kDa Receptor Research Articles

Dexamethasone suppresses release of soluble TNF receptors by human monocytes concurrently with TNF-alpha suppression.

Glucocorticoids suppress many monocyte functions, including endotoxin-stimulated release of TNF-alpha. Monocytes also release soluble receptors for TNF (sTNF-R), which can modulate TNF bioactivity. We therefore examined the effects of the glucocorticoid, dexamethasone, on the release of soluble forms of the 55 kDa and 75 kDa receptors for TNF (sTNF-R55 and sTNF-R75) by human monocytes and the human monocytic Mono Mac 6 cell line. Peripheral blood mononuclear cells (PBMC) spontaneously released 406 +/- 181 pg/10(6) cells of sTNF-R75 over 18 h in culture and Mono Mac 6 cells released 554 +/- 29 pg/10(6) cells. Lipopolysaccharide (LPS) exposure increased release of sTNF-R75 by 54 and 217%, respectively. Dexamethasone suppressed both spontaneous and LPS-stimulated release. The effect of dexamethasone was concentration dependent. At 1 mumol/L, dexamethasone suppressed the LPS-stimulated release of sTNF-R75 by 86% in PBMC and by 40% in Mono Mac 6 cells. Neither PBMC nor Mono Mac 6 cells released measurable amounts of sTNF-R55, but spontaneous release of sTNF-R55 from purified human monocytes (55 +/- 2 pg/10(6) cells over 18 h) was reduced by 45% in the presence of dexamethasone. Dexamethasone reduced bioactive TNF in PBMC cultures, as well as immunoassayable TNF-alpha, which indicates that suppression of TNF-alpha release was biologically more important than suppressed release of soluble inhibitors. Similar concurrent suppression of IL-1 beta and IL-1ra release occurred in PBMC and Mono Mac 6 cultures exposed to dexamethasone.

Homologous down-regulation of luteinizing hormone/chorionic gonadotropin receptors by increasing the degradation of receptor transcripts in human uterine endometrial stromal cells.

We investigated the possible homologous down-regulation of LH/hCG receptors in human uterine endometrial stromal cells. The cells contained a major 4.3-kilobase (kb) and minor 3.6-kb, 2.4-kb, 1.8-kb, and 1.0-kb transcripts of receptors and an 80-kDa receptor protein that can bind [125I]hCG. Culturing these cells with increasing concentrations of highly purified hCG resulted in a dose-dependent significant decrease in steady-state levels of all the receptor transcripts, the 80-kDa receptor protein, and [125I]hCG binding as compared to the control values. The hCG effect was hormone specific and required the conformation of native hormone. The decrease in steady-state receptor transcript levels by hCG was not due to a decrease in the transcription rate of the LH/hCG receptor gene. It was rather due to a significant decrease in the half-life of receptor transcripts from 40.1 +/- 12.4 h in the control to 13.3 +/- 3.6 h after treatment. The homologous down-regulation observed in the present study may potentially explain low endometrial receptor levels in the postmenopausal human endometrium.

Peyer's patch organogenesis is intact yet formation of B lymphocyte follicles is defective in peripheral lymphoid organs of mice deficient for tumor necrosis factor and its 55-kDa receptor.

Targeted inactivation of genes in the tumor necrosis factor (TNF)/lymphotoxin (LT) ligand and receptor system has recently revealed essential roles for these molecules in lymphoid tissue development and organization. Lymphotoxin-alphabeta (LTalphabeta)/lymphotoxin-beta receptor (LTbeta-R) signaling is critical for the organogenesis of lymph nodes and Peyer's patches and for the structural compartmentalization of the splenic white pulp into distinct B and T cell areas and marginal zones. Moreover, an essential role has been demonstrated for TNF/p55 tumor necrosis factor receptor (p55TNF-R) signaling in the formation of splenic B lymphocyte follicles, follicular dendritic cell networks, and germinal centers. In contrast to a previously described essential role for the p55TNF-R in Peyer's patch organogenesis, we show in this report that Peyer's patches are present in both TNF and p55TNF-R knockout mice, demonstrating that these molecules are not essential for the organogenesis of this lymphoid organ. Furthermore, we show that in the absence of TNF/p55TNF-R signaling, lymphocytes segregate normally into T and B cell areas and a normal content and localization of dendritic cells is observed in both lymph nodes and Peyer's patches. However, although B cells are found to home normally within Peyer's patches and in the outer cortex area of lymph nodes, organized follicular structures and follicular dendritic cell networks fail to form. These results show that in contrast to LTalphabeta signaling, TNF signaling through the p55TNF-R is not essential for lymphoid organogenesis but rather for interactions that determine the cellular and structural organization of B cell follicles in all secondary lymphoid tissues.

Recycling of the urokinase receptor upon internalization of the uPA:serpin complexes.

The GPI-anchored urokinase plasminogen activator receptor (uPAR) does not internalize free urokinase (uPA) but readily internalizes and degrades uPA:serpin complexes in a process that requires the alpha2-macroglobulin receptor/low density lipoprotein receptor-related protein (alpha2MR-LRP). This process is accompanied by the internalization of uPAR which renders it resistant to phosphatidylinositol-specific phospholipase C (PI-PLC). In this paper we show that during internalization of uPA:serpins at 37 degrees C, analysed by FACScan, immunofluorescence and immunoelectron microscopy, an initial decrease of cell surface uPAR was observed, followed by its reappearance at later times. This effect was not due to redistribution of previously intracellular receptors, nor to the surface expression of newly synthesized uPAR. Recycling was directly demonstrated in cell surface-biotinylated, uPA:PAI-1-exposed cells in which biotinylated uPAR was first internalized and subsequently recycled back to the surface upon incubation at 37 degrees C. In fact, uPAR was resistant to PI-PLC after the 4 degrees C binding of uPA:PAI-1 to biotinylated cells, but upon incubation at 37 degrees C PI-PLC-sensitive biotinylated uPAR reappeared at the cell surface. Binding of uPA:PAI-1 by uPAR, while essential to initiate the whole process, was, however, dispensable at later stages as both internalization and recycling of uPAR could be observed also after dissociation of the bound ligand from the cell surface.

55- and 75-kilodalton tumor necrosis factor receptors mediate distinct actions in regard to human immunodeficiency virus type 1 replication in primary human macrophages.

We report in this study that repeated tumor necrosis factor alpha (TNF-alpha) pretreatment, starting before and continued after infection by human immunodeficiency virus type 1 (HIV-1), inhibits replication of the monocytotropic Ada strain in primary tissue culture-differentiated macrophages (TCDM), as assessed by sixfold lower levels of reverse transcriptase (RT) activity than that in untreated cells and absence of syncytium formation in TCDM cultures. In order to determine the pathways involved in inhibition of HIV-1 replication in primary TCDM pretreated with TNF-alpha, we tested TNF-alpha mutants T55 and T75, which recognize either the 55-kDa (TNF-R1) or the 75-kDa (TNF-R2) TNF receptor, respectively. Pretreatment of TCDM with the T75 mutant decreased the RT activity compared with that in untreated infected control cells fivefold and almost totally inhibited syncytium formation. In contrast, when TCDM were pretreated with the T55 mutant alone, syncytia were observed and RT activity was decreased about one-half. These results suggest that the inhibition of HIV-1 replication in TCDM pretreated with TNF-alpha might be mediated mainly through the 75-kDa TNF receptor (TNF-R2) rather than through the 55-kDa receptor (TNF-R1). Inhibition of HIV-1 replication in TCDM was observed with both T75 mutant pretreatment and posttreatment, starting at 1 h or 3 days after infection, whereas posttreatment with the T55 mutant, but not pretreatment, stimulated HIV-1 growth in primary TCDM. Both pre- and posttreatment with TNF-alpha inhibited HIV-1 replication in primary TCDM. The stimulation of HIV-1 replication by TNF-alpha in a chronically infected promonocytic cell line, U1, which contains two copies of integrated provirus, was mediated through the 55-kDa TNF-R1 alone and not through the 75-kDa TNF-R2. These results demonstrate that the 55-kDa TNF-R1 is involved in postintegration stimulation of HIV-1 while the 75-kDa TNF-R2 is involved in the inhibition of an early step of the viral life cycle in primary human TCDM.

Neurons from fetal rat brains contain functional luteinizing hormone/chorionic gonadotropin receptors.

Adult and neonatal rat brains contain functional LH/hCG receptors. These findings have led us to hypothesize that the fetal rat brain may also contain these receptors. To test this hypothesis, we isolated neurons from 19-day-old fetal rat brains and cultured them in chemically defined serum-free medium. The reverse transcription polymerase chain reaction amplified an expected 256-base pair size LH/hCG receptor fragment that could hybridize with a full-length LH/hCG receptor cDNA in Southern blotting. Northern blotting demonstrated that neurons contained a major 2.6 kilobase (kb) and a minor 4.3 kb transcript. Immunocytochemistry demonstrated that the neurons contained LH/hCG receptor immunostaining. Western immunoblotting showed that neurons contained an 80-kDa receptor protein that increased to a maximal level on Day 3 of culture and then gradually decreased until the 9th day of culture. Culturing neurons for 3 days in the presence of highly purified hCG resulted in a dose-dependent increase in the outgrowth of neurite processes and total cellular protein and a decrease in DNA fragmentation as compared to values in the corresponding controls. At the maximally effective hCG concentration, the number of neurite-bearing cells was increased by 53% and the total cellular protein by 60%, and DNA fragmentation decreased by 31%. In summary, this is the first study to demonstrate the presence of LH/hCG receptors and neurotrophic effects of hCG in fetal rat brain neurons. These findings imply that locally produced gonadotropins may possibly play a role in the growth and development of the fetal brain.

USE OF A SOLID-PHASE RANDOM PEPTIDE LIBRARY TO IDENTIFY INHIBITORS OF TNF-α MEDIATED CYTOTOXICITY IN VITRO

Tumour necrosis factor (TNF) is a pleiotropic cytokine which plays a central role in infection, inflammation and autoimmune diseases. Its functions are mediated through binding to high affinity cell surface receptors. An approach to modulate excessive levels of TNF-α in the serum is the use of soluble receptors. In this study the potential of a solid-phase combinatorial peptide library was investigated to identify peptide mimics of the binding site of the TNF-α receptor. One of the identified mimotopes was shown to inhibit TNF-α-mediated cytotoxicity in mouse L929 and in a human KYM-1D4 cell lines in a dose-dependent fashion. Characterization of the mimotope sequence by high-performance liquid chromatography and mass spectroscopy has shown that the inhibitory effect of the mimotope was dependent upon the presence of a protective MTR group on the side chains of the arginine residues in the mimotope sequence. Furthermore, antibodies to the mimotope were shown to recognize the recombinant human TNF 55-kDa receptor in an ELISA assay. These findings highlight the potential of combinatorial peptide libraries in identifying peptide mimics of the binding site of TNF-α receptor, which can be used as competitive inhibitors of ligand–receptor interactions.

Effect of age on the vasodilatory action of elastin peptides

We have recently shown, on young adult rat aorta rings, that elastin peptides induce a dose and endothelium-dependent vasodilation mediated by the 67 kDa subunit of the high affinity elastin–laminin receptor and, at least in part, by EDRF (NO) [1]. Here we have studied the effects of elastin peptides at circulating concentrations and below, on noradrenaline-contracted rat aortic rings, as a function of age. First, we have observed that, unlike 2-month-old (2M), 4–6-month-old (4M) and 12-month-old (12M) rat aorta rings, 30-month-old (30M) rat aorta rings were unable to maintain their contraction in long lasting experiments. Secondly, elastin peptides at physiological circulating concentrations (10 −6–10 −3 mg/ml) induce a dose-dependent vasodilation on 4M rings. By contrast, only higher elastin peptide concentrations (10 −3 mg/ml) were effective on 12M rings, whereas rings from both younger (2M) and older animals (30M) did not respond to elastin peptides. Finally, using lactose and laminin as inhibitors, we have demonstrated that elastin peptide-induced vasodilation on 4M and 12M rings is mediated by the 67 kDa subunit of the elastin–laminin receptor. These experiments suggest that the functional availability of the 67 kDa subunit of the elastin–laminin receptor changes with age. It could be hypothesized that in young animals (0–2M) the reusable shuttle role recently demonstrated for the 67 kDa receptor subunit during elastic fiber formation [2]leads to a major decrease in its availability for signal transduction. On the contrary, in adult animals (4–12M), when developmental elastogenesis is completed, this subunit is essential for extracellular signal transduction [3]. Inefficiency of this receptor in old animals (30M) can be attributed to its uncoupling from its transduction pathway, as previously shown on human cells [4]. Finally, the age-dependent variations of circulating elastin peptide concentration and elastin–laminin receptor responsiveness to elastin peptides are two independent parameters which could influence the vascular tension regulation.

Processing of the Transforming Growth Factor β Type I and II Receptors: BIOSYNTHESIS AND LIGAND-INDUCED REGULATION

Three cell surface transforming growth factor beta (TGFbeta) receptor (R) proteins regulate the effects of TGFbeta isoforms on growth and differentiation. TGFbeta-IR and -IIR are transmembrane serine/threonine kinases directly mediating the signaling across the plasma membrane. Both TGFbeta and its receptors are ubiquitously expressed, hence the fine regulation of the multiplicity of responses most likely involves several levels of control including the regulation of expression, complex formation, and down-regulation of the receptor proteins. In mink lung epithelial cells, TGFbeta-IIR was first synthesized as a approximately 60-kDa endoglycosidase H-sensitive precursor protein, which was converted to a mature approximately 70-kDa protein. The half-life of metabolically labeled mature TGFbeta-IIR was estimated to be 60 min and was further reduced to approximately 45 min in the presence of exogenous TGFbeta1. Minimal internalization of 125I-TGFbeta1 at 37 degrees C was detected suggesting that the rapid turnover was not due to endocytosis and degradation of the ligand-receptor complexes. TGFbeta-IR was synthesized as a approximately 53-kDa precursor protein, which was processed to a mature approximately 55-kDa receptor protein. The half-life of TGFbeta-IR was >12 h. A fraction of tunicamycin-treated type I and II receptors that reach the cell surface was able to associate in the presence of ligand suggesting that heteromeric complexes can form in a post-endoplasmic reticulum compartment before full glycosylation is achieved. These results show differential processing and turnover of TGFbeta-IR and TGFbeta-IIR providing a potential additional mechanism for modulation of cellular responses to TGFbetas.

Novel expression of functional luteinizing hormone/chorionic gonadotropin receptors in cultured glial cells from neonatal rat brains.

Adult rat brains contain LH/hCG receptors, and these receptors are functional in neuroendocrine regulation and behaviors. Since glial cells are important for development, maturation, and functioning of the brain, we tested the hypothesis that these cells from neonatal rat brains may also contain functional LH/hCG receptors. Reverse-transcriptase polymerase chain reaction amplified an expected 256 base-pair LH/hCG receptor fragment from glial cells. This fragment can bind to LH/hCG receptor cDNA in Southern blotting. Northern blotting demonstrated that glial cells contain a major 2.6-kilobase (kb) and a minor 4.3-kb transcript of LH/hCG receptors. Western immunoblotting demonstrated that glial cells also contain an 80-kDa receptor protein and that its levels are significantly higher in secondary and tertiary glial cells than in primary glial cells. Immunocytochemistry confirmed that LH/hCG receptor immunostaining is present in glial cells. Since glial cells are quite active in synthesis of prostaglandins (PGs), we investigated the effect of highly purified hCG on PGD2 and PGE2 levels. The results showed that culturing secondary glial cells for three days with highly purified hCG resulted in a dose-dependent and hormone-specific increase in PGD2 and a decrease in PGE2 levels in the medium as compared to control levels. In summary, we conclude that cultured glial cells from neonatal rat brains contain functional LH/hCG receptors. Through regulation of PG synthesis, LH and hCG may influence glial cell functions that are important for neonatal brain development and function.

Towards a latex molecular diagnostic of yield potential and the genetic engineering of the rubber tree

The latex from Hevea brasiliensis is expelled from specialized cells upon bark tapping. The latex yield is mainly limited by the duration of the latex flow, which is controlled by coagulation processes. Bark treatment with ethylene is known to delay coagulation and increase latex yield. The molecular basis of latex coagulation has been characterized: (1) Hevein, a lectin-like protein, induces latex coagulation by bringing together the rubber particles (RPs). The hevein-RPs bridging is mediated by N-Acetyl-D-glucosamine, and involves a 22 kDa receptor glycoprotein localized on the RPs surface. This process is inhibited by the removal of the sugar moiety from the receptor, through the action of N-acetyl-glucosaminidase and chitinases. (2) Ethylene induces, in the latex cells, an over-expression of the 3 genes coding for hevein and its receptor, and a chitinase. The higher over-expression of one chitinase can explain the partial deglycosylation of the hevein receptor and the resulting delay in coagulation. (3) The level of hevein and chitinase expression in the latex is a clonal characteristic, linked to the characteristics of the latex flow. Expression of these genes might be used as molecular markers for high yield potential. Based on these findings, it would be interesting to improve the rubber tree through the genetic engineering technics, to get new high yielding cultivars with prolonged latex flow.

A 50 kDa membrane protein from bovine kidney cells is a putative receptor for bovine viral diarrhea virus (BVDV).

A 50 kDa cell surface protein from MDBK cells has been identified as a putative receptor for bovine viral diarrhea virus (BVDV) by using a BVDV specific anti-idiotypic antibody (Anti-D89). This study delineates further characterization of the receptor protein. Protease treatment of cultured MDBK cells adversely affected the receptor thus abolishing the binding of anti-D89 to the cells. However, pretreatment of the cells with either phospholipases or glycosidases did not signficantly alter the extent of anti-D89 binding. Additionally, pretreatment of cell monolayers with proteases decreased BVDV attachment and replication in the cells. These results suggested that the receptor for BVDV is a protein in nature, and glycosylation and phosphorylation of the receptor protein may not play a direct role in BVDV attachment to cells. The BVDV receptor protein gradually regenerated on cells when they were maintained in culture following protease treatment. The purified 50 kDa receptor protein also significantly inhibited BVDV infection in a plaque reduction assay.

Hormonal and non-hormonal regulation of glutamine synthetase in the developing neural retina

Two isoforms of the glucocorticoid receptor, with apparent molecular mass of 90 and 95 kDa, are expressed in embryonic chicken neural retina. The 95-kDa receptor represents a hyperphosphorylated form of the 90-kDa receptor. Activation of the glucocorticoid receptor by cortisol results in a dose-dependent increase in receptor phosphorylation, translocation of receptor molecules into the nucleus and a decline in the total amount of the receptor. Activation of the glucocorticoid receptor can also be observed in the developing retinal tissue in ovo. At late embryonic ages, when the systemic level of glucocorticoids increases, a substantial quantity of receptor molecules becomes translocated into the nucleus, the relative level of the 95-kDa isoform increases, and the total amount of receptor declines. Activation of the receptor molecules in ovo correlates directly with an increase in transcription of the glucocorticoid-inducible gene, glutamine synthetase. The close correlation between the increase in systemic glucocorticoids, activation of glucocorticoid receptor molecules and induction of glutamine synthetase gene transcription suggests that glucocorticoids are directly involved in the developmental control of glutamine synthetase expression. Long-term organ culturing of embryonic retinal tissue in the absence of hormone results in an increase in glutamine synthetase expression. This increase, which is only 5 to 10% of that observed in ovo, is not mediated by activated receptor molecules and represents a mechanism for non-hormonal regulation of glutamine synthetase.

TNF-mediated cytotoxicity and resistance in human prostate cancer cell lines.

The contribution of TNF receptor (TNF-R) expression was investigated with respect to TNF sensitivity or insensitivity for androgen-dependent and androgen-independent human prostate cancer (PCA) cell lines, respectively. Flow cytometric analyses using monoclonal antibodies against the 55-kDa receptor (TNF-R1) and the 75-kDa receptor (TNF-R2) indicated that both receptors were expressed on all three cell lines. Moreover, expression of TNF-R1 was greater than expression of TNF-R2 in these PCA cells. All three PCA cell lines produced IL-6. However, IL-6 production was enhanced when TNF-insensitive JCA-1 and PC-3 cells, but not TNF-sensitive LNCaP cells, were treated with rTNF (10(-9) M). These data suggest that the lack of an antiproliferative effect of rTNF on the androgen-independent PCA cell lines PC-3 and JCA-1 is not due to the failure of these cells to express TNF-R, but may be related to the differences in TNF-mediated IL-6 expression by these PCA cell lines.

TNF‐mediated cytotoxicity and resistance in human prostate cancer cell lines

BACKGROUND The contribution of TNF receptor (TNF-R) expression was investigated with respect to TNF sensitivity or insensitivity for androgen-dependent and androgen-independent human prostate cancer (PCA) cell lines, respectively. METHODS Flow cytometric analyses using monoclonal antibodies against the 55-kDa receptor (TNF-R1) and the 75-kDa receptor (TNF-R2) indicated that both receptors were expressed on all three cell lines. RESULTS Moreover, expression of TNF-R1 was greater than expression of TNF-R2 in these PCA cells. All three PCA cell lines produced IL-6. However, IL-6 production was enhanced when TNF-insensitive JCA-1 and PC-3 cells, but not TNF-sensitive LNCaP cells, were treated with rTNF (10−9 M). CONCLUSIONS These data suggest that the lack of an antiproliferative effect of rTNF on the androgen-independent PCA cell lines PC-3 and JCA-1 is not due to the failure of these cells to express TNF-R, but may be related to the differences in TNF-mediated IL-6 expression by these PCA cell lines. © 1996 Wiley-Liss, Inc.

SERUM LEVELS AND IN SITU EXPRESSION OF TNF-α AND TNF-α BINDING PROTEINS IN INFLAMMATORY LIVER DISEASES

Cells respond to tumour necrosis factor-α (TNF-α) via binding to 75-kDa (type A) and 55-kDa (type B) receptors which have different intracellular signalling pathways and can also circulate as soluble molecules. Both receptors are co-expressed in many tissues, but their relative contributions to cellular TNF responses is for most situations unknown. In patients with viral and non-viral inflammatory liver diseases serum TNF-α was determined by an immunoenzymetic assay and soluble type A and B TNF receptors (TNF-αr) by enzyme linked immunological and biological assays (ELIBA). In addition, cellular expression of TNF and its binding proteins were studied in liver biopsies by an indirect immunoperoxidase technique. Secretion of TNF-α and upregulation of TNF-αr-A were particularly prominent in viral hepatitis. Strong TNF-α-in-situ production by mononuclear cells could be demonstrated in liver biopsies from patients with acute viral hepatitis. However, TNF-αr-A was detected only on hepatocytes. Serum TNF-αr-A was elevated two-fold in relative abundance over TNF-αr-B and was correlated to serum TNF-α ( r= 0.6464, P< 0.0001). Soluble TNF-αr levels normalized, when the viral hepatitis was cleared, and successful therapy of hepatitis B was associated with a temporary rise of TNF-αr-A during the initial flare of aminotransferases. Patients with alcoholic hepatitis had also evidence of TNF-α activation but clearly differed from patients with viral induced liver diseases: Soluble TNF-αr-A and TNF-αr-B were highly elevated in equal proportions. In situ analysis in liver biopsies revealed a distinctive pattern of TNF-αr expression with strong cytoplasmic staining for both TNF-αr-A and B on scattered hepatocytes in addition to infiltrating mononuclear cells. The data propose that TNF release during antiviral immune responses is predominantly associated with TNF-αr-A upregulation and shedding, whereas upregulation and shedding of TNF-αr-B is more prominent in alcoholic hepatitis. As cytotoxicity and apoptosis by TNF are mediated mainly via TNF-αr-B, our results are consistent with more severe TNF-α induced liver damage in alcoholic hepatitis as compared to viral hepatitis.

Lipoprotein lipase degradation by adipocytes: receptor-associated protein (RAP)-sensitive and proteoglycan-mediated pathways

Lipoprotein lipase (LPL), the major enzyme responsible for the hydrolysis of triglycerides, is primarily synthesized by adipocytes and myocytes. In addition to synthesis, degradation of cell surface-associated LPL is thought to be important in regulating production of the enzyme. We studied LPL metabolism in the LPL synthesizing adipocyte cell line BFC-1 beta and assessed the contributions of cell surface heparan sulfate proteoglycans (HSPG), low density lipoprotein receptor related protein (LRP), and glycosylphosphatidylinositol (GPI)-linked proteins to LPL uptake and degradation by these cells. Adipocytes degraded 10-12% of total cell surface I-labeled LPL in 2 h and 23-28% in 4 h. In 1 h, 30-54% of the degradation was inhibited by the 39 kDa receptor associated protein (RAP), an inhibitor of ligand binding to LRP. At 4 h, only 19-23% of the LPL degradation was RAP inhibitable. This suggested that two pathways with different kinetics were important for LPL degradation. Heparinase/heparitinase treatment of cells showed that most LPL degradation required the presence of HSPG. Treatment with phosphatidylinositol-specific phospholipase C (PIPLC) inhibited 125I-labeled LPL degradation by 13%. However, neither RAP nor PIPLC treatment of adipocytes significantly increased the amount of endogenously produced LPL activity in the media. To determine whether direct uptake of LPL bound to HSPG could account for the non-RAP sensitive LPL uptake and degradation, proteoglycan metabolism was assessed by labeling cells with 35SO4. Of the total pericellular proteoglycans, 14% were PIPLC releasable; surprisingly, 30% were dissociated from the cells with heparin. The amount of labeled pericellular proteoglycans decreased 26% in 2 h and 50% in 8 h, rapid enough to account for at least half of the degradation of cell surface LPL. We conclude that adipocytes degrade a fraction of the cell surface LPL, and that this process is mediated by both proteoglycans and RAP-sensitive receptors.

Processing of Carcinoembryonic Antigen by Kupffer Cells: Recognition of a Penta-Peptide Sequence

Carcinoembryonic antigen (CEA) binds to an 80-kDa cell surface receptor on Kupffer cells via the peptide sequence PELPK (residues 108–112) located at the hinge region between the N and A1 immunoglobulin-like domains. This study is aimed at analyzing the specificity of the peptide binding, determining biodistribution of 80-kDa receptor, and processing of CEA by this receptor. We synthesized a number of bovine serum albumin (BSA) derivatives carrying PELPK and related sequences. A series of peptides (YPELPK, YPDLPK, YPDLPR, and YPELGK) were conjugated to bovine serum albumin usingN-hydroxysuccinimidyl-4-azidobenzoate. When125I peptide conjugates, CEA, and BSA were injected intravenously into rats CEA and the PELPK–albumin conjugate were cleared rapidly. The other peptide conjugates and BSA cleared at a much slower rate. Activity of125I-labeled CEA and PELPK–albumin conjugate per gram of tissue was highest for the liver and spleen. Clearance of125I-CEA was inhibited by the presence of higher concentrations of the PELPK–albumin conjugate. With isolated rat Kupffer cells, only CEA and the PELPK–albumin conjugate were bound and internalizedin vitroand CEA binding was inhibited by higher concentrations of the PELPK–albumin conjugate. Similarly, binding of the PELPK–albumin conjugate was inhibited by the presence of unlabeled CEA. Use of a heterobifunctional cross linking agent demonstrated reaction of the PELPK–albumin with an 80-kDa protein on the Kupffer cell surface by SDS–polyacrylamide gel electrophoresis (SDS–PAGE). This semisynthetic ligand (PELPK-albumin) allows us to examine the function of the 80-kDa receptor without interference due to other properties of CEA including its ability to bind lectins and to cause homotypic aggregation of cells. The consequences of CEA binding to the 80-kDa receptor may have implications in the development of hepatic metastasis from colorectal cancer.

Elements of Neural Adhesion Molecules and a Yeast Vacuolar Protein Sorting Receptor Are Present in a Novel Mammalian Low Density Lipoprotein Receptor Family Member

Normal cell development depends to a large part on multifunctional proteins that have evolved by recombination of proven modular elements. We now have discovered and characterized in rabbit such a multi-domain protein, and classify it as novel member of the low density lipoprotein (LDL) receptor gene family. The extracellular portion of the approximately 250-kDa membrane protein, termed LR11, contains a cluster of 11 LDL receptor ligand binding repeats, a group of 5 LDL receptor "YWTD" repeats, a large hexarepeat domain of structural elements found in neural cell adhesion molecules, and a domain with similarity to a yeast receptor for vacuolar protein sorting, VPS10. The cytoplasmic domain exhibits features typical of endocytosis-competent coated-pit receptors. The mosaic, and presumably multifunctional, receptor is expressed abundantly in brain, in particular the hippocampus, dentate gyrus, and cerebral cortex, and is present at significant levels in liver, adrenal glands, and testis. Western blotting of tissues and ligand blotting of LR11-transfected cells demonstrated that the novel protein binds apolipoprotein E-containing lipoproteins. In contrast to the LDL receptor, hepatic expression of LR11 is unaffected by hyperlipidemia. The identification of this highly conserved and superbly complex protein offers the opportunity to gain new insights into the emergence of multifunctional mosaic proteins akin to the ever expanding LDL receptor gene family.

Up-regulation of cyclooxygenase-2 gene expression by chorionic gonadotropin in mucosal cells from human fallopian tubes.

The present study investigated the regulation of cyclooxygenase-2 (COX-2) gene by human CG (hCG) in mucosal cells from human fallopian tubes. The mucosal cells contained a major [4.3 kilobase (kb)] and several minor (3.6, 2.4, and 1. 8 kb) messenger RNA (mRNA) transcripts of LH/hCG receptors and also an 80-kDa receptor protein. The receptor protein can bind 125I-hCG. Culturing mucosal cells with increasing concentrations of highly purified hCG resulted in a dose- and time-dependent increase in steady-state levels of a 4.4-kb mRNA transcript and 72-kDa protein of COX-2. Whereas hLH and hCG could mimic each other in increasing COX-2 protein levels, FSH, TSH, PRL, and isolated alpha- and beta-subunits of hCG had no effect, suggesting that the hCG effect is hormone specific and requires the conformation of native hormone. Culturing mucosal cells with increasing concentrations of hCG also resulted in a dose-dependent increase in media PGE2, levels, suggesting that the COX-2 protein increased by hCG is catalytically active. To determine the molecular mechanism of hCG action responsible for increasing the steady-state COX-2 mRNA levels, we measured the transcription rate of the COX-2 gene by nuclear run-on assay and the stability of its transcripts by an actinomycin D blocking method. The results showed that although hCG treatment had no effect on the transcription rate of COX-2 gene, it significantly increased the stability of COX-2 transcripts from 3.7 h in the control to 7.3 h after treatment. In summary, we conclude that tubal mucosal cells contain LH/hCG receptor transcripts and the receptor protein that can bind hCG. Culturing these cells with exogenous hCG and LH can up-regulate the expression of COX-2 gene by increasing the stability of transcripts. Through this up-regulation, LH and hCG may influence tubal functions that are important for early pregnancy in women.