Developmental analysis of the cell recognition mechanism in the ciliate Euplotes raikovi.

Euplotes raikovi, like other ciliates, passes through a postconjugal immaturity, operatively identified by an apparent cell inability to form mating pairs under experimental conditions that are the same as those used for inducing mating at maturity. In cells homozygous for the gene mat-2, which controls the pheromone Er-2, Er-2 mRNA synthesis and mature Er-2 secretion were shown to start from the very beginning of the life cycle and continue throughout immaturity, although to extents estimated to be 5- to 10-fold lower than at maturity. In addition, experiments of 125I-Er-2 binding and crosslinking provided evidence that autocrine pheromone-binding sites, showing values of the dissociation constant of the order of 10(-9) M, are on the surface of immature cells. The number of these sites per cell was estimated to increase from less than 10(6) per cell of 5-7 fissions of age, to about 16 x 10(6) at maturity. These results were taken to suggest that a pheromone-receptor production is stimulated during immaturity by autocrine pheromone binding to cells and that this production might be essential for the development of a pheromone-receptor density high enough to transform the cell from "immature" to "adult," that is competent to respond as well to pheromones of conspecific, genetically different cells.     

Purification and characterization of new mating pheromones of the ciliate Euplotes raikovi

Cell union in mating pairs in the ciliate Euplotes raikovi is controlled by a system of multiple mating types which are inherited with alleles codominant at the genetic locus mat and expressed via diffusible mating pheromones. The mating pheromones Er-2, Er-3, and Er-11 were purified from cells homozygous for the mat-2, mat-3, and mat-11 alleles, respectively. These pheromones are proteins of similar Mr (11,000-12,000) and acidity (pI 3.7-4.0) and are active at a concentration that varies from 2.9 X 10(-12) to 1.2 X 10(-11) M. Data on amino acid composition revealed that an unusually high amount of cysteine (12-15.7%) and poor contents of basic amino acids are common to every pheromone. On the basis of this uniformity in the main biochemical traits, which also holds for the previously purified pheromone Er-1, it was concluded that E. raikovi mating pheromones are members of a family of proteins structurally diversified from each other to varying extents.     

Insulin-like growth factor-II receptors in cultured rat hepatocytes: regulation by cell density

Insulin-like growth factor-II (IGF-II) receptors in primary cultures of adult rat hepatocytes were characterized and their regulation by cell density examined. In hepatocytes cultured at 5 X 10(5) cells per 3.8 cm2 plate [125I]IGF-II bound to specific, high affinity receptors (Ka = 4.4 +/- 0.5 X 10(9) l/mol). Less than 1% cross-reactivity by IGF-I and no cross-reactivity by insulin were observed. IGF-II binding increased when cells were permeabilized with 0.01% digitonin, suggesting the presence of an intracellular receptor pool. Determined by Scatchard analysis and by polyacrylamide gel electrophoresis after affinity labeling, the higher binding was due solely to an increase in binding sites present on 220 kDa type II IGF receptors. In hepatocytes cultured at low densities, the number of cell surface receptors increased markedly, from 10-20,000 receptors per cell at a culture density of 6 X 10(5) cells/well to 70-80,000 receptors per cell at 0.38 X 10(5) cells/well. The increase was not due simply to the exposure of receptors from the intracellular pool, as a density-related increase in receptors was also seen in cells permeabilized with digitonin. There was no evidence that IGF binding proteins, either secreted by hepatocytes or present in fetal calf serum, had any effect on the measurement of receptor concentration or affinity. We conclude that rat hepatocytes in primary culture contain specific IGF-II receptors and that both cell surface and intracellular receptors are regulated by cell density.     

Insulin Binding to Human Astrocytoma Cells and Its Effect on Uridine Incorporation into Nucleic Acid

Binding of [125I]monoiodoinsulin to human astrocytoma cells (U-373 MG) was time dependent, reaching equilibrium after 1 h at 22 degrees C with equilibrium binding corresponding to 2.2 fmol/mg protein: this represents approximately 2,000 occupied binding sites per cell. The t1/2 of 125I-insulin dissociation at 22 degrees C was 10 min; the dissociation rate constant of 1.1 X 10(-2) s-1 was unaffected by a high concentration of unlabeled insulin (16.7 microM). Porcine insulin competed for specific 125I-insulin binding in a dose-dependent manner and Scatchard analysis suggested multiple affinity binding sites (higher affinity Ka = 4.4 X 10(8) M-1 and lower affinity Ka = 7.4 X 10(6) M-1). Glucagon and somatostatin did not compete for specific insulin binding. Incubation of cells with insulin (0.5 microM) for 2 h at 37 degrees C increased [2-14C]uridine incorporation into nucleic acid by 62 +/- 2% (n = 3) above basal. Cyclic AMP, in the absence of insulin, also stimulated nucleoside incorporation into nucleic acid [65 +/- 1% (n = 3)] above basal. Preincubation with cyclic AMP followed by insulin had an additive effect on nucleoside incorporation [160 +/- 4% (n = 3) above basal]. Dipyridamole (50 microM), a nucleoside transport inhibitor, blocked both basal and stimulated uridine incorporation. These studies confirm that human astrocytoma cells possess specific insulin receptors with a demonstrable effect of ligand binding on uridine incorporation into nucleic acid.     

Further evidence for the presence of "septide-sensitive" tachykinin binding sites in tissues possessing solely NK(1) tachykinin receptors

Binding experiments performed with [(125)I]-NKA allowed us to demonstrate the presence of "septide-sensitive" specific binding sites on membranes from rat CHO cells transfected with the NK(1) receptor cDNA (CHO-rat-NK1 cells), human astrocytoma U373 MG, or mouse cortical astrocytes, cells which express NK(1) but neither NK(2) nor NK(3) receptors. In all cases, [(125)I]-NKA was specifically bound with high affinity (2 to 5 nM) to a single population of sites. In the three preparations, pharmacological characteristics of [(125)I]-NKA binding sites were notably different from those of classical NK(1) binding sites selectively labelled with [(125)I]-BHSP. Indeed, the endogenous tachykinins NKA, NPK, and NKB and the septide-like compounds such as septide, SP(6-11), ALIE-124, [Apa(9-10)]SP, or [Lys(5)]NKA(4-10) had a much higher affinity for [(125)I]-NKA than [(125)I]-BHSP binding sites. Interestingly, differences were also found in the ratio of B(max) values for [(125)I]-NKA and [(125)I]-BHSP specific bindings from one tissue to another. These latter observations suggest that these two types of NK(1) binding sites are present on distinct NK(1) receptor isoforms (or conformers). Finally, while several tachykinins and tachykinin-related compounds stimulated cAMP formation or increased inositol phosphate accumulation in CHO-rat-NK1 cells, these compounds only increased the accumulation of inositol phosphates in the two other preparations.     

Insulin-like growth factor II binding to the type I somatomedin receptor. Evidence for two high affinity binding sites

We have previously shown that the antireceptor antibody alpha IR-3 inhibits binding of 125I-somatomedin-C/insulin-like growth factor I (Sm-C/IGF-I) to the 130-kDa alpha subunit of the type I receptor in human placental membranes, but does not block 125I-insulin-like growth factor II (IGF-II) binding to a similar 130-kDa complex in these membranes. To determine whether the 130-kDa 125I-IGF-II binding complex represents a homologous receptor or whether 125I-IGF-II binds to the type I receptor at a site that is not blocked by alpha IR-3, type I receptors were purified by affinity chromatography on Sepharose linked alpha IR-3. The purified receptors bound both 125I-Sm-C/IGF-I and 125I-IGF-II avidly (KD = 2.0 X 10(-10) M and 3.0 X 10(-10) M, respectively). The maximal inhibition of 125I-Sm-C/IGF-I binding by the antibody, however, was 62% while only 15% of 125I-IGF-II binding was inhibited by alpha IR-3. In the presence of 500 nM alpha IR-3, Sm-C/IGF-I bound with lower affinity (KD = 6.5 X 10(-10) M) than IGF-II (KD = 4.5 X 10(-10) M) and IGF-II was the more potent inhibitor of 125I-Sm-C/IGF-I binding. These findings suggest that the type I receptor contains two different binding sites. The site designated IA has highest affinity for Sm-C/IGF-I and is blocked by alpha IR-3. Site IB has higher affinity for IGF-II than for Sm-C/IGF-I and is not blocked by alpha IR-3.     

Type I insulin-like growth factor receptors in human ovarian stroma

Hyperandrogenism observed in a variety of hyperinsulinemic states is thought to be due to an effect of insulin mediated through the type I insulin-like growth factor (IGF) receptors. These receptors, however, have not yet been demonstrated in normal human ovarian cells capable of androgen production. We now report the presence of type I IGF receptors in membrane preparations of human ovarian stroma. The ovarian stromal tissue was obtained from women undergoing indicated oophorectomy. Stromal plasma membranes were prepared. Specific 125I-IGF-I binding was 6.6 +/- 0.2%/100 micrograms protein. The affinity constant estimated by Scatchard analysis was 4.6 X 10(-9) M. 50% inhibition of 125I-IGF-1 binding was observed at 5 ng/ml of IGF-1. Specificity of the 125I-IGF-I-binding sites was confirmed by analogue specificity studies and in experiments utilizing monoclonal antibody to the IGF-I receptor, alpha-IR-3. IGF-II and insulin competed with 125I-IGF-I for the binding sites, but with an affinity significantly lower than that of IGF-I: 50% inhibition was observed at approximately 60 ng/ml of IGF-II or insulin. alpha-IR-3, a monoclonal antibody with high specificity for the type I IGF receptor, effectively inhibited 125I-IGF-I binding in a dose-dependent manner, confirming that the 125I-IGF-I binding was indeed to the type I IGF receptor. We conclude that type I IGF receptors are present in human ovarian stroma. These receptors may mediate effects of insulin on the ovary in hyperinsulinemic insulin-resistant states.     

Receptors for insulin-like growth factors and insulin on murine fetal cortical brain cells

Fetal murine neuronal cells bear somatomedin receptors which can be classified according to their affinities for IGF-I, IGF-II and insulin. Binding of 125I-IGF-I is half-maximally displaced by 7 ng/ml IGF-I while 15- and 700-fold higher concentrations are required for, respectively, IGF-II and insulin. Linear Scatchard plots of competitive-binding data with IGF-I suggest one single class of type I IGF receptors (Ka = 2.6 X 10(9) M-1; Ro = 4500 sites per cell). The occurrence of IGF-II receptors appears from the specific binding of 125I-IGF-II and competition by unlabeled IGF-II; the IGF-II binding sites display a low affinity for IGF-II and no affinity for insulin. IGF-II also interacts with insulin receptors although 50- to 100-fold less potent than insulin in competing for 125I-insulin binding. The presence of distinct receptors for IGF-I, IGF-II and insulin on fetal neuronal cells is consistent with a role of these peptides in neuronal development, although our data also indicate that IGF-I receptors could mediate the growth promoting effects of insulin.     

Cellular receptors for type beta transforming growth factor. Ligand binding and affinity labeling in human and rodent cell lines

Type beta transforming growth factor (beta TGF) purified from human platelets to homogeneity as judged by NH2-terminal amino acid sequence analysis has been labeled with 125I to characterize its interaction with cellular receptors. Binding of 125I-beta TGF to target cells is temperature- and time-dependent, specific, saturable, and reversible. About 1.6-1.9 X 10(4) binding sites/cell with high affinity for beta TGF (Kd = 5.6-7.8 X 10(-11) M and 9.1-14 X 10(-11) M, respectively) are found in NRK-49F and BALB/c 3T3 cells. beta TGF receptors do not appear to undergo acute down-regulation by the ligand. Specific binding of 125I-beta TGF has been observed in several human, rat, and mouse fibroblast lines and in some, but not all, tumor-derived cell lines examined. 125I-beta TGF has been cross-linked to intact cells and isolated membrane preparations using disuccinimidyl suberate. Cells and isolated membranes from human, rat, and mouse origin affinity labeled with 125I-beta TGF exhibit a major labeled species of approximately 280 kilodaltons that has the properties of high affinity and specificity expected from a physiologically relevant beta TGF receptor. Minor labeled species of 70-90 kilodaltons are also labeled by 125I-beta TGF, but they correspond to molecular species with low apparent affinity (Kd approximately 10(-8) M) for 125I-beta TGF.     

Characterization of insulin-like growth factor I receptors on human erythroleukemia cell line (K-562 cells)

Specific insulin-like growth factor I (IGF-I) receptors on a human erythroleukemia cell line (K-562 cells) were identified and characterized. [125I]-IGF-I specifically bound to K-562 cells and the binding was displaced by unlabeled IGF-I in a dose dependent manner, and half maximal inhibition of the binding was observed at 7 ng/ml IGF-I. [125I]IGF-I binding to the cells was displaced by multiplication stimulating activity (MSA) and by porcine insulin, with potencies that were 10, and 100 times less than that of IGF-I, respectively. By an affinity labeling technique, IGF type I receptors were found to be present in the K-562 cells. When the cells were differentiated by hemin (40 microM), specific binding of [125I]IGF-I to the cells was decreased to 56.8 +/- 5.0% of that for undifferentiated cells. Furthermore, at physiological concentration of IGF-I stimulated thymidine incorporation into DNA and increased the number of cells. These data demonstrate that K-562 cells have specific receptors for IGF-I which may be functionally important for these cells, and that the IGF-I binding sites decrease with cell differentiation. This system might be useful in studying the interaction of IGF-I receptors.     

Competition Among Homologous Polypeptide Pheromones of the Ciliate Euplotes raikovi for Binding to Each Other's Cell Receptors

ABSTRACT. Purified preparations of the polypeptide pheromones Er-1 and Er-2 were obtained from type I and II cells of Euplotes raikovi , respectively, radiolabeled, and used to study their ability to compete with each other in binding to the same type I and II cells and to type × cells secreting Er-10, another pheromone homologous to Er-1 and Er-2. It was shown that: 1) These radiolabeled pheromone preparations bind, with similar but not identical affinities, to cells from which they originate as well as to cells of the other types. 2) Every pheromone binding reaction can be completely inhibited by another homologous pheromone added in excess to cells. These results are discussed in relation to phenomena, common in ciliates, of instability of (homotypic) mating pairs formed between genotypically identical cells suspended with a foreign (nonself) pheromone.     

Production and characterization of a murine monoclonal IgM antibody to human C1q receptor (C1qR)

A hybridoma cell line that produces a monoclonal antibody (MAb) to cell surface C1q receptor (C1qR) has been produced by fusion of the P3 X 63-Ag8.653 mouse myeloma cell line with the spleen cells of a CD-1 mouse that had been hyperimmunized with viable Raji cell suspensions (5 X 10(7) cells/inoculum). This MAb, designated II1/D1, is an IgM antibody with lambda-light chain specificity. Radiolabeled or unlabeled, highly purified II1/D1 was used to determine that: this antibody competes for C1q binding sites on C1qR-bearing cells; the molecule recognized by this MAb is the C1qR; and cells that are known to bind C1q also bind II1/D1 in a specific manner. Western blot analysis of solubilized Raji, or U937 cell membranes, showed that the 125I-MAb detected a major protein band of approximately 85,000 m.w. in its unreduced state, indicating that the C1qR is similar, if not identical, in both types of cells. Analyses of 125I-II1/D1 binding experiments revealed that the antibody bound to Raji cells or U937 cells in a specific manner. Uptake of the antibody was saturable, with equilibrium virtually attained within 35 min. Scatchard analysis of the binding data using the intact MAb suggests that the affinity constant KD is 2.9 X 10(-10) M, and at apparent saturation, 24.6 ng of the antibody were bound per 2 X 10(6) cells, giving an estimated 7.8 X 10(3) antibody molecules bound per cell. That the II1/D1 antibody is specifically directed to the C1q was further evidenced by an ELISA in which the ability of C1qR-bearing cells to bind the MAb was abrogated by c-C1q in a specific and dose-dependent manner. These results indicate that the II1/D1 is a specific antibody directed against the C1q and can be a useful tool in studying the biologic interaction of human C1q with its receptors on a variety of cells.     

125I-DPDYN, monoiodo[D-Pro10]dynorphin(1-11): a highly radioactive and selective probe for the study of kappa opioid receptors

The mono- and diiodinated derivatives of the kappa-selective ligand [D-Pro10]dynorphin(1-11), DPDYN, were prepared. Their binding properties at the three opioid receptor types (mu, delta and kappa) were examined and compared to those of the parent peptide. The monoiodo derivative shows a general although moderate decrease in affinity and retains high kappa selectivity (KI mu/KI kappa = 48 and KI delta/KI kappa = 140). The binding properties of the diiodo derivative are found to be dramatically decreased. Radioiodination of DPDYN leads to the monoiodinated peptide with high specific activity (700-800 Ci/mmol). In guinea-pig cerebellum membranes, a kappa-specific tissue, [125I]-labelled monoiodo[D-Pro10]dynorphin(1-11), 125I-DPDYN, interacts specifically and reversibly with a single class of binding sites (Bmax = 118 fmol/mg protein) with a high affinity (KD = 0.12 nM from equilibrium experiments, 0.18 nM from kinetics studies). Therefore, because of its high specific radioactivity, high affinity and reasonably good selectivity, 125I-DPDYN designates itself as the probe of the k-opioid receptor type.     

Characterization of the asialoglycoprotein receptor in a continuous hepatoma line

The asialoglycoprotein receptor has been identified on a continuous human hepatoma cell line, HepG2. This receptor requires Ca2+ for ligand binding and is specific for asialoglycoprotein. There are approximately 150,000 ligand molecules bound/cell at 4 degrees C. These receptors represent a homogeneous population of high affinity binding sites with Kd = 7 X 10(-9) M. From the rate of 125I-ASOR binding at 4 degrees C, kon was 0.95 X 10(6) M-1 min-1. Uptake of 125I-ASOR at 37 degrees C was approximately 0.02 pmol/min/10(6) cells.     

Nerve growth factor receptors. Characterization of two distinct classes of binding sites on chick embryo sensory ganglia cells

Steady state and kinetic studies on the binding of 125I-beta nerve growth factor (NGF) to single cells from sensory ganglia of 8-day-old chick embryos show two distinct, saturable binding sites with dissociation constants of Kd(I) = 2.3 X 10(-11) M and Kd(II) = 1.7 X 10(-9) M. The difference in the affinities is due to different rate constants of dissociation (k-1(I) = 10(-3) s-1, k-1(II) = 2 X 10(-1 s-1). The association to both sites is apparently diffusion controlled (k+1(I) = 4.8 X 10(7) M-1s-1, k+2(II) = 10(7) to 10(8) M-1s-1). The binding of betaNGF to both sites is specific, since none of a number of hormones or proteins tested compete for the binding of 125I-betaNGF to either of those two sites. The heterogeneity of the binding of 125I-betaNGF is not due to heterogeneity of the 125I-betaNGF preparation nor to a negatively cooperative binding. In experiments where the dissociation of 125I-betaNGF is induced by the addition of saturating amounts of unlabeled betaNGF, the ratio of the 125I-betaNGF released with either of the two dissociation rate constants is solely dependent on the occupancy of the two sites before dissociation is started and is independent of the total occupancy of the sites during dissociation. The rate of dissociation of 125I-betaNGF from the higher affinity binding site I is accelerated by unlabeled betaNGF under conditions where the occupancy is both increased and decreased. Although the dissociation characteristics of 125I-beta NGF change with increasing times of exposure of the cells to the ligand, and 125I-beta NGF is degraded after it binds to the cells, these secondary processes do not interfere with the analysis of the binding data. At the lowest concentration of 125I-beta NGF used for the analysis less than 10% of the 125I-beta NGF is degraded. Both kinetic and steady state binding data reveal the two NGF binding sites at 2 degrees C as well as at 37 degrees C.     

Characterization and regulation of alpha-interferon receptor expression in interferon-sensitive and -resistant human lymphoblastoid cells

Interferon-sensitive (IFN-S) and IFN-resistant (IFN-R) Daudi lymphoblastoid cells were studied for IFN-alpha receptor expression and regulation by steady state and kinetic procedures, utilizing a homogeneous 125I-IFN-alpha 2 probe. Heterogeneity in the binding of this probe to IFN-S cells was determined to result from negatively cooperative interactions between an initially homogeneous class of IFN receptor. No such heterogeneity was noted in the IFN-R cells, indicating an apparent difference in the interaction of IFN-alpha 2 with these cells. The apparent dissociation constants (Kd) for IFN-S cell receptors were calculated to be 1 X 10(-10) M and 1 X 10(-8) M, for the high and low affinity sites, respectively. The Kd for sites on the IFN-R cells was estimated to be 4 X 10(-9) M. IFN-R and IFN-S cells expressed 2.4 X 10(4) and 3.5 X 10(4) binding sites per cell, respectively, representing an increase of at least 6-fold over previous reports of IFN-S Daudi IFN receptor density. Both IFN-S and IFN-R cells were capable of down-regulating expression of the IFN-alpha receptor in response to low concentrations of IFN-alpha 2. Furthermore, both cell lines were shown to be capable of internalizing specifically bound 125I-IFN-alpha 2 to an equivalent degree. Accordingly, we propose that the relative insensitivity of the Daudi IFN-R phenotype involves the loss of a high affinity interaction between cellular receptors and IFN-alpha 2, in addition to the reduced level of expressed low affinity binding sites.     

Specific and common epitopes in mating pheromones of Euplotes raikovi revealed by monoclonal antibodies.

Polypeptide mating pheromones Er-1 and Er-2, purified from the supernatant of Euplotes raikovi cultures of mating type I and mating type II, respectively, were used to immunize mice and obtain monoclonal antibodies. Five hybridoma clones producing antibodies specific to the mating pheromones were selected. They were analyzed for immunospecificity by immunoperoxidase assay, immunoblotting, and for their efficacy in inhibition of mating pheromone activity. Monoclonal antibodies from two hybridoma clones recognized only the mating pheromone used as antigen: those from the other three clones reacted, to comparable extents, with both mating pheromones. On the basis of these results it was assumed that two immunogenic sites exist in Er-1 and Er-2, one specific and the other common to both mating pheromones.     

Characterization of transforming growth factor-beta (TGF-beta) receptors on BeWo choriocarcinoma cells including the identification of a novel 38-kDa TGF-beta binding glycoprotein.

Transforming growth factor-beta (TGF-beta) is a potential mediator of placental trophoblast functions, including differentiation, hormone production, endometrial invasion, and immunosuppression. Equilibrium binding and affinity-labeling assays were used to investigate the binding characteristics of TGF-beta 1 and TGF-beta 2 on an established human choriocarcinoma trophoblastic cell line (BeWo). The equilibrium binding experiments indicated that the BeWo cells exhibited similar average affinities and total number of binding sites for TGF-beta 1 and TGF-beta 2. The Kd values obtained from Scatchard analyses were approximately 65 pM for 125I-TGF-beta 1 and approximately 40 pM for 125I-TGF-beta 2, with 70,000 and 85,000 sites per cell, respectively. Competitive equilibrium binding experiments indicated that TGF-beta 1 and TGF-beta 2 were equipotent (apparent half maximal inhibition [IC50] approximately 70 pM) and that all binding sites were capable of recognizing both isoforms. Affinity-labeling studies with 125I-TGF-beta 1 and 125I-TGF-beta 2 and the chemical cross-linking agent bis(sulfosuccinimidyl)suberate (BS3) revealed a predominant type III/betaglycan receptor, a low level of apparently heterogeneous type I and II receptors and an additional novel 38-kDa TGF-beta binding glycoprotein that was present both under reducing and nonreducing conditions on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Affinity-labeling saturation and competition studies indicated that the type III/betaglycan component appears to have a 7-fold higher capacity for TGF-beta 1 than for -beta 2 yet exhibits a 5- to 10-fold higher affinity for TGF-beta 2 than for -beta 1. The 38-kDa TGF-beta binding component, an N-linked glycoprotein, exhibits a higher affinity for TGF-beta 2 than for -beta 1 that is strikingly similar to that of the type III/betaglycan receptor. This 38-kDa binding protein appears to be upregulated after methotrexate-induced differentiation of the BeWo cells.     

Receptor-binding properties of a purified fragment of the 4070A amphotropic murine leukemia virus envelope glycoprotein.

A 208-amino-acid amino-terminal fragment of the 4070A amphotropic murine leukemia virus envelope glycoprotein contains all of the determinants required to recognize cell surface amphotropic receptors. This fragment was fused with a streptavidin-binding tag, expressed in Sf9 insect cells by using a baculovirus vector, and purified to homogeneity. The (125)I-labeled purified fragment (AS208) specifically bound various cell lines susceptible to amphotropic murine leukemia virus infection. The number of AS208-binding sites was in the range of 7 X 10(4) to 17 X 10(4) per cell. Quantitative analysis of binding revealed that AS208-binding sites are heterogeneous with regard to ligand binding affinity or that cooperativity exists between receptors. Competition experiments showed that the concentration of AS208 required to inhibit virus entry was lower than that required to inhibit the binding of virus particles at the cell surface. Taken together, these data suggested that amphotropic envelope-binding sites present at the cell surface do not act independently and do not participate equally in virus infection.