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.     

Immunoaffinity purification of membrane protein constituents of the IM-9 lymphoblast receptor for substance P

Substance P (SP) is an undecapeptide neuromediator that stimulates human T-lymphocyte function by binding to stereospecific membrane receptors. Human IM-9 cultured B-lymphoblasts express approximately 20,000 receptors per cell for [125I]SP with a Kd of 0.3 nM. [125I]SP was specifically crosslinked by disuccinimidyl suberate to IM-9 cell membrane proteins of 78, 58, and 33 kDa. An indirect immunoaffinity purification procedure has now been developed based on immunoabsorption of detergent-solubilized [125I]SP-labeled IM-9 cell membrane proteins to anti-SP antibody that was bound to an epoxide ultraffinity high-performance liquid chromatography column, followed by elution in acidic 8 M urea. The 58- and 33-kDa SP-receptor complexes were purified to apparent homogeneity by immunoaffinity chromatography and identified by autoradiography and silver staining of sodium dodecyl sulfate-polyacryl-amide gels.     

Binding characteristics and affinity labeling of protein constituents of the human IM-9 lymphoblast receptor for substance P

The neuropeptide substance P (SP) stimulates human T-lymphocyte function in vitro. Human blood T-lymphocytes and cultured human IM-9 B-lymphoblasts express 7,000-10,000 and 25,000-30,000 substance P receptors per cell, respectively. The specific binding of 125I-SP is retained in IM-9 lymphoblast membranes solubilized in 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS) at a detergent-to-protein ratio of 1.0. In addition, specific and reversible SP binding to soluble IM-9 cell membrane proteins is demonstrated by gel filtration. The saturation of binding of 125I-SP to both intact and solubilized IM-9 cell membranes attained a steady state after 40-50 min at 4 degrees C. Scatchard analysis of the concentration dependence of 125I-SP binding to IM-9 cell membranes revealed a KD of 0.87 +/- 0.8 nM (mean +/- S.D., n = 4), which is similar to that observed in intact cells, and a density of receptors of 21 +/- 3 fmol/mg of membrane protein (mean +/- S.D.). Binding of 125I-SP to solubilized membranes demonstrated a KD of 0.75 +/- 0.33 nM (mean +/- S.D., n = 3) and a density of receptors of 3.7 +/- 1.5 fmol/mg of membrane protein (mean +/- S.D., n = 3). Affinity cross-linking of 125I-SP by disuccinimidyl suberate to intact IM-9 cells and membranes revealed specifically labeled proteins of Mr 58,000 and 33,000 in cells, and 58,000, 33,000, and 16,000 in membranes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under both reducing and nonreducing conditions. Competitive effects of substituent peptides of SP on cross-linking and 125I-SP binding to membranes demonstrated that the SP receptor recognized the carboxyl-terminal domain of the peptide. Membranes from cells preincubated in vitro for 12 h at 37 degrees C with 10(-8) M SP demonstrated a decrease in SP receptor density to 13 +/- 2 fmol/mg (mean +/- S.D., n = 2), and a parallel diminution in the specific labeling of membrane proteins of Mr 58,000 and 33,000. These observations suggest that solubilization in CHAPS preserves the binding characteristics of the IM-9 lymphoblast receptor for SP, and that affinity cross-linking techniques identify by sodium dodecyl sulfate-polyacrylamide gel electrophoresis membrane proteins that are specifically labeled by SP.     

Stereospecific receptors for substance P on cultured human IM-9 lymphoblasts

The neuropeptide substance P (SP), which has been demonstrated to bind specifically to human blood T lymphocytes and to stimulate their uptake of [3H]thymidine and [3H]leucine, now is shown to bind stereospecifically to cultured human lymphoblasts of the IM-9 line. The specific binding of [3H]SP by IM-9 lymphoblasts increases linearly with the concentration of IM-9 lymphoblasts, achieves a plateau after approximately 15 to 20 min at 4 degrees C and 4 to 6 min at 37 degrees C, and is rapidly reversible at both 4 degrees C and 37 degrees C. The binding of [3H]SP at steady-state conditions demonstrates a dissociation constant (KD) of 0.65 +/- 0.19 nM (mean +/- SD, n = 5) and 22,641 +/- 6143 receptors per IM-9 lymphoblast. Maximal specific binding of [3H]SP to IM-9 lymphoblasts is observed at pH 7.4 and is dependent on the presence of Mg2+, but not Ca2+, in the medium. The peptide structural determinants of the inhibition of binding of [3H]SP to IM-9 lymphoblasts by substituent peptides and homologs of SP indicate that the receptors recognize predominantly the carboxy-terminal portion of SP. The characteristics of the interaction of SP with IM-9 lymphoblasts suggests a receptor-directed mechanism by which neuropeptides may modulate specifically the contributions of lymphocytes to immunity.     

Effect of the 90 kDa heat shock protein, HSP90, on glucocorticoid receptor binding to DNA-cellulose

Glucocorticoid receptors in the IM-9 human lymphoblastoid cell line were affinity labeled with [3H]dexamethasone 21-mesylate and activated to a DNA-binding form by filtration through a Bio-Gel A-1.5m column. The 90 kDa heat shock protein, HSP90, was identified by labeling IM-9 cells with 35S-methionine at both 37 degrees C and 42 degrees C and purified to near homogeneity by sequential chromatography through DE52 and hydroxyapatite. Addition of purified HSP90 to activated, affinity labeled glucocorticoid receptors in a molecular ratio of 16 to 1 inhibited the binding of the receptors to DNA-cellulose. HSP90 did not affect the binding of other proteins to DNA-cellulose, indicating that the inhibitory effect of HSP90 was specific for the glucocorticoid receptor. These results suggest that HSP90 may associate with the glucocorticoid receptor, masking its DNA-binding site and thereby inhibiting receptor interaction with DNA.     

Processing of the human IM-9 lymphoblast substance P receptor. Biosynthetic and degradation studies using a monoclonal anti-receptor antibody

Cellular membrane receptors for the immunostimulatory neuropeptide substance P have been previously identified on the cultured lymphoblast cell line, IM-9. The regulation of this receptor by ligand and the contribution to its molecular weight by N-linked sugars was studied by incubating IM-9 cells for 14 hr in the presence of [35S]met with or without substance P and tunicamycin, respectively. Cells were lysed and the receptor proteins were immunoprecipitated with an anti-receptor monoclonal antibody. SDS-PAGE analysis of untreated cellular lysates revealed specifically precipitated proteins of 38 kD and 33 kD, which were down-regulated by substance P. In tunicamycin-treated cells, whose substance P binding was not affected, the major immunoprecipitated protein had an apparent Mr of 29 kD. The time course of receptor processing was studied by pulse chase analysis. Three proteins of molecular weights 38 kD (mature receptor), 36 kD and 33 kD (receptor precursors) were identified for time periods of 30 min to 4 hr. The half life of the mature receptor and its precursors was approximately 1 hr and 0.5 hr, respectively. Results from the present studies suggest that the lymphocyte substance P receptor is translated as a precursor protein that is glycosylated.     

Effect of glucocorticoid on epidermal growth factor receptor in human salivary gland adenocarcinoma cell line HSG

Human salivary gland adenocarcinoma (HSG) cells treated with 10(-6) M triamcinolone acetonide for 48 h exhibited a 1.7- to 2.0-fold increase in [125I]human epidermal growth factor (hEGF) binding capacity as compared with untreated HSG cells. Scatchard analysis of [125I]EGF binding data revealed that the number of binding sites was 83,700 (+/- 29,200) receptors/cell in untreated cells and 160,500 (+/- 35,500) receptors/cell in treated cells. No substantial change in receptor affinity was detected. The dissociation constant of the EGF receptor was 0.78 (+/- 0.26).10(-9) M for untreated cells, whereas it was 0.93 (+/- 0.31).10(-9)M for treated cells. The triamcinolone acetonide-induced increase in [125I]EGF binding capacity was dose-dependent between 10(-9) and 10(-6)M, and maximal binding was observed at 10(-6)M. EGF receptors on HSG cells were affinity-labeled with [125I]EGF by use of the cross-linking reagent disuccinimidyl suberate (DSS). The cross-linked [125I]EGF was 3-4% of the total [125I]EGF bound to HSG cells. The affinity-labeled EGF receptor was detected as a specific 170 kDa band in the autoradiograph after SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Densitometric analysis revealed that triamcinolone acetonide amplified the intensity of this band 2.0-fold over that of the band of untreated cells. EGF receptor synthesis was also measured by immunoprecipitation of [3H]leucine-labeled EGF receptor protein with anti-hEGF receptor monoclonal antibody. Receptor synthesis was increased 1.7- to 1.8-fold when HSG cells were treated with 10(-8)-10(-6)M triamcinolone acetonide for 48 h. When the immunoprecipitated, [35S]methionine-pulse-labeled EGF receptor was analyzed by SDS-PAGE and fluorography, the newly synthesized EGF receptor was detected at the position of 170 kDa; and treatment of HSG cells with triamcinolone acetonide resulted in a 2.0-fold amplification of this 170 kDa band. There was no significant difference in turnover rate of EGF receptor between treated and untreated HSG cells. These results demonstrate that the triamcinolone acetonide-induced increase in [125I]EGF binding capacity is due to the increased synthesis of EGF receptor protein in HSG cells.     

Insulin-like growth factor binding to the atypical insulin receptors of a human lymphoid-derived cell line (IM-9).

The cells of the IM-9 human lymphocyte-derived line contain a sub-population of insulin-binding sites whose immunological and hormone-binding characteristics closely resemble those of the atypical insulin-binding sites of human placenta. These binding sites, which have moderately high affinity for multiplication-stimulating activity [MSA, the rat homologue of insulin-like growth factor (IGF) II] and IGF-I, are identified on IM-9 cells by 125I-MSA binding. They account for approximately 30% of the total insulin-receptor population, and do not react with a monoclonal antibody to the type I IGF receptor (alpha IR-3). The relative concentrations of unlabelled insulin, MSA and IGF-I required to displace 50% of 125I-MSA from these binding sites (1:4.7:29 respectively) are maintained for cells, particulate membranes, Triton-solubilized membranes precipitated either by poly(ethylene glycol) or a polyclonal antibody (B-10) to the insulin receptor, and receptors purified by insulin affinity chromatography. Because the atypical insulin/MSA-binding sites outnumber the type I IGF receptors in IM-9 cells by approximately 10-fold, they also compete with the latter receptors for 125I-IGF-I binding. Thus 125I-IGF-I binding to IM-9 cells is inhibited by moderately low concentrations of insulin (relative potency ratios for insulin compared with IGF-I are approx. 1/14 to 1/4) and is partially displaced (65-80%) by alpha IR-3. When type I IGF receptors are blocked by alpha IR-3 or removed by B-10 immunoprecipitation or insulin affinity chromatography, the hormone-displacement patterns for 125I-IGF-I binding resemble those of the atypical insulin/MSA-binding sites.     

Insulin receptor expression in the Burkitt lymphoma cells Daudi and Raji

The specific binding of insulin to either intact or Triton-solubilized Daudi cells (a Burkitt lymphoma cell line) was reduced by over 95% compared to that to control IM-9 lymphocytes due to a decrease in receptor number without a change in affinity. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography revealed that 125I-labeled Daudi cells had reduced amounts (approximately 1/20th) of immunoprecipitable binding (alpha) subunit [mol wt (Mr), 130,000] of the receptor and a relatively abundant 210,000 Mr form not seen in IM-9 cells. The transmembranous (beta) subunit (Mr, 90,000) of the receptor, although not detected by 125I surface labeling, could be phosphorylated and, together with the 210,000 Mr form, exhibited the same 2-fold stimulation of phosphorylation by insulin as that in IM-9 cells. Northern blot hybridization revealed a decrease in Daudi cells of all four major species of insulin receptor mRNA. The Raji cell, another Burkitt lymphoma cell line, also exhibited reduced protein and genetic expression of the insulin receptor, indicating that reduced insulin receptor expression may be representative of other Burkitt lymphoma cell lines.     

Complete glycosylation of the insulin and insulin-like growth factor I receptors is not necessary for their biosynthesis and function. Use of swainsonine as an inhibitor in IM-9 cells

Swainsonine, an indolizidine alkaloid which is a potent inhibitor of the Golgi enzyme, mannosidase II, leads to the production of incompletely processed glycoproteins lacking complex type oligosaccharides. This inhibitor has been used to examine the importance of terminal sugar groups in the biosynthesis and function of both the insulin receptor and the insulin-like growth factor I receptor. IM-9 cells were metabolically labeled using [35S]methionine and the two receptors were independently immunoprecipitated using specific monoclonal antibodies. The incompletely processed receptors have slightly lower molecular weights and contain hybrid rather than complex type oligosaccharides as indicated by their sensitivity to endoglycosidase H and neuraminidase. Both receptors made in the presence of swainsonine are still autophosphorylated in the presence of the respective hormone. The insulin receptor made in the presence of the inhibitor can be affinity labeled at the cell surface using 125I-insulin and disuccinimidyl suberate cross-linking; there is also no significant difference in its affinity for insulin. These results suggest that for the insulin and insulin-like growth factor I receptors to be synthesized, processed, and function normally, they do not require all of the sugars which are normally added in the terminal stages of glycosylation.     

Characterization of functional calcitonin gene-related peptide receptors on rat lymphocytes.

Calcitonin gene-related peptide (CGRP), a vasoactive neuropeptide present in peripheral neurons, is released at local sites of inflammation. In these studies specific high affinity adenylyl cyclase linked CGRP receptors were characterized on rat lymphocytes. The distribution, affinity, and specificity of CGRP receptors was analyzed by radioligand binding. 125I-[His10]CGRP binding to rat lymphocytes was rapid, reaching equilibrium by 20 to 30 min at 22 degrees C, and dependent on cell concentration. The dissociation constants, Kd, for the CGRP receptor on purified T and B lymphocytes are 0.807 +/- 0.168 nM and 0.387 +/- 0.072 nM and the densities are 774 +/- 387 and 747 +/- 244 binding sites/cell, respectively. Competition binding studies determined that rat CGRP inhibits 125I-[His10]CGRP binding to lymphocytes with the highest affinity (Ki = 0.192 +/- 0.073) followed by human CGRP and the CGRP receptor antagonist CGRP8-37. 125I-[His10]CGRP binding to rat lymphocytes was not inhibited by the neuropeptides substance P, calcitonin, or neuropeptide Y. Lymphocyte CGRP receptor proteins were identified by affinity labeling by using disuccinimidyl suberate to covalently cross-link 125I-[His10]CGRP to its receptor. Specifically labeled CGRP binding proteins visualized by SDS-PAGE analysis had molecular masses of 74.5 and 220 kDa. A third high molecular mass protein band which did not penetrate the gel was also observed. In functional studies, CGRP stimulated a rapid, sustained increase in cAMP with an ED50 of approximately 8 pM. In experiments comparing optimal concentrations of isoproterenol, a beta 2-adrenergic agonist, and CGRP, intracellular cAMP elevation after isoproterenol treatment returned to basal levels by 30 min, whereas cAMP was still elevated at 60 min after CGRP treatment. The response to CGRP was specific in that it could be completely blocked by CGRP8-37. The presence of high affinity functional CGRP receptors on T and B lymphocytes provides evidence for a modulatory role for CGRP in regulating lymphocyte function.     

The growth hormone-like factor produced by the tapeworm Spirometra mansonoides specifically binds receptors on cultured human lymphocytes

Plerocercoid larvae of the tapeworm Spirometra mansonoides produce a factor with activities similar to those of growth hormone (GH). Highly selective receptors for GH have been described on cultured human lymphocytes (IM-9 cells) and these cells have been used as a model of binding essentially restricted to human GH (hGH). We compared the displacement of [125I]hGH by hGH and partially purified plerocercoid growth factor (PGF) in assays using rabbit hepatic membranes and IM-9 cells. PGF displaced [125I]hGH from both rabbit hepatic membranes and IM-9 cells in a dose-dependent manner (r greater than 0.98). These results show that PGF specifically binds to hGH receptors on human IM-9 cells and suggest the possibility that PGF will have somatotropic activity in humans.     

Human lymphocytes lack substance P receptors.

Substance P (SP), a neuropeptide found in high concentrations in the gut, is reported to have many potent immunomodulatory actions. This study evaluated some effects of SP on human peripheral blood lymphocytes (PBL) and jejunal intraepithelial lymphocytes (IEL) and the expression of SP receptors on these and other lymphocytes types. In contrast to previous studies, SP (10(-8) or 10(-12) M) did not affect the proliferation (spontaneous or mitogen-induced) nor spontaneous cytotoxicity by PBL or IEL. To determine whether this unresponsiveness was due to an absence of SP receptors, the SP binding potential of these and other human lymphocyte types was determined by Scatchard analysis of radioligand binding. The IM-9 B lymphoblastoid cell line, used as a positive control, demonstrated 4838 +/- 603 or 3131 +/- 832 receptors per cell, with a Kd of 0.21 +/- 0.01 or 0.18 +/- 0.09 nM, using [3H]SP or 125I-SP, respectively. No receptors were found on PBL, polymorphonuclear leukocytes, splenocytes, IEL, or jejunal lamina propria lymphocytes using either radioligand. These findings dispute the presence of large numbers of SP receptors on lymphocytes in peripheral blood, spleen, or intestinal mucosa, and argue against any major effect of SP on T cell proliferation or spontaneous cytotoxicity.     

Direct evidence for intra- and intermolecular disulfide bond formation in the human glucocorticoid receptor. Inhibition of DNA binding and identification of a new receptor-associated protein

We have investigated the potential for the steroid affinity-labeled human glucocorticoid receptor to form both intramolecular and intermolecular disulfide bonds. Glucocorticoid receptors labeled in intact HeLa S3 cells with the covalent affinity label [3H]dexamethasone mesylate ([3H]DM) were analyzed on denaturing 5-12% polyacrylamide gels under both nonreducing and reducing conditions. Under nonreducing conditions the affinity-labeled receptor migrated as a heterogeneous species having an average molecular mass of approximately 96 kDa whereas, under reducing conditions, the receptor migrated as a more discrete form. These data suggest that a reducing environment can influence the structure of the glucocorticoid receptor monomer and further imply that sulfhydryl groups within the affinity-labeled receptor are available for modification. To pursue this observation in greater detail, we tested the effect of oxidizing conditions on the structure of the glucocorticoid receptor. The presence of low concentrations (0.125-0.5 mM) of three oxidizing reagents (sodium tetrathionate, disulfiram, and iodosobenzoate) altered the migration of the affinity-labeled receptor resulting in forms of apparent lower molecular mass (as low as 78 kDa). This altered migration, not seen with most other cytosolic proteins, is consistent with the formation of intramolecular disulfide bonds within the receptor which presumably cause it to assume a folded conformation and migrate faster through the gel. At higher concentrations of these reagents (up to 5.0 mM), we also detect a saturably labeled [3H]DM band which has a higher molecular mass (approximately 140 kDa), indicating the formation of intermolecular disulfide bonds between the [3H]DM-labeled receptor and another closely associated protein(s) having a molecular mass of approximately 40 kDa. The effects which these oxidizing reagents have on glucocorticoid receptor structure are completely reversed upon the addition of dithiothreitol, indicating that the observed changes in migration do not reflect receptor proteolysis but rather a folding and unfolding within the receptor monomeric protein. We have also analyzed the effect of this oxidation/reduction on the function of the glucocorticoid receptor. Oxidation of the [3H]DM-labeled receptor complex with 0.5 mM sodium tetrathionate inhibited activation of receptor to a form capable of binding to DNA-cellulose. This inhibition can be reversed with dithiothreitol at 25 degrees C but not at 0 degrees C, suggesting that these oxidizing reagents are inhibitory at the transformation and/or activation steps.(ABSTRACT TRUNCATED AT 400 WORDS)     

Monoclonal antibody to the human glucose transporter that differentiates between the glucose and nucleoside transporters

A monoclonal antibody to the glucose transporter has been prepared with band 4.5 (Mr 45,000-65,000) from human erythrocyte ghosts as antigen. This antibody, designated 7F7.5, is of the IgG2b type. The antibody bound exclusively to proteins in the band 4.5 region of immunoblots of human erythrocyte ghosts separated on sodium dodecyl sulfate-polyacrylamide gels. Immobilized 7F7.5 antibody removed glucose transport activity from solubilized alkaline-treated ghosts. The material that was eluted from the immobilized antibody matrix migrated primarily in the band 4.5 region of electrophoretic gels and bound the antibody in immunoblots. To test the specificity of the antibody, glucose and nucleoside transporters in alkaline-treated human erythrocyte ghosts were affinity labeled with [3H]cytochalasin B and [3H]-S-(nitrobenzyl)thioinosine (NBMPR), respectively. Both of these transporters are band 4.5 proteins and "copurify" by DEAE-cellulose chromatography. A filter paper assay was developed to assess the presence of the labeled transporters. Immobilized 7F7.5 antibody bound 99% of the labeled glucose transporter. In contrast, only 3% of the specifically labeled nucleoside transporter bound to the immobilized antibody. Furthermore, the antibody did not remove nucleoside transport or NBMPR binding activities from detergent solution. The antibody recognized two tryptic fragments, Mr 23,000 and 18,000, which contain the cytochalasin B binding site of the glucose transporter. By immunoblot, the monoclonal antibody recognized the glucose transporter in cultured human IM9 lymphocytes, synovial cells, and HBL 100 mammary cells but not cells of murine or rat origin. These results indicate that the glucose and nucleoside transporters are distinct proteins which can be distinguished by monoclonal antibody 7F7.5. The method developed to quantitate covalently labeled glucose and nucleoside transporters should have broad applicability as a rapid and easy method for determining the recovery of affinity-labeled membrane proteins in detergent solution during purification. Because of the location of the epitope, the antibody itself should prove to be a valuable tool in establishing the molecular basis for the function and regulation of the glucose transporter.     

Identification of a 27-kDa protein with the properties of type II iodothyronine 5'-deiodinase in dibutyryl cyclic AMP-stimulated glial cells

Type II iodothyronine 5'-deiodinase (5'D-II) catalyzes the intracellular conversion of thyroxine (T4) to 3,5,3'-triiodothyronine (T3), producing greater than 90% of the bioactive thyroid hormone in the cerebral cortex. In cultured glial cells, expression of this enzyme is cAMP dependent. Exploiting the cAMP-dependent nature of this enzyme in these cells and utilizing N-bromoacetyl-L-3'- or 5'-[125I]thyroxine (BrAc[125I]T4) to affinity label cellular proteins, a 27-kDa protein with the properties of this enzyme was identified. Intact cells labeled with BrAc[125I]T4 showed three prominent radiolabeled bands of proteins of Mr 55,000, 27,000, and 18,000 (p55, p27, p18, respectively) which incorporated approximately 80% of the affinity label. All three affinity-labeled proteins were membrane associated. One protein (p27) increased 5-6-fold after treating the cells for 16 h with dibutyryl cAMP; maximal specific incorporation of affinity label into the stimulated p27 was approximately 2 pmol/mg of cell protein in intact cells. Alterations in the steady-state levels of 5'D-II resulted in parallel changes in the quantity of p27. In cell sonicates, the rate of enzyme inactivation by BrAcT4 equaled the rate of affinity label incorporation into stimulated p27, whereas p55 and p18 showed little or no specific dibutyryl cAMP-stimulated labeling. Enzyme substrates T4 and 3,3'5'-triiodothyronine (rT3) specifically blocked p27 labeling, whereas T3 and the competitive 5'D-II inhibitor EMD 21388 (a synthetic flavonoid) were much less effective. Iopanoate, an inhibitor of all deiodinase isozymes, was ineffective in blocking p27 labeling. Inhibition kinetics revealed that iopanoate was a noncompetitive inhibitor of dibutyryl cAMP-stimulated glial cell 5'D-II, suggesting that it interacts at a site distant from the substrate-binding site. These data identify a cAMP-inducible membrane-associated protein (p27) that has many of the properties of 5'D-II.     

Reduced peptide bond pseudopeptide analogues of substance P. A new class of substance P receptor antagonists with enhanced specificity

Each of the last 6 peptide bonds in the COOH terminus of [Leu11]substance P [( Leu11]SP) and [Nle11]spantide were replaced with [CH2NH], and each analogue was tested for SP agonist or antagonist activity by determining its ability to interact with SP receptors on dispersed acini from guinea pig pancreas. Each of the 6 spantide and 5 of the 6 SP analogues had no agonist activity, whereas [psi 9-10]SP was an agonist. For the spantide pseudopeptides, the psi 10-11 analogue (Ki,2.8 microM) was equipotent as an antagonist to spantide itself, whereas the psi 9-10, psi 8-9, psi 7-8, and psi 6-7 analogues were 2.5, 7, 5, and 3 times less potent. For the SP pseudopeptides, the psi 10-11 analogue was the most potent antagonist (Ki, 6.2 microM), whereas the psi 8-9, psi 7-8, and psi 6-7 analogues were 7-, 36-, and 39-fold less potent. There was a close correlation between the ability of each pseudopeptide to inhibit binding of 125I-Bolton-Hunter-SP and to affect amylase secretion. [psi 10-11]SP inhibited SP-stimulated amylase release in a competitive manner, and its inhibitory ability was specific for the SP receptor. Despite [psi 10-11]SP, spantide, and [psi 10-11]spantide having similar affinities for the SP receptor (Ki, 2-6 microM), for inhibition of binding of 125I-[Tyr4]bombesin, the analogues differed with [psi 10-11]SP having a 50-fold lower affinity than for the SP receptor, whereas [psi 10-11]spantide had a 4-fold lower affinity and spantide a 1.5-fold lower affinity for the SP receptor. These results demonstrate that SP pseudopeptides represent a new class of SP receptor antagonists and, in contrast to the currently described SP receptor antagonists, are more specific for SP receptors.     

Chemical modification to reduce renal uptake of disulfide-bonded variable region fragment of anti-Tac monoclonal antibody labeled with 99mTc.

The anti-Tac disulfide-bonded variable region fragment (dsFv) is a genetically engineered, 25 kDa, murine monoclonal antibody fragment that recognizes the alpha subunit of the interleukin-2 receptor (IL-2Ralpha). The dsFv radiolabeled with the tetrafluorophenyl ester (TFP) of [99mTc]mercaptoacetyltriglycine ([99mTc]MAG3-TFP) showed rapid tumor uptake and fast blood clearance in mice, resulting in high tumor-to-nontumor background ratios. However, its high renal uptake was a problem. In this study, we tested the effect of lowering the isoelectric point (pI) of dsFv to <9.3 on renal and tumor uptake. To lower the pI, dsFv was acylated simultaneously with both [99mTc]MAG3-TFP and TFP-glycolate. The acylation of dsFv decreased its pI and its immunoreactivity inversely proportional to the molar ratio of TFP-glycolate to dsFv, whereas the conjugation of [99mTc]MAG3-TFP alone did not. When biodistribution studies were performed in nude mice, the effect of the lowered pI was reflected primarily in decreased kidney uptake and whole-body retention, with its highest effect seen at the earliest time point (15 min) after injection. In tumor-bearing nude mice, glycolated [99mTc]MAG3-dsFv with a pI range of 4.9 to 6.5 accumulated selectively into IL-2 receptor-positive SP2/Tac tumor similar to that of the control [125I]dsFv labeled by the Iodo-Gen method, whereas its renal uptake was 25% of [125I]dsFv at 15 min. At 90 min, the ratios of tumor to receptor-negative SP2/0 tumor, liver, kidney, stomach, and blood had peaked at 10.9, 8.5, 0.3, 5.0, and 6.2, respectively, for the glycolated [99mTc]MAG3-dsFv. The corresponding ratios for [125I]dsFv were 3.7, 5.0, 0.1, 1.5, and 2.1, respectively.     

A single Mr approximately 103,000 125I-beta-nerve growth factor-affinity-labeled species represents both the low and high affinity forms of the nerve growth factor receptor

Both high and low affinity receptors for nerve growth factor (NGF) have been described, but only the former appear to mediate NGF actions and uptake. To specifically characterize the molecular identity of the high affinity site and to compare it with the low affinity site, the water-soluble carbodiimide EDC was used to cross-link 125I-NGF to NGF receptors on: rat PC12 cells, PC12nnr5 cells (PC12 mutants that have only low affinity NGF binding), SH-SY5Y human neuroblastoma cells (which have only high affinity binding sites), and cultured rat sympathetic ganglion cells. A variety of criteria were used to distinguish the two classes of affinity-labeled receptors: competition with unlabeled NGF, dissociation rate, and selective solubilization by 0.1% Triton X-100. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that cross-linking generated only a single Mr approximately 103,000 125I-NGF affinity-labeled species which represents both the low and high affinity forms of the receptor. The 125I-NGF X receptor complexes formed with both affinity classes of the receptor were quantitatively immunoprecipitated by the monoclonal anti-NGF-receptor antibody 192-IgG and both showed identical shifts in mobility when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions. These findings indicate that both high and low affinity NGF receptors possess apparently identical NGF-binding moieties. The differences between the kinetic and functional properties of the two receptor types may therefore result from their interactions with other membrane components or with cytoplasmic proteins.     

Comparison of the nuclear and cytosolic forms of the Ah receptor from Hepa 1c1c7 cells: charge heterogeneity and ATP binding properties.

2-[125I]iodo-7,8-dibromo-p-dioxin ([125I]Br2DpD) and 2-[125I]iodo-3-azido-7,8-dibromo-p-dioxin ([125I]N3Br2-DpD) are both capable of binding to the Ah receptor (AhR) with a high degree of specificity in cultured Hepa 1c1c7 cells. After incubation with either [125I]N3Br2DpD or [125I]Br2DpD Hepa 1c1c7 cytosolic and high salt nuclear extracts were analyzed by sucrose density gradient analysis with the following results: (i) With both radioligands an approximately 9 S form of the AhR was observed in cytosolic extracts. (ii) Nuclear extracts labeled with [125I]N3Br2DpD revealed both approximately 6 S and approximately 9 S forms of the AhR. (iii) In contrast, analysis of nuclear extracts labeled with [125I]Br2DpD revealed only an approximately 6 S form of the AhR. The approximately 9 S [125I]N3Br2DpD-labeled AhR was preferentially extracted with 100 mM KCl from a nuclear fraction and mixed with monoclonal antibody 8D3, an anti-90-kDa heat shock protein antibody. Monoclonal antibody 8D3 was able to bind to the approximately 9 S nuclear form of the AhR and caused the receptor to sediment as a heavier complex on sucrose density gradients. This would indicate that the AhR can reside in the nucleus bound to 90-kDa heat shock protein. The [125I]N3Br2DpD-labeled approximately 6 S peak fractions were collected and subjected to denaturing two-dimensional gel electrophoresis. A comparison of [125I]N3Br2DpD-labeled cytosolic (9 S) AhR preparations with the nuclear (6 S) AhR by 2-D gel electrophoresis was performed. The cytosolic form of the AhR was present in the apparent pI range of 5.2-5.7; the nuclear form focused between 5.5 and 6.2. The [125I]N3Br2DpD-labeled nuclear extracts were incubated with ATP-agarose and 43% of the photoaffinity-labeled AhR bound to the affinity gel. In contrast, approximately threefold lower binding of [125I]N3Br2DpD-labeled receptor was obtained when GTP-, AMP-, or ADP-agarose was used. Only 2% of the [125I]N3Br2DpD-labeled cytosolic AhR was able to bind to ATP-agarose. These results suggest that after the AhR translocates into the nucleus the following biochemical changes occur: (i) The sedimentation value for the AhR changes from an approximately 9 S to an approximately 6 S species. (ii) The AhR attains the ability to bind with specificity to ATP. (iii) The AhR undergoes a shift to a more basic pI.