Cleavage of a 135 kD cell surface glycoprotein correlates with loss of fibroblast adhesion to fibronectin

We have previously described a group of three plasma membrane glycoproteins that are recognized by an adhesion-disrupting antiserum and that are involved in fibronectin-mediated BHK cell adhesion. A peculiar property of these molecules is their resistance to tryptic digestion. We have now extended this study in the attempt to identify the active component within this group of molecules. SR/BALB mouse fibroblasts, used in this work, expose at their surface only two trypsin-resistant glycoproteins, gp1 (150 K) and gp2 (135 K), that are recognized by the adhesion-disrupting anti-BHK serum. Controlled proteolysis of the cell surface in the presence of a reducing agent results in the loss of cell adhesion to fibronectin-coated substratum. gp2 is selectively cleaved under these conditions. Moreover, cells treated with trypsin and reducing agent can no longer adsorb the adhesion-relevant antibodies from the anti-BHK serum. These data indicate that gp2 plays a critical role in the adhesion of SR/BALB fibroblasts to fibronectin-coated substratum, and that disulfide bonds are important in the conformation and function of this molecule.     

Rapid cell surface appearance of endocytic membrane proteins in Chinese hamster ovary cells.

Lactoperoxidase was used to selectively radiolabel endocytic membrane. CHO cells were incubated with enzyme at 37 degrees C for 10 min to permit lactoperoxidase internalization. Radioiodination was done at 4 degrees C. About 90% of the radioiodinated products pelleted at 100,000 X g. From 12 to 15 different electrophoretic species were detected by one-dimensional gel electrophoresis. When cells labeled by internalized lactoperoxidase were warmed to 37 degrees C, the incorporated radioactivity was lost in a biphasic manner with an overall t1/2 of approximately 20 h. Upon warming cells to 37 degrees C, the labeled species became sensitive to pronase or trypsin digestion. The increase in protease sensitivity was progressive over a 10- to 20-min period. Maximally 45% of the initially intracellular radiolabel could be released. A digest of exterior-radioiodinated cells released 50% of the incorporated radioiodine. These observations strongly suggest a rapid shuttling of approximately 90% of the radioiodinated membrane species initially present within the cell to the cell surface.     

Binding of plasma fibronectin to the surfaces of BHK cells in suspension at 4 °C

Plasma fibronectin is shown by several different criteria to bind to suspended BHK cells if the binding incubations are carried out at 4 °C. In indirect immunofluorescence experiments, fibronectin bound to suspended BHK cells at 4 °C in a punctate distribution over the entire cell surfaces. Little binding, however, was detected on cells incubated with fibronectin at 37 °C. The fibronectin bound to the cells at 4 °C was functionally active, since these cells subsequently were able to spread on tissue culture dishes in protein-free medium, unlike cells preincubated with fibronectin at 37 °C or in the absence of fibronectin. Also, the cell surface receptors for soluble fibronectin and fibronectin-coated beads appeared to be similar, since cells preincubated with fibronectin at 4 °C subsequently bound fewer fibronectin-coated beads than control cells. In biochemical studies with radiolabeled fibronectin, binding of fibronectin to the cells was shown to increase with incubation time up to 4 h. In competition experiments with unlabeled fibronectin, 30% of the binding of radiolabeled fibronectin could be inhibited.     

Selective adhesion of mast cells to tracheal epithelial cells in vitro

In allergic and nonallergic lung diseases, if intraluminal mast cells adhere to airway epithelium, inflammatory mediators released from activated mast cells may reach high local concentrations and thus greatly affect airway function. To determine whether mast cells adhere to airway epithelial cells, radiolabeled or unlabeled dog mastocytoma cells were incubated with cultured dog tracheal epithelial cells, with extracellular matrix substrates, and with cryostat-cut sections of dog trachea. Mast cells adhered well to cultured epithelial cells (35 +/- 13% adhesion, mean +/- 1 SD, n = 23) but adhered poorly to types I and IV collagen or to fibronectin (less than 7.5% mean adhesion in all cases). Similarly, in tracheal tissue sections, mast cells adhered preferentially to epithelial cells in surface epithelium or in submucosal glands but not to basal membrane or connective tissue. Adhesion to cultured epithelial cells was a characteristics of a subpopulation of mast cells, could persist for more than 48 h, did not require energy or the presence of divalent cations, and was not mediated by a known family of leukocyte-associated adhesion glycoproteins. Adhesion was completely abolished by pretreatment of mast cells with pronase E or proteinase K but not with trypsin (up to 10 micrograms/ml at 37 degrees C for 20 min each). In contrast, pretreatment of cultured epithelial cells with any of these proteinases had no effect on adhesion. It is concluded that dog mastocytoma mast cells adhere to dog tracheal epithelial cells and do so selectively. It is suggested that mast cell adhesion to airway epithelium may play a role in the effectiveness of mast cell-epithelial cell interactions, and thus, in certain lung diseases, airway function may be affected by intraluminal mast cells more than is currently appreciated.     

Collagen can modulate cell interactions with fibronectin

We have examined the effects of soluble collagen on the function of fibronectin in baby hamster kidney (BHK) cells. Collagen and its purified alpha1(l) chain noncompetitively inhibited cell spreading on substrates precoated with fibronectin or a 75,000-D cell-binding fragment of fibronectin. Neither preincubation of cells with collagen followed by washing nor the addition of collagen to previously spread cells had any inhibitory effect on cell spreading, which indicates a requirement for the concurrent presence of collagen during the process of spreading. Treatment of collagen or alpha1(l) chain with collagenase abolished the inhibitory effect on fibronectin-mediated cell spreading. However, direct attachment of BHK cells to fibronectin-coated or 75,000-D fragment-coated substrates was not inhibited by collagen or by the alpha1(l) chain. Moreover, the binding of [3H]fibronectin or the 3'-75,000-D fragment to cell surfaces was not inhibited by the presence of soluble collagen, whereas soluble fibronectin inhibited binding. Although the binding of [3H]fibronectin-coated beads to BHK cell surfaces was also not inhibited by collagen, the phagocytosis of such beads was inhibited by the presence of collagen. On the other hand, soluble fibronectin partially inhibited the binding of fibronectin-coated beads but did not inhibit phagocytosis of the beads that did bind. The mechanism of the inhibition of fibronectin function by collagen and the possible interactions of two different kinds of receptors on the cell surface are discussed.     

Expression and function of a putative cell surface receptor for fibronectin in hamster and human cell lines

We have previously reported the use of monoclonal antibodies to identify a 140-kD cell surface glycoprotein in mammalian cells that is specifically involved in fibronectin-mediated cell adhesion. We now report the purification of this molecule using immunoaffinity chromatography and the subsequent generation of polyclonal antibodies that selectively immunoprecipitate 140-kD putative fibronectin receptor glycoprotein (gp140) extracted from rodent or human cells; these antibodies also specifically block fibronectin-mediated cell adhesion but not adhesion mediated by other factors in serum. Expression of gp140-like molecules was detected on the surfaces of several adherent human cell lines (HDF, WISH, and EFC) but not on erythrocytes; however, gp140 was also detected on a nonadherent human lymphoid line (DAUDI). Analysis of gp140 on nonreducing SDS gels revealed two closely migrating bands. Protease digestion and peptide mapping suggests that the two bands are closely related polypeptides.     

Thiol-sensitive sites in cell adhesion. Decreased entry of SH-binding reagents into attached BHK cells.

Studies were carried out to learn more about the critical SH groups involved in cell spreading. Pretreatment of suspended baby hamster kidney (BHK) cells with 3 mM-iodoacetate or iodoacetamide for 10 min at 4 degrees C completely inhibited the ability of the cells to spread on fibronectin-coated substrata. If, however, BHK cells were permitted to attach and spread before being treated with the SH-binding reagents, and then harvested by trypsinization and assayed for spreading on fibronectin-coated substrata, there was no inhibition of cell spreading. The extent of prior attachment required before the cells became insensitive to the SH-binding reagents was tested and was found to occur early during the cell adhesion process, before any cell spreading was observed. In analytical experiments, there did not appear to be any difference in the total number of SH groups between suspended or spread cells as determined with 5,5'-dithiobis-(2-nitrobenzoic acid). The uptake of radiolabelled iodoacetate into intact spread cells, however, was found to be 3.5 times less than that found with suspended cells. On the other hand, the distribution of incorporated radioactivity into suspended and spread cells was similar. Most of the radioactivity (approximately 70%) was incorporated into small molecules (e.g. glutathione and cysteine), less (approximately 20%) was incorporated into cytoplasmic proteins, and the least incorporation (approximately 10%) was into the cell cytoskeleton. The data are interpreted to indicate there is a decreased permeability of spread cells to the SH-binding reagents.     

Fibronectin-mediated binding and phagocytosis of polystyrene latex beads by baby hamster kidney cells

The binding and phagocytosis of fibronectin (pFN)-coated latex beads by baby hamster kidney (BHK) cells was studied as a function of fibronectin concentration and bead diameter. Cells were incubated with radioactive pFN-coated beads, and total bead binding (cell surface or ingested) was measured as total radioactivity associated with the cells. Of the bound beads, those that also were phagocytosed were distinguished by their insensitivity to release from the cells by trypsin treatment. In continuous incubations, binding of pFN-coated beads to cells occurred at 4 degrees C or 37 degrees C, but phagocytosis was observed only at 37 degrees C. In addition, degradation of 3H-pFN from ingested beads occurred at 37 degrees C, as shown by the release of trichloroacetic acid-soluble radioactivity into the incubation medium. When the fibronectin density on the beads was varied, binding at 4 degrees C and ingestion at 37 degrees C were found to have the same dose-response dependencies, which indicated that pFN densities that permitted bead binding were sufficient for phagocytosis to occur. The fibronectin density for maximal binding of ingestion was approximately 250 ng pFN/cm2. When various sized beads (0.085-1.091 micron), coated with similar densities of pFN, were incubated with cells at 4 degrees C, no variation in binding as a function of bead size was observed. Under these conditions, the absolute amount of pFN ranged from less than 100 molecules on the 0.085-micron beads to greater than 15,000 molecules on the 1.091-micron beads. Based upon these results it can be concluded that the critical parameter controlling fibronectin-mediated binding of latex beads by BHK cells is the spacing of the pFN molecules on the beads. Correspondingly, it can be suggested that the spacing between pFN receptors on the cell surface that is optimal for multivalent interactions to occur is approximately 18 nM. When phagocytosis of various sized beads was compared, it was found that the largest beads were phagocytosed slightly better (two fold) than the smallest beads. This occurred both in continuous incubations of cells with beads and when the beads were prebound to the cells. Finally, the kinetic constants for the binding of 0.085 microM pFN-coated beads to the cells were analyzed. There appeared to be approximately 62,000 binding sites and the KD was 4.03 X 10(-9) M. Assuming a bivalent interaction, it was calculated that BHK cells have approximately 120,000 pFN receptors/cell and the binding affinity between pFN and its receptor is approximately 6 X 10(-5) M.     

Monensin and FCCP inhibit the intracellular transport of alphavirus membrane glycoproteins

Temperature-sensitive mutants of semliki forest virus (SFV) and sindbis virus (SIN) were used to study the intracellular transport of virus membrane glycoproteins in infected chicken embryo fibroblasts. When antisera against purified glycoproteins and (125)I- labeled protein A from staphylococcus aureus were used only small amounts of virus glycoproteins were detected at the surface of SFV ts-1 and SIN Ts-10 infected cells incubated at the restrictive temperature (39 degrees C). When the mutant-infected cells were shifted to the permissive temperature (28 degrees C), in the presence of cycloheximide, increasing amounts of virus glycoproteins appeared at the cell surface from 20 to 80 min after the shift. Both monensin (10muM) and carbonylcyanide-p- trifluoromethoxyphenylhydrazone (FCCP; 10-20 muM) inhibited the appearance of virus membrane glycoproteins at the cell surface. Vinblastine sulfate (10 μg/ml) inhibited the transport by approximately 50 percent, whereas cytochalasin B (1 μg/ml) had only a marginal effect. Intracellular distribution of virus glycoproteins in the mutant-infected cells was visualized in double-fluorescence studies using lectins as markers for endoplasmic reticulum and Golgi apparatus. At 39 degrees C, the virus membrane glycoproteins were located at the endoplasmic reticulum, whereas after shift to 28 degrees C, a bright juxtanuclear reticular fluorescence was seen in the location of the Golgi apparatus. In the presence of monensin, the virus glycoproteins could migrate to the Golgi apparatus, although transport to the cell surface did not take place. When the shift was carried out in the presence of FCCP, negligible fluorescence was seen in the Golgi apparatus and the glycoproteins apparently remained in the rough endoplasmic reticulum. A rapid inhibition in the accumulation of virus glycoproteins at the cell surface was obtained when FCCP was added during the active transport period, whereas with monensin there was a delay of approximately 10 min. These results suggest a similar intracellular pathway in the maturation of both plasma membrane and secretory glycoproteins.     

Intermediate steps in cellular iron uptake from transferrin. Detection of a cytoplasmic pool of iron, free of transferrin.

The uptake of transferrin-bound iron by receptor-mediated endocytosis has been the subject of extensive experimental investigation. However, the path followed by iron (Fe) after release from transferrin (Tf) remains obscure. Once Fe is released from Tf within the endosome, it must be transported across the endosomal membrane into the cell. The present investigation describes the presence of a cytoplasmic Tf-free Fe pool which is detectable only when cells are detached from their culture dishes at low temperature, after initial incorporation of diferric transferrin at 37 degrees C. This cellular iron pool was greatly reduced if incubation temperatures were maintained at 37 degrees C or if cells were treated with pronase. Human melanoma cells (SK-MEL-28) in culture were prelabeled by incubation with human 125I-59Fe-transferrin for 2 h, washed, and reincubated at 4 degrees C or 37 degrees C in balanced salt solution in the presence or absence of pronase. The cells were then mechanically detached from the plates and separated into "internalized" and supernatant fractions by centrifugation. Approximately 90% of cellular 59Fe and 20% of 125I-Tf remained internalized when this reincubation procedure was carried out in balanced salt solution at 37 degrees C. However, at 4 degrees C, cellular internalized iron was reduced to approximately 50% of the initial value. The release of this component of cellular 59Fe (approximately 40% of total cell 59Fe) at 4 degrees C was completely inhibited in the presence of pronase and other general proteinases at 4 degrees C and at 37 degrees C, without affecting internalized transferrin levels. Similar results were obtained in fibroblasts and hepatoma cells, indicating that this phenomenon is not unique to melanoma cells. The characterization of this Tf-free cellular Fe pool which is detectable at low temperature may yield valuable insights into the metabolic fate of iron following its transport across the membrane of the endocytotic vesicle.     

The effect of short-term temperature changes on the adhesivity of nerve cells. Participation of DNA molecules on non-specific cellular adhesion

Brain cells from 16 to 18-day-old mice embryos were dissociated by mild trypsinization and sieving. Immediately after dissociation the cells were preincubated in a PBS solution at -6 to +54 degrees C for 3 and 20 min. After this preincubation cells were rotated for 60 min at 37 degrees C in the PBS solution. Cellular adhesivity was estimated during this time period and EM pictures of organized in vitro aggregates after 24-28 h were taken. In a separate series of experiments, freshly dissociated were treated with DNAase before the rotation procedure. Preincubation in a cold or a warm medium did not alter the inhibition of cellular adhesivity significantly. Distinct inhibition of cellular adhesion was observed in cells preincubated above 53 degrees C. Adhesion was also inhibited below -5 degrees C, however, this effect was mainly dependent on the rate of freezing and thawing. Digestion of dissociated cells with DNAase (20 micrograms/ml) decreased cell adhesion. At 37 degrees C the adhesivity decreased by about 20%. Aggregates of cells preincubated at 0 degrees C for 20 min did not exhibit marked EM changes after 24-28 h in vitro. The present results have shown the rather high resistance of molecules responsible for cellular adhesion and its reversibility to temperature changes. Furthermore, non-specific cellular adhesion was shown on physically active DNA molecules.     

The cell surface glycoprotein Mac-1 (CD11b/CD18) mediates neutrophil adhesion and modulates degranulation independently of its quantitative cell surface expression

It has previously been shown that during degranulation Mac-1 (CD11b/CD18)--a glycoprotein that plays a central role in neutrophil adhesion-is up-regulated on PMN surfaces. It has been assumed that this quantitative change in adhesion Ag expression on the cell surface would in turn lead to increased cellular adhesiveness. In contrast, we found that at an incubation temperature of 16 degrees C, stimulated neutrophil adhesion to plastic tissue culture dishes in the presence of FMLP (2.5 x 10(-6) M), TNF (10 ng/ml), or PAF (1 x 10(-4) M) occurred without cellular degranulation or Mac-1 surface up-regulation as measured cytofluorometrically. As shown by functional inhibition studies employing monoclonal antibodies 60.3 (anti-CD18) and 60.1 (anti-CD11b), adhesion at 16 degrees C, where no CD11b/CD18 up-regulation was seen, is mediated by CD11b/CD18 just as it is at 37 degrees C, where degranulation and CD11b/CD18 up-regulation could be demonstrated. The physiologic importance of these findings was underscored by experiments done on endothelial monolayers, which showed that PMN association with endothelial cells is absolutely independent from the quantitative up-regulation of Mac-1 on PMN surfaces. When neutrophils were stimulated at 37 degrees C by endotoxin, an agent that does not induce aggregation (a form of intercellular adhesion), Mac-1 surface expression increased only after cells had become adherent, whereas cells held in suspension to prevent cell-substrate adhesion neither degranulated nor up-regulated their Mac-1 surface expression. Thus, not only is adherence independent of degranulation and Mac-1 cell surface up-regulation, but both degranulation and Mac-1 surface up-regulation appear to depend on the process of adhesion. Correspondingly, incubation of neutrophils with antibodies 60.1 and 60.3 inhibited not only adhesion of cells stimulated with FMLP at 37 degrees C but degranulation as well. These results indicate that Mac-1 influences degranulation as well as it controls adhesion not by its mere quantity on the cell surface, but rather by an yet undefined molecular modulation.     

BHK21 fibroblast aggregation inhibited by glycopeptides from the cell surface.

Glycopeptides were removed by trypsinization from the surface of baby hamster kidney cells (line BHK21-C13), digested by pronase and separated into 2 fractions by exclusion chromatography. The addition of small amounts of either glycopeptide fraction to shaken suspensions of lightly trypsinzied cells inhibited their rapid aggregation, but one fraction was more active than the other and in higher concentrations it was able to inhibit aggregation completely. After this fraction was purified by high-voltage electrophoresis one subfraction also inhibited aggregation. The effect of the glycopeptides increased following their pretreatment with neuraminidase, but preincubation with periodiate or galactose oxidase destroyed all activity. Galactose oxidase also inhibited cell aggregation directly. Similar glycopeptides from virus-transformed BHK21 cells, oligosaccharides and intact and desialysed human urinary glycoproteins had comparatively little or no effect on BHK21 cell aggregation. The results suggest terminal beta-galactosides and possible alpha-galactosides, and to some extent a particular substructure of cell surface heteroglycans are necessary for their inhibitory activity. The parent, plasma membrane of glycoproteins might serve as adhesive binding sites in cell cohesion, but some evidence indicates cell surface sialyl- and galactosyltransferases may not ordinarily act as their complementary binding receptors.     

The interaction of plasma fibronectin with fibroblastic cells in suspension

We have examined the interaction of soluble plasma [3H]fibronectin with fibroblastic cells in suspension. Fibronectin labeled by reductive methylation binds to baby hamster kidney cells in serum-free medium in a time-dependent manner at 4, 22, and 37 degrees C, with half-maximal binding occurring in 12-15 min at 22 degrees C. The binding is saturable and reversible. At least 90% of the cell-associated fibronectin is external to the plasma membrane, as judged by trypsin susceptibility of the bound radioactivity. Scatchard analysis of the concentration dependence of binding indicates the presence of a single class of binding sites, even at low input concentrations of fibronectin. There are approximately 5 +/- 1 X 10(5) sites/cell with an apparent dissociation constant of 8.0 +/- 0.5 X 10(-7) M; thus, the binding of soluble fibronectin to these cells is of moderate affinity. This putative fibroblast fibronectin receptor is resistant to trypsin in the presence of physiological concentrations of divalent cations but is susceptible to trypsin in the presence of 5 mM EDTA. Binding of 0.1 mg/ml [3H]fibronectin is 60-80% inhibited by 8 mg/ml unlabeled fibronectin and 95% inhibited by 1 mg/ml purified 75-kDa fibronectin cell-binding domain, but is unaffected by 1 mg/ml 44-kDa collagen-binding domain or 5 mg/ml ovalbumin. The binding parameters determined in this study further define the fibroblast cell-surface fibronectin receptor.     

Inhibition of fibronectin receptor function by antibodies against baby hamster kidney cell wheat germ agglutinin receptors

Previous studies suggest that the baby hamster kidney (BHK) cell fibronectin receptor is also a wheat germ agglutinin receptor (WGA-R). To analyze this possibility further, IgG and Fab fragments of antibodies produced against a BHK cell WGA-R preparation were tested to determine their effects on cell adhesion mediated by fibronectin, wheat germ agglutinin, concanavalin A, and polycationic ferritin. The WGA-R preparation was isolated by octylglucoside extraction of BHK cells followed by chromatography of the extract on WGA-agarose. The antibodies against the WGA-R preparation reacted primarily with polypeptides of molecular weights 48, 61, 83, 105, 120, 165, 210, and 230 kilodaltons (kdaltons). It was concluded that the antibodies interfered with BHK cell fibronectin receptors on the basis of the ability of anti-WGA-R IgG or Fab fragments to (a) inhibit cell spreading on fibronectin-coated substrata; (b) cause rounding and detachment of cells previously spread on fibronectin-coated substrata; and (c) inhibit binding of fibronectin-coated latex beads to the cells. Antibody activity was blocked by treatment of anti-WGA-R with the WGA-R preparation or by absorption of anti-WGA-R with intact BHK cells. The antibodies also appeared to prevent coupling of ligand-receptor complexes (involving concanavalin A or polycationic ferritin) with the cytoskeleton. Finally, cell rounding and detachment caused by the antibodies were found to require metabolic energy since it did not occur in the presence of azide or at 4 degrees C.     

Temperature-dependent internalization of virus glycoproteins in cells infected with a mutant of Semliki Forest virus.

When the ts-1 mutant of Semliki Forest virus (SFV) was grown in chick embryo or BHK 21 cells at the restrictive temperature (39 degrees C), its membrane glycoproteins were arrested in the endoplasmic reticulum, but started to migrate to the cell surface once the cultures were shifted to the permissive temperature (28 degrees C). If the temperature of infected cells was raised back to 39 degrees C, ts-1 glycoproteins disappeared from the cell surface as evidenced by loss of surface immunofluorescence and by radioimmunoassay based on the binding of 125I-labeled protein A. This phenomenon was specific for ts-1 at 39 degrees C as it was observed neither in cells infected with wild-type SFV at 39 degrees C nor with ts-1 at 28 degrees C. The disappearance of the ts-1 glycoproteins was due to internalization. The internalized proteins were digested, as shown by specific decrease of virus glycoproteins labelled with [35S]methionine at 39 degrees C before shift to 28 degrees C, and by concomitant release of acid soluble 35S-activity into the culture medium. Ts-1 infected cells were treated before shift back to 39 degrees C with Fab' fragments, prepared from IgG against the viral membrane glycoproteins. After shift back to 39 degrees C, the Fab' fragments disappeared from the cell surface. In the presence of chloroquine, they could be visualized in vesicular structures, using an anti-IgG-fluorescein isothiocyanate conjugate. The internalization of ts-1 glycoproteins was not inhibited by carbonylcyanide p-trifluoromethoxy phenylhydrazone, chloroquine, cytochalasin B, vinblastine, colcemid, or monensin.     

Adipocyte insulin receptor. Generation of a cryptic domain of the alpha-subunit during internalization of hormone-receptor complexes.

The dynamics of the internalization of photoaffinity-labelled insulin-receptor complexes was investigated in isolated rat adipocytes by using tryptic proteolysis to probe both the orientation and cellular location of the labelled complexes. In cells that were labelled at 16 degrees C and not prewarmed, 150 micrograms of trypsin/ml rapidly degraded the labelled 125 kDa insulin-receptor subunit into a major proteolytic fragment of 70 kDa and minor amounts of 90- and 50-kDa fragments. With milder trypsin treatment conditions (100 micrograms of trypsin/ml, 15 s at 37 degrees C), the 90 kDa peptide (different from the 90 kDa beta-subunit of the insulin receptor) appeared as a major intermediate proteolytic product, but this species was rapidly and completely converted into the 70- and 50-kDa fragments with continued exposure to trypsin, such that it did not accumulate to appreciable amounts in cells that were not prewarmed before trypsin exposure. By contrast, trypsin treatment of cells prewarmed to 37 degrees C for various times showed that: first, a proportion of the labelled 125 kDa receptors was internalized (became trypsin-insensitive); secondly, the 90 kDa tryptic peptide was formed in large amounts, with proportionate decreases occurring in the amounts of the 70- and 50-kDa tryptic peptides. The increased accumulation of the 90 kDa tryptic peptide from cells preincubated at 37 degrees C, but not at 16 degrees C, indicated that trypsin cleavage sites within the 90 kDa segment of the insulin-receptor alpha-subunit that were exposed at 16 degrees C were made inaccessible by incubation at 37 degrees C, a finding that is consistent with generation of a cryptic domain of the receptor subunit. The tryptic generation of the 90 kDa peptide at 37 degrees C was rapid, becoming half-maximal in 4.4 +/- 0.6 min and maximal in 15-20 min, preceded the intracellular accumulation of labelled receptors (half-maximal in 12.6 +/- 0.7 min and maximal in 30-40 min), was highly correlated with receptor internalization, and was not observed in cultured IM-9 lymphocytes, a cell line in which photolabelled insulin receptors are primarily lost by shedding into the incubation media. These results show that, in adipocytes incubated at 37 degrees C, rapid masking of a previously (at 16 degrees C) accessible domain of the insulin-receptor alpha-subunit occurs and that this dynamic process happens at an early stage in the internalization of insulin-receptor complexes.     

Calcium ions protect cell-substratum adhesion receptors against proteolysis : Evidence from immunoabsorption and electroblotting studies

An antibody preparation against BHK cell wheat germ agglutinin receptors (anti-WGA-R), which inhibits fibronectin-mediated cell adhesion, was characterized by immunoabsorption and electroblotting experiments. The results indicated that a 48 kD cell surface WGA-R receptor is protected from proteolysis by Ca²⁺ ions and is important in fibronectin-mediated cell adhesion.     

Formation of tight junctions in epithelial cells. I. Induction by proteases in a human colon carcinoma cell line

The experimental modulation of tight junctions (TJ) was studied in the human adenocarcinoma cell line HT 29 by freeze-fracture electron microscopy. The cell line has virtually no TJ when grown in culture. TJ could be induced by mild treatment with a variety of endopeptidases (trypsin, chymotrypsin, collagenase, elastase, plasmin, thrombin, papain, and pronase). Pronase induced the formation of TJ at low (but not at high) concentrations. All exopeptidases studied were unable to induce the formation of TJ. At 0 degree C the trypsin-induced formation of TJ was greatly slowed down although not entirely inhibited. However, when cells were briefly treated with trypsin at 0 degree C and subsequently transferred to 37 degrees C in the presence of protease inhibitors, TJ were rapidly assembled. Thus an induction phase at low temperature and an assembly phase at high temperature could be experimentally separated. When cells were briefly trypsinized at 0 degrees and subsequently kept at 0 degree C without trypsin for several hours, TJ still formed abundantly upon incubation at 37 degrees C. It appears therefore that the effect produced by the protease is retained for long periods in the cold.     

Study of the interaction between germ cells and Sertoli cells in vitro

The nature of membrane components involved in the binding between Sertoli cells and pachytene spermatocytes in culture and the metabolic requirements for the binding to occur have been studied. Mild proteolytic digestion of germ cells by trypsin completely inhibited adhesion of germ cells to somatic monolayer. Protein synthesis and glycosylation were required to restore the adhesive properties of trypsin-treated germ cells, showing that surface molecules involved in the binding are glycoproteins. Trypsinization of germ cells after labelling causes a great reduction of several bands which become detectable again after 12 h of recovery from trypsin digestion. Among these, two bands with apparent molecular weight (MW) of 78 000 and 51 000 could be candidate components in cell adhesion.