Binding of recombinant-produced interferon beta ser to human lymphoblastoid cells. Evidence for two binding domains

Human interferon beta (IFN beta ser), produced by recombinant DNA technology, was radiolabeled to approximately one atom of iodine-125/molecule of interferon without detectable loss of antiviral activity. At 37 degrees C, binding of 125I IFN beta ser occurred rapidly (t1/2max less than or equal to 15 min) followed by internalization and degradation of bound ligand. Kinetic analysis at 4 degrees C indicated diffusion-limited association kinetics independent of 125I IFN beta ser concentration. Dissociation of bound 125I IFN beta ser from Daudi cells was slow (t1/2 = 1.2 h) of bound radiolabeled ligand was observed in the presence of unlabeled IFN beta ser, naturally produced IFN beta, and IFN alpha 6, but was not observed with unlabeled IFN gamma or nonspecific proteins. Concomitantly, equilibrium analysis indicated heterogeneous binding of 125I IFN beta ser to six cell lines of lymphoid origin consistent with either negative cooperativity or two populations of receptors. Analysis of binding of 125I IFN beta ser to Daudi cell receptors in the presence of unlabeled IFN alpha 6 suggested that one receptor served both ligands. The latter conclusion was supported by results of chemical cross-linking experiments in which an 125I IFN beta ser/receptor complex (Mr 120,000-130,000) was observed following sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This complex was absent when binding occurred in the presence of either excess unlabeled IFN beta ser or IFN alpha 6.     

Interferon-alpha down-regulates insulin receptors in lymphoblastoid (Daudi) cells. Relationship to inhibition of cell proliferation

The Daudi line of human lymphoblastoid cells requires insulin and transferrin for growth in serum-free medium and is highly sensitive to the inhibitory effect of human leukocyte interferon (IFN-alpha) on cell proliferation. A variant subline of Daudi cells, which is resistant to the antiproliferative action of IFN-alpha, also has been grown in serum-free medium containing insulin and transferrin. The proliferation of IFN-sensitive and -resistant Daudi cells is dependent on the occupancy of insulin receptors, with optimal cell proliferation observed at high receptor occupancy (nearly 100%). No evidence was found for receptors for insulin-like growth factor I on Daudi cells. IFN treatment of IFN-sensitive cells decreased the capacity of the cells to bind 125I-insulin. The altered binding capacity was due to diminished specific, lower affinity insulin binding, as detected at high 125I-insulin concentrations. Higher affinity insulin binding was not altered by IFN. Insulin binding was also reduced in detergent-solubilized extracts from IFN-treated sensitive Daudi cells and the magnitude of the effect was comparable to that observed in intact cells. This indicates that the total number of insulin binding sites (surface + internal) is decreased in IFN-treated sensitive cells. Insulin binding to IFN-sensitive cells decreased linearly with time between 6 and 48 h from the addition of IFN. The effect on lower affinity insulin binding developed more rapidly than the inhibitory effect of IFN on cell proliferation. The insulin-binding capacity of Daudi cells resistant to the antiproliferative effect of IFN was unaffected by IFN, despite the fact that these cells contain as many cell surface IFN receptors as sensitive cells. These observations raise the possibility that lower affinity insulin binding is important in the growth-promoting actions of insulin.     

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.     

Identification and characterization of cellular receptors for the growth regulator, oncostatin M

Oncostatin M is a polypeptide growth regulator produced by activated T cells and phorbol ester-treated U937 cells. To identify specific cellular receptors for this factor, we have characterized the binding of 125I-labeled oncostatin M to a variety of normal and malignant mammalian cells. Recombinant oncostatin M was labeled with 125I with full retention of growth inhibitory activity on A375 melanoma cells. 125I-Oncostatin M bound to sensitive cells in a time- and temperature-dependent fashion. Binding was specifically inhibited by unlabeled native or recombinant oncostatin M, but not by other polypeptide growth factors tested. Binding to human leukemic and normal blood cells was generally less than to nonhematopoietic cells. With four different cell lines, maximal growth inhibition by oncostatin M was achieved at less than maximal binding site occupancy. Scatchard graphs of direct binding data were curvilinear and indicated that 125I-oncostatin M bound with higher apparent affinity at lower 125I-oncostatin M concentrations. Using a two binding site model, affinity constants of Kd1 = 11 +/- 11 pM and Kd2 = 1000 +/- 380 pM were extrapolated from binding data with A375 cells, and values of Kd1 = 3 +/- 2 pM and Kd2 = 400 +/- 44 pM from A549 cells. The major 125I-oncostatin M binding species in a number of mammalian cell lines was identified by chemical cross-linking as a specific protein(s) of Mr = 150,000-160,000. 125I-Oncostatin M was internalized (t1/2 = 30 min) and degraded subsequent to binding to a responsive cell line.(ABSTRACT TRUNCATED AT 250 WORDS)     

Treatment of lymphoblastoid cells with interferon decreases insulin binding

Lymphoblastoid Daudi cells, which are highly sensitive to growth inhibition by interferon (IFN), can be grown in a defined serum-free medium containing insulin, transferrin, and albumin as the only proteins. We examined whether the growth inhibition by IFN could be in part due to a change in receptors for insulin or transferrin. Cells treated for at least 2 days with 100 units/ml of IFN-alpha 2 bound less 125I-insulin and after 3 days of treatment this binding was reduced by more than 50%. No change in the binding of 125I-transferrin was observed. Treatment with IFN of Raji cells, which are resistant to growth inhibition by IFN, resulted in a similar decrease in 125I-insulin binding. Growth inhibition of Daudi cells by serum deprivation had no effect on 125I-insulin binding. Therefore, the IFN-induced loss of insulin binding sites is not a consequence of growth inhibition.     

A soluble binding protein specific for interleukin 1 beta is produced by activated mononuclear cells

Soluble interleukin 1 (IL 1) binding proteins were identified by gel filtration and covalent cross-linking of 125I IL 1 in normal human serum and inflammatory exudate. High molecular weight 125I IL 1 protein complexes occurred with both IL 1 alpha and IL 1 beta, however, high molecular weight binding appeared to be non-specific. One specific IL 1 beta binding protein was observed to elute at approximately 100 kDa on gel filtration when bound to 125I IL 1 beta. This complex migrated as a broad band at 60 kDa when covalently cross-linked and analyzed by SDS-PAGE. The protein did not bind 125I IL 1 alpha and 125I IL 1 beta binding was only displaceable by excess cold IL-1 beta. The production of the specific IL 1 beta binding protein was assessed in a number of cell populations. Unstimulated peripheral blood mononuclear cells (PBMNC) did not produce the binding protein, but stimulation with phytohemagglutinin (PHA) caused production within 24 hr and binding protein levels remained elevated for up to 7 days. Stimulation with lipopolysaccharide (LPS) and IL 1 alpha did not consistently induce synthesis of the binding protein. Ligand-binding studies were performed to compare solubilized EL 4 NOB.1 cell membrane IL 1 receptor (sIL 1R) with semi-purified IL 1 beta binding protein from pooled synovial fluid. The sIL 1R preparation bound ligand with an affinity of 168 pM while the IL 1 beta binding protein bound 125I IL 1 beta with an affinity of 370 pM. This protein may function as an important carrier molecule for IL 1 beta and determine its distribution and kinetics in vivo.     

Interleukin 1 receptors on rabbit articular chondrocytes: relationship between biological activity and receptor binding kinetics

This study gives an account of the biologic and kinetic binding properties of interleukin 1 alpha (IL 1 alpha), interleukin 1 beta (IL 1 beta), and Glu-4 (an NH2-terminal mutant of IL 1 beta) to interleukin 1 (IL 1) receptors in rabbit articular chondrocytes. All three IL 1's demonstrated full agonist properties in their ability to stimulate prostaglandin E2 (PGE2) synthesis. IL 1 alpha was 23-fold more biologically active than IL 1 beta, which was around 110-fold more active than Glu-4 based on the concentration of IL 1 required for half-maximal stimulation of PGE2. The binding of all three ligands was concentration-dependent and saturable at 4 degrees C. Scatchard analysis of receptor binding data showed that the dissociation constant (KD) of IL 1 alpha was 46 +/- 12 pM, and the receptor density was 3120 sites/cell. The association of IL 1 alpha at 4 degrees C did not attain equilibrium until after 10 h at 100 pM of 125I-labeled IL 1 alpha. The dissociation of bound IL 1 alpha was very slow, t1/2 of 21 h, although only one class of high-affinity receptors was detected. The KD of IL 1 beta binding was 72 +/- 3 pM with a receptor density of 800 +/- 40 sites/cell. Dissociation of bound 125I-labeled IL 1 beta at 4 degrees C appeared to indicate the presence of two receptor subsets, a fast and a slower component with a t1/2 of 2 min and 5 h, respectively. The receptor binding affinity of Glu-4 was 324 +/- 3 pM, in line with its reduced biologic activity. Both IL 1 alpha and IL 1 beta are rapidly internalized in chondrocytes in a time- and temperature-dependent manner.     

Role of protein kinase C in induction of gene expression and inhibition of cell proliferation by interferon alpha.

Recent studies have suggested that protein kinase C (PKC) may be involved in the mechanism of signal transduction by which members of the interferon (IFN) family regulate gene expression and cell phenotype. We have investigated the role of PKC in the control of cell growth and gene expression by IFN alpha in Daudi cells. Treatment of these cells with two analogues of staurosporine, which are potent inhibitors of PKC, completely blocked the induction by IFN alpha of the mRNA for 2',5'-oligoadenylate synthetase and the 6-16 gene. These compounds also inhibited cell proliferation and thymidine incorporation in this system. In contrast, the protein kinase inhibitor 1-(5-isoquinolinylsulphonyl)-2-methylpiperazine (H7) did not significantly inhibit the induction of these genes by IFN alpha and had no effect on Daudi cell growth or thymidine incorporation in the presence or absence of IFN alpha. No effect of IFN alpha on total PKC activity could be observed, and there were no significant changes in the overall levels of individual PKC isoforms or their mRNA following IFN alpha treatment. In contrast, treatment of Daudi cells with the phorbol ester 12-O-tetradecanoylphorbol 13-acetate, which also inhibits cell proliferation, strongly down-regulated PKC. These data suggest that the activity of a PKC species, or a closely related enzyme, may be required both for continued cell proliferation and the response to IFN alpha in Daudi cells, but that IFN-induced growth inhibition does not involve overall down-regulation or change in activity of PKC.     

Receptor-induced degradation of atrial natriuretic peptide by a rabbit carotid artery smooth muscle cell

We have used a smooth muscle cell line isolated from rabbit carotid artery (RCA) as a model system with which to study the expression of atrial natriuretic peptide (ANP) receptors and, in addition, the receptor-mediated degradation of ANP. RCA cells bind rat alpha ANP-(1-28) reversibly at 37 C, apparently to a single class of high affinity (Kd approximately equal to 50 pM) binding sites (approximately equal to 20,000 sites per cell). Binding of rat alpha ANP-(1-28) elicits rapid accumulation of intracellular cGMP. However, the concentrations of rat alpha ANP-(1-28) and related peptides, abbreviated at the N- and C-terminals, required to stimulate cGMP synthesis are substantially greater than those required for binding. Analysis by HPLC of 125I-labeled rat alpha ANP-(1-28) bound to RCA cells at 37 C illustrates the rapid and continuous degradation of the radioiodinated rat alpha ANP-(1-28) to two radiolabeled products, one of which, 125I-labeled tyrosine is the major radiolabeled component that dissociates from the cells. Measurement of rat alpha ANP-(1-28) interaction with RCA cells by radioligand binding techniques therefore subsumes several processes. One of these processes is the rapid and continuous degradation of specifically bound ANP by these cells and perhaps also other target cells that respond to ANP.     

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.     

Identification of somatostatin receptors by covalent labeling with a novel photoreactive somatostatin analog

We have synthesized two photoreactive derivatives of somatostatin, namely [125I-Tyr11,azidonitrobenzoyl (ANB)-Lys4]somatostatin and [125I-Tyr11,ANB-Lys9]somatostatin, and used them to characterize somatostatin receptors biochemically in several cell types. Saturation binding experiments carried out in the dark demonstrated that [125I-Tyr11,ANB-Lys4]somatostatin bound with high affinity (KD = 126 +/- 39 pM) to a single class of binding sites in GH4C1 pituitary cell membranes. The affinity of this analog was similar to that of the unsubstituted peptide [125I-Tyr11]somatostatin (207 +/- 3 pM). In contrast, specific binding was not observed with [125I-Tyr11,ANB-Lys9]somatostatin. The binding of both [125I-Tyr11,ANB-Lys4]somatostatin and [125I-Tyr11]somatostatin was potently inhibited by somatostatin (EC50 = 300 pM) whereas at 100 nM unrelated peptides had no effect. Furthermore, both pertussis toxin treatment and guanyl-5'yl imidophosphate (Gpp(NH)p) markedly reduced [125I-Tyr11,ANB-Lys4]somatostatin binding. Thus, [125I-Tyr11,ANB-Lys4]somatostatin binds to G-protein coupled somatostatin receptors with high affinity. To characterize these receptors biochemically, GH4C1 cell membranes were irradiated with ultraviolet light following the binding incubation, and the labeled proteins were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. A major band of 85 kDa was specifically labeled with [125I-Tyr11,ANB-Lys4]somatostatin but not with [125I-Tyr11,ANB-Lys9]somatostatin or [125I-Tyr11]somatostatin. The binding affinity of the 85-kDa protein for [125I-Tyr11,ANB-Lys4]somatostatin was very high (Kd = 34 pM). Labeling of this protein was inhibited competitively by somatostatin (EC50 = 140 +/- 80 pM) but not by unrelated peptides. Furthermore, this band was not labeled in pertussis toxin-treated membranes or in untreated membranes incubated with Gpp(NH)p. Finally, [125I-Tyr11,ANB-Lys4]somatostatin specifically labeled bands of 82, 75, and 72 kDa in membranes prepared from mouse pituitary AtT-20 cells, rat pancreatic acinar AR4-2J cells, and HIT hamster islet cells, respectively. Thus, [125I-Tyr11,ANB-Lys4]somatostatin represents the first photolabile somatostatin analog able to bind to receptors with high affinity. Our studies demonstrate that this novel peptide covalently labels specific somatostatin receptors in a variety of target cell types.     

Binding of human recombinant 125I-interferon gamma to receptors on human cells

Recombinant human interferon gamma (rIFN-gamma) produced in Escherichia coli was labeled with 125I to study its binding to receptors of HeLa and lymphoblastoid cells. All the cell lines examined had receptors for rIFN-gamma, although the binding varied considerably among different cell lines. The binding of 125I-rIFN-gamma was competed up to 90% by the addition of unlabeled rIFN-gamma, although not by the addition of IFN-alpha or -beta. By adding increasing concentrations of unlabeled rIFN-gamma to binding assays containing a constant amount of 125I-rIFN-gamma, we determined a KD of 3.7 and 6.3 X 10(-10) M for its binding to Daudi and HeLa cells, respectively. About 13,000 receptors per cell were present in Daudi and 5,000 in HeLa cells. The Mr of the IFN-gamma/receptor complex was determined by cross-linking experiments to be about 125,000. This complex is smaller than the IFN-alpha/receptor complex that has a Mr of about 140,000. The rIFN-gamma receptor was down-regulated when HeLa cells were treated with this interferon, but not when these cells were treated with IFN-beta. These findings suggest that the receptors for IFN-alpha and -gamma differ in several characteristics. The turnover of the rIFN-gamma receptor was measured by inhibiting protein synthesis with cycloheximide and the half-life of this receptor was found to be 2 h. The unglycosylated rIFN-gamma was bound to cellular receptors with an affinity similar to that previously reported for natural IFN-gamma. The lymphoblastoid cell lines examined had high affinity receptors for rIFN-gamma, but did not respond to treatment with this IFN with an induction of the synthesis of the enzyme (2'-5')oligo(A) synthetase, whereas HeLa cells responded to rIFN-gamma. The reason for the lack of response of lymphoblastoid cells is presently unknown.     

Vascular smooth muscle cells express distinct transforming growth factor-beta receptor phenotypes as a function of cell density in culture

Transforming growth factor-beta (TGF-beta) is a bifunctional, density-dependent regulator of vascular smooth muscle cell (SMC) proliferation in vitro (at sparse densities SMC are growth-inhibited by the peptide, whereas at confluent densities TGF-beta potentiates their growth). We have used affinity labeling and ligand binding techniques to characterize cell surface receptors for TGF-beta under sparse and confluent culture conditions. Confluent SMC, whose growth are promoted by TGF-beta, exhibited a single class of high affinity TGF-beta binding sites (Kd = 6 pM, 3,000 sites/cell). In contrast, sparse SMC (whose growth are inhibited by TGF-beta) expressed two distinct classes of high affinity binding sites with binding constants of 6 pM (3,000 sites/cell) and 88 pM (11,000 sites/cell). By affinity labeling using 125I-TGF-beta and disuccinimidyl suberate cross-linking, confluent cells were found to express a major Mr = 280,000 TGF-beta receptor as well as trace amounts of low molecular weight (Mr = 85,000 and 65,000) receptor subtypes. All three of these receptors were determined, by ligand competition, to show similar affinity for TGF-beta. The predominant receptor subtypes expressed by sparse SMC exhibited apparent Mr = 75,000 and 65,000. In ligand competition experiments, the Mr = 75,000 receptor subtype (never present in confluent cultures) exhibited lower relative affinity for TGF-beta than did the Mr = 65,000 form. The ability of TGF-beta to inhibit SMC proliferation, therefore, correlates with the expression of a unique TGF-beta-binding protein on the SMC surface. The data suggest that TGF-beta may exert opposite biological effects on the same cell type via an interaction with distinct, selectively expressed receptor subtypes.     

The kinetics of S phase entry by FMP2.1: effect of IL-3 and GM-CSF receptor expression and ligand affinity

FMP2.1, a cloned cell line which has morphological characteristics of mast/basophil cells, requires either interleukin 3 (IL-3) or granulocyte-macrophage colony-stimulating factor (GM-CSF) for both survival and proliferation. IL-3 and GM-CSF were equally effective as proliferative stimuli. FMP2.1 cells were sensitive to growth factor stimulation in the G1 phase, which has a duration of 9.5 h. G1 cells were selected from FMP2.1 in log phase growth on the basis of Hoechst 33324 staining using a fluorescence activated cell sorter (FACS). It was found that G1 phase cells had to be exposed to either IL-3 or GM-CSF for approximately 1 h for cells to enter S (greater than 20%); without growth factor, FMP2.1 remained in G1 unable to progress into S. Receptor expression was analyzed to further understand this rapid activation of FMP2.1 into cycle. Autoradiography using either 125I-IL-3 or 125I-GM-CSF showed that most cells express both receptor types. In the presence of saturating concentrations of IL-3, FMP2.1 have a relatively high number of IL-3 receptors (42,000/cell) compared to other cell lines (e.g., 32D cl23; 13,000 receptors/cell), and far outnumber GM-CSF receptors on the same cells (600 receptors/cell). Although average IL-3 receptor expression differed for FMP2.1- and IL-3-dependent 32D cl23, the concentration-dependent proliferative response to IL-3 was essentially identical for both cell types. Scatchard plot analysis for 125I-IL-3 and 125I-GM-CSF binding to FMP2.1 cells at 4 degrees C revealed a single type of binding site for both ligands, with dissociation constants (Kd) of approximately 1 nM for GM-CSF and 8 pM for IL-3. The relatively high affinity IL-3 binding to a large number of available IL-3 receptors was associated with a shallow dose response of the FMP2.1 cells to IL-3, compared to the steep GM-CSF dose response which was mediated through fewer receptor sites of relatively low affinity. Mitogenic stimulation of G1 phase cells was observed with either IL-3 or GM-CSF, and appeared to be unaffected by differences in receptor number or binding affinity.     

Radiolabeling of the interferon-alpha receptor

The action of alpha interferon (IFN-alpha) is initiated by its binding to a specific cell-surface glycoprotein, the IFN-alpha receptor, which is not well characterized. IFN-alpha A was reacted with an 125I-labeled, cleavable, heterobifunctional reagent. The derivatized IFN-alpha A was bound to human Daudi cells and photoactivated, forming a covalent IFN/receptor complex of apparent molecular weight 130,000-140,000 by SDS-polyacrylamide gel electrophoresis. Cleavage of the complex produced a new 125I-labeled 110 kDa band, representing the 125I-labeled IFN-alpha receptor free of IFN-alpha. This result provides a better estimate of the apparent molecular weight of the IFN-alpha receptor, and also provides a tool for tracking the migration of the free receptor in SDS-polyacrylamide gel electrophoresis.     

Norepinephrine-induced down regulation of alpha 1 adrenergic receptors in cultured rabbit aorta smooth muscle cells

Drug-induced refractoriness of alpha-adrenergic receptor-mediated vasoconstriction may be a clinically important phenomenon. We have investigated the possible molecular mechanisms underlying this phenomenon in cultured vascular smooth muscle cells derived from the rabbit aorta. alpha 1-Adrenergic receptors were identified in membranes prepared from these cells by [125I]HEAT binding. The radioligand bound to a high affinity site (Kd = 140 pM) in a saturable fashion (202 fmol/mg protein). Adrenergic agonists and antagonists competed for binding of [125I]HEAT with the expected order of potency for an alpha 1-receptor, (-)epinephrine greater than or equal to (-) norepinephrine greater than (+)epinephrine greater than isoproterenol and prazosin greater than phentolamine greater than yohimbine. Exposure of cells for 26 hours to 10 microM norepinephrine resulted in a 70% decrease in the number of alpha 1-receptors as measured by [125I]HEAT binding without any significant change in the affinity of the receptor for the ligand. When the alpha-receptors were blocked with 10 microM phentolamine the loss of receptors induced by norepinephrine was completely prevented. Similar down-regulation of the [125I]HEAT binding sites was observed when the alpha 1-agonist phenylephrine was used instead of norepinephrine. It is concluded that alpha-agonists induce down-regulation of aortic smooth muscle alpha 1-receptors. This reduction of alpha-receptors could be important in the mechanisms by which vascular smooth muscle develops refractoriness to alpha-adrenergic stimulation.     

Identification and characterization of endothelin binding sites in rat renal papillary and glomerular membranes

The present study was designed to identify and characterize specific endothelin binding sites in membranes of rat renal papillae and glomeruli which appear to be target tissues for this new peptide hormone. Saturation binding studies indicate that the sites have a high and uniform affinity. The dissociation constants averaged 662 +/- 151 and 1309 +/- 123 pM and the receptor densities 7666 +/- 920 and 5831 +/- 348 fmol/mg protein for papillary and glomerular membranes, respectively. Endothelin 1, endothelin 3 and sarafotoxin all inhibited [125I]-endothelin binding with IC50's in the 100-300 pM range, whereas unrelated peptides, namely angiotensin II, atrial natriuretic peptide, and platelet-derived growth factor failed to compete for [125I]-endothelin binding. Deletion of the carboxyterminal tryptophan in endothelin 1 reduced its affinity for glomerular binding sites by 2 orders of magnitude. Specific endothelin binding to these membranes was maximal at pH 4 and was markedly inhibited as the pH was raised above 8. When [125I]-endothelin is covalently linked to glomerular membrane binding sites, SDS-PAGE of these solubilized membranes followed by autoradiography reveals a predominant specifically labeled band of 45 kDa. Whether this band represents a subunit of the endothelin receptor(s), the receptor proper, or an intracellular endothelin binding protein remains to be determined.     

Expression of IFN-alpha and IFN-gamma receptors on normal human small resting T lymphocytes and large granular lymphocytes

The expression of interferon (IFN) receptors was studied on freshly isolated human lymphocytes from normal donors. Highly enriched populations of small resting T lymphocytes and large granular lymphocytes (LGL) were found to constitutively express high-affinity receptors for IFN-alpha and IFN-gamma. Both types of lymphocytes also had lower-affinity IFN-alpha binding sites. T lymphocytes had a mean of 230 IFN-alpha and 520 IFN-gamma high-affinity receptors per cell, whereas LGL had 520 IFN-alpha and 760 IFN-gamma receptors. However, because LGL were larger than the T lymphocytes, the IFN receptor density was similar on the two types of lymphocytes. The affinity of binding was similar on the two types of normal lymphocytes and on the cultured lymphoblastoid cell line Daudi. The number of IFN receptors per cell and the affinities of the IFN-receptor interactions varied little among the normal donors. Both the freshly isolated normal lymphocytes and the cultured cell line Daudi had separate receptors for type I (alpha and beta) and type II (gamma) IFN. Taken together, our data indicate that two types of freshly isolated normal lymphocytes constitutively express IFN receptors that are similar to those present on the lymphoblastoid cell line Daudi derived from a patient with Burkitt's lymphoma.     

Comparative antiproliferative and receptor binding activities of interferons alpha and beta on lymphoblastoid and melanoma cells

The relative antiproliferative and receptor binding characteristics of the hitherto little-characterized interferon alpha 4a on cells of lymphoid and epithelial origin are compared with two other type I interferons, alpha 2 a and beta. Using the lymphoblastoid cell line, Daudi, interferons alpha 4 a and alpha 2 b had similar antiproliferative activity, and were about 10-fold more active than IFN beta. By contrast, using the melanoma cell line Sk-Mel-28, IFN beta was the most active, whereas IFN alpha 2b and IFN alpha 4a were respectively 60-fold and greater than 1000-fold less active than on Daudi cells. Receptor binding did not correlate with antiproliferative sensitivities, but confirmed a shared receptor component for these three interferons. These results indicate that the antiproliferative activities of three type I IFNs differs markedly on different cell types and that this is unlikely to be due to receptor binding, but more likely a post receptor binding event.