Characterization of the IgG-Fc receptor on human platelets

To determine quantitatively the number and avidity of receptors for the Fc portion of IgG on human platelets, we have measured the binding to platelets of human monomeric monoclonal IgG, and of small covalently crosslinked polymers of IgG1 labeled with 125I. The binding of labeled IgG1 monomers to platelets is too weak to permit quantitation. The binding of dimers or larger polymers of IgG1 is much more avid (greater at 4 degrees C than 37 degrees C), is readily reversible, and is saturable. The number of receptor sites ranges from 400 to 2000 per platelet and the mean equilibrium association constant (Ka) for the binding of dimers at 4 degrees C is 2.2 x 10(7) M-1 +/- 0.9 x 10(7) M- 1. The binding is specific for the Fc portion of IgG, and IgG1 and IgG3 bind to the receptors much more avidly than IgG2 or IgG4. Unlabeled IgG1 dimers are about 7--8-fold more potent in inhibiting binding than are IgG1 monomers, and larger polymers are even more potent than dimers. Thus, the Fc receptors on platelets bind human IgG1 with the same specificity and similar avidity as Fc receptors on polymorphonuclear leukocytes (PMNs), but PMNs have about 300-fold more receptors per unit of surface area than platelets.     

Size and morphology of toxic oligomers of amyloidogenic proteins: a case study of human stefin B

Amyloid-induced toxicity is a well-known phenomenon but the molecular background remains unclear. One hypothesis relates toxicity to amyloid–membrane interactions, predicting that amyloid oligomers make pores into membranes. Therefore, the toxicity and membrane interaction of prefibrillar aggregates and individual oligomers of a non-pathological yet highly amyloidogenic protein human stefin B (cystatin B) was examined. By monitoring caspase-3 activity and by testing cell viability, we showed that the lag phase aggregates obtained at pH 5 and 3 were toxic to neuroblastoma cells. Of equal toxicity were the higher-order oligomers prepared at pH 7 by freeze–thaw cycles. The higher-order oligomers eluted on size-exclusion chromatography (SEC) as a broad peak comprising hexamers, octamers, 12- and 16-mers, well separated from monomers, dimers and tetramers. Only oligomers higher than the tetramers (Rh >3.5 nm) proved toxic, in contrast to dimers and tetramers. In accordance with data from SEC, dynamic light scattering and atomic force microscopy data indicate that the toxic oligomers have diameters larger than 4 nm. Critical pressure measurements showed that the toxic higher-order oligomers inserted more effectively into model lipid monolayers than dimers and tetramers. They also bound, similarly to prefibrillar aggregates, to the plasma membrane and became internalized. Taken together, our results confirm the importance of membrane interaction and perforation in the phenomenon of cytotoxicity.     

Dimerization of the class A G protein-coupled neurotensin receptor NTS1 alters G protein interaction

G protein-coupled receptors (GPCRs) have been found as monomers but also as dimers or higher-order oligomers in cells. The relevance of the monomeric or dimeric receptor state for G protein activation is currently under debate for class A rhodopsin-like GPCRs. Clarification of this issue requires the availability of well defined receptor preparations as monomers or dimers and an assessment of their ligand-binding and G protein-coupling properties. We show by pharmacological and hydrodynamic experiments that purified neurotensin receptor NTS1, a class A GPCR, dimerizes in detergent solution in a concentration-dependent manner, with an apparent affinity in the low nanomolar range. At low receptor concentrations, NTS1 binds the agonist neurotensin with a Hill slope of approximately 1; at higher receptor concentrations, neurotensin binding displays positive cooperativity with a Hill slope of approximately 2. NTS1 monomers activate G alpha q beta(1)gamma(2), whereas receptor dimers catalyze nucleotide exchange with lower affinity. Our results demonstrate that NTS1 dimerization per se is not a prerequisite for G protein activation.     

Cell-type-specific receptors for alpha-fetoprotein in a mouse T-lymphoma cell line.

Binding and uptake of alpha-fetoprotein (AFP) by mouse T-lymphoma YAC-1 cells exhibited the characteristics of receptor-mediated endocytosis. The binding saturation curve obtained by incubating YAC-1 cells at 4 degrees C with 125I-labeled AFP at different concentrations (50 ng/ml to 2.5 mg/ml) showed three saturation plateaus. AFP binding was inhibited by unlabeled mouse, rat, or bovine AFP and, to a lesser extent, by rat or bovine serum albumin. No significant competition was observed with transferrin, alpha 2-macroglobulin, IgG, or ovalbumin. Scatchard analysis suggested the presence of three types of receptor sites with a Kd of 2.2 X 10(-9) M (approximately equal to 700 sites per cell), 8.6 X 10(-7) M (approximately equal to 210,000 sites per cell), and 5.7 X 10(-6) M (approximately equal to 910,000 sites per cell), respectively. At 37 degrees C, AFP was rapidly internalized and could be localized in the cytoplasm after incubation of cells with fluoresceinated AFP. After a short residence time, AFP was released undegraded from the cells. Normal adult thymocytes and T lymphocytes, which are counterparts of YAC-1 cells, did not show any significant uptake of AFP. On the other hand, a small subpopulation of fetal and newborn thymocytes was labeled by fluoresceinated AFP.     

Receptor distribution and the endothelial uptake of transcobalamin II in liver cell suspensions

To determine the nature of binding of transcobalamin II (TC-II) to liver cells, we covalently coupled purified holo-TC-II to submicron latex minibeads using glutaraldehyde. Incubation of the probe with liver cell suspensions at 4 degrees C led to its binding by endothelial cells but not by hepatocytes or Kupffer cells, as visualized by scanning electron microscopy. At 37 degrees C, the probe was internalized by the endothelium through a system of coated pits and vesicles as shown by transmission electron microscopy. Inhibition studies by pre-incubation with excess native TC-II demonstrated the specificity of binding. Fractionation of these cell suspensions on metrizamide gradients yielded large cell (hepatocyte-rich) and small cell (endothelium-rich) fractions. The binding of the minibead probe occurred again exclusively on endothelial cells in the small cell fraction. 125I-labeled holo-TC-II also bound to the small cell but not to the large cell fraction. Binding was saturable (Ka, 0.225 X 10(9) mol/L-1) and receptor number was calculated to be 1.33 X 10(3) per cell. Time-dependent incubation of 125I-labeled TC-II with the endothelium-rich fraction led to its uptake, reaching a steady-state plateau at 4 degrees C. At 37 degrees C, however, the initial uptake was followed by gradual release of the label into the medium. We conclude that in the liver, holo-TC-II binds initially to endothelium, where it is internalized and is subsequently released probably to the interstitial space. Thus, the endothelium may play a fundamental role in the regulation of the uptake of TC-II by the liver.     

Thyrotropin modulation of epidermal growth factor (EGF) binding to receptors on cultured thyroid cells.

Previous studies had shown that epidermal growth factor (EGF) will stimulate growth of cultured thyroid cells in vitro, and TSH will stimulate total assayable EGF receptor in cultured porcine thyroid cells. In this study, we report the effect of TSH on EGF binding to human thyroid cells. Addition of bTSH (1 mU/mL) in binding buffer during receptor assay stimulated specific EGF binding to cells, with an increase of 44% observed over the control after 1 h incubation at 37 degrees C. Affinity crosslinking of the [125I]EGF-receptor complex showed a single labeled band with molecular size of 170 kD. No additional band was detected in the presence of TSH. Preincubation of cells with chloroquine, which inhibits lysosomal degradative enzyme activity, caused a continuous accumulation of bound EGF over a 4 h study period at 37 degrees C, and TSH stimulated an increase in internalized EGF. In the presence of chloroquine, total specific bound EGF was linearly correlated to incubation time up to 4 h and can be expressed as Bound = slope*time+intercept (time0) Addition of TSH during the binding assay significantly increased the value of the slope when compared to control (p < 0.002). The rate at which prebound [125I]EGF was released into medium was not affected by the presence of TSH, indicating that TSH-enhanced binding may not be attributed to a reduction in EGF degradation. Coincubation of thyroid cells with EGF at 0 and 1 ng/mL and increasing concentrations of TSH (0-10 mU/mL) indicated that EGF stimulated thymidine incorporation, although TSH failed to synergistically enhance EGF-stimulated cell growth.(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding and internalization of recombinant human erythropoietin in murine erythroid precursor cells

Erythropoietin (EPO) biosynthetically labelled with [35S]cysteine was produced from Chinese hamster ovary (CHO) cells containing amplified copies of human EPO cDNA. The glycosylated recombinant [35S]EPO, purified to virtual radiochemical homogeneity, was biologically active. We studied the interaction of this labeled recombinant EPO with erythroid precursor cells from mice made anemic with phenylhydrazine. The [35S]-labeled molecule bound to erythroid precursors in a time- and temperature-dependent manner. The binding was specific for EPO, and neither insulin, transferrin, epidermal growth factor, nor multiplication stimulating activity could compete for EPO binding sites. In the presence of 0.2% sodium azide, which blocks 80% to 90% of internalization, the recombinant molecule bound with an apparent Kd of 750 pmol/L and 100 to 200 binding sites per cell at 37 degrees C. Asialo-EPO was a more effective competitor than sialated EPO for the available binding sites. Thus, the enhanced biological specific activity of asialo-EPO could result from its enhanced binding affinity. We also studied recombinant human EPO labeled with 125I and found that it also bound to the erythroid cells in a saturable and specific manner. After 90 minutes of incubation at 37 degrees C, most of the bound [35S]EPO was internalized, whereas most of the [125I]EPO remained on the cell surface. The reduced internalization of the iodinated molecule could account for the previously reported functional deficit associated with iodination.     

Insulin internalization into monocytes is decreased in patients with type II diabetes mellitus

We studied the internalization of [125I]insulin into circulating human monocytes, a cell type widely used for insulin binding studies. The internalization of [125I]insulin was assessed by both an acid extraction technique, which removes surface-bound insulin but not intracellular insulin, and by a trypsinization technique, which removes cell surface-bound hormone. After 5 h of incubation at 22 C, over 40% of the total cell-associated [125I]insulin was internalized into monocytes of normal subjects. This internalization was temperature dependent; the fraction of internalized hormone was progressively decreased when the incubation temperature was reduced from 37 to 4 C. Treatment of monocytes with increasing concentrations of 2,4-dinitrophenol also decreased [125I]insulin internalization, whereas dansylcadaverine, an inhibitor of transglutaminase, had no effect. Analysis by gel filtration of the internalized labeled hormone after 4 h of incubation at 22 C indicated that 50-60% of the label was degraded insulin, but detectable intact insulin was still present. Internalization of insulin was then studied in monocytes from eight obese patients (161% of ideal body weight) with type II diabetes mellitus. After 4 h of incubation at 22 C, the specific total monocyte-associated [125I]insulin was decreased compared to that in cells from 7 normal subjects [6.02 +/- 0.38% (+/- SE) vs. 3.91 +/- 0.31% of the total; P less than 0.001]. Moreover, the percentage of hormone that was internalized was also decreased from 41.4 +/- 1.2% of the total to 28.9 +/- 1.8% (P less than 0.001). In 20 nondiabetic obese subjects, specific cell-associated [125I]insulin was reduced to 3.9 +/- 0.3% (P less than 0.001). However, compared to that in normal subjects, the percentage of hormone that was internalized was not decreased (39.7 +/- 3.51% of the total). The present findings indicate that human circulating monocytes internalize [125I]insulin; this process is temperature and energy dependent; and monocytes from obese type II diabetic patients have a significantly decreased ability to internalize insulin. This decreased internalization may play a role in the cellular resistance to insulin that occurs in these patients.     

Platelet-derived growth factor binds specifically to receptors on vascular smooth muscle cells and the binding becomes nondissociable.

Radioiodinated platelet-derived growth factor (125I-PDGF) was used in studies of PDGF binding sites on vascular smooth muscle cells. There was an excellent correlation between the ability of 125I-PDGF to stimulate cell proliferation and to bind specifically to cultured vascular smooth muscle cells. The half-maximal concentration for both processes was 0.1 nM. There were 50,000 binding sites per cell. Reduced PDGF, prepared by treatment of PDGF with 20 mM dithiothreitol, had neither the ability to bind to smooth muscle cells nor to stimulate cellular proliferation. Epidermal growth factor, nerve growth factor, fibroblast growth factor, and histone B did not compete for the binding sites at a concentration of 10 nM. 125I-PDGF binding was slowly reversible at 4 degrees C and was rapidly and totally reversible after a 1-min incubation at 37 degrees C. After continued incubation at 37 degrees C, the binding became irreversible. The half-time for formation of the nondissociable state of 125I-PDGF binding was approximately equal to 5 min at 37 degrees C. The nondissociable state of binding was not formed at 4 degrees C even after 1 hr of incubation. These data suggest that the sites we labeled are the PDGF receptors that mediate PDGF's mitogenic action and that a nondissociable state of PDGF binding is formed at 37 degrees C. It is likely that nondissociable PDGF represents internalized ligand or binding to sites that are converted to a high-affinity state after the ligand binds.     

Intracellular transport of high-density lipoprotein 3 in intestinal epithelial cells (Caco-2) is tubulin associated

BACKGROUND: A retroendocytotic pathway for high-density lipoprotein 3 (HDL(3)) in cultured intestinal epithelial cell lines has been described. In small intestinal crypt cells and Caco-2, HDL(3) is internalized, transported to lipid droplets and, after solubilization of these lipid droplets, resecreted. In the present study we examined the mechanisms of intracellular transport of HDL(3) in the Caco-2 cell line. METHODS: Apolipoprotein E free HDL(3 )was gold-labeled for transmission electron microscopy and 1, 1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine iodide [DiI(3)] labeled for fluorescence and confocal laser scanning microscopy. For tubulin desintegration Caco-2 cells were incubated with taxol, colchicine and beta- and gamma-lumicolchicine. Tubulin staining was performed using a FITC labeled antibody. Uptake of HDL(3) was quantified by FACS analysis. RESULTS: HDL(3) was rapidly internalized and found to be in contact with lipid droplets in the perinuclear region after 10 min. By transmission electron microscopy a frequent colocalization of HDL(3)-containing vesicles and tubular structures was demonstrated. The close association of HDL(3)-containing vesicles with fluorescence stained tubulin could be confirmed by confocal laser scanning microscopy. Preincubation of the cells with taxol and colchicine did not completely prevent internalization but reduced it during a 2-hour incubation period to less than 50% of the control cells. The transport of DiI(3)-labeled HDL(3) to the lipid droplets in the perinuclear region was almost completely blocked by taxol and colchicine. CONCLUSION: Internalization and intracellular transport of HDL(3) in intestinal epithelial cells (Caco-2) is dependent on a tubulin-mediated mechanism.     

Direct observation of substance P-induced internalization of neurokinin 1 (NK1) receptors at sites of inflammation.

Substance P (SP) can cause plasma leakage at sites of inflammation by binding to neurokinin type 1 (NK1) receptors on the surface of endothelial cells. Internalization after ligand binding could reduce the number of NK1 receptors on the cell surface and thus participate in the desensitization and resensitization of the inflammatory response to SP. By using an antibody to the receptor, we directly observed SP-induced internalization of NK1 receptors into endosomes in endothelial cells of postcapillary venules in the rat tracheal mucosa. In the absence of SP, an average of 15 immunoreactive endosomes were present per endothelial cell. After an intravenous injection of SP, the number of immunoreactive endosomes peaked at 107 per cell at 3 min and gradually returned to the baseline by 120 min. In parallel experiments we observed that when cultured cells transfected with the NK1 receptor were exposed to rhodamine-SP and an antibody to an extracellular Flag epitope of the NK1 receptor, the SP was internalized with the receptor antibody. Both in the cultured cells and in the endothelial cells of intact animals, the prompt SP-induced internalization was accompanied by rapid, long-lasting desensitization to SP. These studies suggest that internalization of NK1 receptors by endothelial cells may be one of the mechanisms that limit the amount of plasma leakage at sites of inflammation.     

Selective internalization of granule membrane after secretion in mast cells.

[3H]Galactose, covalently bound to cell surface glycoconjugates of rat peritoneal mast cells, was used to study internalization of labeled plasma membrane and granule membrane constituents before or after secretion stimulated by compound 48/80. Internalized label was distinguished quantitatively from label on the cell surface by its inaccessibility to enzymatic removal. Three different situations were compared. (i) With label only on the plasma membrane, and in the absence of secretion, incubation at 37 degrees C (but not at 0 degree C) resulted in a gradual decrease of label on the cell surface until, after approximately equal to 2 hr, a steady state was reached with 93% of all cell-bound label remaining on the cell surface. Recycling of internalized label was demonstrated. (ii) When cells were labeled on the plasma membrane and then stimulated to secrete, subsequent retrieval of (unlabeled) granule membrane did not affect the rate or extent of simultaneous internalization of labeled plasma membrane. (iii) When both plasma membrane and exposed granule membrane were labeled after secretion, subsequent incubation at 37 degrees C (but not at 0 degrees C) resulted in approximately equal to 33% of all cell-bound label becoming internalized during 4 hr, indicating additional internalization of label due to retrieval of labeled granule membrane. In all three cases, loss of label into the medium occurred with a half-life of 8-11 hr, showing that no extensive shedding of granule membrane occurred after secretion. The results suggest either that no mixing of labeled membrane constituents occurred between the plasma membrane and granule membrane or that during retrieval of granule membrane, sorting of membrane was taking place at the cell surface.     

The binding behavior and functional significance of Fc receptors for IgG on T lymphocytes of the mouse

In consequence of allogenic stimulation of mouse spleen lymphocytes the binding of fluorescein isothiocyanate conjugated aggregated IgG (FITC-aggr. IgG) per cell is increased (higher affinity of receptors and/or higher number of receptors). A higher specificity of the FITC-aggr. IgG binding to allogeneically stimulated lymphocytes in comparison to fresh prepared resting and syngeneically cultivated lymphocytes could be only detected on the higher fluorescing allogeneically stimulated cells. Increased amount of Fc-receptors for IgG (Fc gamma R) is mainly found on T-lymphocytes with the Lyt-1 + 2 +-phenotype. The manipulation of that Fc gamma R on T-lymphocytes, which were expressed in consequence of allogeneic stimulation by binding of respective ligands, did not change the original effect of nonmanipulated, allogeneically activated cells on a primary MLR. But if the cells were negatively selected by means of monoclonal antibodies and complement, the originally suppressive effect of allogeneically stimulated Lyt-2 positive cells on a primary MLR was abolished by manipulating the respective Fc gamma R.     

Expression of insulin-like growth factor receptors in primary human thyroid neoplasms

The presence of IGF-I receptors was demonstrated in normal and neoplastic tissues of human thyroid. Binding of (125I)IGF-I to thyroid membranes was dependent on time and temperature of incubation, and maximal binding was achieved at 4 degree C and 18 h of incubation. (125I)IGI-I binding was dose-dependently displaced by unlabelled IGF-I; half-maximal inhibition occurred at concentrations of 10-20 milligrams. IGF-II and insulin had relative potencies of 5 and 1% compared with IGF-I. Scatchard analysis of binding data revealed a single class of IGF-I receptors with high affinity (Ka: 1.2-8.6 x 10(9) 1/mol) in normal thyroid tissues. Affinity cross-linking and autoradiography demonstrated the type IIGF receptors. Specific binding of (125I)IGF-1 in thyroid cancer tissues (9.69 +/- 2.07% per 200 micrograms protein; mean +/- SEM, N = 8) was significantly (p less than 0.05) higher than that in the surrounding normal tissues (3.03 +/- 0.35%, N = 8). In contrast there was no difference in the binding between adenoma tissues (4.19 +/- 0.53%, N = 5) and the adjacent normal tissues (2.94 +/- 0.24%, N = 5). The higher IGF-I binding in cancer tissues was due to an increase in the binding capacity without any change in the affinity. The presence of IGF-I receptors suggests a possible role of IGF-I and its receptors in the growth of thyroid cancer cells.     

Nuclear thyroid hormone receptors in cultured human fibroblasts: improved method of isolation, partial characterization, and interaction with chromatin

In order to characterize the nuclear thyroid hormone receptors in human tissue, an improved method for isolation of nuclei from cultured human fibroblasts was developed. This method provided nuclei with a protein/DNA ratio of 2.8 and recovery of 42%. The purity of nuclei was verified by phase contrast and electron microscopy, which showed normal appearance of chromatin structure. Nuclear binding assay was performed by incubation of whole cells at 37 degrees C or isolated nuclei at 22 degrees C with L-triiodothyronine (T3). In both cases, an affinity constant (Ka) of 2.0-3.0 X 10(10) M-1 and an average binding capacity of 41 femtomoles of T3/100 micrograms DNA (3,100 binding sites/nucleus) were obtained. During incubation of the nuclei, 13% to 16% of receptors that had an identical Ka was released into the medium. Salt extraction recovered 85% to 90% of the receptors, which had a Ka of 4.5 X 10(10) M-1 and the capacity of 0.13 pmol of T3/mg protein. The Ka fo. L-thyroxine (T4) was seven to 18 times lower than that for T3, but the capacity was the same in isolated nuclei, receptors released during incubation of nuclei, and in salt-extracted receptors. Of the iodothyronines examined, affinity for triiodothyroacetic acid was the highest, followed by L-T3, D-T3, L-T4. Isokinetic glycerol gradient analysis revealed that salt-extracted receptors had a sedimentation coefficient of 3.4 S, whereas micrococcal nuclease digested receptors showed two major (6.0 to 6.5 and 12.5 S), and two minor (17 and 19 S) peaks. These results were virtually identical to those obtained with rat liver nuclei analyzed in parallel studies.(ABSTRACT TRUNCATED AT 250 WORDS)     

Receptor-mediated endocytosis and degradative processing of growth hormone by rat adipocytes in primary culture

At 37 degrees C, cultured rat adipocytes bound [125I]human GH ([125I]hGH) rapidly, with binding being detectable within 1 min of incubation. The bound [125I]hGH was then internalized (within 10 min) and accumulated in the cell interior until a steady state was reached (by 60 min). At this time, where the rates of GH internalization, processing, and release are equivalent, 55% of total cell-associated [125I]hGH was intracellular. Internalization of [125I]hGH by acutely isolated (noncultured) adipocytes was preceded by a 20-min lag phase indicative of a temporary postbinding defect. The lag phase was not seen with cultured adipocytes. After preloading of [125I]hGH into the cell interior, cultured cells rapidly released [125I]hGH (t1/2 = 20-30 min) into the extracellular medium as both intact (25%) and degraded (75%) GH. The release of intact vs. degraded GH was distinguishable on the basis of kinetics and temperature dependence. In order to determine when internalized [125I]hGH entered a catabolic compartment, cultured adipocytes were incubated with [125I]hGH and the composition of intracellular GH was determined as a function of time. All [125I]hGH internalized during the first 20 min was intact. Between 20 and 30 min some of the internalized [125I]hGH entered a catabolic compartment and degradation products began accumulating within the adipocytes. Release of degraded [125I]hGH from cultured adipocytes began at 60 min. The processing of GH through the complete degradative pathway (binding, internalization, degradation, release) required a period of 1 h at 37 degrees C.     

Biallelic neutrophil Na-antigen system is associated with a polymorphism on the phospho-inositol-linked Fc gamma receptor III (CD16)

Neutrophils express two distinct types of receptor for the Fc region of IgG, FcRII and FcRIII, in amounts of 10,000 to 20,000 FcRII (40 Kd) and 100,000 to 200,000 FcRIII (50 to 80 Kd) per neutrophil. We showed that the FcRIII exhibits genetically determined heterogeneity, detectable by differences in electrophoretic mobility with sodium dodecyl sulfate (SDS) as well as by reaction with antibodies against the biallelic neutrophil-specific antigen system NA. FcRIII was precipitated with an FcRIII-specific monoclonal antibody (MoAb) from the neutrophils of 35 donors. NA1NA1 donors expressed an FcRIII with a molecular weight (mol wt) of 50 to 65 Kd, NA1NA2 donors expressed an FcRIII with a mol wt of 50 to 80 Kd, and NA2NA2 donors expressed an FcRIII with a mol wt of 65 to 80 Kd. Statistical analysis showed that the electrophoretic heterogeneity corresponds with the NA polymorphism (k = 1). Sequential immunoprecipitation with a MoAb against NA1 and a MoAb against anti- FcRIII, followed by SDS-polyacrylamide gel electrophoresis (PAGE), showed that NA1-FcRIII is distinct from NA2-FcRIII. Moreover, immunoprecipitation with a MoAb against NA1 yielded a protein of 50 to 65 Kd, and immunoprecipitation with human anti-NA2 sera or an MoAb against NA2 yielded a protein of 65 to 80 Kd. Preincubation of NA1NA2 neutrophils with F(ab')2 fragments of an MoAb against anti-NA1 reduced binding of IgG dimers to these cells with about 50%, whereas it completely prevented binding of the dimers to NA1NA1 neutrophils. Inhibition experiments with the MoAb against NA2 yielded the same results for NA1NA2 cells, whereas binding of IgG dimers to NA2NA2 cells was completely prevented. Thus, the products of both NA alleles bind IgG. Immunoprecipitation from the medium of neutrophils either stimulated with formyl- methionyl-leucyl-phenylalanine (FMLP) or treated with glycosyl-phosphatidyl-inositol-specific phospholipase C (GPI- PLC) showed that both the NA1-FcRIII and the NA2-FcRIII are released from the cell surface, indicating that both forms of FcRIII have some structural features in common. Deglycosylation of FcRIII from homozygous donors yielded material that showed several bands on SDS- PAGE. GPI-PLC treatment of neutrophils indicated that all of this material is phosphatidyl-inositol linked.     

Inhibition of prostaglandin biosynthesis in human adipose tissue by glucocorticosteroids

Cultured stromal cells derived from human sc adipose tissue synthesized and secreted into the medium prostaglandin E2 (PGE2), PGF2 alpha, and 6-keto-PGF1 alpha, a metabolite of prostacyclin. PGE2 was quantitatively the major PG formed by these cells. Dexamethasone inhibited PG biosynthesis in a concentration- and time-dependent manner; dexamethasone (2.5 X 10(-10) M) caused greater than 50% inhibition of PGE2 synthesis after 24 h of incubation and 90% inhibition after 48 h. The effect of dexamethasone to inhibit PGE2 synthesis was blocked by simultaneous incubation of cells with cortisol-21-mesylate, an antagonist of the binding of glucocorticosteroids to cytosolic receptors. (Bu)2cAMP (1 mM), forskolin (10 microM), and 1-methyl-3-isobutylxanthine (0.1 mM) markedly stimulated PGE2 biosynthesis in cultured adipose stromal cells. The inhibitory effect of dexamethasone on PGE2 synthesis was attenuated by simultaneous incubation of cells with (Bu)2cAMP. The results of this study suggest that glucocorticosteroids inhibit PG biosynthesis in adipose tissue stromal cells, whereas cAMP and certain analogs thereof stimulate PG biosynthesis and overcome the inhibitory action of glucocorticosteroids.     

Biologically active steroids activate receptor-bound human sex hormone-binding globulin to cause LNCaP cells to accumulate adenosine 3',5'-monophosphate

The binding of human sex hormone-binding globulin (SHBG) to a human prostatic cancer cell line (LNCaP) and the results of that binding were examined. Membranes derived from LNCaP cells bound unliganded SHBG on two sets of sites whose affinities were: Ka1 = 3.1 +/- 1.6 x 10(10) M-1 and Ka2 = 8.7 +/- 4.3 x 10(6) M-1. Intact cells also bound SHBG, but even after 6 h, less than 10% of specifically bound SHBG was internalized. This observation speaks against a role for the membrane binding of SHBG in steroid transport across cell membranes. When LNCaP cells were prebound with SHBG, addition of dihydrotestosterone or estradiol resulted in a dose-dependent increase in intracellular cAMP. SHBG in the absence of steroids or dihydrotestosterone in the absence of SHBG was without effect. 2-Methoxyestradiol, a steroid metabolite without biological activity, but which binds to SHBG more tightly than does estradiol, was also without effect. These observations demonstrate a potentially important role for SHBG as a regulator of cell function. They also demonstrate an additional mode of action of steroid hormones, one that does not require that the steroid interact with a steroid receptor.     

Radioiodinated nondegradable gonadotropin-releasing hormone analogs: new probes for the investigation of pituitary gonadotropin-releasing hormone receptors.

Studies of pituitary plasma membrane gonadotropin-releasing hormone (GnRH) receptors using [125I]-iodo-GnRH suffer major disadvantages. Only a small (less than 25%) proportion of specific tracer binding is to high affinity sites, with more than 70% bound to low affinity sites (Ka = 1 x 10(6) M-1). [125I]Iodo-GnRH is also inactivated during incubation with pituitary plasma membrane preparations. Two superactive analongs of GnRH, substituted in positions 6 and 10, were used as the labeled ligand to overcome these problems. Both analogs bound to the same high affinity sites as GnRH on bovine pituitary plasma membranes, though the affinity of the analogs was higher than that of the natural decapeptide (Ka = 2.0 x 10(9), 6.0 x 10(9), and 3.0 x 10(8) M-1 for [D-Ser(TBu)6]des-Gly10-GnRH ethylamide, [D-Ala6]des-Gly10-GnRH ethylamide, and GnRH, respectively. The labeled analogs bound to a single class of high affinity sites with less than 15% of the specific binding being to low affinity sites (Ka approximately equal to 1 x 10(6) M-1). The labeled analogs were not inactivated during incubation with the pituitary membrane preparations. Using the analogs as tracer, a single class of high affinity sites (K1 = 4.0 x 10(9) M-1) was also demonstrated on crude 10,800 x g rat pituitary membrane preparations. Use of these analogs as both the labeled and unlabeled ligand offers substantial advantages over GnRH for investigation of GnRH receptors, allowing accurate determination of changes in their numbers and affinities under various physiological conditions.