Characterization of a receptor for C5a anaphylatoxin on human eosinophils

The complement anaphylatoxin peptide C5a is well known to activate human polymorphonuclear leukocytes through receptor-mediated processes. C5a has also been reported to activate eosinophils for both chemotaxis and hexose uptake. We characterized the receptor molecule for human C5a on human eosinophils and compared it with the receptor on human neutrophils. At 4 degrees C, uptake of 1 nM 125I-C5a reaches equilibrium within 10 min on both cell types. Binding of 125I-C5a occurs over a concentration range comparable to that which stimulates lysosomal enzyme release and hexose uptake in both cell types. Scatchard analyses of the data indicate the presence of two receptor populations on eosinophils; a high affinity receptor with 15,000-20,000 sites/cell and a Kd of 3.1 +/- 0.6 x 10(-11) M, and a low affinity receptor with approximately 375,000 sites/cell and a Kd of 1 x 10(-7) M. Parallel experiments with neutrophils indicate the presence of a single receptor population with approximately 90,000 sites/cell and a Kd of 4.8 +/- 0.1 x 10(-10)M. The eosinophil receptor molecule was further characterized by covalently cross-linking 125I-C5a to cells followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the solubilized material. Autoradiography indicates the presence of a dominant C5a-eosinophil receptor complex with an apparent mass of 60-65 kDa. The corresponding neutrophil-C5a receptor complex has an apparent mass of 50-52 kDa as observed by others. When the cross-linked 125I-C5a-receptor complex was treated with cyanogen bromide, different patterns were observed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis for neutrophils and eosinophils. Thus, human eosinophils have a receptor for C5a anaphylatoxin which appears to be distinct from the C5a receptor present on human neutrophils.     

Demonstration of a specific C3a receptor on guinea pig platelets

Guinea pig platelets reportedly contain receptors specific for the anaphylatoxin C3a based on both ligand-binding studies and functional responses. A portion of the human 125I-C3a that binds to guinea pig platelets is competitively displaced by excess unlabeled C3a; however, the majority of ligand uptake was nonspecific. Uptake of 125I-C3a by guinea pig platelets is maximal in 1 min, and stimulation of guinea pig platelets by thrombin, ADP, or the Ca2+ ionophore A23187 showed little influence on binding of the ligand. Scatchard analysis indicated that approximately 1200 binding sites for C3a exist per cell with an estimated Kd of 8 x 10(-10) M. Human C3a des Arg also binds to guinea pig platelets, but Scatchard analysis indicated that no specific binding occurred. Because the ligand-binding studies were complicated by high levels of nonspecific uptake, we attempted to chemically cross-link the C3a molecule to a specific component on the platelet surface. Cross-linkage of 125I-C3a to guinea pig platelets with bis(sulfosuccinimidyl)suberate revealed radioactive complexes at 105,000 and 115,000 m.w. on SDS-PAGE gels by autoradiographic analysis. In the presence of excess unlabeled C3a, complex formation was inhibited. No cross-linkage could be demonstrated between the inactive 125I-C3a des Arg and the putative C3a-R on guinea pig platelets. Human C3a, but not C3a des Arg induces serotonin release and aggregation of the guinea pig platelets. Human C3a was unable to induce either serotonin release or promote aggregation of human platelets. Uptake of human 125I-C3a by human platelets was not saturable, and Scatchard analysis was inconclusive. Attempts to cross-link 125I-C3a to components on the surface of human platelets also failed to reveal a ligand-receptor complex. Therefore, we conclude that guinea pig platelets have specific surface receptors to C3a and that human platelets appear devoid of receptors to the anaphylatoxin.     

Identification of the polymorphonuclear leukocyte C5a receptor

The peptide C5a is thought to play an important role in the inflammatory response primarily through its action on the polymorphonuclear leukocyte (PMN). The receptor for C5a on human PMN has now been identified by affinity labeling. Cross-linking 125I-C5a to intact PMN with disuccinimidyl suberate produced a species that had a molecular mass on sodium dodecyl sulfate gels of 5.2 X 10(4) daltons. We believe this species represents a complex between C5a and its receptor for the following reasons. The band is eliminated if the cross-linking experiment is performed in the presence of a large excess of unlabeled C5a, but is unaffected by the presence of nonspecific protein or the chemotactic factors N-formyl-Met-Leu-Phe and leukotriene B4. The 5.2 X 10(4)-dalton species is not observed if the cross-linker is omitted. Finally, the dose-response curves for the inhibition of binding of 125I-C5a by unlabeled C5a and the inhibition of cross-linking are similar. Subtraction of the molecular mass of C5a from that of the complex gives a molecular mass for the binding moiety of the C5a receptor of 4.0 X 10(4) daltons.     

Interaction of human monomeric C3b with its receptor (complement receptor type 1, CR1) on neutrophils. Evidence for negative cooperativity

The binding of highly purified monomeric 125I-C3b to its receptor (CR1) on resting human polymorphonuclear neutrophils (PMN) was analyzed under equilibrium conditions, at 4 degrees C and low ionic strength. Scatchard analysis of specific binding data yielded curvilinear concave upward plots, which resulted from the presence of site-site interactions of the negative type among PMN C3b-receptors (negative cooperativity), as shown by dissociation kinetic experiments. Indeed, the dissociation rate of 125I-C3b from PMN was markedly increased in the presence of an excess of unlabeled C3b in the dilution medium and was directly dependent on the degree of initial receptor occupancy with the radioligand. These interactions occurred when 2% of the receptors were occupied with 125I-C3b and resulted in a 4-fold decrease in CR1 affinity when the receptor went from its "empty" to its "filled" conformation. In a disease associated with a continuous production of C3b (factor I deficiency), CR1 on in vivo circulating PMN was found to be in a "low affinity" and "high dissociating" state similar to that of normal CR1 at high occupancy. Finally, negative cooperativity among CR1 sites disappeared after PMN activation with chemotactic peptides.     

Identification of the receptor for atrial natriuretic factor on cultured vascular cells

Binding experiments with 125I-atrial natriuretic factor (ANF) followed by covalent attachment with disuccimidyl suberate show that the peptide binds predominantly to a protein of apparent molecular mass of 66,000 daltons on the cell surface of cultured bovine aortic smooth muscle cells. A minor protein species of 180,000 Mr is also visualized after cross-linking. Endothelial cells, however, whose ANF binding parameters differ substantially from smooth muscle cells, also appear to have qualitatively identical 125I-ANF binding proteins. The identity of these putative proteins, as the ANF receptor, is confirmed by findings that covalent attachment of 125I-ANF is saturable, concentration-dependent, and competed by nanomolar concentrations of unlabeled ANF. Furthermore, other peptide hormones such as angiotensin II, glucagon, or insulin are ineffective in competing for 125I-ANF binding and cross-linking to the receptor.     

Covalent cross-linking of vasoactive intestinal polypeptide to its receptors on intact human lymphoblasts

125I-labeled vasoactive intestinal polypeptide (125I-VIP) was covalently cross-linked with its binding sites on intact cultured human lymphoblasts by each of three bifunctional reagents: disuccinimidyl suberate (DSS), ethylene glycol bis(succinimidyl succinate) (EGS), and N-succinimidyl 6-(4'-azido-2'-nitrophenylamino) hexanoate (SANAH). A fourth cross-linking agent with a shorter chain length, N-hydroxysuccinimidyl 4-azidobenzoate (HSAB), was much less effective in cross-linking 125I-VIP to the site. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography demonstrated a band of Mr approximately equal to 50,000 +/- 3,000, regardless of which cross-linker was used. The labeling of this band was specific in that it was prevented by 10(-6) M unlabeled VIP and was partially blocked by the homologous hormones secretin and glucagon. The relative potencies of these peptides in blocking the cross-linking of 125I-VIP to the Mr approximately equal to 50,000 band of the lymphoblasts (VIP greater than secretin greater than or equal to glucagon) were similar to those previously found for competitive inhibition of 125I-VIP binding to its putative high-affinity receptor on these cells. The covalent cross-linking required a bifunctional reagent; it was dependent on both the number of Molt cells and the concentration of 125I-VIP. The apparent molecular weight of the cross-linked species was unchanged by treatment with dithiothreitol. These observations suggest that the Mr = 50,000 species represents 125I-VIP cross-linked to a specific plasma membrane receptor and that the receptor does not contain interchain disulfide bonds.     

Characterization of a novel low affinity receptor for IFN-gamma on adherent human monocytes by radioligand binding studies and chemical cross-linking

Freshly isolated monocytes in suspension express 2000 to 4000 high affinity receptors for IFN-gamma. Because monocytes change phenotypically as they migrate out of the circulation and adhere to extracellular matrix, modulation of the expression of IFN-gamma receptors may occur. In order to determine if adherence alone modulates the receptor for IFN-gamma, we have studied receptor expression in adherent human peripheral blood monocytes. Elutriation-purified monocytes were allowed to adhere to polystyrene overnight at 37 degrees C. These cells now expressed 1 to 2 x 10(5) low affinity (Ka = 10(8) liters/M) receptors for [125I]rIFN-gamma. Binding to this receptor was specific and saturable. The expression of these receptors occurred rapidly (within 3 h) after adherence and was not inhibited by cycloheximide treatment. Binding to the receptor was abrogated by treating cells with trypsin, but was enhanced after treatment with alkaline protease or proteinase K. mAb against the high affinity receptor did not block binding to the low affinity receptor on adherent cells. The low affinity receptor transduced a signal to the cell as measured by the IFN-gamma-induced enhancement in FcR for human IgG1. The structure of the receptor on adherent cells was investigated by chemical cross-linking techniques. A receptor-[125I]rIFN-gamma complex was observed by SDS-PAGE to have a Mr of 180,000 to 200,000. Reduction of this complex with 2-ME resulted in the loss of the high Mr complex and the appearance of a doublet of lower Mr of 68,000 and 82,000. In contrast, cross-linking of monocytes in suspension yielded a complex of 110,000 to 120,000 Mr, which was unchanged upon reduction. Upon adherence, human monocytes express large numbers of a novel receptor for rIFN-gamma which is capable of stimulating the cell. This receptor appears to be composed of at least two components which are disulfide linked and structurally differs from the high affinity receptor on nonadherent monocytes.     

The human interferon-gamma receptor. Purification, characterization, and preparation of antibodies

The receptor for human interferon-gamma (IFN-gamma) was purified from foreskin fibroblasts. Triton X-100 extracts obtained from either intact cells or membrane preparations were passed through an immobilized interferon-gamma column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of eluted fractions revealed a major band of Mr = 95,000 and minor bands of Mr = 80,000 and 60,000. Further purification was obtained by steric exclusion and by lectin chromatography. The purified receptor retained the ability to bind 125I-IFN-gamma with a Kd of 2.2 X 10(-10) M, a value close to that obtained with intact fibroblasts (5 X 10(-10) M). A complex of Mr = 105,000-125,000 was visualized by immunoprecipitation of 125I-IFN-gamma cross-linked to the purified receptor followed by SDS-PAGE and autoradiography. A similar complex was obtained when 125I-IFN-gamma was cross-linked to intact cells. Immunization of mice with the excised SDS-PAGE band of Mr = 95,000 elicited antibodies that blocked the antiviral activity of IFN-gamma and immunoprecipitated the cross-linked complex of 125I-IFN-gamma and its receptor.     

Molecular characteristics of nerve growth factor receptors on PC12 cells

Cross-linking of 125I-nerve growth factor (NGF) to PC12 cells with the photoreactive heterobifunctional agent N-hydroxysuccinimidyl-4-azidobenzoate results in the labeling of two major bands with Mr 158,000 and 100,000 and a minor band with Mr 225,000 as determined by polyacrylamide gel electrophoresis under denaturing and reducing conditions. Binding of 125I-NGF to and cross-linking into all these species is abolished in the presence of excess unlabeled NGF but not in the presence of unlabeled epidermal growth factor, insulin, or bovine pancreatic trypsin inhibitor. When PC12 cells with bound 125I-NGF are incubated in excess unlabeled NGF at 0 degree C prior to cross-linking, only the Mr 158,000 species remains. In addition, binding of 125I-NGF to the Mr 158,000 complex is trypsin-resistant, whereas binding to the Mr 100,000 complex is not. These experiments identify the Mr 158,000 species as the slow NGF-receptor complex (chase stable at 0 degree C) and the smaller Mr 100,000 species as the fast NGF-receptor complex (trypsin sensitive). Furthermore, 125I-NGF bound to the former but not to the latter species is displaced by very-low concentrations of NGF, showing that at least a significant fraction of the high-molecular-weight slow receptor is also a high-affinity receptor. This identification is supported by the finding that chick sensory neurons which possess both high- and low-affinity receptors exhibit two major labeled bands with Mr 145,000 and 105,000 as a result of cross-linking with 125I-NGF, whereas a cell population enriched in non-neuronal cells, which possess only low-affinity receptors, exhibits only the Mr 105,000 component. A shift in molecular weight of both species after pretreatment with neuraminidase indicates that both complexes contain sialoglycoproteins and rules out the possibility that differences in sialic acid content are responsible for the difference in molecular weight of the two complexes. The relative amount of the labeling of these two complexes is not affected by the presence of protease inhibitors nor by a variation of 5000-fold in cross-linker concentration. These results place some limits on possible models for the NGF receptors and their interconversion.     

Presence of IL-1 receptors on human and murine neutrophils. Relevance to IL-1-mediated effects in inflammation

Inflammatory responses are characterized by the infiltration of polymorphonuclear neutrophils (PMN) at the involved site. IL-1 may have an important role in mediating this response, but whether IL-1 acts directly on PMN is controversial. In this study, we examined PMN for the presence of IL-1R and determined the effect of IL-1 on PMN migration in vivo. Thioglycollate, proteose-peptone, or IL-1 elicited peritoneal exudate cells were found to bind 125I-IL-1 alpha in a specific and saturable manner. This binding was localized to the PMN in the exudate. Scatchard plot analysis indicates the presence of approximately 1700 receptors per PMN and an apparent dissociation constant of 3.0 x 10(-10) M. Binding sites for 125I-IL-1 alpha were also found on human PMN prepared from peripheral blood. There are approximately 900 receptors per cell on human PMN with a dissociation constant similar to that observed for elicited murine PMN. Binding of 125I-IL-1 alpha to the mouse and human PMN is inhibited by both recombinant human IL-1 alpha and IL-1 beta, indicating that both IL-1 proteins bind to the same receptor on these cells. Human PMN were able to internalize radioiodinated IL-1. We conclude that PMN possess receptors for IL-1 and that these binding sites may be important in mediating IL-1 effects on granulocytes that are involved in the inflammatory response.     

The membrane attack complex of complement: relation of C7 to the metastable membrane binding site of the intermediate complex C5b-7

Isolated C7 (m.w. 120,000) in 1% deoxycholate (DOC) forms dimers with an apparent m.w. of 230,000 and a DOC-binding capacity of 82 mol per mol of dimer. Dimerization of C7 also occurs in the presence of DOC-phospholipid mixed micelles and eventuates in the insertion of C7 dimers into the lipid bilayer upon the removal of the detergent. C5b-7 complex formation in the fluid phase or on lipid vesicles likewise involves polymerization. C5b-7 sedimented with 17-40S, which suggests a dimeric to hexameric composition. In avidin-biotin binding experiments in which two differentially labeled forms of C5b,6 (biotinyl 125I-C5b,6, and 131I-C5b,6) were used in equimolar amounts to assemble C5b-7, more than 50% of the biotinyl 125I-C5b,6-containing complexes also contained 131I label; again suggesting that C5b-7 consisted of oligomers rather than monomers. The conformation of C7 in C5b-7 and in dimeric C7 appeared similar by the following criteria. On formation of C5b-7 from C5b,6 and C7, a 20% increase in beta-pleated sheet structure was observed by circular dichroism spectroscopy, and a similar change occurred on dimerization of isolated C7. Tryptic and thermolytic digests of C5b-7 and C7 dimers containing 125I-C7 were analyzed by autoradiography after SDS-polyacrylamide gel electrophoresis and were found to contain similar peptides that were distinct from those in the digests of monomeric C7. Direct evidence showing that the metastable membrane binding site of the C5b-7 complex resides in the C7 subunit was obtained by using the conjugates of C5b,6 and colloidal gold. Viewed in the electron microscope, these conjugates were aggregated upon the addition of isolated C7. In contrast, when conjugates of C7 and colloidal gold were treated with soluble C5b,6, no such aggregates occurred, but instead, individual C5b-7 complexes were observed arranged around single gold particles, resulting in star-like structures. The results strongly suggest that structures of C7 are responsible for the expression of the membrane binding site of metastable C5b-7.     

Complement pores in erythrocyte membranes: Analysis of C8/C9 binding required for functional membrane damage

The number of membrane-bound terminal complement proteins (C5b-9) required to generate a functional pore in the human erythrocyte membrane ghost has been determined. Resealed erythrocyte ghost membranes (ghosts) were treated with human complement proteins C5b6, C7, ¹³¹I-C8, and ¹²⁵I-C9 under non-lytic conditions. Following C5b-9 assembly, sucrose-permeant ghosts were separated from C5b-9 ghosts that remained impermeant to sucrose by centrifugation over density barriers formed of 43% (w/v) sucrose. Analysis of ¹³¹I-C8 and ¹²⁵I-C9 bound to sucrose-permeant and sucrose-impermeant subpopulations of C5b-9 ghosts revealed: 1. Sucrose-permeant C5b-9 ghosts show increased uptake of both ¹³¹I-C8 and ¹²⁵I-C9 as compared to ghosts that remain impermeant to sucrose. Ghosts with less than 300 molecules ¹³¹I-C8 bound remain impermeant to sucrose, irrespective of the total C9 input, or, the multiplicity of C9 uptake by membrane C5b-8. 2. In the presence of excess ¹²⁵I-C9, the ratio of ¹²⁵I-C9/¹³¹I-C8 bound to membrane C5b67 is 3.2 ± 0.8 (mean ± 2 S.D.), suggesting an average stoichiometry of 3 C9 per C5b-8. Under these conditions, the ratio of ¹²⁵I-C9/¹³¹I-C8 bound to sucrose-permeant ghosts (3.3 ± 0.7) does not significantly differ from the ratio bound to sucrose-impermeant ghosts (2.9 ± 0.6). 3. With limiting C9 input, the threshold of total C5b-8 uptake required for sucrose permeability increases significantly above 300 per cell when the ratio of bound ¹²⁵I-C9/¹³¹I-C8 is decreased below unity. In the complete absence of C9, 11 700 C5b-8 complexes are bound to sucrose-permeant ghosts. It is concluded that more than 300 C5b-9 complexes must bind to the human erythrocyte to form a sucrose-permeant lesion. Although the binding of one C9 per C5b-8 is critical to the pore-forming activity of these proteins, the binding of additional molecules of C9 to each complex (C9/C8 > 1) does not significantly alter the threshold of total C5b-9 uptake required for lesion formation.     

Affinity labeling of high-affinity alpha-thrombin binding sites on the surface of hamster fibroblasts

The serine proteinase alpha-thrombin potently stimulates reinitiation of DNA synthesis in quiescent Chinese hamster fibroblasts (CCL39 line). 125I-labeled alpha-thrombin binds rapidly and specifically to CCL39 cells with high affinity (Kd approximately 4 nM). Binding at 37 degrees C was found to remain stable for 6 h or more during which time no receptor down-regulation, ligand internalization and/or degradation could be detected. The structure of alpha-thrombin receptors on CCL39 cells was identified by covalently coupling 125I-alpha-thrombin to intact cells using a homobifunctional cross-linking agent, ethylene glycol bis(succinimidyl succinate). By resolution in sodium dodecyl sulfate polyacrylamide gel electrophoresis we observed the specific labeling of a major alpha-thrombin-binding site of Mr approximately 150 000 revealed as a 125I-alpha-thrombin cross-linked complex of Mr approximately 180 000. Independent of chemical cross-linking, 125I-alpha-thrombin also formed a covalent complex with a minor, 35 000 Mr, membrane component identified as protease nexin. Two derivatives of alpha-thrombin modified at the active site are 1000-fold less than alpha-thrombin for mitogenicity. These two non-mitogenic derivatives bound to cells with similar affinity and maximal binding capacity as native alpha-thrombin, and affinity-labeled the receptor subunit of Mr 150 000. When present in large excess, during incubation of cells with alpha-thrombin, these binding antagonists were ineffective in blocking alpha-thrombin-induced DNA synthesis. These data suggest that the specific 150 000 Mr binding sites that display high affinity for alpha-thrombin do not mediate induction of the cellular mitogenic response.     

Solubilization and characterization of adrenal and uterine angiotensin II receptors after photoaffinity labeling

The physical characteristic of the receptors for angiotensin II in dog adrenal cortex and uterus were determined after affinity labeling. 125I-nitro-5-azido-benzoyl-angiotensin II, a photosensitive angiotensin II analogue which retained aldosterone-stimulating activity, was used to couple the octapeptide specifically and irreversibly to its membrane receptors. After solubilization with Triton X-100, the covalent hormone . receptor complex was analyzed by gel filtration and sucrose density gradient centrifugation. Two radioactive species were consistently observed, with calculated Mr values of 126,000 +/- 10,000 and 64,500 +/- 11,000. the elution profiles of solubilized adrenal and uterine particles were almost identical. When the solubilized complexes were subjected to sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis, a single radioactive band was detected upon autoradiography, with Mr - 65,000 +/- 6,000 for adrenal cortex and 68,000 +/- 7,000 for myometrium. These results indicate that the receptors for angiotensin II in adrenal cortex and uterus are composed of two subunits of similar molecular weight, and that the common functional properties of the receptors from both tissues are probably related to their similar physicochemical characteristics.     

Mammalian beta-adrenergic receptors. Distinct glycoprotein populations containing high mannose or complex type carbohydrate chains

Mammalian beta-adrenergic receptor binding peptides can be visualized by covalently labeling them with the photoaffinity reagent p-azido-m-[125I]iodobenzylcarazolol followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. The receptor peptides migrate as broad bands of Mr approximately equal to 62,000. In the present study, we examined the carbohydrate composition of the mammalian beta receptor through the use of specific exo- and endoglycosidases and lectin affinity chromatography. Treatment of p-azido-m-[125I]iodobenzylcarazolol-labeled beta2-adrenergic receptors from hamster lung or rat erythrocyte with the exoglycosidases neuraminidase and alpha-mannosidase provided evidence for the existence of both high mannose and complex type carbohydrate chains on beta 2-adrenergic receptors. The nonadditivity of the effect of sequential treatments with these enzymes suggested discrete populations of beta-adrenergic receptors containing either complex or high mannose type chains. Deglycosylation of receptor with endoglycosidase F results in a single labeled polypeptide at Mr = 49,000 for both systems. The same two populations of the beta receptors (high mannose or complex type chain) could also be fractionated by lectin affinity chromatography of solubilized p-azido-m-[125I]iodobenzylcarazolol-labeled receptors. The high mannose-containing receptors could be absorbed to and specifically eluted from concanavalin A-agarose. Those containing complex type carbohydrates could be adsorbed to and eluted from wheat germ agglutinin-agarose. Taken together, these data suggest that mammalian beta-adrenergic receptors contain both complex and high mannose type carbohydrate chains and that microheterogeneity of these chains likely explains the broad band pattern typically obtained on sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Identification of a 70-kDa gastrin-binding protein on DLD-1 human colorectal carcinoma cells

Gastrin17gly acts as a growth factor for the colonic mucosa. Studies of the receptor involved have generally been restricted to its binding properties, and no investigation of the structure of gastrin17gly receptors on human colorectal carcinoma cell lines has yet been reported. The aim of this study was to optimise the conditions for binding of gastrin17gly to the human colorectal carcinoma cell line DLD-1, and to investigate the structure of the receptor responsible. Binding of 125I[Met15]gastrin17gly to DLD-1 cells was measured in competition experiments with increasing concentrations of either gastrin17gly or gastrin17, or with single concentrations of gastrin receptor antagonists. The molecular weights of the gastrin17gly binding proteins were determined by gel electrophoresis and autoradiography after covalent cross-linking of 125I[Nle15]gastrin2,17gly to cells or membranes with disuccinimidyl suberate. The IC50 value for binding of gastrin17gly to DLD-1 cells was 2.1+/-0.4 microM. Binding was inhibited by the non-selective gastrin/cholecystokinin receptor antagonists proglumide and benzotript, but not by the cholecystokinin-A receptor antagonist L364,718, or the gastrin/cholecystokinin-B receptor antagonist L365,260. The molecular weight of the major gastrin binding protein on DLD-1 cells or membranes was 70,000. We conclude that the major gastrin17gly binding site on the human colorectal carcinoma cell line DLD-1 is clearly distinct from the cholecystokinin-A and gastrin/cholecystokinin-B receptors, but is similar in some respects to the gastrin/cholecystokinin-C receptor.     

Interleukin-5, interleukin-3, and granulocyte-macrophage colony-stimulating factor cross-compete for binding to cell surface receptors on human eosinophils.

Human interleukin (IL)-5 receptors were characterized by means of binding studies using bioactive 125I-labeled IL-5. Of purified primary myeloid cells, eosinophils and basophils but not neutrophils or monocytes expressed surface receptors for IL-5. Binding studies showed that eosinophils expressed a single class of high affinity receptors (Ka = 1.2 x 10(10) M-1) with the number of receptors being small (less than 1000 receptors/cell) and varying between individuals. Among several cell lines examined only HL-60 cells showed detectable IL-5 receptors which were small in numbers (200 receptors/cell) and also bound 125I-IL-5 with high affinity. The binding of IL-5 was rapid at 37 degrees C while requiring several hours to reach equilibrium at 4 degrees C. Specificity studies revealed that the two other human eosinophilopoietic cytokines IL-3 and granulocyte-macrophage colony-stimulating factor (GM-CSF) inhibited the binding of 125I-IL-5 to eosinophils. No competition was observed by other eosinophil activating or nonactivating cytokines. The inhibition of 125I-IL-5 binding by IL-3 and GM-CSF was partial up to a concentration of competitor of 10(-7) M with GM-CSF consistently being the stronger competitor. Converse experiments using IL-5 as a competitor revealed that this cytokine inhibited the binding of 125I-IL-3 and of 125I-GM-CSF in some but not all the individuals tested, perhaps reflecting eosinophil heterogeneity in vivo. Cross-linking experiments on HL-60 cells demonstrated two IL-5-containing complexes of Mr 150,000 and Mr 80,000 both of which were inhibited by GM-CSF. The competition between IL-5, IL-3, and GM-CSF on the surface of mature eosinophils may represent a unifying mechanism that may help explain the common biological effects of these three eosinophilopoietic cytokines on eosinophil function. This unique pattern of competition may also be beneficial to the host by preventing excessive eosinophil stimulation.     

Dopamine D2 receptors retain agonist high-affinity form and guanine nucleotide sensitivity after removal of sialic acid

The ligand binding subunit of the D2 dopamine receptor (Mr approximately equal to 94,000) can be visualized by autoradiography following photoaffinity labeling with [125I]N-azidophenethylspiperone and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Following removal of sialic acids with the exoglycosidase, neuraminidase, [125I]N-azidophenethylspiperone photoincorporated into a protein of Mr = 54,000 with the appropriate pharmacological profile for D2 receptors. The desialylated D2 receptor bound dopaminergic agonists with high affinity and was capable of coupling to a functional G-protein as indexed by: 1) pertussis-toxin mediated [32P]ADP ribosylation of proteins of Mr = 42,000 and 39,000, and 2) the conversion of the agonist high affinity form of D2 receptors to one displaying low affinity for agonists in the presence of guanine nucleotides. These data suggest that sialic acid residues do not contribute significantly to the ligand binding characteristics of D2 receptors despite the large change produced in the estimated molecular mass of the binding subunit.     

Functional properties of the asialo-fifth component of human complement

Removal of exposed, terminal sialic acid (SA) from carbohydrate chains N-glycosidically linked to asparagine residues of highly pure human C5 with bacterial sialidase increased C-mediated hemolysis of antibody-sensitized sheep E maximally 2.77-fold. Sialidase-treated C5 used as a reagent for the titration of C6, C7, C8, and C9 resulted in increased titers of all these components compared to buffer-treated C5. As determined by a fluorometric method, ca. 65% of the SA was enzymically hydrolyzed under optimal conditions. Endoglycosidase F incubated with C5 followed by monosaccharide analyses by anion exchange chromatography with pulsed amperometric detection revealed both high mannose and complex (terminate in SA) oligosaccharides were hydrolyzed; no effect was found on the functional activity of C5. Approximately 4% of the complex oligosaccharides were hydrolyzed from C5. Comparison of sialidase- and buffer-treated C5 decay rates from EAC1gp(4b,oxy2a,3b)hu resulted in two linear components of the decay curve with sialidase-treated C5, but one linear component with buffer-treated C5. Of the sialidase-treated 125I-C5 15% was bound to EAC1gp(4b,oxy2a,3b)hu compared to 9.3% of buffer-treated 125I-C5. Furthermore, 27% of sialidase-treated 125I-C5 was bound to EAC1gp,4bhu compared to 16.6% of buffer-treated 125I-C5, but no lysis occurred after the addition of C6-C9. The mechanism of increased hemolytic activity after removal of SA from C5 is: the Tmax is prolonged at 30 degrees C (ca. 15 min vs 9 min), and a higher percentage of C5 binds to cellular intermediates compared to buffer-treated C5.