Glycosylation of the basic fibroblast growth factor receptor. The contribution of carbohydrate to receptor function

We have examined the glycosylation of the basic fibroblast growth factor (bFGF) receptor to determine whether carbohydrates contribute to receptor structure and function. Using a combination of cross-linking and radioreceptor assays, we demonstrated that the two bFGF receptors in baby hamster kidney cells have protein cores of 100 and 125 kDa. They are glycosylated to high mannose forms of 115 and 140 kDa and further processed to their mature forms of 130 and 150 kDa. Because peptide:N-glycosidase F, but not endo-alpha-N-acetylgalactosamidase can reduce the size of the bFGF receptors, the carbohydrate residues of the receptor appear all N-linked. The inability of deglycosylated receptors to bind 125I-bFGF supports the notion that the carbohydrate residues are required for receptor function. Furthermore, the capacity of the wheat germ agglutinin lectin to inhibit 125I-bFGF binding and the biological activity of bFGF suggests that N-acetylglucosamine residues are functionally significant components of the receptor.     

Natriuretic peptide receptors in cultured rat diencephalon

To characterize the type of cell expressing natriuretic peptide receptors in the brain and the nature of these receptors, we conducted studies in primary cultured glial and neuronal cells derived from fetal rat diencephalon. The glial predominant cultures (95% of total cells and glial fibrillary acidic protein positive) expressed nearly a 10-fold greater specific binding of the natriuretic peptides to cell surface receptors compared with the neuron-predominant cultures. Scatchard analysis of binding studies with 125I-atrial natriuretic peptide (ANP) and 125I-brain natriuretic peptide (BNP) revealed a single class of receptors with dissimilar affinities (0.25 +/- 0.09 and 0.74 +/- 0.07 nM, respectively, n = 3 experiments p less than 0.01) but similar numbers of binding sites for both peptides (93 and 88 fmol/mg of protein, respectively). Cross-linking of 125I-ANP and BNP to cultured glia followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography identified distinct bands at either approximate Mr 130,000, or 102,000 and 66,000, corresponding to two high molecular weight (B) receptors and one low molecular weight (C) receptor described in other tissues. Different subtypes of astrocytes appeared to express different B receptors. Binding and cross-linking of radiolabeled ANP or BNP were competitively inhibited equally by unlabeled ANP or BNP, indicating that ANP and BNP probably bind the same receptors. The glial cultures functionally expressed a receptor(s) with guanylate cyclase activity; BNP was less potent than ANP in stimulating cGMP at lower concentrations. These results indicate that both high and low molecular weight natriuretic peptide receptors are expressed in astrocyte-predominant cultures from the fetal diencephalon and suggest that glia participate in several actions of ANP which are probably mediated through this area of the brain.     

Isolation and partial characterization of high affinity laminin receptors in neural cells

Neural cells in culture (NG-108, PC12, chick dorsal root ganglion, chick spinal cord, and rat astrocytes) bind laminin with an apparent Kd of congruent to 10(-9) M. Laminin affinity chromatography of chick brain membranes washed with 150 mM NaCl and eluted with 0.2 M glycine buffer, pH 3.5, yields a single protein with an apparent molecular mass of 67 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Isoelectric focusing and peptide mapping indicate that the 67-kDa protein is distinct from bovine serum albumin (68 kDa) but indistinguishable from high affinity laminin receptors isolated from skeletal muscle. After electroblotting onto nitrocellulose paper and probing with 125I-laminin, this putative laminin receptor binds laminin specifically (100 ng/ml). A second protein (congruent to 120-140 kDa) is also detected with 125I-laminin (100 ng/ml) in the laminin affinity-purified membrane proteins. Both 67- and congruent to 120-140-kDa proteins can be laminin affinity-purified from cultures enriched for neurons (greater than 90%) following metabolic labeling with [35S]methionine. Our data suggest that neural cells (dorsal root ganglion, central nervous system neurons, astrocytes, and several neural cell lines) have high affinity binding sites for laminin and that two membrane proteins, 67- and congruent to 120-140-kDa, are responsible at least in part for this binding.     

Characterization of a neuronal subtype of insulin-like growth factor I receptor

Primary neuronal cultures from fetal rat brain were utilized to investigate the possible role of insulin-like growth factor I (IGF-I) in neuronal growth and differentiation. 125I-IGF-I binding to intact cultured neurons was specific and saturable with an apparent Kd of 7.0 +/- 1.2 nM and a Bmax of 1.8 +/- 0.3 pmol/mg protein. Binding of 125I-IGF-I to neurons was inhibited by IGF-I, followed by IGF-II and insulin. 7 S nerve growth factor, but not beta-nerve growth factor, also inhibited 125I-IGF-I binding. A similar binding site was detected on brain membranes. Affinity cross-linking of 125I-IGF-I to intact cultured neurons revealed, under reducing conditions, a major binding moiety with an Mr of 115,000 and a minor component at Mr 260,000. The former represents a neuronal type of the IGF-I receptor alpha subunit, whereas the latter probably represents an alpha dimer. The Mr = 115,000 binding component for 125I-IGF-I was also present in membranes prepared from postnatal whole brain. In contrast, the binding moiety in cultured glial cells was of Mr = 135,000, which was identical to the IGF-I receptor alpha subunit of placenta. Thus mature brain, despite its cellular heterogeneity, expresses a structural subtype of IGF-I receptor which appears to be unique to differentiated neurons. Moreover, glial and neuronal cultures secreted a polypeptide which specifically bound IGF-I; the apparent Mr of this binding protein was determined by affinity cross-linking to be approximately 35,000. The presence of neuronal IGF-I receptors and binding proteins suggested that IGF-I may exert neurotrophic effects on developing neurons. This possibility was supported by the observation that IGF-I markedly stimulated neuronal RNA synthesis.     

In situ photolabelling of the human androgen receptor

In situ photoaffinity labelling of the human androgen receptor has been performed in the LNCaP (Lymph Node Carcinoma of the Prostate) cell line. The covalently labelled receptors were identified by SDS-PAGE. Intact LNCaP cells, incubated with [3H]-R1881 and subsequently irradiated with u.v. light and directly solubilized in SDS-buffer, revealed two photolabelled protein bands at 110 and 50 kDa. Irradiation of intact cells and subsequent isolation of nuclei followed by extraction with 0.5 M NaCl resulted in one major photolabelled protein band at 110 kDa. The labelling of this band could be completely suppressed by a 100-fold molar excess of non-radioactive R1881. Photolabelling of androgen receptors in a cytosolic preparation of LNCaP cells after anion exchange chromatography resulted in a much lower labelling efficiency compared with the in situ labelling procedure, although the androgen receptor was purified 100-fold. The steroid binding domain of the human androgen receptor has been partially mapped with chymotrypsin and S. aureus V8 protease digestion. Proteolytic digestion with chymotrypsin of purified photoaffinity-labelled 110 kDa human androgen receptor resulted in the generation of a 15 kDa peptide which still contains the covalently linked hormone. It is concluded that the in situ photoaffinity labelling technique can be applied successfully for characterization of the steroid binding domain of androgen receptors in prostate cancer cells and in other androgen target cells. Furthermore, it was demonstrated that the human androgen receptor is a monomer with a molecular mass of 110 kDa, of which the steroid binding site is confined to a 15 kDa domain.     

Insulin-like growth factor I receptors in neuronal and glial cells. Characterization and biological effects in primary culture

Primary cultures of neuronal and glial cells from 1-day-old neonatal rats contain high affinity receptors for insulin-like growth factor I (IGF-I). The IC50 for displacement of 125I-IGF-I binding by unlabeled IGF-I was 3 nM for neuronal cells and 4 nM for glial cells. Unlabeled insulin was 20-50 times less potent. Apparent molecular mass of the alpha subunits of the IGF-I receptor was 125 kDa in neuronal and 135 kDa in glial cells. IGF-I induced autophosphorylation of the IGF-I receptor beta subunit in lectin-purified membrane preparations in a dose-dependent manner. The major phosphoamino acid of the beta subunit in both cell types was tyrosine in the IGF-I-stimulated state and serine in the basal state. Apparent molecular mass of the beta subunits of the IGF-I receptors was 91 kDa for neuronal and 95 kDa for glial cells. Tyrosine kinase activity of the IGF-I receptors was demonstrated by IGF-I-induced phosphorylation of the exogenous substrate poly(Glu, Tyr) 4:1 in both cell types. IGF-I had no effect on 2-deoxyglucose uptake in neuronal cells. In contrast, in glial cells, IGF-I stimulated 2-deoxyglucose uptake at very high doses, presumably acting via the insulin receptor. The effect of IGF-I as a neurotrophic growth factor in both neuronal and glial cells was demonstrated by its stimulation of [3H]thymidine incorporation. These findings suggest the IGF-I is an important growth factor in nervous tissue-derived cells.     

Identification of heparan sulfate proteoglycan as a high affinity receptor for acidic fibroblast growth factor (aFGF) in a parathyroid cell line.

We have characterized two high affinity acidic fibroblast growth factor (aFGF) receptors in a rat parathyroid cell line (PT-r). Affinity labeling with 125I-aFGF showed that these two receptors, apparent molecular masses, 150 and 130 kDa, respectively, display higher affinity for aFGF than for bFGF. The 150-kDa receptor bears a heparan sulfate chain(s), demonstrated by a decrease in size of 15-20 kDa with heparitinase digestion after affinity labeling. Heparitinase digestion before affinity labeling markedly reduced the intensity of the 150 kDa species. Scatchard analysis showed two different high affinity binding sites (Kd of 3.9 pM with 180 sites/cell and Kd of 110 pM with 5800 sites/cell). The higher affinity site was completely eliminated by digestion with heparitinase before adding labeled aFGF; the lower affinity site was unaffected. In ion exchange chromatography after metabolic labeling of the cells with [3H]glucosamine and affinity labeling with 125I-aFGF, the larger receptor-ligand complex, 165 kDa, eluted with approximately 0.5 M NaCl, typical eluting conditions for heparan sulfate proteoglycans. Both of the receptor-ligand complexes were smaller on sodium dodecyl sulfate-polyacrylamide gel electrophoresis than two major heparan sulfate proteoglycans, HSPG I and II, which we characterized in this cell line previously (Yanagishita, M., Brandi, M. L., and Sakaguchi, K. (1989) J. Biol. Chem. 264, 15714-15720). Both receptors have similar N-linked oligosaccharide and sialic acid contents, shown by analysis of affinity-labeled receptors upon digestion with glycopeptidase F and with neuraminidase. All together, these results suggest that PT-r cells bear two distinct high affinity receptors for aFGF, a 150-kDa receptor which is a heparan sulfate proteoglycan and another that is a glycoprotein. The heparan sulfate glycosaminoglycan moiety of the 150- kDa receptor is critical for high affinity binding of aFGF. These findings contrast with current concepts derived from other systems, suggesting that heparan sulfate glycosaminoglycans/proteoglycans function as a reservoir source for FGF or as a group of low affinity binding sites.     

Purification of basic fibroblast growth factor receptors from bovine brain

Fibroblast growth factors are proteins which play a major role, in vitro and in vivo, in the control of cellular growth and differentiation of a large number of cells. Biological activities of these factors are mediated by the interaction with specific membrane receptors. Previous studies indicated that the apparent molecular weight of a family of these receptors for the basic form of Fibroblast Growth Factor (bFGF), ranges from 125 to 165 kDa according to cell species and types. We have purified this family of receptors from bovine brain. We first set up a radioreceptor assay to detect receptors throughout the purification by measuring its ability to inhibit the fixation of radiolabeled bFGF to insolubilized membranes from bovine brain. The purification was also monitored by using cross-linking reagents in order to allow the visualization of radiolabeled bFGF bound to its receptor. The first purification steps involved 2 anion-exchange chromatographic steps, DEAE Trysacryl and FPLC Mono Q, and yielded an enrichment over 500 fold. Affinity chromatography with bFGF immobilized on Sepharose 4B was then performed. Covalent fixation of bFGF to the Sepharose matrix was carried out in presence of N-acetylated heparin in order to protect the recognition site for bFGF on its receptor. These 3 chromatographic steps yielded only 2 bands of apparent molecular weight of 100 kDa and 135 kDa as detected by electrophoresis. These 2 bands are also detected after chromatography on immobilized wheat germ agglutinin hence confirming the presence of carbohydrates on bFGF receptors.     

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.     

The Mycobacterium tuberculosis membrane protein Rv2560--biochemical and functional studies

The characterization of membrane proteins having no identified function in Mycobacterium tuberculosis is important for a better understanding of the biology of this pathogen. In this work, the biological activity of the Rv2560 protein was characterized and evaluated. Primers used in PCR and RT-PCR assays revealed that the gene encoding protein Rv2560 is present in M. tuberculosis complex strains, but transcribed in only some of them. Sera obtained from rabbits inoculated with polymer peptides from this protein recognized a 33 kDa band in the M. tuberculosis lysate and a membrane fraction corresponding to the predicted molecular mass (33.1 kDa) of this protein. Immunoelectron microscopy analysis found this protein on the mycobacterial membrane. Sixteen peptides covering its entire length were chemically synthesized and tested for their ability to bind to A549 and U937 cells. Peptide 11024 (121VVALSDRATTAYTNTSGVSS140) showed high specific binding to both cell types (dissociation constants of 380 and 800 nm, respectively, and positive receptor-ligand interaction cooperativity), whereas peptide 11033 (284LIGIPVAALIHVYTYRKLSGG304) displayed high binding activity to A549 cells only. Cross-linking assays showed the specific binding of peptide 11024 to a 54 kDa membrane protein on U937. Invasion inhibition assays, in the presence of shared high-activity binding peptide identified for U937 and A549 cells, presented maximum inhibition percentages of 50.53% and 58.27%, respectively. Our work highlights the relevance of the Rv2560 protein in the M. tuberculosis invasion process of monocytes and epithelial cells, and represents a fundamental step in the rational selection of new antigens to be included as components in a multiepitope, subunit-based, chemically synthesized, antituberculosis vaccine.     

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.     

Characterization of the receptor for keratinocyte growth factor. Evidence for multiple fibroblast growth factor receptors

Keratinocyte growth factor (KGF) is a member of the fibroblast growth factor (FGF) family. KGF exhibits potent mitogenic activity for a variety of epithelial cell types but is distinct from other known FGFs in that it is not mitogenic for fibroblasts or endothelial cells. We report saturable specific binding of 125I-KGF to surface receptors on intact Balb/MK mouse epidermal keratinocytes. 125I-KGF binding was completed efficiently by acidic FGF (aFGF) but with 20-fold lower efficiency by basic FGF (bFGF). The pattern of 125I-acidic FGF binding and competition on Balb/MK keratinocytes and NIH/3T3 fibroblasts suggests that these cell types possess related but distinct FGF receptors. Scatchard analysis of 125I-KGF binding suggested major and minor high affinity receptor components (KD = 400 and 25 pM, respectively) as well as a third high capacity/low affinity heparin-like component. Covalent affinity cross-linking of 125I-KGF to its receptor on Balb/MK cells revealed two species of 115 and 140 kDa. KGF also stimulated the rapid tyrosine phosphorylation of a 90-kDa protein in Balb/MK cells but not in NIH/3T3 fibroblasts. Together these results indicate that Balb/MK keratinocytes possess high affinity KGF receptors to which the FGFs may also bind. However, these receptors are distinct from the receptor(s) for aFGF and bFGF on NIH/3T3 fibroblasts, which fail to interact with KGF.     

Binding of human C-reactive protein to mouse macrophages is mediated by distinct receptors

Human C-reactive protein (CRP) is known to activate mouse macrophages (M phi) to a tumoricidal state and to serve as an opsonin for M phi. Therefore, cell surface receptors for CRP on mouse M phi were characterized and their relationship to the IgG FcR determined. The specific binding of 125I-CRP to resident or elicited mouse M phi was saturable, reversible, and involved both a high and a low affinity receptor population. Binding of CRP to the mouse M phi cell lines PU5 1.8 and J774 was nearly identical to that observed with peritoneal M phi. The high affinity receptor population had a calculated K of 10 nM and a receptor density of approximately 10(5) sites per cell. Mouse Ig of the IgG2a, IgG2b, or IgG1 isotypes inhibited binding of 125I-CRP to PU5 1.8 cells at concentrations five-fold greater than that of the homologous ligand. In the converse experiment, unlabeled CRP failed to inhibit specific binding of 125I-labeled IgG2a, IgG2b or IgG1. Isolation of CRP binding proteins from surface iodinated PU5 1.8 cells by ligand-affinity chromatography or chemical cross-linking yielded a major protein band of 57 to 60 kDa which appeared to be distinct from the IgG1/IgG2b FcR (FcR-II) membrane proteins. Removal of radiolabeled IgG2b/IgG1 binding membrane proteins by affinity chromatography did not remove CRP-binding proteins. The rat mAb 2.4G2 which inhibits binding of radiolabeled mouse IgG2b, did not inhibit the binding of CRP. A rat polyclonal antiserum to CRP-binding membrane proteins of PU5 1.8 cells inhibited 125I-CRP binding, but not 125IgG2b binding. The rat polyclonal antibody reacted with two 57 to 60 kDa membrane proteins from PU5 1.8 cells that appear to be of a similar size on Western blots. The 125I-CRP was internalized via endosomes and intact CRP subunits could be detected intracellularly. The findings suggest that binding of CRP occurs through a receptor that is distinct from the IgG FcRs, but that CRP-R activity may be influenced by an association with an IgG FcR.     

The inhibitory neuronal glycine receptor

Glycine is a major inhibitory neurotransmitter in the spinal cord and in the brain stem, where it acts by activating a chloride conductance. The postsynaptic glycine receptor has been purified and contains two transmembrane subunits of 48 kDa (α) and 58 kDa (β), and a peripheral membrane protein of 93 kDa. cDNA sequencing of the α and β subunits has revealed a common structural organization and a strong homology between these polypeptides and the nicotinic acetylcholine and GABA_A receptor proteins. The glycine receptor exhibits a heterogeneity resulting from the existence of several α subtypes with distinct functional properties and different developmental expressions. When present in the central nervous system in situ, as well as in primary cultures of spinal cord neurons, these receptors are localized at the postsynaptic membrane adjacent to the presynaptic release sites, thus forming functional microdomains at the neuronal surface. This distribution raises the question of the formation and the maintenance of the heterogeneity of the somato-dendritic plasma membrane.     

Ontogenesis of the insulin receptor in the rabbit brain

We delineated the ontogeny of the brain insulin binding, insulin receptor number and affinity using plasma membranes isolated from the rabbit. Specific 125I-insulin binding and receptor number expressed per milligram of protein increased from the 20 day gestation fetus to the 1-day-old newborn, declining thereafter to attain adult values by day 6 of postnatal life. Specific 125I-insulin binding and the receptor number in the adult brain was less than the fetal and neonatal (1 day) brain receptors. Although a similar trend was observed specifically during fetal development, the changes in receptor number expressed per microgram DNA were not significant in the neonatal period. The adult brain insulin receptor number was higher than the 20- to 27-day fetus and similar to that of the 30-day fetus and the 1- to 5-day newborns. The total receptor number correlated linearly with the brain plasma membrane protein increment velocity. The affinity of the receptors increased during early fetal development (20-27 days) and remained constant thereafter in the postnatal period. We conclude that the ontogenic changes of the brain insulin receptors are similar to the ontogenic changes of brain plasma membrane protein. The developmental changes are more pronounced when the receptor number is expressed per milligram protein versus microgram DNA.     

A 20 amino acid synthetic peptide of a region from the 55 kDa human TNF receptor inhibits cytolytic and binding activities of recombinant human tumour necrosis factor in vitro.

Tumour Necrosis Factor (TNF) and Lymphotoxin (LT) can exert a wide range of effects on cells and tissues and they are important effector molecules in cell mediated immunity. All these effects are induced subsequent to the binding of these cytokines to specific membrane receptors. Recently, two of these membrane receptors of 55 and 75 kDa, have been identified which share some amino acid (AA) homology in their N-terminal extracellular domains but differ in their intracellular domains. We synthesized two synthetic 20 AA peptides from hydrophilic regions of the N-terminal extracellular domains of the 55 kDa receptor; peptide A shares homology with both 55 and 75 kDa receptors, peptide B is unique. We found peptide B inhibits both the binding and cytolytic activity of recombinant human TNF when tested on murine L929 cells in vitro. Polyclonal antiserum generated against peptide B will block binding of 125I-labelled TNF to these cells in vitro. However, peptide A and antiserum prepared against peptide A are without effect in these same assay systems. These data suggest that the 20 AA sequences from AA 175 to 194 in the N-terminal extracellular domain of the 55 kDa TNF receptor are expressed on the cell surface and are involved in the binding of TNF.     

Basic fibroblast growth factor expression in human omental microvascular endothelial cells and the effect of phorbol ester.

The human omentum contains a potent, not yet identified angiogenic activity. The omentum is very vascularized. Therefore, we investigated whether human omental microvascular endothelial cells (HOME cells) express the angiogenic peptide basic fibroblast growth factor (bFGF). Cytosol prepared from HOME cells stimulated DNA synthesis in bovine epithelial lens cells (BEL cells). The mitogenic activity could be neutralized by an anti-bFGF antibody. Basic FGF-like material from the HOME cell cytosol was bound onto a heparin-Sepharose column at 0.6 M and was eluted at 3 M NaCl. The 3 M NaCl eluted material reacted with the specific anti-bFGF antibody in an ELISA and stimulated DNA synthesis. It did not react with a specific anti-acidic fibroblast growth factor (aFGF) antibody. Western blotting experiments using the same bFGF antibody showed the presence of a major band of 17 Kd and a doublet of 20-22 Kd. Northern blotting of non-stimulated HOME cells using a specific 1.4 kb bFGF probe showed the presence of 5 molecular species of 6.6, 3.7, 2.2, 2.0, and 1.0 kb. No aFGF mRNA was detected with a specific previously characterized 4.04 kb probe. 12-O-tetradecanoylphorbol 13-acetate (TPA) did not influence significantly the expression of bFGF at the protein and mRNA level in HOME cells. Thus, protein kinase C activation by TPA did not appear to modulate significantly the expression of bFGF for that cell type. Contrastingly, human umbilical vein endothelial cells (HUVE cells), which expressed no bFGF and aFGF mRNA at a basal level, were induced to express bFGF but not aFGF mRNA when stimulated by TPA. These results suggest that the described angiogenic activity could be the bFGF-like mitogen contained in HOME cells and that these cells are different from endothelial cells derived from large vessels (HUVE cells) regarding the expression of bFGF.     

Identification of retinal insulin receptors using site-specific antibodies to a carboxyl-terminal peptide of the human insulin receptor alpha-subunit. Up-regulation of neuronal insulin receptors in diabetes

Insulin receptor-specific polyclonal antipeptide serum was generated against a synthetic pentadecapeptide (residues 657-670) of the deduced amino acid sequence of human insulin proreceptor cDNA for use in the analysis of insulin receptors in the retina. The affinity-purified antibodies recognized peptide antigen but not keyhole limpet hemocyanin as determined by dot blot analysis and solid phase radioimmunoassay. Addition of either synthetic peptide or the affinity-purified serum had no effect on 125I-insulin binding to placental membranes or to cells in culture. alpha-Subunits of approximately 125 kDa from human placental membranes and liver membranes were labeled by immunoblot analysis with this antiserum. In membranes isolated from human retina and brain, two classes of alpha-subunits of approximately 125 and 115 kDa were detectable. The 115-kDa subunit was neuraminidase resistant whereas the 125-kDa subunit was digested to a band of 115 kDa, indicating that these bands represent peripheral and neuronal receptors, respectively. Analysis of human retinas obtained from type I diabetic donors revealed an increased level of neuronal receptor as compared with normal retinas. These data indicate that human retina expresses neuronal insulin receptor subtypes that are up-regulated in diabetes.     

Heparan sulfate primed on β-D-xylosides restores binding of basic fibroblast growth factor

Heparan sulfate proteoglycans (HSPG) are obligatory for receptor binding and mitogenic activity of basic fibroblast growth factor (bFGF). Mutant Chinese hamster ovary cells (pgsA-745) deficient in xylosyltransferase are unable to initiate glycosaminoglycan synthesis and hence can not bind bFGF to low- and high-affinity cell surface receptors. Exposure of pgsA-745 cells to β-D-xylopyranosides containing hydrophobic aglycones resulted in restoration of bFGF binding in a manner similar to that induced by soluble heparin or by heparan sulfate (HS) normally associated with cell sulfate. Restoration of bFGF binding correlated with the ability of the β-D-xylosides to prime the synthesis of heparan sulfate. Thus, both heparan sulfate synthesis and bFGF receptor binding were induced by low concentrations (10–30 μM) of estradiol-β-D-xyloside and naphthyl-β-D-xyloside, but not by cis/trans-decahydro-2-naphthyl-β-D-xyloside, which at low concentration primes mainly chondroitin sulfate. The obligatory involvement of xyloside-primed heparan sulfate in restoration of bFGF-receptor binding was also demonstrated by its sensitivity to heparinase treatment and by the lack of restoration activity in CHO cell mutants that lack enzymatic activities required to form the repeating disaccharide unit characteristic of heparan sulfate. Xyloside-primed heparan sulfate binds to the cell surface. Restoration of bFGF receptor binding was induced by both soluble and cell bound xyloside-primed heparan sulfate and was abolished in cells that were exposed to 0.5–1.0 M NaCl prior to the bFGF binding reaction. These results indicate that heparan sulfate chains produced on xyloside primers behave like heparan sulfate chains attached to cellular core proteins in terms of affinity for bFGF and ability to function as low-affinity sites in a dual receptor mechanism characteristic of bFGF and other heparin-binding growth promoting factors.     

Internalization of basic fibroblast growth factor at the mouse blood-brain barrier involves perlecan,a heparan sulfate proteoglycan

In this study, the internalization mechanism of basic fibroblast growth factor (bFGF) at the blood-brain barrier (BBB) was investigated using a conditionally immortalized mouse brain capillary endothelial cell line (TM-BBB4 cells) as an in vitro model of the BBB and the corresponding receptor was identified using immunohistochemical analysis. The heparin-resistant binding of [125I]bFGF to TM-BBB4 cells was found to be time-, temperature-, osmolarity- and concentration-dependent. Kinetic analysis of the cell-surface binding of [125I]bFGF to TM-BBB4 cells revealed saturable binding with a half-saturation constant of 76 +/- 24 nm and a maximal binding capacity of 183 +/- 17 pmol/mg protein. The heparin-resistant binding of [125I]bFGF to TM-BBB4 was significantly inhibited by a cationic polypeptide poly-L-lysine (300 micro m), and compounds which contain a sulfate moiety, e.g. heparin and chondroitin sulfate-B (each 10 micro g/mL). Moreover, the heparin-resistant binding of [125I]bFGF in TM-BBB4 cells was significantly reduced by 50% following treatment with sodium chlorate, suggesting the loss of perlecan (a core protein of heparan sulfate proteoglycan, HSPG) from the extracellular matrix of the cells. This type of binding is consistent with the involvement HSPG-mediated endocytosis. RT-PCR analysis revealed that HSPG mRNA and FGFR1 and FGFR2 (tyrosine-kinase receptors for bFGF) mRNA are expressed in TM-BBB4 cells. Moreover, immunohistochemical analysis demonstrated that perlecan is expressed on the abluminal membrane of the mouse brain capillary. These results suggest that bFGF is internalized via HSPG, which is expressed on the abluminal membrane of the BBB. HSPG at the BBB may play a role in maintaining the BBB function due to acceptance of the bFGF secreted from astrocytes.