Membrane vesicles of A431 cells contain one class of epidermal growth factor binding sites

Epidermoid carcinoma A431 cells exhibit two classes of epidermal growth factor (EGF) receptors as deduced from Scatchard analysis. Steady-state binding of EGF to isolated A431 membranes indicated, however, the presence of only one class of EGF binding sites. The apparent dissociation constant (Kd) of these sites was approx. 0.45 nM which is similar to that of the high-affinity receptor of intact A431 cells. These results suggest that the vesicle receptor population consists only of high-affinity receptors. However, further studies indicated that the binding sites were similar to the low-affinity class, since binding of EGF could be blocked entirely by 2E9, a monoclonal anti-EGF receptor antibody which is able to inhibit specifically EGF binding to low-affinity receptors in A431 cells. The difference in affinity of the receptors in membrane vesicles as compared to intact cells may be explained by differences in biophysical parameters such as diffusion-limited EGF binding and receptor distribution. Based upon these considerations, it is concluded that membrane vesicles of A431 cells contain one class of EGF receptors which are apparently identical to the low-affinity receptors of intact cells.     

Analysis of interleukin 5 receptors on murine eosinophils: a comparison with receptors on B13 cells

The interleukin 5 receptor (IL-5R) on murine eosinophils and a mouse B cell line (B13) was investigated using iodinated murine IL-5 produced in the baculovirus system. Electrophoretic analysis of this recombinant protein identified a range of bands Mr 26,000 to 32,000 resulting from differential glycosylation. The specific activity and binding kinetics of the iodinated IL-5 (125I-IL-5) were essentially identical to unlabeled material. Both high-affinity (Kd approximately 50 pM) and low-affinity (Kd approximately 1 nM) receptor populations were identified on murine eosinophils. Approximately 50 high-affinity receptors and 10,000 low-affinity receptors were present. This was compared with approximately 2,000 high-affinity (Kd approximately 80 pM) and about 8,000 low-affinity (Kd approximately 3 nM) sites on B13 cells. An antibody that inhibits IL-5 binding to, and proliferation of, B13 cells (R52.120) was also shown to inhibit eosinophil proliferation, suggesting that eosinophils and B cells bear the equivalent IL-5 binding proteins.     

Cross-linking of epidermal growth factor receptors in intact cells: detection of initial stages of receptor clustering and determination of molecular weight of high-affinity receptors

A method was developed to label epidermal growth factor (EGF) receptors with 125I-EGF in whole cells using chemical cross-linking reagents. Polyacrylamide gel electrophoresis resolved an Mr approximately 180,000 EGF-receptor complex and larger Mr greater than or equal to 360,000 aggregates. The formation of the larger complexes was time and temperature dependent and appeared to represent the initial events of EGF receptor clustering. Alteration of the ratio of 125I-EGF-labeled high- (Kd approximately 0.16 nM) and low- (Kd approximately 1.5 nM) affinity complexes by competition with unlabeled EGF or by induction of additional high-affinity sites with dexamethasone suggested that both sites were represented by the Mr approximately 180,000 125I-EGF-receptor complexes. Digestion of cells before cross-linking detected a small population of trypsin-resistant Mr approximately 180,000 receptors, which could represent previously described cryptic and/or high-affinity receptors. Few of the Mr approximately 360,000 receptors were trypsin resistant. Glucocorticoid induction of high-affinity EGF receptors failed to induce detectable changes in the microclustering of EGF receptors but did result in a 50% increase in EGF-induced receptor phosphorylation in HeLa S3 cell membranes at 4 degrees C. Thus, glucocorticoids increase high-affinity EGF binding sites, EGF-induced receptor phosphorylation, and cell growth.     

Analysis of binding sites and efficacy of a species-specific peptide at rat and human neurotensin receptors.

We have developed a neurotensin analog, L-[3,1'-naphthylalanine11]NT(8-13), NT34, that can distinguish between rat and human neurotensin receptors, and exhibits more than a 100-fold difference in binding affinities and a 60-fold difference in functional coupling to phosphatidylinositol turnover. Using cells transfected with different numbers of the appropriate receptors, we measured the changes in phosphatidylinositol production, and then evaluated the efficiency of receptor-effector coupling based on Furchgott's design. The binding of NT34 at both rat and human neurotensin receptors stably expressed in CHO-K1 cells was to two sites, while the binding of NT was to one site. At the rat receptor the equilibrium dissociation constant (Kd) for NT34 at the high-affinity site was 0.058 nM, while that at the low-affinity site was 3.1 nM. For the human receptor at the high-affinity site, the Kd for NT34 was 18 nM, while that at the low-affinity site was 180 nM. For both species the percentage of receptors representing the high-affinity site was approximately 60-70% with 30-40% at the low-affinity site. We derived agonist dissociation constants (Ka) for NT and NT34, which suggest that for NT34, the low-affinity site is functionally coupled to phosphatidylinositol turnover. Finally, we compared the relative efficacies of both compounds and found that NT34 was about 2-fold and 4-fold more efficacious than NT in stimulating phosphatidylinositol turnover in rat and human NT receptors, respectively.     

Receptor-mediated endocytosis of epidermal growth factor by hepatocytes in the perfused rat liver: ligand and receptor dynamics

We have used biochemical and morphological techniques to demonstrate that hepatocytes in the perfused liver bind, internalize, and degrade substantial amounts of murine epidermal growth factor (EGF) via a receptor-mediated process. Before ligand exposure, about 300,000 high-affinity receptors were detectable per cell, displayed no latency, and co-distributed with conventional plasma membrane markers. Cytochemical localization using EGF coupled to horseradish peroxidase (EGF-HRP) revealed that the receptors were distributed along the entire sinusoidal and lateral surfaces of hepatocytes. When saturating concentrations of EGF were perfused through a liver at 35 degrees C, ligand clearance was biphasic with a rapid primary phase of 20,000 molecules/min per cell that dramatically changed at 15-20 min to a slower secondary phase of 2,500 molecules/min per cell. During the primary phase of uptake, approximately 250,000 molecules of EGF and 80% of the total functional receptors were internalized into endocytic vesicles which could be separated from enzyme markers for plasma membranes and lysosomes on sucrose gradients. The ligand pathway was visualized cytochemically 2-25 min after EGF-HRP internalization and a rapid transport from endosomes at the periphery to those in the Golgi apparatus-lysosome region was observed (t 1/2 approximately equal to 7 min). However, no 125I-EGF degradation was detected for at least 20 min. Within 30 min after EGF addition, a steady state was reached which lasted up to 4 h such that (a) the rate of EGF clearance equaled the rate of ligand degradation (2,500 molecules/min per cell); (b) a constant pool of undegraded ligand was maintained in endosomes; and (c) the number of accessible (i.e., cell surface) receptors remained constant at 20% of initial values. By 4 h hepatocytes had internalized and degraded 3 and 2.3 times more EGF, respectively, than the initial number of available receptors, even in the presence of cycloheximide and without substantial loss of receptors. All of these results suggest that EGF receptors are internalized and that their rate of recycling to the surface from intracellular sites is governed by the rate of entry of ligand and/or receptor into lysosomes.     

Role of TGF alpha and its receptor in proliferation of immortalized rat tracheal epithelial cells: studies with tyrphostin and TGF alpha antisera

We have shown in the present study and in studies reported previously that preneoplastic and neoplastic rat tracheal epithelial (RTE) cell lines express TGF alpha and do so regardless of the mechanism by which they were transformed. In order to determine whether TGF alpha is an autocrine growth regulator of immortalized RTE cells, we have examined the function of TGF alpha/EGF receptors and the growth requirements for TGF alpha in these cells. The level of immunoprecipitated TGF alpha/EGF receptor protein in immortalized RTE cells was similar to or less than levels in primary RTE cells, indicating that chemically induced transformation of RTE cells does not involve overexpression of TGF alpha/EGF receptors. Scatchard analysis of TGF alpha/EGF receptors in the neoplastic EGV5T cell line revealed the presence of high-affinity (Kd = 0.4 nM) and low-affinity (Kd = 9.8 nM) binding sites. A tyrphostin TGF alpha/EGF receptor tyrosine kinase inhibitor decreased in a dose-dependent manner the proliferation as well as EGF-induced autophosphorylation of the TGF alpha/EGF receptor of transformed RTE cells. The inhibitory effect of tyrphostin on proliferation and receptor kinase activity was attenuated in late log and plateau phase cultures. The phosphotyrosine content of several other EGF-dependent and independent phosphoproteins was also decreased by the tyrphostin. Proliferation of transformed RTE cells was also inhibited when TGF alpha antisera was added to the media of growing cells. These data are consistent with the hypothesis that proliferation of transformed RTE cells involves autocrine regulation by TGF alpha and its receptor.     

Two receptor classes for epidermal growth factor on pheochromocytoma cells, distinguishable by temperature, lectins, and tumor promoters

Rat pheochromocytoma cells (clone PC12) display cell surface receptors for both nerve growth factor (NGF) and epidermal growth factor (EGF) and therefore provide a useful model system with which to study the role of these receptors in the regulation of proliferation and differentiation. In this paper PC12 cells are demonstrated to possess two classes of EGF receptors, a high-affinity class with 7,600 sites per cell and an apparent dissociation constant (Kd) of 0.05 nM, and a low-affinity class with 62,000 sites per cell and a Kd of 14.1 nM. These findings are contrary to literature data (Huff et al., 1981; Vale and Shooter, 1983) but can be explained in part by differences in experimental conditions. Binding studies at 37 degrees C compared with room temperature demonstrated similar affinities of both classes, but during prolonged incubation at 37 degrees C, the binding capacities of both classes decreased. Furthermore the high-affinity class was sensitive to lectins, such as concanavalin A (Con A), and to the tumor-promoting phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). Both compounds caused a decrease of the affinity of the high-affinity class without affecting the low-affinity class. At high concentrations of Con A or TPA, a decrease of the apparent number of binding sites of the low-affinity class was also observed. The similarities between the characteristics of EGF binding and NGF binding in PC12 cells are striking and will be discussed.     

High- and low-affinity interleukin 2 receptors: distinctive effects of monoclonal antibodies

We previously established several mouse hybridoma cell lines producing monoclonal antibodies against the human interleukin 2 (IL 2) receptor molecule. As they bind to both high- and low-affinity IL 2 receptors, their effects on binding of 125I-labeled IL 2 to high- and low-affinity receptors were examined by Scatchard plot analysis. Two of these monoclonal antibodies, HIEI and H-47, reduced the IL 2 binding affinity of high-affinity receptors from a Kd of 14 to 20 pM to a Kd of 110 to 140 pM, but slightly raised that of low-affinity receptors. These two antibodies scarcely affected the numbers of high- and low-affinity receptors. On the other hand, H-31 completely blocked IL 2 binding to both high- and low-affinity receptors, and H-A26 slightly reduced the affinities of both high- and low-affinity receptors, from 17 pM to 28 pM and from 28 nM to 54 nM, respectively. H-48 had little affect on IL 2 binding to high- or low-affinity receptors. By use of these monoclonal antibodies, the inhibitory effect of IL 2 on growth of an HTLV-I-immortalized T cell line was demonstrated to be transmitted from high-affinity, but not low-affinity, receptors.     

Rapid and efficient purification of plasma membrane from cultured cells: characterization of epidermal growth factor binding

We have devised a rapid and simple protocol for the purification of the plasma membrane from several lines of transformed cultured cells. A431 or KB plasmalemma was purified in 90 min with a two-step centrifugation cycle after selectively inducing microsomal aggregation by the addition of calcium to homogenized cells. Relative specific activity analysis using membrane marker enzymes on the various fractions indicated that the isolated plasmalemma was purified 8-12-fold over the starting homogenate and contained a high density of epidermal growth factor (EGF) receptors. Transmission electron microscopy showed the final membrane suspension consisted of unilamellar vesicles with an average diameter of approximately 100 A. The purified membrane vesicles avidly bound to 125I-EGF and reached equilibrium within 30 min. Microfiltration assays indicated more than 90% of the total binding can be displaced by excess unlabeled ligand. Equilibrium binding analysis showed a single class of high-affinity 125I-EGF binding site, with Kd = 0.14 nM and Bmax = 0.1 pmol/mg of protein for purified KB membrane and Kd = 1.2 nM and Bmax = 5.26 pmol/mg of protein for purified A431 membrane. Gel electrophoresis of 125I-EGF cross-linked to membrane EGF receptors showed a distinct autoradiographic band at 170 kilodaltons, which could be displaced with excessive amounts of unlabeled EGF. Finally, EGF-dependent autophosphorylation of the EGF receptor was clearly demonstrated with the purified membrane preparation. Membrane vesicles purified in this manner can be stored in liquid nitrogen for several months without losing their biological activity.     

High-affinity epidermal growth factor binding is specifically reduced by a monoclonal antibody, and appears necessary for early responses

We have tested the effects of an mAb directed against the protein core of the extracellular domain of the human EGF receptor (mAb108), on the binding of EGF, and on the early responses of cells to EGF presentation. We used NIH 3T3 cells devoid of murine EGF receptor, transfected with a cDNA encoding the full-length human EGF receptor gene, and fully responsive to EGF. The binding to saturation of mAb108 to the surface of these cells at 4 degrees C and at other temperatures specifically reduced high-affinity binding of EGF, but did not change the dissociation constant or the estimated number of binding sites for low-affinity binding of EGF. The kinetics of EGF binding to the transfected cells were measured to determine the effects of the mAb on the initial rate of EGF binding at 37 degrees C. Interestingly, high-affinity EGF receptor bound EGF with an intrinsic on-rate constant 40-fold higher (9.8 x 10(6) M-1.s-1) than did low-affinity receptor (2.5 x 10(5) M-1.s-1), whereas the off-rate constants, measured at 4 degrees C were similar. Cells treated with the mAb or with phorbol myristate acetate displayed single on-rate constants similar to that for the low-affinity receptors. At low doses of EGF ranging from 0.4 to 1.2 nM, pretreatment of cells with mAb108 inhibited by 50-100% all of the early responses tested, including stimulation of tyrosine-specific phosphorylation of the EGF receptor, turnover of phosphatidyl inositol, elevation of cytoplasmic pH, and release of Ca2+ from intracellular stores. At saturating doses of EGF (20 nM) the inhibition of these early responses by prebinding of mAb108 was overcome. On the basis of these results, we propose that the high-affinity EGF receptors are necessary for EGF receptor signal transduction.     

Two classes of receptor specific for sperm-activating peptide III in sand-dollar spermatozoa.

We characterized receptors specific for sperm-activating peptide III (SAP-III: DSDSAQNLIQ) in spermatozoa of the sand dollar, Clypeaster japonicus, using both binding and cross-linking techniques. Analyses of the data obtained from the equilibrium binding of a radiolabeled SAP-III analogueto C. japonicus spermatozoa, using Klotz, Scatchard and Hill plots, showed the presence of two classes of receptors specific for SAP-III in the spermatozoa. One of the receptors (high-affinity) had a Kd of 3.4 nM and 3.4 x 10(4) binding sites/spermatozoon. The other receptor (low-affinity) had a Kd of 48 nM, with 6.1 x 10(4) binding sites/spermatozoon. The Kd of the high-affinity receptor was comparable to the median effective concentration of the intracellular-pH-increasing activity of SAP-III and that of the low-affinity receptor was comparable to the median effective concentration of the cellular-cGMP-elevating activity of the peptide. In addition, Scatchard and Hill plots of the data suggested the existence of positive cooperativity between the high-affinity members. Similar results were also obtained from a binding experiment using a sperm-membrane fraction prepared from C. japonicus spermatozoa. The incubation of intact spermatozoa or sperm plasma membranes with the radioiodinated SAP-III analogue and a chemical cross-linking reagent, disuccinimidyl suberate, resulted in the radiolabeling of three proteins with molecular masses of 126, 87 and 64 kDa, estimated by SDS/PAGE under reducing conditions.     

Ligand-induced association of epidermal growth factor receptor to the cytoskeleton of A431 cells

Recently, we have obtained evidence in favor of a structural interaction between the epidermal growth factor (EGF) receptor and the Triton X-100-insoluble cytoskeleton of epidermoid carcinoma A431 cells. Here we present a further analysis of the properties of EGF receptors attached to the cytoskeleton. Steady-state EGF binding studies, analyzed according to the Scatchard method, showed that A431 cells contain two classes of EGF-binding sites: a high-affinity site with an apparent dissociation constant (KD) of 0.7 nM (7.5 x 10(4) sites per cell) and a low-affinity site with a KD of 8.5 nM (1.9 x 10(6) sites per cell). Non-equilibrium binding studies revealed the existence of two kinetically distinguishable sites: a fast-dissociating site, with a dissociation rate constant (k-1) of 1.1 x 10(-3) s-1 (1.0-1.3 x 10(6) sites per cell) and a slow-dissociating site, with a k-1 of 3.5 x 10(-5) s-1 (0.6-0.7 x 10(6) sites per cell). The cytoskeleton of A431 cells was isolated by Triton X-100 extraction. Scatchard analysis revealed that approximately 5% of the original number of receptors were associated with the cytoskeleton predominantly via high-affinity sites (KD = 1.5 nM). This class of receptors is further characterized by the presence of a fast-dissociating component (k-1 = 2.0 x 10(-3) s-1) and a slow-dissociating component (k-1 = 9.1 x 10(-5) s-1). The distribution between fast and slow sites of the cytoskeleton was similar to that of intact cells (65% fast and 35% slow sites). Incubation of A431 cells for 2 h at 4 degrees C in the presence of EGF resulted in a dramatic increase in the number of EGF receptors associated to the cytoskeleton. These newly cytoskeleton-associated receptors appeared to represent low-affinity binding sites (KD = 7 nM). Dissociation kinetics also revealed an increase of fast-dissociating sites. These results indicate that at 4 degrees C EGF induces the binding of low-affinity, fast-dissociating sites to the cytoskeleton of A431 cells.     

Evidence for separate receptors for insulin and insulin-like growth factor-I in choroid plexus of rat brain by quantitative autoradiography

Binding of insulin and insulin-like growth factor-I (IGF-I) to the choroid plexus was quantitatively characterized using autoradiography and computer densitometry. Slide-mounted brain slices were incubated in 0.1 nM [125I]-insulin or [125I]-[Thr59]IGF-I. To determine specificity of the binding sites, the labeled peptides were mixed with unlabeled analogues. Autoradiography was done with LKB Ultrofilm and analyzed with a computer image analysis system and program for densitometry. Results showed that binding was time and temperature dependent and reversible. Binding of the iodinated insulin and IGF-I was inhibited by unlabeled peptides in a dose-dependent manner. The rank order of potency of these peptides in competing for the choroid plexus iodoinsulin binding sites was: chicken insulin greater than porcine insulin greater than desoctapeptide insulin greater than IGF-I. IGF-I was more potent than porcine insulin in competing for the choroid plexus iodolGF-I binding sites. Somatostatin was ineffective. Non-linear regression analysis revealed the presence of high- (Kd 1.3 +/- 0.2 nM) and low-affinity (Kd 36 +/- 1.4 nM) binding sites for insulin and a single high-affinity binding site (Kd 3.1 +/- 0.3 nM) for IGF-I in the choroid plexus. There were approximately 50 times more binding sites (Bmax) for IGF-I than for insulin high-affinity sites, whereas the number of low-affinity sites for insulin was about equal to the number of IGF-I high-affinity sites. The results of these binding studies with iodinated insulin and [Thr59]IGF-I support the conclusion that the rat choroid plexus has separate high-affinity receptors for insulin and IGF-I, and that the IGF-I receptors outnumber the insulin receptors.     

Chondrodysplasia in transgenic mice harboring a 15-amino acid deletion in the triple helical domain of pro alpha 1(II) collagen chain

This report describes analysis of factors which regulate the binding of EGF to EGF receptor, receptor internalization, and receptor recycling. Three different methods were used to inhibit high-affinity EGF binding as measured at equilibrium: treatment of cells with an active phorbol ester (PMA), binding of a mAb directed against the EGF receptor (mAb108), and truncation of most of the cytoplasmic domain of the receptor. These treatments reduced the rate at which low concentrations of EGF bound to cells, but did not affect the rate of EGF dissociation. We conclude that high-affinity EGF binding on living cells results from a difference in the apparent on rate of EGF binding. We then used these conditions and cell lines to test for the rate of EGF internalization at different concentrations of EGF. We demonstrate that internalization of the EGF receptor is stimulated roughly 50-fold at saturating concentrations of EGF, but is stimulated an additional two- to threefold at low concentrations (less than 1 nM). Four treatments reduce the rate of internalization of low concentrations of EGF to the rate seen at saturating EGF concentrations. Phorbol ester treatment and mAb108 binding to "wild type" receptor reduce this rate (and reduce high-affinity binding). Point mutation at Lys721 (kinase negative EGF receptor) and point mutation at Thr654 (removing a major site of protein kinase C phosphorylation) reduce the internalization rate, without affecting high-affinity binding. We suggest that while EGF stimulates endocytosis for all receptors, high-affinity receptors bind and are internalized more quickly than low-affinity receptors. Tyrosine kinase activity and the Thr654 region appear necessary for this response.     

Basic fibroblast growth factor (bFGF): mitogenic activity and binding sites in human breast cancer.

We investigated binding characteristics of basic fibroblast growth factor (bFGF) on membranes prepared from 4 human breast cancer cell lines and 38 primary BC biopsies. Competitive binding experiments were performed and analyzed using the "Ligand" program. Furthermore bFGF mitogenic activity was measured by [3H]thymidine incorporation into DNA from breast cancer cell lines. The presence of high-affinity binding sites was demonstrated in each cell type (MCF-7: Kd = 0.60 nM; T-47D: Kd = 0.55 nM; BT-20: Kd = 0.77 nM; MDA-MB-231: Kd = 0.34 nM). The presence of these high-affinity binding sites was confirmed with saturation experiments. A second class of low-affinity binding sites was detected in the 2 hormone-independent cells (BT-20: Kd = 2.9 nM; MDA-MB-231: Kd = 2.7 nM). bFGF stimulated the proliferation of MCF-7, T-47D, BT-20 but not MDA-MB-231 cell lines. With competition experiments, binding sites were detectable in 36/38 breast cancers; high-affinity binding sites (Kd less than 1 nM) were present in 19/36 cases and low-affinity binding sites (Kd greater than 2 nM) were present in 29/36 cases (the two classes of binding sites were present in 12 breast cancers). No relation between bFGF binding sites and node involvement, histologic type or grading of the tumor was evidenced. There were negative correlations (Spearman test) between total bFGF binding sites and estradiol receptor (P = 0.05) or progesterone receptor (P = 0.009). The demonstration of (1) bFGF specific binding sites in breast cancer membranes, and (2) bFGF growth stimulation of some breast cancer cell lines indicates that this factor may be involved directly in the growth of some breast cancers.     

Characterization of nerve growth factor binding to cultured neural crest cells: evidence of an early developmental form of the NGF receptor

Cultured neural crest cells undergoing differentiation have been shown to contain a subpopulation of cells with specific receptors for nerve growth factor (NGF). These cells are the potential targets of NGF during differentiation and development. This study was done to pharmacologically characterize the binding of NGF to long-term (1- to 3-week) cultures of quail neural crest cells. The data indicate that 125I-NGF binding was specific and saturable, with less than 20% nonspecific binding. Scatchard analysis revealed the presence of one type (class) of receptors with a binding constant (Kd) similar to that of the low-affinity binding site described for embryonic dorsal root and sympathetic ganglia (approximately 3.2 nM). This was corroborated by displacement experiments (Kd of 1.3 nM), in which 125I-NGF binding was measured in the presence of increasing concentrations of nonradioactive NGF. In addition, affinity labeling revealed that the 125I-NGF-receptor complex had a molecular weight of about 93K, characteristic of the low-affinity NGF receptor of PC12 cells. The NGF receptor of cultured neural crest cells was trypsin-sensitive, as is typical of the low-affinity NGF binding sites. These findings indicate that differentiating neural crest cells lack high-affinity 125I-NGF binding sites. In contrast, embryonic dorsal root and sympathetic ganglia cells, known NGF targets, have both high- and low-affinity receptors. Measurements of the differential release of surface-bound 125I-NGF indicated that a relatively small amount (about 14%) of NGF is internalized over a 1-hr period. Cultured neural crest cells which bear NGF receptors were also shown by light microscopic radioautographic techniques to incorporate [3H]thymidine. I suggest, therefore, that cultured neural crest cells which have not terminally differentiated, as judged by morphological criteria and continued proliferation, may express an early developmental form of the NGF receptor.     

Binding mode of cytochalasin B to F-actin is altered by lateral binding of regulatory proteins

The binding of cytochalasin B (CB) to F-actin was studied using a trace amount of [3H]-cytochalasin B. F-Actin-bound CB was separated from free CB by ultracentrifugation and the amount of F-actin-bound CB was determined by comparing the radioactivity both in the supernatant and in the precipitate. A filament of pure F-actin possessed one high-affinity binding site for CB (Kd = 5.0 nM) at the B-end. When the filament was bound to native tropomyosin (complex of tropomyosin and troponin), two low-affinity binding sites for CB (Kd = 230 nM) were created, while the high-affinity binding site was reserved (Kd = 3.4 nM). It was concluded that the creation of low-affinity binding sites was primarily due to binding of tropomyosin to F-actin, as judged from the following two observations: (1) a filament of F-actin/tropomyosin complex possessed one high-affinity binding site (Kd = 3.9 nM) plus two low-affinity binding sites (Kd = 550 nM); (2) the Ca2(+)-receptive state of troponin C in F-actin/native tropomyosin complex did not affect CB binding.     

Europium-labeled epidermal growth factor and neurotensin: novel probes for receptor-binding studies.

We investigated the possibility of labeling two biologically active peptides, epidermal growth factor (EGF) and neurotensin (NT), with europium (Eu)-diethylenetriaminepentaacetic acid. More specifically, we tested them as probes in studying receptor binding using time-resolved fluorescence of Eu3+. The relatively simple synthesis yields ligands with acceptable binding characteristics similar to isotopically labeled derivatives. The binding affinity (Kd) of labeled Eu-EGF to human A431 epidermal carcinoid cells was 3.6 +/- 1.2 nM, similar to the reported Kd values of EGF, whereas the Kd of Eu-NT to human HT29 colon cancer cells (7.4 +/- 0.5 nM) or to Chinese hamster ovary (CHO) cells transfected with the high-affinity NT receptor (CHO-NT1) were about 10-fold higher than the Kd values of NT. The bioactivity of the Eu-labeled EGF as determined by stimulation of cultured murine D1 hematopoietic cell proliferation was nearly the same as that obtained with native EGF. The maximal stimulation of Ca2+ influx with NT and Eu-NT in CHO-NT1 cells was similar, but the respective K0.5 values were 20 pM and 1 nM, corresponding to differences in the binding affinities previously described. The results of these studies indicate that Eu labeling of peptide hormones and growth factor molecules ranging from 10(3) to 10(5) Da can be conveniently accomplished. Importantly, the Eu-labeled products are stable for approximately 2 years and are completely safe for laboratory use compared to the biohazardous radioligands. Thus, Eu-labeled peptides present an attractive alternative for commonly used radiolabeled ligands in biological studies in general and in receptor assays in particular.     

Binding of selenoprotein P to heparin: characterization with surface plasmon resonance

The binding of selenoprotein P to glycosaminoglycans using heparin as a model compound was studied by surface plasmon resonance. It was found that heparin contains two binding sites for selenoprotein P, a high-affinity, low-capacity site (Kd approximately 1 nM) and a low-affinity, high-capacity site (Kd approximately 140 nM). Binding at both sites is sensitive to pH and ionic strength, and the high-affinity site is abolished by histidine carbethoxylation with diethylpyrocarbonate. The pH and salt dependence of binding suggests electrostatic interactions with heparin. The concentrations of selenoprotein P in plasma (approximately 50 nM) are sufficiently high to facilitate binding of selenoprotein P to proteoglycans on the vascular endothelium, and this may contribute to the formation of a protective barrier against oxidants such as peroxynitrite or hydroperoxides.     

Interaction of epidermal growth factor receptors with the cytoskeleton is related to receptor clustering

Recently it has been established that cytoskeleton-associated epidermal growth factor (EGF) receptors are predominantly of the high-affinity class and that EGF induces a recruitment of low-affinity receptors to the cytoskeleton. The nature of this EGF-induced receptor-cytoskeleton interaction, however, is still unknown. Therefore, we have studied the association of mutated EGF receptors with the cytoskeleton. Receptor deletion mutants lacking almost all intracellular amino acid residues displayed no interaction with the cytoskeleton, demonstrating that the cytoplasmic receptor domain is involved in this interaction. Further analysis revealed that receptor-cytoskeleton interaction is independent of receptor kinase activity and the C-terminal 126 amino acid residues, which include the auto-phosphorylation sites. Furthermore, it is shown that the high-affinity receptor subclass is not essential for association of low-affinity receptors to the cytoskeleton. EGF receptor-cytoskeleton interaction was increased, however, by treatment with sphingomyelinase, an enzyme known to induce membrane protein clustering, indicating that EGF receptor clustering may cause the association to the cytoskeleton.