共查询到20条相似文献,搜索用时 15 毫秒
1.
P Ragno A Estreicher A Gos A Wohlwend D Belin J D Vassalli 《Experimental cell research》1992,203(1):236-243
Numerous epithelial cell types produce and secrete plasminogen activators (PAs) and/or PA inhibitors (PAIs). When epithelial cells were grown on polycarbonate filters and their apical and basolateral secretion products analyzed, PA activity accumulated in a highly polarized fashion; depending upon the cell line, the compartment of PA accumulation was either apical (MDCK I cells and HBL-100 cells) or basolateral (LLC-PK1, CaCo-2, and HeLa cells). By contrast, PAI-1 was recovered in roughly equal amounts in both compartments. Basolateral accumulation of urokinase-type plasminogen activator (uPA), but not its apical targeting, required an acidic compartment and the integrity of the cytoskeleton. Polarity of uPA accumulation did not result from removal of the free enzyme from the opposite compartment through its binding to the cell surface. Transfection with wild-type or mutated murine uPA demonstrated that neither the "growth factor" domain nor the kringle domain is required for the appropriate sorting of the protein. We propose that polarized secretion of PAs is one mechanism whereby cells spatially control extracellular proteolysis. 相似文献
2.
Proliferative effects of apical, but not basal, matrix metalloproteinase-7 activity in polarized MDCK cells 总被引:2,自引:0,他引:2
Harrell PC McCawley LJ Fingleton B McIntyre JO Matrisian LM 《Experimental cell research》2005,303(2):308-320
Matrix metalloproteinase-7 (MMP-7) is primarily expressed in glandular epithelium. Therefore, its mechanism of action may be influenced by its regulated vectorial release to either the apical and/or basolateral compartments, where it would act on its various substrates. To gain a better understanding of where MMP-7 is released in polarized epithelium, we have analyzed its pattern of secretion in polarized MDCK cells expressing stably transfected human MMP-7 (MDCK-MMP-7), and HCA-7 and Caco2 human colon cancer cell lines. In all cell lines, latent MMP-7 was secreted to both cellular compartments, but was 1.5- to 3-fold more abundant in the basolateral compartment as compared to the apical. However, studies in the MDCK system demonstrated that MMP-7 activity was 2-fold greater in the apical compartment of MDCK-MMP-7(HIGH)-polarized monolayers, which suggests the apical co-release of an MMP-7 activator. In functional assays, MMP-7 over-expression increased cell saturation density as a result of increased cell proliferation with no effect on apoptosis. Apical MMP-7 activity was shown to be responsible for the proliferative effect, which occurred, as demonstrated by media transfer experiments, through cleavage of an apical substrate and not through the generation of a soluble factor. Taken together, our findings demonstrate the importance of MMP-7 secretion in relation to its mechanism of action when expressed in a polarized epithelium. 相似文献
3.
Kinetics of the activation of plasminogen by natural and recombinant tissue-type plasminogen activator 总被引:6,自引:0,他引:6
The kinetics of the activation of plasminogen by tissue-type plasminogen activator were studied in the presence and the absence of CNBr-digested fibrinogen as a soluble cofactor. Michaelis-Menten kinetics applied and the kinetic parameters obtained were very similar to those previously reported for the activation in the presence of solid phase fibrin (Hoylaerts, M., Rijken, D. C., Lijnen, H. R., and Collen, D. (1982) J. Biol. Chem. 257, 2912-2919). The affinity of the enzyme for plasminogen dramatically increases in the presence of the soluble cofactor while the catalytic rate constant does not change significantly (KM drops from 83 to 0.18 microM and kcat increases from 0.07 to 0.28 s-1 for tissue-type plasminogen activator of melanoma origin). Fragments containing the lysine-binding sites of plasminogen compete with plasminogen for interaction with CNBr-digested fibrinogen. The dissociation constant of this interaction was found to be 4.5 microM for the high affinity lysine-binding site. No difference was found in the kinetic parameters for the activation of plasminogen by either tissue-type plasminogen activator of melanoma origin or by glycosylated forms of tissue-type plasminogen activator obtained by recombinant DNA technology. The present findings obtained in a homogenous liquid milieu support the previously proposed mechanism of the activation of plasminogen by tissue-type plasminogen activator in the presence of fibrin. This mechanism involves binding of both tissue-type plasminogen activator and plasminogen to fibrin. 相似文献
4.
Corticosteroid binding globulin, a member of the serpin family, was previously shown to be secreted mainly apically from MDCK cells in an N-glycan independent manner [Larsen et al. (1999) FEBS Lett. 451, 19-22]. Apart from N-glycosylation, serpins are not known to carry any other posttranslational modifications, suggesting the presence of a proteinaceous apical sorting signal. In the present study we have expressed four other members of the serpin family: alpha1-antitrypsin, C1 inhibitor, plasminogen activator inhibitor-1 and antithrombin in MDCK cells. Tight monolayers of transfected cells were grown on filters and the amounts of recombinantly expressed serpins in the apical and the basolateral media were determined. alpha1-Antitrypsin and C1 inhibitor were found mainly in the apical medium whereas plasminogen activator inhibitor-1 and antithrombin were found in roughly equal amounts in the apical and basolateral media. Control experiments showed that all four serpins are transported along the exocytotic pathway in an uncomplicated way that does not involve transcytosis or differences in stability on the two sides of the cells. We conclude that some members of the serpin family including corticosteroid binding globulin, alpha1-antitrypsin and C1 inhibitor are secreted mainly apically from MDCK cells whereas plasminogen activator inhibitor-1 and antithrombin are secreted in a non-polarized manner. 相似文献
5.
A mutant single chain urokinase plasminogen activator (scu-PA) was constructed by the addition of an apical membrane targeting signal from decay accelerating factor to the scu-PA carboxyl terminus. Bovine aortic endothelial cells (EC) were transduced with the mutant scu-PA. Metabolic labeling, immunoprecipitation, and gel electrophoresis revealed that the mutant scu-PA was present in a single-chain form at the EC surface. Immunohistochemistry and enzyme-linked immunosorbent assay before and after treatment of EC with phosphotidylinositol-specific phospholipase C confirmed that scu-PA was attached to the EC surface by a glycosyl-phosphotidylinositol anchor. Approximately 10(6) anchored scu-PA molecules/cell were present; however, anchoring was not 100% efficient, with scu-PA released into the medium as well. Selective biotinylation of the apical and basolateral surfaces revealed that anchored scu-PA was polarized to the apical surface. Apically anchored scu-PA could be converted by plasmin to two-chain urokinase, with a normal specific activity (140,000 IU/mg) as measured with the chromogenic substrate S-2444. Expression of anchored scu-PA resulted in an increase in EC surface plasminogen activator activity, as compared with the activity of either untransduced EC or EC transduced with a wild type scu-PA. These experiments demonstrate: 1) apical membrane targeting can be accomplished in EC; 2) scu-PA can be anchored to the EC surface with preservation of enzymatic activity; 3) EC surface plasminogen activator activity is significantly increased by the presence of anchored scu-PA. Cell surface targeted plasminogen activators may eventually be useful in the prevention and treatment of intravascular thrombosis. 相似文献
6.
Nocodazole, a microtubule-active drug, interferes with apical protein delivery in cultured intestinal epithelial cells (Caco-2) 总被引:33,自引:15,他引:18
下载免费PDF全文
![点击此处可从《The Journal of cell biology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
The polarized delivery of membrane proteins to the cell surface and the initial secretion of lysosomal proteins into the culture medium were studied in the polarized human intestinal adenocarcinoma cell line Caco-2 in the presence or absence of the microtubule-active drug nocodazole. The appearance of newly synthesized proteins at the plasma membrane was measured by their sensitivity to proteases added either to the apical or the basolateral surface of cells grown on nitrocellulose filters. Nocodazole was found to reduce the delivery to the cell surface of an apical membrane protein, aminopeptidase N, and to lead to its partial missorting to the basolateral surface, whereas the drug had no influence on the delivery of a basolateral 120-kD membrane protein defined by a monoclonal antibody. Furthermore, nocodazole selectively blocked the apical secretion of two lysosomal proteins, cathepsin D and acid alpha-glucosidase, whereas the drug had no influence on their basolateral secretion. These results suggest that in Caco-2 cells an intact microtubular network is important for the transport of newly synthesized proteins to the apical cell surface. 相似文献
7.
Extracellular nucleotides such as ATP have been shown to regulate ion transport processes in a variety of epithelia. This effect is mediated by the activation of plasma membrane P2Y receptors, which leads to Ca(2+) signaling cascade. Ion transport processes (e.g. activation of apical calcium-dependent Cl(-) channels) are then stimulated via an increase in [Ca(2+)](i). Many polarized epithelia express apical and/or basolateral P2Y receptors. To test whether apical and basolateral stimulation of P2Y receptors elicit polarized Ca(2+) signaling and anion secretion, we simultaneously measured the two parameters in polarized epithelia. Although activation of P2Y receptors located at both apical and basolateral membranes evoked an increase in [Ca(2+)](i), only apical P2Y receptors-coupled Ca(2+) release stimulated an increase in anion secretion. Moreover, the calcium influx evoked by apical and basolateral P2Y receptor stimulation is predominately via the basolateral membrane domain. It appears that the apical P2Y receptor-regulated Ca(2+) release and activation of apical Cl(-) channels is compartmentalized in polarized epithelia with basolateral P2Y-stimulated Ca(2+) release failing to activate anion secretion. These data suggest that there may be two distinct ATP-releasable Ca(2+) pools, each coupled to apical and basolateral membrane receptor but linked to the same calcium influx pathway located at the basolateral membrane. 相似文献
8.
We have grown polarized epithelial Madin-Darby canine kidney II (MDCK II) cells on filters in the presence of [(35)S]sulfate, [(3)H]glucosamine, or [(35)S]cysteine/[(35)S]methionine to study proteoglycan (PG) synthesis, sorting, and secretion to the apical and basolateral media. Whereas most of the [(35)S]sulfate label was recovered in basolateral PGs, the [(3)H]glucosamine label was predominantly incorporated into the glycosaminoglycan chains of apical PGs, indicating that basolateral PGs are more intensely sulfated than their apical counterparts. Expression of the PG serglycin with a green fluorescent protein tag (SG-GFP) in MDCK II cells produced a protein core secreted 85% apically, which was largely modified by chondroitin sulfate chains. Surprisingly, the 15% of secreted SG-GFP molecules recovered basolaterally were more heavily sulfated and displayed a different sulfation pattern than the apical counterpart. More detailed studies of the differential modification of apically and basolaterally secreted SG-GFP indicate that the protein cores have been designated to apical and basolateral transport platforms before pathway-specific, post-translational modifications have been completed. 相似文献
9.
Microtubular organization and its involvement in the biogenetic pathways of plasma membrane proteins in Caco-2 intestinal epithelial cells 总被引:7,自引:0,他引:7
We characterized the three-dimensional organization of microtubules in the human intestinal epithelial cell line Caco-2 by laser scanning confocal microscopy. Microtubules formed a dense network approximately 4-microns thick parallel to the cell surface in the apical pole and a loose network 1-micron thick in the basal pole. Between the apical and the basal bundles, microtubules run parallel to the major cell axis, concentrated in the vicinity of the lateral membrane. Colchicine treatment for 4 h depolymerized 99.4% of microtubular tubulin. Metabolic pulse chase, in combination with domain-selective biotinylation, immune and streptavidin precipitation was used to study the role of microtubules in the sorting and targeting of four apical and one basolateral markers. Apical proteins have been recently shown to use both direct and transcytotic (via the basolateral membrane) routes to the apical surface of Caco-2 cells. Colchicine treatment slowed down the transport to the cell surface of apical and basolateral proteins, but the effect on the apical proteins was much more drastic and affected both direct and indirect pathways. The final effect of microtubular disruption on the distribution of apical proteins depended on the degree of steady-state polarization of the individual markers in control cells. Aminopeptidase N (APN) and sucrase-isomaltase (SI), which normally reach a highly polarized distribution (110 and 75 times higher on the apical than on the basolateral side) were still relatively polarized (9 times) after colchicine treatment. The decrease in the polarity of APN and SI was mostly due to an increase in the residual basolateral expression (10% of control total surface expression) since 80% of the newly synthesized APN was still transported, although at a slower rate, to the apical surface in the absence of microtubules. Alkaline phosphatase and dipeptidylpeptidase IV, which normally reach only low levels of apical polarity (four times and six times after 20 h chase, nine times and eight times at steady state) did not polarize at all in the presence of colchicine due to slower delivery to the apical surface and increased residence time in the basolateral surface. Colchicine-treated cells displayed an ectopic localization of microvilli or other apical markers in the basolateral surface and large intracellular vacuoles. Polarized secretion into apical and basolateral media was also affected by microtubular disruption. Thus, an intact microtubular network facilitates apical protein transport to the cell surface of Caco-2 cells via direct and indirect routes; this role appears to be crucial for the final polarity of some apical plasma membrane proteins but only an enhancement factor for others. 相似文献
10.
Dietary fatty acids (FAs) crossing the apical plasma membrane of small intestinal enterocytes are targeted to different metabolic pathways than serum FAs crossing the basolateral membrane. This apparent compartmentalization of FA metabolism in enterocytes was further investigated using a model human enterocyte-like intestinal cell line. [3H]Oleic acid bound to bovine serum albumin (BSA) was added to the apical or basolateral surfaces of confluent monolayers of Caco-2 cells growing on uncoated polycarbonate filters. In other experiments, [3H]oleic acid incorporated into micelles with taurocholate (+/- 2-monoacylglycerol) was added apically. Caco-2 cells absorbed oleic acid bound to BSA from both the apical and basolateral surfaces at the same rate. Oleic acid in micellar solution was absorbed more efficiently than oleic acid bound to BSA. Regardless of its site or mode of presentation, the majority of the incorporated oleic acid was found in triglycerides. Only a small fraction was subjected to beta-oxidation or esterification into phospholipids. Most of the incorporated oleic acid was still retained intracellularly at 24 h. The polarity of triglyceride secretion was influenced by the experimental conditions. Triglyceride secretion was not significantly polarized when oleic acid-BSA was presented apically. However, the ratio of basolateral to apical secretion at 24 h was 9:1 for oleic acid-BSA presented basolaterally. For oleic acid in taurocholate micelles there was a trend toward polarity of secretion to the apical media (apical to basolateral ratio = 2:1). The inclusion of 2-monoacylglycerol in oleic acid-taurocholate micelles did not augment triglyceride synthesis or secretion. These differences indicate that compartmentation of FA metabolism in Caco-2 cells is influenced by the site of FA presentation. Northern and Western blot hybridization studies indicated that the liver fatty acid-binding protein but not the intestinal fatty acid-binding protein gene is expressed in these cells. The absence of this latter 15 kDa protein indicates that it is not required by Caco-2 cells for the synthesis of triglycerides or for the polarized export of triglyceride. These studies indicate that the Caco-2 cell line will be a useful model system for studying the polarization of FA trafficking/metabolism in enterocytes and defining the role of intracellular fatty acid binding proteins in these processes. 相似文献
11.
In human endothelial cell conditioned medium a fast-acting inhibitor of tissue-type plasminogen activator and urokinase has been detected. Moreover, an inactive inhibitor of these plasminogen activators is present, that can be activated by denaturing agents such as sodium dodecyl sulphate (SDS). The mutual relationship between these inhibitors was studied. The fast-acting plasminogen activator inhibitor from human endothelial cell conditioned medium was purified in a complex with tissue-type plasminogen activator by immune adsorption, using an immobilized anti-tissue-type plasminogen activator antibody. With the complex as an antigen, specific antibodies were raised against this inhibitor in rabbits. The antiserum immunoreacted with both the inactive and the fast-acting plasminogen activator inhibitor. Endothelial cell conditioned medium (containing the inactive plasminogen activator inhibitor) was treated with SDS and the inhibitory activity that emerged was purified. The SDS-generated product formed complexes with tissue-type plasminogen activator with the same molecular mass as those formed with the fast-acting inhibitor. Moreover, the inhibitory activity generated by SDS treatment showed the same kinetic behaviour with tissue-type plasminogen activator as did the fast-acting inhibitor. These data show that the fast-acting and the inactive plasminogen activator inhibitor are immunologically and functionally related to each other, and probably represent different molecular forms of the same protein. 相似文献
12.
Spontaneously released plasminogen activator after perfusion with 0.9% NaCl of isolated pig ear was purified by affinity chromatography on Heparin-Sepharose. The molecular weight of the plasminogen activator is about 60,000 Daltons, the isoelectric point lies at pH 7.6. The enzyme is most stable at neutral pH. At 37 degrees C it is stable for two hours. The activator did not show lytic activity either on heated or on PAMBA fibrin plates. The activity of the activator was inhibited by exposure to DFP and PMSF but not by exposure to TLCK and TPCK, suggesting that it is an enzyme with an active-site residue which belongs to the tissue-type activators. 相似文献
13.
Thyrotropin regulation of plasminogen activator activity in primary cultures of ovine thyroid cells 总被引:1,自引:0,他引:1
W W Mak M C Eggo G N Burrow 《Biochemical and biophysical research communications》1984,123(2):633-640
In primary cultures of ovine thyroid cells, a high level of plasminogen activator (PA) activity was detected in the culture media. This level is much higher than in primary cultures of Sertoli cells, granulosa cells, and pituitary cells. PA activity increased with time in culture and was regulated by TSH and insulin. Activity gel analysis of the culture media revealed a major band of 43,000 daltons and a minor one of 70,000 daltons, suggesting the presence of both of the urokinase-type and the tissue-type PA in the media. 相似文献
14.
The interaction of streptokinase.plasminogen activator complex, tissue-type plasminogen activator, urokinase and their acylated derivatives with fibrin and cyanogen bromide digest of fibrinogen. Relationship to fibrinolytic potency in vitro. 总被引:1,自引:1,他引:0
下载免费PDF全文
![点击此处可从《The Biochemical journal》网站下载免费的PDF全文](/ch/ext_images/free.gif)
The effects of purified soluble fibrin and of fibrinogen fragments (fibrin mimic) on the activation of Lys-plasminogen (i.e. plasminogen residues 77-790) to plasmin by streptokinase.plasminogen activator complex and by tissue-type plasminogen activator were studied. Dissociation constants of both activators were estimated to lie in the range 90-160 nM (fibrin) and 16-60 nM (CNBr-cleavage fragments of fibrinogen). The kinetic mechanism for both types of activator comprised non-essential enzyme activation via a Rapid Equilibrium Ordered Bireactant sequence. In order to relate the fibrin affinity of plasminogen activators to their fibrinolytic potency, the rate of lysis of supported human plasma clots formed in the presence of unmodified or active-centre-acylated precursors of plasminogen activators was studied as a function of the concentration of enzyme derivative. The concentrations of unmodified enzyme giving 50% lysis/h in this assay were 0.9, 2.0 and 11.0 nM for tissue-type plasminogen activator, streptokinase.plasmin(ogen) and urokinase respectively. However, the potencies of active-centre-acylated derivatives of these enzymes suggested that acylated-tissue plasminogen activator and streptokinase.plasminogen complexes of comparable hydrolytic stability were of comparable potency. Both types of acyl-enzyme were significantly more potent than acyl-urokinases. 相似文献
15.
Neutral endopeptidase-24.11 (NEP; EC 3.4.24.11) is an abundant metalloendopeptidase of the brush border membrane of kidney proximal tubules. We have recently shown that NEP is delivered directly to the apical domain of the plasma membrane when expressed in polarized Madin-Darby canine kidney (MDCK) cells in culture (Jalal, F., Lemay, G., Zollinger, M., Berteloot, A., Boileau, G., and Crine, P. (1991) J. Biol. Chem. 266, 19826-19832). Here, a soluble form of NEP consisting of the signal peptide of pro-opiomelanocortin fused in-frame with the ectodomain of NEP has been expressed in MDCK cells. Enzymatic assays performed on apical and basolateral culture media of MDCK cells grown on semi-permeable supports indicated that the recombinant enzyme was predominantly released at the apical surface. In contrast, when the chimeric protein was expressed in NIH 3T3 cells or when pro-opiomelanocortin was expressed in MDCK cells, non-polarized secretion was observed into both the apical and basolateral compartments of the culture chamber. Our results suggest that the ectodomain of NEP is sufficient for directing the targeting of this protein to the apical membrane of polarized MDCK epithelial cells. 相似文献
16.
17.
Analysis of the signals for polarized transport of influenza virus (A/WSN/33) neuraminidase and human transferrin receptor, type II transmembrane proteins. 总被引:4,自引:2,他引:2
下载免费PDF全文
![点击此处可从《Journal of virology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
In polarized MDCK cells influenza virus (A/WSN/33) neuraminidase (NA) and human transferrin receptor (TR), type II glycoproteins, when expressed from cloned cDNAs, were transported and accumulated preferentially on the apical and basolateral surfaces, respectively. We have investigated the signals for polarized sorting by constructing chimeras between NA and TR and by making deletion mutants. NATR delta 90, which contains the cytoplasmic tail and transmembrane domain of NA and the ectodomain of TR, was found to be localized predominantly on the apical membrane, whereas TRNA delta 35, containing the cytoplasmic and transmembrane domains of TR and the ectodomain of NA, was expressed preferentially on the basolateral membrane. TR delta 57, a TR deletion mutant lacking 57 amino acids in the TR cytoplasmic tail, did not exhibit any polarized expression and was present on both apical and basolateral surfaces, whereas a deletion mutant (NA delta 28-35) lacking amino acid residues from 28 to 35 in the transmembrane domain of NA resulted in secretion of the NA ectodomain predominantly from the apical side. These results taken together indicate that the cytoplasmic tail of TR was sufficient for basolateral transport, but influenza virus NA possesses two sorting signals, one in the cytoplasmic or transmembrane domain and the other within the ectodomain, both of which are independently able to transport the protein to the apical plasma membrane. 相似文献
18.
Purification of epidermal plasminogen activator inhibitor 总被引:1,自引:0,他引:1
A plasminogen activator inhibitor was purified from human cornified cell extract by DEAE-Sepharose, Sephacryl S-200, and high-performance liquid chromatographies on hydroxyapatite HPHT and anion-exchanger Mono Q at pH 7.2 and 8.0. The purified inhibitor showed Mr 43,000 and pI 5.2 50% inhibition of fibrinolytic activity (1.5 IU) of urokinase and tissue-type plasminogen activator was attained by 0.60 ng and 11.0 ng purified inhibitor, respectively. Synthetic substrate assay demonstrated slow tight-binding inhibition to both urokinase and tissue-type plasminogen activator. The inhibitor did not inactivate plasmin, thrombin, glandular kallikrein or trypsin. 相似文献
19.
The secretion of tissue-type plasminogen activator (t-PA) from melanoma cells (Bowes) was investigated with or without monensin treatment. Monensin inhibited secretion of t-PA from the cells to the medium in a dose-and time-dependent manner. The inhibition was accompanied by an intracellular accumulation of t-PA. Electrophoretic enzymography of the cell homogenate showed the main lytic zone at 72 kDa, which reacted with the IgG of anti-t-PA. Analysis of the cell organelles using ultracentrifugation with a discontinuous sucrose density gradient revealed that the activity and the antigen of t-PA were observed near the discontinuous phase of the sucrose gradient. Analysis of 3H-mannose- and 35S-methionine-labeled t-PA in the cell organelles revealed that the radioactivity of each was increased by monensin treatment, and that such treatment increased the ratio of 3H-mannose-related glycoprotein to 35S-methionine-related protein. The sugar chain of intracellular t-PA was analyzed with endoglycosidase H and N-glycanase, which reduced the molecular weight of t-PA by 4.5-10 kDa, indicating the intracellular presence of a high-mannose type sugar chain and a complex-type sugar chain of t-PA. t-PA secreted from the monensin-treated cells possesses a high-mannose type sugar chain only. Therefore, monensin alters the secretion of t-PA by abnormal glycosylation. 相似文献
20.
Apical and basolateral transferrin receptors in polarized BeWo cells recycle through separate endosomes 总被引:4,自引:2,他引:2
下载免费PDF全文
![点击此处可从《The Journal of cell biology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Contrary to most other epithelia, trophoblasts in the human placenta, which form the physical barrier between the fetal and the maternal blood circulation, express high numbers of transferrin receptors on their apical cell surface. This study describes the establishment of a polarized trophoblast-like cell line BeWo, which exhibit a high expression of transferrin receptors on the apex of the cells. Cultured on permeable filter supports, BeWo cells formed a polarized monolayer with microvilli on their apical cell surface. Across the monolayer a transepithelial resistance developed of approximately 600 omega.cm2 within 4 d. Depletion of Ca2+ from the medium decreased the resistance to background levels, showing its dependence on the integrity of tight junctions. Within the same period of time the secretion of proteins became polarized. In addition, the compositions of integral membrane proteins at the apical and basolateral plasma membrane domains were distinct as determined by domain-selective iodination. Similar to placental trophoblasts, binding of 125I-labeled transferrin to BeWo monolayers revealed that the transferrin receptor was expressed at both plasma membrane domains. Apical and basolateral transferrin receptors were found in a 1:2 surface ratio and exhibited identical dissociation constants and molecular weights. After uptake, transferrin recycled predominantly to the domain of administration, indicating separate recycling pathways from the apical and basolateral domain. This was confirmed by using diaminobenzidine cytochemistry, a technique by which colocalization of endocytosed 125I-labeled and HRP-conjugated transferrin can be monitored. No mixing of the two types of ligands was observed, when both ligands were simultaneously internalized for 10 or 60 min from opposite domains, demonstrating that BeWo cells possess separate populations of apical and basolateral early endosomes. In conclusion, the trophoblast-like BeWo cell line can serve as a unique model to compare the apical and basolateral endocytic pathways of a single ligand, transferrin, in polarized epithelial cells. 相似文献