High affinity human IgE receptor (Fc epsilon RI). Analysis of functional domains of the alpha-subunit with monoclonal antibodies.

The binding of IgE to the high affinity Fc epsilon receptor (Fc epsilon RI) on mast cells and basophils is mediated by the alpha-subunit of the tetrameric receptor complex. Based on sequence homologies, the 50-kDa alpha-subunit is a member of the immunoglobulin superfamily of proteins and has two predicted disulfide-bonded loops. Monoclonal antibodies specific for the human alpha-subunit have been identified and separated into two major classes: inhibitory and noninhibitory antibodies. Inhibitory antibodies (i.e. 15A5) block 125I-IgE binding to a recombinant chimeric alpha-subunit (ch-alpha-protein) expressed on Chinese hamster ovary cells and immunoprecipitate 125I-labeled purified ch-alpha-protein. Noninhibitory antibodies (i.e. 22E7) immunoprecipitate both 125I-labeled ch-alpha-protein and the soluble complex of 125I-IgE cross-linked to ch-alpha-protein but do not block 125I-IgE binding to the ch-alpha-protein expressed on Chinese hamster ovary cells. Both classes of antibodies bind to natural Fc epsilon RI present on human basophils and induce histamine release from these cells. Inhibitory antibody 15A5 specifically binds to a peptide corresponding to amino acids 125-140 of the putative second domain of the alpha-subunit sequence. All the inhibitory antibodies compete with 125I-15A5 for binding to the ch-alpha-protein, indicating that these antibodies recognize inhibitory epitopes that are either identical or sterically overlapping. Noninhibitory antibodies (i.e. 22E7) do not block 125I-15A5 binding to the ch-alpha-protein. These data suggest that antibodies binding to the predicted second domain of the alpha-subunit can inhibit IgE binding to the alpha-subunit, while antibodies binding at a distance from this site do not inhibit IgE binding. These inhibitory antibodies may block IgE binding to the ch-alpha-protein by direct overlap, steric inhibition, or induced conformational changes of the receptor contact points for IgE.     

Identification of Serotonin Receptors Recognized by Anti-Idiotypic Antibodies

Anti-idiotypic antibodies were generated by immunizing rabbits with affinity-purified antibodies to serotonin (5-hydroxytryptamine; 5-HT). Anti-5-HT activity was removed from the resulting antisera by chromatography through a 5-HT affinity column. The anti-idiotypic antibodies were demonstrated by enzyme-linked immunosorbent assay to bind to affinity-purified whole anti-5-HT antibodies and their Fab fragments. Anti-idiotypic antibodies, purified by affinity chromatography on columns to which antibodies to 5-HT were coupled, competed with 5-HT (covalently bound to protein) for the binding sites on anti-5-HT antibodies and serotonin binding protein. The anti-idiotypic antibodies antagonized the binding of [3H]5-HT to membranes isolated from the cerebral cortex, striatum, and raphe area more than to membranes from hippocampus or cerebellum. The anti-idiotypic antibodies also blocked the binding of the 5-HT1B-selective ligand (-)-[125I]iodocyanopindolol (in the presence of 30 microM isoproterenol) to cortical membranes. In contrast, anti-idiotypic antibodies failed to inhibit binding of the 5-HT1A-selective ligand 8-hydroxy-2-(di-n-[3H]propylamino)-tetralin [( 3H]8-OH-DPAT) to raphe area membranes or hippocampal membranes. These observations suggested that the anti-idiotypic antibodies may recognize some 5-HT receptor subtypes but not others. This hypothesis was tested by ascertaining the ability of anti-idiotypic antibodies to immunostain cells transfected in vitro with cDNA encoding the 5-HT1C or 5-HT2 receptor or with a genomic clone encoding the 5-HT1A receptor. Punctate sites of immunofluorescence were found on the surfaces of fibroblasts that expressed 5-HT1C and 5-HT2 receptors, but not on the surfaces of HeLa cells that expressed 5-HT1A receptors. Immunostaining of cells by the anti-idiotypic antibodies was inhibited by appropriate pharmacological agents: immunostaining of cells expressing 5-HT1C receptors was blocked by mesulergine (but not ketanserin, 8-OH-DPAT, or spiperone), whereas that of cells expressing 5-HT2 receptors was blocked by ketanserin or spiperone (but not mesulergine or 8-OH-DPAT). The anti-idiotypic antibodies failed to inhibit the uptake of [3H]5-HT by serotonergic neurons. It is concluded that the anti-idiotypic antibodies generated with anti-5-HT serum recognize the 5-HT1B, 5-HT1C, and 5-HT2 receptor subtypes; however, neither 5-HT1A receptors nor 5-HT uptake sites appear to react with these antibodies.     

Loss of the transferrin receptor during the maturation of sheep reticulocytes in vitro. An immunological approach.

Sheep reticulocyte-specific antiserum absorbed with mature sheep red cells has been used to isolate and identify reticulocyte-specific plasma-membrane proteins and to monitor their loss during incubation in vitro. Specific precipitation of labelled plasma-membrane proteins is obtained when detergent-solubilized extracts of 125I-labelled reticulocyte plasma membranes are incubated with this antiserum and Staphyloccus aureus, but not when mature-cell plasma membranes are treated similarly. During maturation of reticulocytes in vitro (up to 4 days at 37 degrees C), there is a marked decrease in the immunoprecipitable material. The anti-reticulocyte-specific antibodies have been identified as anti-(transferrin receptor) antibodies. By using these antibodies as a probe, the transferrin receptor has been shown to have a subunit molecular weight of 93 000. The data are consistent with reported molecular weights of this receptor and with the proposal that the receptor may exist as a dimer, since [125I]iodotyrosyl-peptide maps of the 93 000- and 186 000-mol.wt. components isolated are shown to be identical. Evidence is presented for the transmembrane nature of the receptor and for the presence of different binding sites for transferrin and these antibodies on the receptor.     

Monoclonal antibodies to either domain of ovotransferrin block binding to transferrin receptors on chick reticulocytes

Monoclonal antibodies produced to both chicken ovotransferrin and to the isolated N- and C-terminal half-molecule domains of ovotransferrin have been used to probe the interaction of ovotransferrin with its specific receptor on chick embryo red blood cells. Two antibodies to epitopes on the N-terminal domain and one antibody to an epitope on the C-terminal domain were able to block the binding of 125I-labeled diferric ovotransferrin to the receptor. When the cellular surface receptors were first saturated with ovotransferrin at 0 degrees C, none of these antibodies bound to the cell-associated ovotransferrin. This suggests that the antibodies are to epitopes which lie very near to, or in the regions of, the two domains which interact with receptor. The same three antibodies also blocked the binding to the receptor of ovotransferrin associated in situ from the isolated N- and C-terminal half-molecule domains. A fourth antibody did not block binding to receptor of 125I-labeled diferric ovotransferrin or the associated domains; furthermore, it was able to bind to ovotransferrin bound to the cell surface at 0 degrees C. This antibody thus appears to recognize an epitope remote from the receptor binding region of ovotransferrin. Additional evidence for the requirement of the presence of both domains of ovotransferrin to effect binding to the transferrin receptor on chick reticulocytes was obtained with a fifth antibody which recognized only the N-terminal half-molecule domain but not holo-ovotransferrin. Although this antibody had no effect on the binding of 125I-labeled ovotransferrin to cells, it blocked binding to receptor of the associated domains of ovotransferrin, presumably by inhibiting the association of the two domains.     

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.     

Monoclonal antibodies to the low density lipoprotein receptor as probes for study of receptor-mediated endocytosis and the genetics of familial hypercholesterolemia

Monoclonal antibodies directed against the low density lipoprotein (LDL) receptor have been prepared by immunization of mice with a partially purified receptor from bovine adrenal cortex. Spleen cells from the mice were fused with the Sp2/0-Ag14 line of mouse myeloma cells. The most extensively studied monoclonal antibody, designated immunoglobulin-C7, reacts with the human and bovine LDL receptor, but not with receptors from the mouse, rat, Chinese hamster, rabbit, or dog. 125I-labeled monoclonal antibody binds to human fibroblasts in amounts that are equimolar to 125I-LDL. In fibroblasts from 6 of 8 patients with the receptor-negative form of homozygous familial hypercholesterolemia, which have less than 5% of normal LdL binding, the amount of monoclonal antibody binding was also less than 5% of normal. Fibroblasts from the other two receptor-negative homozygotes bound an amount of monoclonal antibody that was much greater than expected on the basis of LDL binding, suggesting that these two patients produce a structurally altered receptor that binds the antibody, but not LDL. In normal fibroblasts, the receptor-bound monoclonal antibody was taken up and degraded at 37 degrees C at rapid rate similar to that for LDL. Fibroblasts from a patient with the internalization defective form of familial hypercholesterolemia bound the monoclonal antibody, but did not internalize or degrade it. The current data demonstrate the usefulness of monoclonal antibodies as probes for the study of the cellular and genetic factors involved in receptor-mediated endocytosis.     

Structural domains of the extracellular domain of human nerve growth factor receptor detected by partial proteolysis.

Using partial proteolytic cleavage, the nerve growth factor (NGF) binding site and the epitopes for two anti-NGF receptor (NGFR) monoclonal antibodies were localized on the recombinant extracellular domain (RED) of the NGFR. The RED was prepared in the baculovirus-insect cell system and was purified by immunoaffinity and ion-exchange chromatography. The four cysteine-rich repeat domains and some additional C-terminal sequences were resistant to proteolysis with papain or proteinase K. The Mr 32,000 papain-resistant fragment (P32) and the Mr 30,000 proteinase K-resistant fragment (K30) share the same N terminus as the intact RED and have C termini in the vicinity of residue 170. Even though P32 and K30 have the same N terminus and probably differ by only a small number of amino acids at the C terminus, P32, but not K30, binds 125I-NGF. As judged by Western blot analysis, two anti-NGFR antibodies (ME20.4 and NGFR5) bind to P32 but have a lesser affinity for K30. Since antibody ME20.4 inhibits NGF binding but antibody NGFR5 does not, these antibodies bind to distinct epitopes. However, these epitopes apparently are closely spaced since these antibodies compete with each other for binding to biotinylated RED. NGF, but not the control protein cytochrome c, protects RED from papain digestion. Therefore, the P32 C terminus is important for the expression of the NGF binding site and the antibody-defined epitopes, even though the NGF binding site and antibody-defined epitopes probably are not encoded by the P32 C terminus. These data suggest that complex interactions occur between different regions of the RED, and that optimum NGF binding requires the integrity of multiple RED domains, including a short sequence to the C terminus of residue 170.     

Isolation and function of a human endothelial cell C1q receptor

It has been shown previously that cultured human venous and arterial endothelial cells (EC) bind C1q in a time- and dose-dependent manner. Cultured human endothelial cells express an average number of 5.2 x 10(5) binding sites/cell. In the present study the putative receptor for C1q (C1qR) was isolated from the membranes of 1-5 x 10(9) human umbilical cord EC by affinity chromatography on C1q-Sepharose. During isolation, C1qR was detected by its capacity to inhibit the lysis of EAC1q in C1q-deficient serum. The eluate from C1q-Sepharose was concentrated, dialysed and subjected to QAE-A50 chromatography and subsequently to gel filtration on HPLC-TSK 3000. C1qR filtered at an apparent molecular weight of 60 kDa. Purified C1qR exhibited an apparent molecular weight of 55-62 kDa in the unreduced state and a molecular weight of 64-68 kDa in reduced form. Two IgM monoclonal antibodies (mAb) D3 and D5 were raised following immunization of mice with purified receptor preparations. Both monoclonal antibodies increased the binding of (125)I-C1q to endothelial cells but F(ab')(2) anti-C1qR mAb inhibited the binding of a(125)I-C1q to EC in a dosedependent manner. The D3 mAb recognized a band of 54-60 kDa in Western blots of membranes of human EC and polymorphonuclear leukocytes. Previously, the authors showed that C1q induces the binding of IgM-containing immune complexes to EC. Therefore, it was hypothesized that during a primary immune response generation of IgM-IC may occur, resulting in binding and activation of C1, dissociation of activated C1 by C1 inhibitor and subsequent interaction of IgM-IC bearing C1q with EC-C1qR.     

Stoichiometric binding of low density lipoprotein (LDL) and monoclonal antibodies to LDL receptors in a solid phase assay

The current paper describes a solid phase ligand binding assay for the low density lipoprotein (LDL) receptor that takes advantage of the domain structure of the protein. An antibody directed against one domain, e.g. the cytoplasmic tail, is adsorbed to a microtiter well. A detergent solution containing the LDL receptor is added, and the receptor is allowed to bind to the antibody. The wells are then washed, and one of the following radioiodinated ligands is added: 125I-LDL or an 125I-labeled monoclonal antibody directed against a different domain than the antibody adsorbed to the well. Under these conditions, the human LDL receptor shows high affinity for 125I-LDL and for 125I-IgG-HL1, a monoclonal antipeptide antibody directed against a 10-amino-acid "linker" between repeats 4 and 5 in the ligand binding domain. The binding affinity is the same at 4 degrees C and 37 degrees C. The binding of 125I-LDL and 125I-IgG-HL1 occurs with 1:1 molar stoichiometry, suggesting that the human LDL receptor binds 1 mol of LDL per mol of receptor. The acid-dependent dissociation of 125I-LDL and 125I-labeled monoclonal antibody from LDL receptors that is observed in intact cells was also shown to occur in the solid phase binding assay. We used the solid phase assay to demonstrate the secretion of LDL receptors from monkey cells that have been transfected with a cDNA encoding a truncated form of the human receptor that lacks the membrane-spanning domain. This assay may be useful in measuring the relative amounts of the intact LDL receptor in tissue extracts and the secreted receptor in transfected cells.     

Characterization of monoclonal antibodies to the beta-adrenergic antagonist alprenolol as models of the receptor binding site

Antibodies to receptor ligands have been valuable in understanding the nature of receptor-ligand interactions. We have developed four monoclonal antibodies to the beta-adrenergic receptor antagonist alprenolol by immunizing A/J mice with (-)-alprenolol coupled to keyhole limpet hemocyanin. The antisera from these mice displayed specific [3H]dihydroalprenolol ([3H]DHA) binding that was inhibited by alprenolol, propranolol, and isoproterenol. Somatic cell fusion of spleen cells from the immunized mice to SP2/0 myeloma cells, followed by limited dilution subcloning, resulted in the isolation of four hybridomas (1B7, 5B7, 5D9, and 2G9) demonstrating three different classes of ligand binding characteristics. 1B7 had the highest binding affinity for antagonists based on Scatchard analysis (Kd [125I]- CYP = 1.4 X 10(-10) M; Kd [3H]DHA = 6.5 X 10(-9) M), and was the only antibody to demonstrate agonist-inhibition of [3H]DHA binding. Ki values computed from competitive inhibition curves of [3H]DHA binding to 1B7 resulted in a rank order of potency similar to that of beta-2-adrenergic receptors: (-)-propranolol greater than acebutolol amine greater than isoproterenol greater than (+)-propranolol greater than epinephrine greater than norepinephrine. 5B7 and 5D9 exemplified a second class of antibody. This pair had lower antagonist binding affinities (Kd [3H]DHA = 2 X 10(-8) M and 2.5 X 10(-7) M, respectively) and was stereoselective in binding receptor antagonists: (-)-propranolol greater than (+)-propranolol greater than acebutolol amine. Agonist inhibition of [3H]DHA binding to these antibodies could only be observed at very high concentrations (greater than 10(-4) M agonist), and was not dose-dependent. Finally, the class of anti-alprenolol monoclonal antibodies represented by 2G9 had the lowest antagonist binding affinity of all (IC50 alprenolol = 1 X 10(-5) M), did not demonstrate ligand stereoselectivity, and did not recognize agonists. We propose that antibodies raised against beta-adrenergic receptor ligands demonstrating stereoselective agonist binding will also demonstrate high affinity antagonist binding, and that they will closely parallel the binding characteristics of the receptor. According to this "agonist best-fit hypothesis," anti-idiotypic antibodies raised against the binding site of these idiotypes might contain true mirror images of the beta-adrenergic receptor binding site.     

Effect of monoclonal antibodies on human insulin receptor autophosphorylation, negative cooperativity, and down-regulation

Three major functional characteristics of the insulin receptor are negative cooperativity, down-regulation, and beta-subunit tyrosine kinase activity. To investigate the inter-relationships among these functions we studied four antibodies to the insulin receptor alpha-subunit. These monoclonal antibodies competitively inhibited 125I-insulin binding to the insulin receptor of human IM-9 and HEP-G2 cells. When the antibodies were radiolabeled, insulin competed strongly with two antibodies (MA-10 and MA-51) for binding to the insulin receptor, but competed weakly with the two others (MA-5 and MA-20). Antibodies MA-10 and MA-51, like insulin, accelerated the dissociation of bound 125I-insulin from receptors; in contrast, MA-5 and MA-20 strongly inhibited 125I-insulin dissociation. Antibodies MA-10 and MA-51 induced down-regulation of insulin receptors with a potency similar to that of insulin. In contrast, MA-5 and MA-20 were more potent than insulin. None of the antibodies either alone or in combination influenced autophosphorylation of the insulin receptor beta-subunit. These data indicate, therefore, that two major epitopes can be identified on the alpha-subunit of the insulin receptor by the use of monoclonal antibodies. One epitope, recognized by antibodies MA-10 and MA-51, is close to or near the insulin-binding site and mimics insulin-induced negative cooperatively and down-regulation. The other epitope, recognized by antibodies MA-5 and MA-20, is at some distance from the insulin-binding site, and only mimics down-regulation. These data suggest, therefore, that: negative cooperativity and down-regulation may not be inter-related and both processes are independent of insulin receptor tyrosine kinase activity.     

Immunolabeling of Central Serotonin 5-HT1Dβ Receptors in the Rat, Mouse, and Guinea Pig with a Specific Anti-Peptide Antiserum

Abstract: A synthetic peptide (25 amino acids) corresponding to a specific portion of the third intracytoplasmic loop of the rat serotonin 5-HT_1B/1Dβ receptor was coupled to keyhole limpet hemocyanin and injected monthly into rabbits. Anti-peptide antibodies were detected by enzyme-linked immunosorbent assay and characterized by immunoprecipitation of the 5-HT_1B/1Dβ receptor in CHAPS-solubilized extracts from rat striatal membranes. Up to 60% of solubilized striatal serotonin- O -carboxymethylglycyl[¹²⁵I]iodotyrosinamide ([¹²⁵I]GTI; a selective 5-HT_1B/1D radioligand) binding sites were immunoprecipitated and subsequently pharmacologically identified as 5-HT_1B receptors. The remaining 40% of [¹²⁵I]GTI binding sites were shown to be 5-HT_1D receptors. In addition, these antibodies were successfully used in immunofluorescence experiments to detect the 5-HT_1B/1Dβ, but not the 5-HT_1D/1Dα, receptor in transiently transfected LLC-PK1 cells. Immunoautoradiographic experiments performed with brain sections from the rat, mouse, and guinea pig showed that the substantia nigra and globus pallidus contained the highest densities of 5-HT_1Dβ receptor-like immunoreactivity. Comparison of the regional distribution of immunolabeling with that of the specific binding of [¹²⁵I]GTI in the brain of these species further confirmed that the anti-peptide antibodies selectively recognized only the 5-HT_1Dβ component of [¹²⁵I]GTI specific receptor binding sites.     

Complement proteins C5b-9 initiate secretion of platelet storage granules without increased binding of fibrinogen or von Willebrand factor to newly expressed cell surface GPIIb-IIIa

The functional and conformational activation of cell surface glycoproteins IIb-IIIa (GPIIb-IIIa) was probed in platelets stimulated to secrete by complement proteins C5b-9. Gel-filtered human platelets exposed to the purified human C5b-9 proteins exhibited non-lytic secretory release of both alpha- and dense granule storage pools with only a small increase in total binding of 125I-fibrinogen (less than 3000 molecules/cell) to the cell surface. By contrast to ADP- or thrombin-activated platelets, increased 125I-fibrinogen bound to C5b-9 platelets was not inhibited by Arg-Gly-Asp-containing peptides, suggesting that the high affinity membrane receptor for fibrinogen is not expressed under these conditions. C5b-9-stimulated platelets also failed to bind 125I-von Willebrand factor (less than 1 ng/10(8) platelets), confirming that the adhesive protein receptor function of cell surface GPIIb-IIIa is not expressed in these cells. Although specific binding of 125I-fibrinogen or 125I-von Willebrand factor did not significantly increase after C5b-9 assembly, these proteins elicited de novo expression of the GPIIb-IIIa activation-associated epitope recognized by monoclonal antibody PAC-1, and binding of this antibody to C5b-9 platelets was fully competed by Arg-Gly-Asp-containing peptides. These data suggest that the metabolic events which trigger granule secretion after C5b-9 insertion into the plasma membrane cause cell surface GPIIb-IIIa to be expressed in an activation-associated but functionally incompetent conformation.     

Chemical modification of surfactant protein A alters high affinity binding to rat alveolar type II cells and regulation of phospholipid secretion

Alveolar type II cells express a high affinity receptor for pulmonary surfactant protein A (SP-A), and the interaction of SP-A with these cells leads to inhibition of surfactant lipid secretion. We have investigated the binding of native and modified forms of SP-A to isolated rat alveolar type II cells. Native and deglycosylated forms of SP-A readily competed with 125I-SP-A for cell surface binding. Alkylation of SP-A with excess iodoacetamide yielded forms of SP-A that did not inhibit surfactant lipid secretion and did not compete with 125I-SP-A for cell surface binding. Reductive methylation of SP-A with H2CO and NaCNBH3 yielded forms of SP-A with markedly reduced receptor binding activity that also exhibited significantly reduced capacity to inhibit lipid secretion. Modification of SP-A with cyclohexanedione reversibly altered cell surface binding and the activity of SP-A as an inhibitor of lipid secretion. Two monoclonal antibodies that block the function of SP-A as an inhibitor of lipid secretion completely prevented the high affinity binding of SP-A to type II cells. A monoclonal antibody that recognizes epitopes on SP-A but failed to block the inhibition of secretion also failed to completely attenuate high affinity binding to the receptor. Concanavalin A inhibits phospholipid secretion of type II cells by a mechanism that is reversed in the presence of excess alpha-methylmannoside. Concanavalin A did not block the high affinity binding of 125I-SP-A to the receptor. Neither the high affinity binding nor the inhibitor activity of SP-A was prevented by the presence of mannose or alpha-methylmannoside. The SP-A derived from humans with alveolar proteinosis is a potent inhibitor of surfactant lipid secretion but failed to completely displace 125I-SP-A binding from type II cells. From these data we conclude that: 1) cell surface binding activity of rat SP-A is directly related to its capacity to inhibit surfactant lipid secretion; 2) monoclonal antibodies directed against SP-A can be used to map binding domains for the receptor; 3) the lectin activity of SP-A against mannose ligands does not appear to be essential for cell surface binding; 4) concanavalin A does not compete with SP-A for receptor binding; and 5) the human SP-A derived from individuals with alveolar proteinosis exhibits different binding characteristics from rat SP-A.     

Monoclonal antibodies reacting with multiple epitopes on the human insulin receptor.

Monoclonal antibodies for the human insulin receptor were produced following immunization of mice with IM-9 lymphocytes and/or purified placental receptor. Four separate fusions yielded 28 antibodies, all of which reacted with receptor from human placenta, liver and IM-9 cells. Some antibodies cross-reacted to varying degrees with receptor from rabbit, cow, pig and sheep, but none reacted with rat receptor. At least 10 distinct epitopes were recognized as indicated by species specificity and binding competition experiments. All of these epitopes appeared to be on extracellular domains of the receptor as shown by binding of antibodies to intact cells. In some cases the epitopes were further localized to alpha or beta subunits by immunoblotting. Several antibodies inhibited binding of 125I-insulin to the receptor, some had no effect on binding, and others enhanced the binding of 125I-insulin. It is concluded that these antibodies will be valuable probes of receptor structure and function.     

Characterization of two monoclonal antibodies reactive with the external domain of the platelet-derived growth factor receptor

Two monoclonal antibodies against the receptor for platelet-derived growth factor (PDGF) were obtained by immunizing mice with pure PDGF receptor preparations derived from porcine uterus. The antibodies, denoted PDGFR-B1 and PDGFR-B2, both bound to the external domain of the receptor, as demonstrated by indirect immunofluorescence and binding of 125I-labeled antibodies to intact human fibroblasts. Both antibodies precipitated pure 175-kDa 32P-labeled autophosphorylated porcine PDGF receptor as well as a Mr 175,000 glycoprotein from metabolically labeled cells. The monoclonal antibodies did not inhibit binding of 125I-PDGF to human fibroblasts and did not stimulate these cells to undergo mitosis. Both antibodies induced clustering and down-regulation of their antigen. However, this resulted in only a partial loss of cell surface binding sites for PDGF itself, consistent with the conclusion that the monoclonals recognized only one of two or several receptors for PDGF. Clustering and down-regulation were not seen when the cells were incubated with monovalent Fab' fragments of the PDGFR-B2 antibody. The antibodies also stimulated autophosphorylation of pure PDGF receptor, and PDGFR-B2 was shown to stimulate phosphorylation of phosphofructokinase, an exogenous substrate for the PDGF receptor kinase. High concentrations of PDGFR-B2 antibody, or Fab' fragments thereof, failed to enhance the PDGF receptor kinase activity, compatible with the possibility that dimerization was of importance in the antibody-stimulated kinase activity of purified PDGF receptors.     

Insulin-like and insulin-inhibitory effects of monoclonal antibodies for different epitopes on the human insulin receptor.

Monoclonal antibodies previously shown to react with five distinct epitopes on the human insulin receptor were tested for their metabolic effects on isolated human adipocytes. Two antibodies which reacted with receptor alpha-subunit and completely inhibited 125I-insulin binding mimicked the actions of insulin to stimulate lipogenesis from [14C]glucose and to inhibit catecholamine-induced lipolysis. On a molar basis, these antibodies were comparable in potency with insulin itself. Two other antibodies which decreased insulin binding only slightly or not at all also mimicked these metabolic effects of insulin. One of these antibodies reacted with receptor beta-subunit. In contrast, a further antibody which reacted with alpha-subunit and inhibited insulin binding did not affect basal lipogenesis or catecholamine-induced lipolysis, but was able to antagonize the effects of insulin on these processes. The same antibody antagonized the insulin-like effect of another antibody with which it competed in binding to insulin receptor, but not the effect of an antibody which bound independently to the receptor. It is concluded that binding of ligand at or close to the insulin-binding site is neither necessary nor sufficient to trigger insulin-like metabolic effects, which may rather depend on some general property of antibodies, such as their ability to cross-link and aggregate receptor molecules.     

Antipeptide antibodies that recognize a lymphocyte substance P receptor

In an effort to investigate the presence of substance P (SP) receptors on lymphocytes, polyclonal antibodies against SP receptors were developed. The immunogen used to generate these antibodies was a peptide encoded by an RNA complementary to the mRNA for SP. The rationale for using this SP complementary peptide (termed SP CP) as an immunogen resulted from the observation that 3H-SP bound to microtiter wells coated with SP CP in a dose dependent and saturable fashion. Furthermore, binding was blocked with excess unlabeled SP or SP antagonist, D-Pro2-D-Phe7-D-Trp9-SP. Inasmuch as the peptide, SP CP, specifically bound 3H-SP, we hypothesized that antibodies against this peptide might recognize a SP receptor binding site. Using the SP receptor positive lymphoblast cell line, IM-9, affinity-purified antibodies against SP CP but not antibodies against keyhole limpet hemocyanin recognized a molecule on the surface of IM-9 cells. Anti-SP CP binding to IM-9 cells was blocked with excess SP antagonist, suggesting that the antibody and the SP antagonist were competing for the same binding site. In support of this possibility, anti-SP CP antibodies blocked 3H-SP binding to IM-9 cells. An immunoaffinity column coupled with antibodies against SP CP bound protein from solubilized IM-9 cells. This isolated protein bound 125I-Tyr8-SP and binding was specifically blocked with SP as well as by SP antagonist, neurokinin A, and eledoisin. Passthrough material did not bind SP suggesting that a SP receptor had been purified. Western blot analysis of solubilized IM-9 cell proteins using anti-SP CP antibodies but not preimmune IgG recognized a single protein of 58,000 D. Taken together, these results demonstrate that antibodies against SP CP recognize a SP receptor present on the lymphocyte cell line, IM-9.     

Brain somatostatin receptor-G protein interaction. G alpha C-terminal antibodies demonstrate coupling of the soluble receptor with Gi(1-3) but not with Go.

Somatostatin (SST) receptors activate potassium channels, stimulate protein phosphatases, inhibit adenylate cyclase and close calcium channels. These multiple effects are controlled by guanine nucleotide binding (G) proteins of the pertussis toxin-sensitive Gi and Go types. In the present study we have identified the G proteins coupling with brain SST receptors. To this end, brain SST receptors were solubilized in G-protein coupled form. Binding of the SST analogue MK 678 to the solubilized receptor was completely inhibited by guanosine 5'-O-thiotriphosphate (IC50 = 100 nM), reflecting decreased receptor affinity for agonist following uncoupling of the receptor and G protein(s). Antibodies raised against specific COOH-terminal peptides of the G proteins Gi(1-3), Go, and Gz were used to probe for SST receptor-G protein coupling in this system. Antibodies binding to the COOH-terminal regions of Gi1 and Gi2 (antibody AS) and Gi3 (antibody EC) inhibited binding of 125I-MK 678 (75 pM) by 57 +/- 4% and 48 +/- 5%, respectively. The effects of these antibodies were concentration-dependent and additive, such that in combination AS and EC completely inhibited binding. Antibodies binding to the COOH-terminal region of Go (GO) and Gz (QN) did not affect binding of 125I-MK 678, indicating that neither Go nor Gz are associated with the brain SST receptor. Prelabeling of the receptor with 125I-MK 678 prior to addition of antibody induced the formation of a "locked conformation" of the agonist-bound receptor-G protein complex which was insensitive to antibody. In conclusion, Gi1 and/or Gi2 and Gi3 are coupled in approximately equal proportions to the brain 125I-MK 678-binding SST receptor, accounting for all of the G protein coupling of this receptor.     

Association of a lower molecular weight protein to the mu-opioid receptor demonstrated by (125)I-beta-endorphin cross-linking studies

Cross-linking experiments using the (125)I-beta-endorphin revealed the presence of several receptor-related species in cell lines expressing endogenous opioid receptors, including a small molecular mass protein (approximately 22 kDa). Previous reports have suggested that this 22-kDa (125)I-beta-endorphin cross-linked protein could be the degradative product from a higher molecular mass species, i.e., a fragment of the receptor. To determine if this protein is indeed a degraded receptor fragment, (125)I-beta-endorphin was cross-linked to the (His)(6) epitope-tagged mu-opioid receptor (His-mu) stably expressed in the murine neuroblastoma Neuro(2A) cells. Similar to earlier reports with cell lines expressing endogenous receptors, two major bands of 72- and 25-kDa proteins were specifically cross-linked. Initial cross-linking experiments indicated the absolute requirement of the high-affinity (125)I-beta-endorphin binding to the mu-opioid receptor prior to the appearance of the low molecular weight species, suggesting that the 22-kDa protein could be a degraded fragment of the receptor. However, variations in the ratios of these protein bands being cross-linked by several homo- or heterobifunctional cross-linking agents were observed. Although neither the carboxyl terminus mu-opioid receptor-specific antibodies nor the antibodies against the epitope at the amino terminus of the receptor could recognize the 22-kDa protein, this (125)I-beta-endorphin cross-linked species could be coimmunoprecipitated with the receptor antibodies or could be isolated with a nickel resin affinity chromatography. The direct physical association of the 22-kDa protein with the receptor was demonstrated also by the observation that the 22-kDa protein could not bind to the nickel resin alone, but that its binding to the nickel resin was restored in the presence of the His-mu. Taken together, these results suggest that the 22-kDa protein cross-linked by (125)I-beta-endorphin is not a degradative product, but a protein located within the proximity of the mu-opioid receptor, and that it is tightly associated with the receptor.