Isolation of a receptor for acidic and basic fibroblast growth factor from embryonic chick

A receptor for acidic and basic fibroblast growth factors (aFGF and bFGF, respectively) was isolated from 7-day embryonic chick. Chromatography of solubilized membrane proteins on wheat germ agglutininagarose and aFGF-Sepharose yielded three major polypeptides migrating at 150, 70, and 45 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These polypeptides were eluted from aFGF-Sepharose with either 1.0 M NaCl or 100 micrograms/ml heparin, but were not retained on underivatized Sepharose. Cross-linking of 125I-aFGF or 125I-bFGF to either crude membrane preparations or to purified fractions yielded a 165-kDa complex, suggesting the existence of a 150-kDa FGF receptor after subtraction of approximately 15 kDa for 125I-FGF. Addition of excess aFGF or bFGF competed for binding of either 125I-aFGF or 125I-bFGF to FGF receptor preparations. Purified FGF receptor fractions were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to Immobilon membranes, and incubated with 125I-aFGF or 125I-bFGF in order to identify FGF-binding polypeptides. Bound 125I-aFGF and 125I-bFGF were displaced by aFGF and bFGF, but not epidermal growth factor, consistent with the identification of the 150-kDa polypeptide as a receptor for acidic and basic FGF. Treatment of purified FGF receptor fractions with N-glycanase demonstrated that the 150-kDa polypeptide contained approximately 10 kDa of N-linked oligosaccharide. The apparent molecular mass of the 150-kDa polypeptide was unaffected by treatment with heparitinase, indicating that the 150-kDa polypeptide is not a heparan sulfate proteoglycan. Together, these data suggest that the 150-kDa polypeptide is a FGF receptor that may mediate the biological activities of aFGF and bFGF.     

Cell type and tissue distribution of the fibroblast growth factor receptor

A receptor for fibroblast growth factor (aFGF, bFGF) was partially characterized in intact cell cultures, cell plasma membranes, and tissue plasma membrane preparations. Analysis of 24 different cell types from four species identified a 165-kDa FGF receptor present on the cell surface of most mesodermal and neuroectodermal cells. Chemical crosslinking of 125I-aFGF to its cell surface receptor was specifically blocked by a 100-fold molar excess of either aFGF or bFGF. In contrast to the similar molecular weight of FGF receptors, different cell types exhibited significant variations in binding of 125I-aFGF to intact cultures with low values of 8 pM and 700, to high values of 60 pM and 30,000, for the Kd and receptor number per cell, respectively. A binding assay was developed for quantitation of 125I-aFGF binding to cell- and tissue-derived membrane preparations. Membranes prepared from baby hamster kidney cells exhibited a Kd of 55 pM, while a similar Kd of 67 pM was determined for intact baby hamster kidney cells. Although ten different adult bovine tissue membrane preparations and human term placental membranes exhibited no specific binding of 125I-aFGF, FGF receptor was detected in embryonic murine tissues (17 days gestation). These results support the existence, in a variety of cells, of either a common FGF receptor that binds both aFGF and bFGF or closely related FGF receptors that cannot be distinguished by molecular weight. The differential binding of FGF to its receptor in embryonic vs. adult tissues suggests a potentially broad role for FGF in embryonic development and a more restrictive role in the adult.     

Acidic and basic fibroblast growth factor bind with differing affinity to the same heparan sulfate proteoglycan on BALB/c 3T3 cells: Implications for potentiation of growth factor action by heparin

Heparan sulfate proteoglycans on the cell surface act as low affinity binding sites for acidic and basic fibroblast growth factor (FGF) [Moscatelli (1887): J Cell Physiol 131:123–130] and play an important role in the interaction of FGF with the FGF receptor (FGFR). In this study, several aspects of the interaction of FGFs with cell surface heparan sulfate proteoglycans were examined. Reciprocal cross blocking studies demonstrated that acidic FGF (aFGF) and basic FGF (bFGF) bind to identical or closely associated heparan sulfate motifs on BALB/c 3T3 cell surface heparan sulfate proteoglycans. However, the binding affinity of the two growth factros for these heparan sulfate proteoglycans differs considerably, competition binding data indicating that aFGF has a 4.7-fold lower affinity than bFGF for 3T3 heparan sulfate proteoglycan. Subsequent studies of dissociation kinetics demonstrated that bFGF dissociates form the FGFR at least 10-fold slower than aFGF, whereas, following removal of cell surface heparan sulfate proteoplycan. Subsequent studies of dissociation kinetic demonstrated that bFGF dissociates from the FGFR at least 10-fold slwer than aFGF, whereas, following removal of cell surface heparan sulfate proteoglycans by heparinase treatment, the dissociation rate of both FGFs is similar and rapid. These results support the concept that cell surface heparan sulfate proteoglycans stabilize the interactio fo FGF with FGFR, possibly by the formatin of a ternary complex. © Wiley-Liss, Inc.     

Acidic fibroblast growth factor autocrine system as a mediator of calcium-regulated parathyroid cell growth.

Both parathyroid hormone secretion and cell growth are negatively regulated by extracellular calcium in parathyroid cells. The mechanism of growth regulation by calcium has been unknown. Previously, we reported that clonal parathyroid cells (PT-r cells) bear two high affinity receptors for acidic fibroblast growth factor (aFGF) and that at least a subpopulation of the receptors with a higher molecular mass carries heparan sulfate (HS) glycosaminoglycan chains which give the receptor higher affinity (Sakaguchi, K., Yanagishita, M., Takeuchi, Y., and Aurbach, G. D. (1991) J. Biol. Chem. 266, 7270-7278). Here, I have found that the parathyroid cells expressed aFGF and that aFGF receptors with lower affinity apparently translocated in response to changing extracellular calcium concentrations. Expression of both aFGF mRNA and peptide was suppressed by calcium. Cells had more ligand-accessible receptors on the cell surface at lower calcium concentrations. This apparent translocation was temperature-dependent but independent of de novo protein synthesis. Heparin or HS glycosaminoglycans are a prerequisite for the FGF receptor encoded by flg gene to bind basic FGF (Yayon, A., Klagsbrun, M., Esko, J. D., Leder, P., and Ornitz, D. M. (1991) Cell 64, 841-848). In PT-r cells, major cellular HS proteoglycans redistribute between intracellular and extracellular compartments with more HS proteoglycans expressed on the cell surface at lower calcium concentrations (Takeuchi, Y., Sakaguchi, K., Yanagishita, M., Aurbach, G. D., and Hascall, V. C. (1990) J. Biol. Chem. 265, 13661-13668). However, this redistribution of HS proteoglycans cannot explain the difference in bindability of radiolabeled aFGF to its receptors in different calcium concentrations, since addition of heparin did not change the binding of radiolabeled aFGF to the receptors either at high or low calcium conditions. In concordance with the apparent translocation of aFGF receptors, thymidine incorporation was stimulated by decreasing extracellular calcium concentrations with further stimulation by added aFGF. Anti-aFGF antibody inhibited thymidine incorporation by more than 32% in the cells exposed to 0.05 mM Ca2+ shortly before adding [3H]thymidine, whereas the incorporation was not significantly affected by the antibody at 0.7 mM Ca2+. Cell growth was also stimulated by low calcium. Anti-aFGF antibody inhibited cell growth significantly only at low calcium concentrations. From these observations, an aFGF autocrine system including the apparent translocation of aFGF receptors may explain, if not entirely, the mechanism by which calcium regulates parathyroid cell growth.     

Affinity Purification of Proteoglycans that Bind to the Amyloid Protein Precursor of Alzheimer's Disease

Abstract: The binding of the amyloid protein precursor (APP) to heparan sulfate proteoglycans has been shown to stimulate the neurite-promoting activity of APP. In this study, proteoglycans that bind with high affinity to APP were characterized. Conditioned medium from cultures of postnatal day 3 mouse brain cells was applied to an affinity column containing a peptide homologous to a heparin-binding domain of APP. A fraction 17-fold enriched in proteoglycans was recovered by elution with a salt gradient. APP bound saturably and with high affinity to the affinity-purified proteoglycan fraction. Scatchard analysis of the binding showed that APP bound to high- and low-affinity sites with equilibrium dissociation constants of 1.4 × 10⁻¹¹ and 6.5 × 10⁻¹⁰ M , respectively. APP, in conjunction with the affinity-purified proteoglycan fraction, promoted neurite outgrowth. The affinity-purified proteoglycan fraction contained a heparan sulfate proteoglycan and a chondroitin sulfate proteoglycan. Digestion of the affinity-purified fraction with heparitinase I revealed a core protein of 63–69-kDa molecular mass, whereas digestion with chondroitinase ABC revealed a core protein of 100–110 kDa. The results suggest that expression of specific APP-binding proteoglycans may be an important step in the regulation of the neurite outgrowth-promoting activity of APP.     

Basic fibroblast growth factor high and low affinity binding sites in developing mouse brain, hippocampus and cerebellum.

Fibroblast growth factors (FGFs), first extracted from brain and retina, are potent neurotrophic factors. They stimulate neuroblast proliferation and neuron differentiation and survival. In order to study the spatial and temporal distribution of the target cells in the mouse brain we studied by autoradiography and quantified by image analysis 125I-bFGF binding sites as a function of development. We have revealed the presence of two types of specific bFGF receptors. One is heparitinase sensitive and is co-localized with heparan sulfate proteoglycans of the basement membranes (meninges, choroid plexus and blood vessels). It is not developmentally regulated and corresponds to the low affinity receptors. It may be a storage form. The second type is heparitinase resistant and is modified during development, matching, in the adult, layering of the hippocampus and cerebellum. At 13 days of embryonic development there is a preferential distribution of silver grains on the ecto- and neuroectodermal tissues. In the adult, the labeling is localized on the neural process layers. It likely corresponds to the specific binding to cell high affinity receptors. Binding patterns according to the developmental stages of the brain can be correlated with mitotic, migration and differentiation phases of the neuronal cells.     

Fibroblast growth factor phosphorylation and receptors in rod outer segments.

Acidic and basic fibroblast growth factors (aFGF and bFGF) have been isolated and purified from rod outer segments (ROS). aFGF is tightly bound to ROS membranes and can be specifically released by ATP. We show that this mechanism is dependent on the phosphorylation of aFGF itself. Phorbol 12-myristate 13-acetate (PMA) enhances this phenomenon independently of rhodopsin phosphorylation. This demonstrates that aFGF release from ROS membranes is dependent on its phosphorylation by endogenous kinase C. In addition specific binding sites for exogenous FGFs have been identified on ROS and disc membranes. A single high affinity site with a Kd of 40 pM was present in intact ROS while an additional low affinity site with a Kd of 300-600 pM was present in leaky ROS or in disc membranes. Light or ATP modified neither these Kd nor the apparent number of sites. The presence of specific receptors for FGFs and the kinase C dependent release of endogenous membrane bound aFGF suggest an autocrine mechanism which may be involved in photoreceptor cell biology.     

Acidic fibroblast growth factor is present in regenerating limb blastemas of axolotls and binds specifically to blastema tissues

The growth of regenerating limbs of amphibians depends upon proliferation of the blastema cells that accumulate beneath the epidermal cap. The epidermal cap is known to be mitogenic for the blastema cells. We have extracted a mitogenic activity from both the mesenchymal and epidermal (epidermal cap) components of cone stage blastemas which is retained on heparin-Sepharose and elutes with 1.15 M NaCl. This fraction stimulates neurite outgrowth of PC12 cells and [3H]thymidine incorporation into CCL 39 cells and is potentiated by heparin. The 2 M fraction was inactive. The heparin-Sepharose-purified growth factor cross-reacts with bovine acidic FGF polyclonal antibodies and shows a Mr of 16,000 on Western blots. Blastema membranes contain specific high affinity binding sites (Kd = 25 pM; capacity = 30 fmole/mg protein) and low affinity binding sites (Kd = 18 nM; capacity = 30 pmole/mg protein) for aFGF as revealed by Scatchard analysis. 125I-aFGF which is bound specifically by both the epidermal cap and mesenchyme of blastema frozen sections is displaced by an excess of unlabeled factor and inhibited by heparin. Heparinase treatment and 2 M NaCl washing which decreased the binding was fourfold more efficient for epidermal cap than for mesenchyme suggesting the presence of high affinity receptors in the latter tissue. The presence of aFGF (or a closely related molecule) in blastemas is consistent with our earlier results that showed stimulation of proliferation of cultured blastema cells by acidic or basic FGF or heparin alone. These results suggest the possibility that aFGF is stored in the epidermal cap during limb regeneration and that it stimulates the proliferation of the underlaying mesenchyme.     

Ovarian carcinoma cells synthesize both chondroitin sulfate and heparan sulfate cell surface proteoglycans that mediate cell adhesion to interstitial matrix

Metastatic ovarian carcinoma metastasizes by intra-peritoneal, non-hematogenous dissemination. The adhesion of the ovarian carcinoma cells to extracellular matrix components, such as types I and III collagen and cellular fibronectin, is essential for intra-peritoneal dissemination. The purpose of this study was to determine whether cell surface proteoglycans (a class of matrix receptors) are produced by ovarian carcinoma cells, and whether these proteoglycans have a role in the adhesion of ovarian carcinoma cells to types I and III collagen and fibronectin. Proteoglycans were metabolically labeled for biochemical studies. Both phosphatidylinositol-anchored and integral membrane-type cell surface proteoglycans were found to be present on the SK-OV-3 and NIH:OVCAR-3 cell lines. Three proteoglycan populations of differing hydrodynamic size were detected in both SK-OV-3 and NIH:OVCAR-3 cells. Digestions with heparitinase and chondroitinase ABC showed that cell surface proteoglycans of SK-OV-3 cells had higher proportion of chondroitin sulfate proteoglycans (75:25 of chondroitin sulfate:heparan sulfate ratio), while NIH:OVCAR-3 cells had higher proportion of heparan sulfate proteoglycans (10:90 of chondroitin sulfate:heparan sulfate ratio). RT-PCR indicated the synthesis of a unique assortment of syndecans, glypicans, and CD44 by the two cell lines. In adhesion assays performed on matrix-coated titer plates both cell lines adhered to types I and III collagen and cellular fibronectin, and cell adhesion was inhibited by preincubation of the matrix with heparin, heparan sulfate, chondroitin sulfate, dermatan sulfate, or chondroitin glycosaminoglycans. Treatment of the cells with heparitinase, chondroitinase ABC, or methylumbelliferyl xyloside also interfered with adhesion confirming the role of both heparan sulfate and chondroitin sulfate cell surface proteoglycans as matrix receptors on ovarian carcinoma cells.     

Characterization of human interleukin-3 receptors on a multi-factor-dependent cell line

Recombinant human interleukin-3 (hIL-3) was radioiodinated by Bolton-Hunter method with maintenance of biological activity. Using 125I-hIL-3, hIL-3 receptors were characterized on a multi-factor-dependent cell line TF-1. Equilibrium binding studies revealed the existence of a single class of binding sites (667 +/- 306 sites/cell) with a Kd of 173 +/- 25 pM. Affinity labeling of TF-1 cells with 125I-IL-3 yielded two bands of 150 kDa and 85 kDa, implying molecular weights of 135 kDa and 70 kDa for the hIL-3 receptors.     

Specific binding of basic fibroblast growth factor to basement membrane-like structures and to purified heparan sulfate proteoglycan of the EHS tumor

The binding of iodinated basic fibroblast growth factor (bFGF) to low-density heparan sulfate proteoglycan purified from the Engelbreth Holm Swarm (EHS) sarcoma was investigated using different techniques. The tumor clearly contained bFGF, the level being comparable to that found in other tissues such as human or bovine brain. 125I bFGF strongly bound to the basement membrane-like matrix of EHS frozen sections as revealed by autoradiography. Iodinated bFGF bound to purified heparan sulfate proteoglycan but not to laminin or collagen type IV, three components isolated from the same tumor. In contrast, acidic fibroblast growth factor (aFGF) displayed negligible binding to heparan sulfate proteoglycan. Binding of bFGF to frozen sections and to purified proteoglycan could be strongly inhibited by heparin and was displaced by an excess of unlabeled factor and completely suppressed after heparitinase and heparinase treatments. Binding was a function of the salt concentration and was abolished at 0.6 M NaCl. Scatchard analysis indicated the affinity site had a Kd of about 30 nM, a value 10-15 higher than that recently reported by Moscatelli (J. Cell. Physiol., 131:123-130, 1987) in the case of the low-affinity binding sites present on the surface of baby hamster kidney (BHK) cells.     

Proteoheparan sulfate from human skin fibroblasts. Evidence for self-interaction via the heparan sulfate side chains

We have studied the affinity between fibroblast proteoheparan sulfate (medium- and cell surface-derived species) and heparan sulfate-agaroses by affinity chromatography. The evidence for an interaction between the heparan sulfate side chains of the proteoglycans and the immobilized heparan sulfate are as follows: (a) the individual side chains released from the proteoglycan by papain bind to the affinity matrix, (b) the bound proteoglycans are desorbed by a solution of cognate heparan sulfate chains, and (c) the core protein obtained by heparan sulfate-lyase digestion of the proteoglycan does not bind to the affinity matrix. The proteoglycans interact only with one subtype of heparan sulfate. The binding of free heparan sulfate chains to the affinity matrix is completely abolished by heparan sulfate oligosaccharides provided they are composed of both iduronate- and glucuronate-containing disaccharide sequences.     

The core protein of the matrix-associated heparan sulfate proteoglycan binds to fibronectin

The extracellular matrix of cultured human lung fibroblasts contains one major heparan sulfate proteoglycan. This proteoglycan contains a 400-kDa core protein and is structurally and immunochemically identical or closely related to the heparan sulfate proteoglycans that occur in basement membranes. Because heparitinase does not release the core protein from the matrix of cultured cells, we investigated the binding interactions of this heparan sulfate proteoglycan with other components of the fibroblast extracellular matrix. Both the intact proteoglycan and the heparitinase-resistant core protein were found to bind to fibronectin. The binding of 125I-labeled core protein to immobilized fibronectin was inhibited by soluble fibronectin and by soluble cold core protein but not by albumin or gelatin. A Scatchard plot indicates a Kd of about 2 x 10(-9) M. Binding of the core protein was also inhibited by high concentrations of heparin, heparan sulfate, or chrondroitin sulfate and was sensitive to high salt concentrations. Thermolysin fragmentation of the 125I-labeled proteoglycan yielded glycosamino-glycan-free core protein fragments of approximately 110 and 62 kDa which bound to both fibronectin and heparin columns. The core protein-binding capacity of fibronectin was very sensitive to proteolysis. Analysis of thermolytic and alpha-chymotryptic fragments of fibronectin showed binding of the intact proteoglycan and of its isolated core protein to a protease-sensitive fragment of 56 kDa which carried the gelatin-binding domain of fibronectin and to a protease-sensitive heparin-binding fragment of 140 kDa. Based on the NH2-terminal amino acid sequence analyses of the 56- and 140-kDa fragments, the core protein-binding domain in fibronectin was tentatively mapped in the area of overlap of the two fragments, carboxyl-terminally from the gelatin-binding domain, possibly in the second type III repeat of fibronectin. These data document a specific and high affinity interaction between fibronectin and the core protein of the matrix heparan sulfate proteoglycan which may anchor the proteoglycan in the matrix.     

Repression of myogenic differentiation by aFGF, bFGF, and K-FGF is dependent on cellular heparan sulfate

We have proposed a model in which fibroblast growth factor (FGF) signalling requires the interaction of FGF with at least two FGF receptors, a heparan sulfate proteoglycan (HSPG) and a tyrosine kinase. Since FGF may be a key mediator of skeletal muscle differentiation, we examined the synthesis of glycosaminoglycans in MM14 skeletal muscle myoblasts and their participation in FGF signalling. Proliferating and differentiated MM14 cells exhibit similar levels of HSPG, while differentiated cells exhibit reduced levels of chondroitin sulfate proteoglycans and heparan sulfate chains. HSPGs, including syndecan, present in proliferating cells bind bFGF, while the majority of chondroitin sulfate and heparan sulfate chains do not. Treatment of skeletal muscle cells with chlorate, a reversible inhibitor of glycosaminoglycan sulfation, was used to examine the requirement of sulfated proteoglycans for FGF signalling. Chlorate treatment reduced glycosaminoglycan sulfation by 90% and binding of FGF to high affinity sites by 80%. Chlorate treatment of MM14 myoblasts abrogated the biological activity of acidic, basic, and Kaposi's sarcoma FGFs resulting in terminal differentiation. Chlorate inhibition of FGF signalling was reversed by the simultaneous addition of sodium sulfate or heparin. Further support for a direct role of heparan sulfate proteoglycans in fibroblast growth factor signal transduction was demonstrated by the ability of heparitinase to inhibit basic FGF binding and biological activity. These results suggest that activation of FGF receptors by acidic, basic or Kaposi's sarcoma FGF requires simultaneous binding to a HSPG and the tyrosine kinase receptor. Skeletal muscle differentiation in vivo may be dependent on FGFs, FGF tyrosine kinase receptors, and HSPGs. The regulation of these molecules may then be expected to have important implications for skeletal muscle development and regeneration.     

Follistatin, an activin-binding protein, associates with heparan sulfate chains of proteoglycans on follicular granulosa cells

Follistatin, an activin-binding protein secreted by cultured rat granulosa cells, was shown to associate with the cell surface by affinity labeling with 125I-activin. Addition of follistatin to the cultured cells demonstrated a typical ligand-binding saturation curve, suggesting that granulosa cells have a specific binding site for follistatin. This binding was markedly inhibited by heparin and heparan sulfate, but not by chondroitin sulfates A and C, keratan sulfate, and dermatan sulfate. When granulosa cells were treated with glycosaminoglycan-degrading enzymes before or after addition of follistatin to the cultures, heparinase and heparitinase treatments resulted in significant suppression of the binding, whereas treatment with chondroitinase ABC had no effect. A competition study of the binding using heparin derivatives demonstrated that follistatin seemed to recognize O-sulfate groups in the heparin molecule. Heparitinase-treated granulosa cells exhibited almost full responsiveness to activin, indicating that the enzyme treatment had no effect on activin and receptor interaction. These results suggest that follistatin/activin-binding protein binds to heparan sulfate side chains of proteoglycans on the granulosa cell surface to regulate the various actions of activin.     

Isolation and identification of the major heparan sulfate proteoglycans in the developing bovine rib growth plate

Heparan sulfate proteoglycans are thought to mediate the action of growth factors. The heparan sulfate-containing proteoglycans in extracts of the bovine fetal rib growth plate were detected using the monoclonal antibody 3G10, which recognizes a neoepitope generated by heparitinase digestion (David, G., Bai, X. M., Van der Schueren, B., Cassiman, J. J., and Van den Berghe, H. (1992) J. Cell Biol. 119, 961-975). The heparan sulfate proteoglycans that react with this antibody were identified using antisera to known proteoglycans; purified using CsCl density gradient centrifugation, molecular sieve, and ion exchange chromatography; and then characterized. The major heparan sulfate proteoglycans in the growth plate had core proteins of 200 kDa and larger and were identified as perlecan and aggrecan. These two heparan sulfate proteoglycans could be effectively separated from each other by CsCl density gradient centrifugation alone. Perlecan contained 25% heparan sulfate and 75% chondroitin sulfate. The heparan sulfate chains on growth plate perlecan were considerably smaller than the chondroitin sulfate chains, and the heparan sulfate disaccharide content was different than that found for heparan sulfate from either kidney, tumor tissue, or growth plate aggrecan. Aggrecan contained only 0.1% heparan sulfate, which was localized to the CS-1 domain of aggrecan. These results indicate that perlecan and aggrecan would be the principal candidate proteoglycans involved in the action of heparan sulfate-binding proteins in the developing growth plate.     

The transforming growth factor-beta receptor type III is a membrane proteoglycan. Domain structure of the receptor

The transforming growth factor-beta (TGF-beta) receptor type III is a low abundance cell surface component that binds TGF-beta 1 and TGF-beta 2 with high affinity and specificity, and is present in many mammalian and avian cell types. Type III TGF-beta receptors affinity-labeled with 125I-TGF-beta migrate in sodium dodecyl sulfate-polyacrylamide electrophoresis gels as diffuse species of 250-350 kDa. Here we show that type III receptors deglycosylated by the action of trifluoromethanesulfonic acid yield affinity-labeled receptor cores of 110-130 kDa. This marked decrease in molecular weight is also achieved by combined treatment of type III receptors with heparitinase and chondroitinase ABC. Digestion of receptor-linked glycosaminoglycans by treatment of intact cell monolayers with heparitinase and chondroitinase does not prevent TGF-beta binding to the type III receptor core polypeptide and does not release the receptor polypeptide from the membrane. The type III TGF-beta receptor binds tightly to DEAE-Sephacel and coelutes with cellular proteoglycans at a characteristically high salt concentration. Thus, the type III TGF-beta receptor has the properties of a membrane proteoglycan that carries heparan and chondroitin sulfate glycosaminoglycan chains. The binding site for TGF-beta appears to reside in the 100-120-kDa core polypeptide of this receptor. The type III receptor is highly sensitive to cleavage by trypsin. Trypsin action releases the glycosaminoglycan-containing domain of the receptor leaving a 60-kDa membrane-associated domain that contains the cross-linked ligand. A model for the domain structure of the TGF-beta receptor type III is proposed based on these results.     

High affinity interactions between the Alzheimer's beta-amyloid precursor proteins and the basement membrane form of heparan sulfate proteoglycan

High affinity interactions were studied between the basement membrane form of heparan sulfate proteoglycan (HSPG) and the 695-, 751-, and 770-amino acid Alzheimer amyloid precursor (AAP) proteins. Based on quantitative analyses of binding data, we identified single binding sites for the HSPG on AAP-695 (Kd = 9 x 10(-10) M), AAP-751 (Kd = 10 x 10(-9) M), and AAP-770 (Kd = 9 x 10(-9) M). It is postulated that the "Kunitz" protease inhibitor domain which is present in AAP-751 and -770 reduces the affinity of AAPs for the HSPG through steric hindrance and/or conformational alteration. HSPG binding was inhibited by heparin and dextran sulfate, but not by dermatan or chondroitin sulfate. HSPG protein core, obtained by heparitinase digestion, also bound to the beta-amyloid precursor proteins with high affinity, indicating that the high affinity binding site is constituted by the polypeptide chain rather than the carbohydrate moiety. The effects of various cations on these interactions were also studied. Our results suggest that specific interactions between the AAP proteins and the extracellular matrix may be involved in the nucleation stages of Alzheimer's disease type amyloidogenesis.     

Isolation and characterization of heparan sulfate proteoglycans produced by cloned rat microvascular endothelial cells.

We have isolated heparan sulfate proteoglycans (HSPGs) from cloned rat microvascular endothelial cells using a combination of ion-exchange chromatography, affinity fractionation with antithrombin III (AT III), and gel filtration in denaturing solvents. The anticoagulantly active heparan sulfate proteoglycans (HSPGact) which bind tightly to AT III bear mainly anticoagulantly active heparan sulfate (HSact) whereas the anticoagulantly inactive heparan sulfate proteoglycans (HSPGinact) possess mainly anticoagulantly inactive heparan sulfate (HSinact). HSact and HSinact were also isolated by a combination of ion-exchange chromatography, treatment with protease and chondroitin ABC lyase, and affinity fractionation with AT III. HSact and HSinact have molecular sizes of about 25-30 kDa with the same overall composition of monosaccharides except that HSact exhibits about nine glucuronsyl 3-O-sulfated glucosamines/chain whereas HSinact possesses about three glucuronsyl 3-O-sulfated glucosamines/chain. Direct isolation of the AT III-binding site of HSact by exposing carbohydrate chains to Flavobacterium heparitinase in the presence of protease inhibitor revealed only a single interaction site which contained two to three glucuronsyl 3-O-sulfated glucosamine residues. The core proteins of HSPGact and HSPGinact were isolated by treatment with Flavobacterium heparitinase and purification by ion-exchange chromatography. The molecular sizes of the core proteins were established by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and their primary structures were examined by cleavage with trypsin or endopeptidase Glu-C as well as separation of peptides by reverse-phase high performance liquid chromatography. The results showed that both sets of core proteins exhibited three major components with molecular sizes of 50, 30, and 25 kDa, respectively. The 25-kDa species appears to be a proteolytic degradation product of the 30-kDa species. The peptide mapping revealed that HSPGact and HSPGinact possess extremely similar core proteins.     

Vasoactive intestinal peptide receptors on AR42J rat pancreatic acinar cells.

VIP receptors on AR42J rat pancreatic cells were analyzed by competition binding, affinity labeling and by N-glycanase digestion analyses. These studies revealed the presence of specific, high affinity (Kd approximately 1 nM) VIP receptors with a mass of 67 kDa or 59 kDa under reducing or non-reducing conditions, respectively. N-glycanase digestion of affinity labeled membranes generated a core receptor protein of approximately 44 kDa and evidence for at least two N-linked glycans on the mature receptor. The receptor lacked O-linked oligosaccharides but contained terminal sialic acid residues on its N-linked glycan(s) based on digestions with O-glycanase and neuraminidase. The similarity of the AR42J VIP receptor to the recently cloned cDNA for human VIP receptors makes this cell line an attractive model for further analysis of VIP receptor signal transduction events.