrahydrobiopterin (H4biopterin) modulates the binding of interleukin 2 to high-affinity sites of the cloned mouse cytotoxic T-lymphocyte clone CTLL-2. Scatchard plot analysis of the equilibrium binding data reveals increased affinity when the cells are exposed simultaneously to interleukin 2 and to the pterin. The Kd values are statistically significantly reduced from 1.4 x 10(-11) M to 0.78 x 10(-11) M interleukin 2. The dissociation kinetics of the ligand were followed at 4 degrees C after equilibrium binding under high-affinity conditions (1.2 x 10(-10) M interleukin 2). In the presence of H4 biopterin, the dissociation rate constant (k-1) decreases from 6.2 x 10(-3) min-1 to 3.0 x 10(-3) min-1 and the half-time for dissociation increases from 106.8 min to 218.0 min. As a third approach interleukin 2 was bound to the surface of cells under high-affinity conditions by incubation in the cold and the internalization kinetics upon warming were determined. Sigmoidal-shaped kinetics of endocytosis in control cells indicate that the internalization rates increase only gradually. The presence of H4 biopterin causes an apparent immediate transition from higher-order kinetics to a linear response so that maximum internalization rates are reached immediately upon warming. The data show that lymphocyte-derived H4 biopterin in vitro at concentrations ranging from 2-8 x 10(-7) M modulates interleukin 2 high-affinity binding and that H4 biopterin potentially participates in the control of interleukin 2 receptor assembly.     

If pemphigus vulgaris IgG are the cause of acantholysis, new IgG-independent mechanisms are the concause

Pemphigus vulgaris (PV) is a disease of epidermal adhesion. Its pathogenesis is currently traced back to the action of autoantibodies against antigens located within the intercellular substance of keratinocytes, such as desmogleins and acetylcholine receptors. In the present paper, we sought to elucidate the non-IgG-mediated effects of PV sera on keratinocytes. Results showed that PV sera depleted of IgG were able to induce well-defined changes on keratinocyte morphology and metabolic activity. Indeed, PV IgG-free sera determined marked alterations on cell shape, accompanied by partial loss of keratinocyte-keratinocyte interactions within 48 h after treatment. Furthermore, PV IgG-depleted sera caused a sharp reduction of cell viability along with a less sustained weakening of intercellular adhesion strength. In light of the above findings, loss of cell-cell adhesion in PV occurs as a result of the cooperating action of both IgG and non-IgG-mediated mechanisms. These data have remarkable consequences on experimental models of PV and might open new "biological" approaches to its therapy. Thus, researchers are well advised that PV pathophysiology cannot be faithfully reproduced by leaving non-IgG serum factors out of consideration.     

The Arg-Gly-Asp-containing, solvent-exposed loop of Ptr ToxA is required for internalization

Internalization of the proteinaceous host-selective toxin, Ptr ToxA (ToxA), into sensitive wheat mesophyll cells is correlated with toxin activity. The solvent-exposed, Arg-Gly-Asp (RGD)-containing loop of ToxA is a candidate for interaction with the plasma membrane, which is a likely prerequisite to toxin internalization. Based on the percentage of cells affected by a given number of ToxA molecules in a treatment zone, the number of ToxA molecules bound to high-affinity sites was estimated at 3 x 10(6) per cell and the Kd for binding was estimated to be near 1 nM. An improved heterologous expression method of proteins that contain mutations in ToxA, coupled with a newly developed semiquantitative bioassay, revealed that some amino acids in the RGD-containing loop contribute more to toxin activity than others. Protease protection assays that detect internalized protein and inhibition of toxin uptake indicated that, for each ToxA variant tested, the extent of toxin activity correlates with the amount of internalized protein. RGD-containing peptide inhibition of both activity and internalization supported these findings. These data support the hypothesis that ToxA interacts with a high-affinity binding site on wheat mesophyll cells through the RGD-containing, solvent-exposed loop, resulting in toxin internalization and eventual cell death. The inability to detect phosphorylation of ToxA in vitro and in vivo suggests that a putative CKII phosphorylation site in the RGD-containing loop is required for internalization, not phosphorylation.     

Pemphigus vulgaris IgG-induced desmoglein-3 endocytosis and desmosomal disassembly are mediated by a clathrin- and dynamin-independent mechanism

Pemphigus vulgaris (PV) is a life-threatening autoimmune disease characterized by oral mucosal erosions and epidermal blistering. The autoantibodies generated target the desmosomal cadherin desmoglein-3 (Dsg3). Previous studies demonstrate that upon PV IgG binding, Dsg3 is internalized and enters an endo-lysosomal pathway where it is degraded. To define the endocytic machinery involved in PV IgG-induced Dsg3 internalization, human keratinocytes were incubated with PV IgG, and various tools were used to perturb distinct endocytic pathways. The PV IgG.Dsg3 complex failed to colocalize with clathrin, and inhibitors of clathrin- and dynamin-dependent pathways had little or no effect on Dsg3 internalization. In contrast, cholesterol binding agents such as filipin and nystatin and the tyrosine kinase inhibitor genistein dramatically inhibited Dsg3 internalization. Furthermore, the Dsg3 cytoplasmic tail specified sensitivity to these inhibitors. Moreover, inhibition of Dsg3 endocytosis with genistein prevented disruption of desmosomes and loss of adhesion in the presence of PV IgG. Altogether, these results suggest that PV IgG-induced Dsg3 internalization is mediated through a clathrin- and dynamin-independent pathway and that Dsg3 endocytosis is tightly coupled to the pathogenic activity of PV IgG.     

Detection and comparison of specific hemin binding by Porphyromonas gingivalis and Prevotella intermedia.

A radioligand assay was designed to detect and compare specific hemin binding by the periodontal anaerobic black-pigmenting bacteria (BPB) Porphyromonas gingivalis and Prevotella intermedia. The assay included physiological concentrations of the hemin-binding protein rabbit serum albumin (RSA) to prevent self-aggregation and nonspecific interaction of hemin with cellular components. Under these conditions, heme-starved P. intermedia cells (two strains) expressed a single binding site species (4,100 to 4,600 sites/cell) with a dissociation constant (Kd) of 1.0 x 10(-9) M. Heme-starved P. gingivalis cells (two strains) expressed two binding site species; the higher-affinity site (1,000 to 1,500 sites/cell) displayed a Kd of between 3.6 x 10(-11) and 9.6 x 10(-11) M, whereas the estimated Kd of the lower-affinity site (1.9 x 10(5) to 6.3 x 10(5) sites/cell) ranged between 2.6 x 10(-7) and 6.5 x 10(-8) M. Specific binding was greatly diminished in heme-replete cells of either BPB species and was not displayed by iron-replete Escherichia coli cells, which bound as much hemin in the absence of RSA as did P. intermedia. Hemin binding by BPB was reduced following treatment with protein-modifying agents (heat, pronase, and N-bromosuccinimide) and was blocked by protoporphyrin IX and hemoglobin but not by Congo red. Hemopexin also inhibited bacterial hemin binding. These findings indicate that both P. gingivalis and P. intermedia express heme-repressible proteinaceous hemin-binding sites with affinities intermediate between those of serum albumin and hemopexin. P. gingivalis exhibited a 10-fold-greater specific binding affinity and greater heme storage capacity than did P. intermedia, suggesting that the former would be ecologically advantaged with respect to heme acquisition.     

A monoclonal antibody recognizing 68- to 75-kilodalton protein(s) associated with the human IL-1 receptor

We produced an IgM mAb termed 4.9 against an EBV-containing lymphoblastoid cell line, termed 3B6. This mAb reacted with both various B and T cell lines such as HSB2 cells, with an NK-like cell line YT-C3 cells, and with human fibroblast MCR-5 cells. It also reacted with normal resting peripheral B lymphocytes, monocytes, and anti-CD2- or anti-CD3-activated T lymphocytes. The 4.9 mAb immunoprecipitated two bands estimated to be of Mr 68 and 75 kDa from iodinated 3B6 cells. The 4.9 mAb inhibited the proliferation of peripheral T lymphocytes induced either by anti-CD3 mAb or anti-CD2 mAb. The 4.9 mAb inhibited also the proliferation of murine thymocytes both in the presence of PHA and IL-1 and the proliferation of human fibroblasts in the presence of IL-1. Radiolabeled IL-1 binding on 3B6 cells revealed two types of IL-1 binding sites with high and low affinity for IL-1 (300 sites/cell with a Kd of 6 x 10(-11)M and 6000 sites/cell with a Kd of 3 x 10(-9)M). On both 3B6 and YT-C3 cells, mAb 4.9 inhibited specifically the binding of 125I-labeled rIL-1, alpha or beta, whereas the irrelevant IgM mAb did not. Conversely, rIL-1, alpha or beta, could inhibit specifically the binding of radioiodinated 4.9 mAb to 3B6 or YT-C3 cells, whereas rIL-2, rIFN, or the irrelevant IgM mAb were ineffective. 125I-4.9 mAb bound 3B6 cells with an association constant (Ka) of 2 x 10(8)/M and demonstrated 6000 binding sites/cell. We thus conclude that mAb 4.9 recognizes a protein complex (68 to 75 kDa) closely associated with the IL-1R.     

Cycle of the vasoactive intestinal peptide and its binding site in a human adenocarcinoma cell line (HT 29)

The disappearance of vasoactive-intestinal-peptide (VIP) binding sites at the cell surface of a cultured target cell, originating from a human colonic adenocarcinoma (HT 29 cell line), was studied, after preexposition of the cell to the peptide, as a function of time, VIP concentration and temperature. Maximum effect (60-80% loss of binding capacity) was obtained after a 5-10 min exposure of the cells at 37 degrees C with a VIP concentration of 100 nM. The t1/2 of maximum disappearance was less than 2 min and the concentration of native VIP giving half-maximum decrease in 125I-VIP binding was 6 nM. The affinity of remaining binding sites for VIP was not affected compared to that of control cells (Kd = 0.3 nM). Disappearance of VIP binding sites was specific since, with the same conditions of preincubation, the specific binding of 125I-labeled epidermal growth factor to HT 29 cells was not modified. The phenomenon was reversible and 90% of binding capacity could be restored in less than 60 min by incubating cells in VIP-free medium. Correlatively we showed, by two independent experimental procedures, that 125I-VIP, initially bound to HT 29 cells, was maximally internalized after 10 min of incubation at 37 degrees C. All the data strongly suggest that: internalization of VIP is receptor-mediated; upon exposure to native VIP, VIP receptors are down-regulated or at least sequestered within HT 29 cells.     

Differentiation of ob 17 preadipocytes to adipocytes. Effect of insulin on the levels of insulin receptors and on the transport of alpha-aminoisobutyrate.

Cells of a clonal cell line (ob 17) isolated from the epididymal fat pad of ob/ob mouse possess insulin receptors. Their number was increased 1.5-fold after growth arrest, with no significant change in the Kd values of the "high affinity" sites determined by extrapolation of the high affinity portion of the curvilinear Scatchard plots. With chronic insulin exposure for 3 to 11 days after confluence, ob 17 cells showed a decrease in insulin receptor concentrations from 8,000 to 1,600 high affinity sites/cell (Kd from 0.45 to 1.10(-9) M) while similar levels of "low affinity" sites were found (80,000 to 100,000 sites/cell; Kd from 10(-8) to 3 x 10(-8) M). The loss of the high affinity binding sites is accompanied by the disappearance of the stimulatory effect by insulin of alpha-aminoisobutyrate uptake. Therefore, in contrast to 3T3-L1 fibroblasts, the ob 17 cells present, in culture, a self-modulation of insulin receptors and a loss of insulin sensitivity after chronic exposure to insulin.     

Qualitative and quantitative interactions of lectins with untreated and neuraminidase-treated normal, wild-type, and temperature-sensitive polyoma-transformed fibroblasts.

The lectin receptors of confluently grown hamster BHK, wild type polyoma virus transformed PyBHK, and temperature-sensitive polyoma transformed ts3-PyBHK fibroblasts were investigated using cell agglutination, quantitative (125I)lectin binding, and ferritin-lectin labeling. PyBHK and permissively grown ts3-PyBHK cells agglutinated more strongly with Ricinus communis I agglutinin (RCA-I)compared to BHK and nonpermissively grown ts3-PyBHK, although saturation binding of (125I)RCA-I to these cells at 4 degrees resulted in a twofold difference in lectin-binding sites on BHK and nonpermissively grown ts3-PyBHK cells (1.0-1.3 x 10 7 sites/cell) compared to PyBHK and permissively grown ts3-PyBHK (0.4-0.6 x 10 7 sites/cell). These cells bound equivalent amounts of (125I)concanavalin A (0.8-1 x 10 7 sites/cell) and (125I)wheat germ agglutinin (1-2.2 x 10 7 sites/cell). Under these binding conditions little endocytosis occurred, as judged by the subsequent release of greater than 90% cell-bound (125I)RCA-I by the RCA-I inhibitor lactose and localization of ferritin-RCA-I exclusively to the extracellular plasma membrane surface. However, if the binding is performed at 22 degrees, only 50% of the bound lectin can be removed by lactose, and ferritin-RCA-I is localized inside the cell within endocytotic vesicles. The relative mobility of RCA-I receptors was examined on ts3-PyBHK cells by the ability of ferritin-RCA-I to induce clustering of its receptors at 22 degrees. RCA-I receptors on permissively grown ts3-PyBHK cells appeared to be more mobile than on nonpermissively grown cells. BHK and PyBHK cells were treated with neuraminidase, and the resulting enzyme-treated cells were assayed for lectin agglutinability and quantitative binding of RCA-I, concanavalin A, and wheat germ agglutinin. Neuraminidase treatment resulted in decreased concanavalin A and wheat germ agglutinability and a slight increase in RCA-I agglutinability. The enzyme-treated BHK and PyBHK cells bound less (125I)wheat germ agglutinin (2.8 x 10 6 and 2.2 x 10 6 sites/cell, respectively) and 2.5 and 6.2 times more (125I)RCA-I (2.5-3 x 10 7) and 3.5-4 x 10 7 sites/per cell, respectively). There was no change in the number of concanavalin A binding sites after neuraminidase treatment. The increase in RCA-I binding sites approximated the decrease in wheat germ agglutinin binding sites indicating that the predominant penultimate oligosaccharide residue to sialic acid on these cells is D-Gal.     

Fc gamma-receptor-mediated changes in the plasma membrane potential induce prostaglandin release from human fibroblasts

Binding of aggregated human immunoglobulin G (IgG) on diploid human fibroblasts leads to a rapid depolarization of the cells within 1-2 min. We resolved this membrane potential change into its plasma membrane and mitochondrial membrane components by measuring the transmembrane distribution of the lipophilic tritium-labelled cation tetraphenylphosphonium, [3H]Ph4P+. The responsibility of the plasma membrane for the membrane potential change, induced by binding of IgGs, is demonstrated. The IgG-induced membrane depolarization leads to the induction of prostaglandin E2 synthesis. Aggregated immunoglobulins (IgG) are specifically bound via the Fc portion because only binding of Fc fragments, in contrast to (Fab')2 fragments, leads to a stimulation of prostaglandin E2 synthesis comparable to that mediated by IgGs. Depolarization of the plasma membrane by short incubation of the fibroblasts in high-K+ buffer (5 min) results in a stimulation of prostaglandin E2 synthesis comparable to that mediated by either aggregated human IgGs or Fc fragments. Our previous results on Fc gamma-receptor-mediated antigen-IgG-antibody complex internalization showed that a maximum uptake of these complexes could be detected 60-90 min after binding. Therefore, we conclude that not internalisation but binding of aggregated IgGs to the Fc gamma receptors on human fibroblasts is the stimulus for plasma membrane depolarization leading to an enhanced prostaglandin E2 release.     

Selective distinction at equilibrium between the two alpha-neurotoxin binding sites of Torpedo acetylcholine receptor by microtitration

The binding of the monoiodinated alpha-neurotoxin I from Naja mossambica mossambica to the membrane-bound acetylcholine receptor from Torpedo marmorata was investigated using a new picomolar-sensitive microtitration assay. From equilibrium binding studies a non-linear Scatchard plot demonstrated two populations of binding sites characterized by the two dissociation constants Kd1 = 7 +/- 4 pM and Kd2 = 51 +/- 16 pM and having equal binding capacities. These two populations differed in their rate of dissociation (k-1.1 = 25 x 10(-6) s-1 and k-1.2 = 623 x 10(-6) s-1 respectively), but not in their rate of formation of the toxin-receptor complex (k + 1 = 11.7 x 10(6) M-1 s-1). From these rate constants the same two values of dissociation constant were deduced (Kd1 = 2 pM and Kd2 = 53 pM). All the specific binding was prevented by the cholinergic antagonists alpha-bungarotoxin and d-tubocurarine. In addition, a biphasic competition phenomenon allowed us to differentiate between two d-tubocurarine sites (Kda = 103 nM and Kdb = 13.7 microM respectively). Evidence is provided indicating that these two sites are shared by d-tubocurarine and alpha-neurotoxin I, with inverse affinities. Fairly conclusive agreement between our equilibrium, kinetic and competition data demonstrates that the two high-affinity binding sites for this short alpha-neurotoxin are selectively distinguishable.