The glycoprotein hormones: recent studies of structure-function relationships

The structural features of the heterodimeric glycoprotein hormones (LH, FSH, TSH, and hCG) are briefly reviewed. Removal of carbohydrate chains does not reduce binding of the hormones to membrane receptors, but markedly reduces biological responses. The glycopeptides from the hormone do not reduce binding of native hormone to receptors but do reduce biological responses. Newer data concerned with replication of different regions of the peptide chains of these molecules using synthetic peptides are reviewed and presented. These studies indicate that two regions on the common alpha subunit are involved with receptor binding of the LH, hCG, and TSH molecules. These regions are alpha 26 to 46 and alpha 75-92. Two synthetic disulfide loop peptides from the hCG beta subunit beta 38-57 and beta 93-100 also block binding of hCG to its receptor. In addition, the beta 38-57 peptide stimulates testosterone production by Leydig cells. These data indicate that glycoprotein hormone binding to plasma membrane receptors involves a discontinuous site on the hormone that spans both the alpha and beta subunits, and that the alpha subunit sites are similar for several hormones.     

Inhibition of human choriotropin binding to receptor by human choriotropin alpha peptides. A comprehensive synthetic approach

Synthetic overlapping peptides of the alpha-subunit of human chorionic gonadotropin (hCG) were made by solid-phase peptide synthesis employing a comprehensive synthetic approach. The entire primary structure of the alpha-subunit was synthesized as a series of nine consecutive peptides, each 15 residues in length, and overlapping with its two adjacent neighbors by 5 residues on each side. Receptor binding activity of each synthetic peptide was measured by the inhibition of binding of 125I-labeled hCG to rat ovarian receptor. Peptides alpha 21-35, alpha 31-45, alpha 71-85, and alpha 81-92 were shown to compete for binding with native hCG, thus demonstrating that at least two regions on the alpha-subunit may be part of the binding site(s) of the hormone. The low affinity of the peptides (10(-5)-10(-6) M) compared to native hormone (10(-10) M) for receptor is not unexpected due to the probability of discontinuous and multiple sites involved in receptor binding. An ultrapure preparation of hCG alpha-subunit also had low affinity (10(-5), suggesting that conformational changes upon combination with beta-subunit to form dimer or changes in conformation after binding are necessary for high affinity interaction. These results correlate with previous predictions of binding sites based on studies employing chemical and enzymatic modifications of intact hormone and show that synthetic peptide strategies are helpful in the elucidation of protein structure and function.     

Mapping the receptor binding regions of human chorionic gonadotropin (hCG) using disulfide peptides of its beta-subunit: possible involvement of the disulfide bonds Cys(9)-Cys(57) and Cys(23)-Cys(72) in receptor binding of the hormone.

Human chorionic gonadotropin (hCG) is a heterodimeric glycoprotein hormone essential for the establishment and maintenance of pregnancy. The alpha- and beta-subunits of hCG are highly cross-linked internally by disulfide bonds which seem to stabilize the tertiary structures required for the noncovalent association of the subunits to generate hormonal activity. The purpose of this study was to delineate the role of the disulfide bonds of hCGbeta in receptor binding of the hormone. Six disulfide peptides incorporating each of the six disulfide bonds of hCGbeta were synthesized and screened, along with their linear counterparts, for their ability to competitively inhibit the binding of [125I] hCG to sheep ovarian corpora luteal LH/CG receptor. Disulfide peptide Cys (9-57) was found to be approximately 4-fold more potent than the most active of its linear counterparts in inhibiting radiolabeled hCG from binding to its receptor. Similarly, disulfide peptide Cys (23-72) exhibited receptor binding inhibition activity, whereas the constituent linear peptides were found to be inactive. The results suggest the involvement of the disulfide bonds Cys(9)-Cys(57) and Cys(23)-Cys(72) of the beta-subunit of hCG in receptor binding of the hormone. This study is the first of its kind to use disulfide peptides rather than linear peptides to map the receptor binding regions of hCG.     

Synthetic hFSH peptide constructs in the evaluation of previous studies on the hFSH receptor interaction

The human follicle-stimulating hormone (hFSH) belongs to a family of glycoprotein hormones which contains two non-identical subunits. This paper describes the design and synthesis of a series of synthetic hFSH constructs as putative ligands for the receptor. The design of these constructs is based on the crystal structure of hCG and molecular modelling using the program package Insight II/Discover. The designed constructs contain peptides ranging from 7 to 48 amino acid residues, disulphide bridges and glycan residues. All the synthetic peptides were synthesized by the stepwise solid-phase method using Fmoc chemistry. Two of the synthetic peptides contain the glycosylated amino acid, Asn(GlcNAc-GlcNAc) and both were prepared using fully protected glycosylated building blocks in the solid-phase peptide synthesis. The disulphide bridges were formed from acetamidomethyl-protected glycopeptides and peptides by a direct deprotection/oxidation method using thallium(III) trifluoroacetate. Mass spectroscopy and amino acid analysis were used for characterization of the synthetic hFSH glycopeptides and peptides. The synthetic hFSH constructs were tested for binding activity on FSH receptor assays but none showed improved binding properties compared with the naturally occurring hormone. It was finally demonstrated that non-related peptides showed non-specific binding at the same level as reported for specific peptides. © 1997 European Peptide Society and John Wiley & Sons, Ltd.     

Overlapping synthetic peptides as a tool to map protein-protein interactions ̶ FSH as a model system of nonadditive interactions

In earlier work, we used partially overlapped synthetic peptides as a tool to find regions of interaction between the human FSH hormone and its receptor, aiming to find possible antagonists or agonists. Years later, the FSH and FSH receptor 3D structures were reported by other laboratories. The 3D results were in close agreement with the interacting regions predicted by using synthetic peptides. These earlier studies are reviewed here, and the predicted regions of interaction compared to the FSH and FSH receptor 3D structures to illustrate the usefulness of the synthetic peptide strategy to find binding regions. Different contact regions contribute multiplicatively to the high affinity of the entire ligand; thus, peptides covering a fraction of the anchor sites and with low free energy density cannot reach the affinity of the entire molecule. The earlier use of multiple linear regression to find the relevant predictors for effective binding, and a new way to estimate ΔG° and nonadditive interactions for the synthetic peptides in solution, by using the buried surface area (BSA), will be discussed.     

Inhibition of thyrotropin binding to receptor by synthetic human thyrotropin beta peptides

In order to study the structure and function relationships of the thyrotropin (TSH)-specific beta-subunit, we produced 11 synthetic overlapping peptides containing the entire 112-amino acid sequence of human beta TSH and tested them for activity in TSH radioreceptor assay using both human and porcine thyroid membranes. Synthetic peptides representing four regions of the beta-subunit demonstrated the ability to inhibit binding of 125I-bovine TSH to crude thyroid membranes. The peptide representing the -COOH terminus of the subunit (beta 101-112) possessed highest binding activity, inhibiting binding of labeled TSH with an EC50 of 80 microM. The remaining active peptides were: beta 71-85 (104 microM), beta 31-45 (186 microM), beta 41-55 (242 microM), and beta 1-15 (331 microM). Specificity of the binding activity was shown by the inability of the peptides representing the remainder of the subunit to inhibit binding of label and by the inability of any of the peptides to inhibit binding of 125I-epidermal growth factor to the same thyroid membranes. The low affinity of the peptides as compared with native hormone is in agreement with previous studies of synthetic alpha-subunit peptides and, further, suggests that the interaction of beta TSH with receptor is multifaceted, requiring cooperative binding of these sites for the observed high affinity of the whole hormone. These studies are in agreement with previous predictions of active regions by chemical modification but add two regions to the list, showing the utility of the synthetic peptide strategy in the study of peptide hormone structure-activity relationships.     

Structure-function relationship of lapemis toxin: a synthetic approach

The synthetic approach to the structure-function relationship of lapemis toxin has been very useful in clarifying the important binding regions. To identify the neurotoxic binding domain(s) of lapemis toxin, several peptides were synthesized using the 9-fluorenylmethoxycarbonyl protocols. These peptides were based on the sequence of lapemis toxin, a 60-amino-acid, short-chain postsynaptic neurotoxin found in sea snake (Lapemis hardwickii) venom. The peptides were purified using high-performance liquid chromatography and sequenced to verify the correct synthesis, isolation, and purity. The synthetic peptide names and single letter sequences were Peptide A1 (15 mer) CCNQQSSQPKTTTNC Peptide B1 (18 mer) CYKKTWSDHRGTRIERGC Peptide B2 (16 mer) YKKTWSDHRGTRIERG Peptide C1 (12 mer) CPQVKPGIKLEC Peptide NS (20 mer) EACDFGHIKLMNPQRSTVWY. The peptide NS (nonsense peptide) sequence was arbitrarily determined and used as a control peptide. Biological activities of the synthetic peptides were determined by in vivo as well as by in vitro assay methods. For the in vivo assay, lethality was determined by intravenous injection in mice (Swiss Webster). For the in vitro assay, peptide binding to the Torpedo californica nicotinic acetylcholine receptor was determined. The peptides were found to be nontoxic at approximately 114 times the known LD50 of lapemis toxin. Binding studies with 125I-radiolabeled lapemis toxin and tyrosine-containing peptides indicated that lapemis toxin and peptide B1 bound the receptor, while the other peptides had no detectable binding. The central loop domain of lapemis toxin (peptide B1) plays a dominate role in the toxin's binding ability to the receptor. These results and the hydrophilicity analysis predict peptide B1 may serve as an antagonist or antigen to neutralize the neurotoxin effects in vivo.     

α-Bungarotoxin Binds to Human Acetylcholine Receptor α-Subunit Peptide 185–199 in Solution and Solid Phase but Not to Peptides 125–147 and 389–409

The nicotinic acetylcholine receptor (AChR) of human skeletal muscle has a reducible disulfide bond near the neurotransmitter binding site in each of its alpha-subunits. By testing a panel of overlapping synthetic peptides encompassing the alpha-subunit segment 177-208 (containing cysteines 192 and 193) we found that specific binding of 125I-labelled alpha-bungarotoxin (alpha-BTx) was maximal in the region 185-199. Binding was inhibited by unlabelled alpha-BTx greater than d-tubocurarine greater than atropine greater than carbamylcholine. Peptide 193-208 did not bind alpha-BTx, whereas 177-192 retained 40% binding activity. Peptides corresponding to regions 125-147 (containing cysteines 128 and 142) and 389-409, or peptides unrelated to sequences of the AChR failed to bind alpha-BTx. No peptide bound 125I-alpha-labelled parathyroid hormone. The apparent affinity (KD) of alpha-BTx binding to immobilized peptides 181-199 and 185-199 was approximately 25 microM and 80 microM, respectively, in comparison with alpha-BTx binding to native Torpedo ACh receptor (apparent KD approximately 0.5 nM). In solution phase, both peptides effectively competed with solubilized native human AChR for binding of alpha-BTx, and peptide 185-199 showed little evidence of dissociation after 24 h. Peptides that bound alpha-BTx did so when sulfhydryls were reduced. Cysteine modification, by N-ethylmaleimide or acetamidomethylation, abolished alpha-BTx-binding activity. The data implicate the region of cysteines 192 and 193 in the binding of neurotransmitter to the human receptor.     

Residues in the alpha subunit of human choriotropin that are important for interaction with the lutropin receptor

Synthetic peptides were used to probe the structure-function relationships between human choriotropin (hCG) and the lutropin (LH) receptor. Previously, a peptide region of the alpha subunit of hCG, residues 26-46, had been shown to inhibit binding of 125I-hCG to the LH receptor in rat ovarian membranes (Charlesworth, M.C., McCormick, D.J., Madden, B., and Ryan, R.J. (1987) J. Biol. Chem. 262, 13409-13416). To determine which residues are important for this inhibitory activity, peptides were truncated from either the amino or carboxyl terminus, or individual residues were substituted with alanine. The amino-terminal boundary was determined to be Gly-30 and the carboxyl-terminal boundary, Lys-44. This core peptide contained all the residues needed for full activity of the parent peptide 26-46. Arg-35 and Phe-33 were particularly important residues; when they were substituted with alanine, the peptide inhibitory potencies were decreased. Ser-43, Arg-42, Cys-32, and Cys-31 were also important but to a lesser degree. These results are consistent with predictions based on chemical and enzymatic modification studies and provide insight into which residues are important for interaction between hCG and the LH receptor.     

Nine Residues Influence the Binding of α-Bungarotoxin in α-Subunit Region 185–200 of Human Muscle Acetylcholine Receptor

Abstract: Identification of residues in the skeletal muscle nicotinic acetylcholine receptor (AChR) that bind snake venom a-neurotoxin antagonists of acetylcholine [e.g., α-bungarotoxin (α-BTx)] provides structural information about the neurotransmitter binding region of the receptor. Using synthetic peptides of the human AChR α-subunit region 177–208, we previously localized a pharmacologically specific binding site for α-BTx in segment 185–199. To define in more detail the residues that influence the binding of α-BTx to this region, we prepared 16 peptide analogues of the α-subunit segment 185–200, with the amino acid Lalanine sequentially replacing each native amino acid. Circular dichroism spectroscopy did not reveal changes in the secondary structure of the peptides except for the analogue in which Pro¹⁹⁴ was substituted with alanine. This implies that any change in α-BTx binding could be attributed to replacement of the native residue's side chain by alanine's methyl group, rather than to a change in the structure of the peptide. The influence of each substitution with alanine was determined by comparing the analogue to the parental sequence α 185–200 in solution-phase competition with native human AChR for binding of ¹²⁵I-labeled α-BTx. The binding of α-BTx by analogue peptides with alanine substituted for Tyr¹⁹⁰, Cys¹⁹², or Cys¹⁹³ was greatly diminished. Binding of α-BTx to peptides containing alanine replacements at Val¹⁸⁸, Thr¹⁸⁹, Pro¹⁹⁴, Asp¹⁹⁵, or Tyr¹⁹⁸ was also reduced significantly (p < 0.003). An unanticipated finding was that substitution of alanine for Ser¹⁹¹ significantly increased α-BTx binding (p < 0.003). The data imply that these nine amino acids influence the binding of the antagonist, α-BTx, to the nicotinic acetylcholine receptor of human skeletal muscle, and confirm previous reports for certain contact residues for α-BTX that were found in region α181-200 of the Torpedo AChR.     

Binding of Human GM-CSF to Synthetic Peptides of the Alpha Subunit of Its Receptor

Abstract

We have synthesized a series of peptides corresponding to portions of the extracellular domain of human granulocyte-macrophage colony stimulating factor receptor α subunit (hGM-CSFRα). The sequences were chosen according to the homology between hGM-CSFRα and the growth hormone receptor (GHR) and correspond to the regions reported to form the binding site of the latter receptor. The peptides were examined for their binding activity to hGM-CSF by affinity chromatography on resin-immobilized hGM-CSF and by a solid phase binding assay. Four peptides endowed with hGM-CSF binding activity were identified and the postulated homology between the binding sites of hGM-CSFRα and GHR was confirmed.     

Molecular complementarity III. peptide complementarity as a basis for peptide receptor evolution: a bioinformatic case study of insulin,glucagon and gastrin

Dwyer has suggested that peptide receptors evolved from self-aggregating peptides so that peptide receptors should incorporate regions of high homology with the peptide ligand. If one considers self-aggregation to be a particular manifestation of molecular complementarity in general, then it is possible to extend Dwyer's hypothesis to a broader set of peptides: complementary peptides that bind to each other. In the latter case, one would expect to find homologous copies of the complementary peptide in the receptor. Thirteen peptides, 10 of which are not known to self-aggregate (amylin, ACTH, LHRH, angiotensin II, atrial natriuretic peptide, somatostatin, oxytocin, neurotensin, vasopressin, and substance P), and three that are known to self-aggregate (insulin, glucagon, and gastrin), were chosen. In addition to being self-aggregating, insulin and glucagon are also known to bind to each other, making them a mutually complementary pair. All possible combinations of the 13 peptides and the extracellular regions of their receptors were investigated using bioinformatic tools (a total of 325 combinations). Multiple, statistically significant homologies were found for insulin in the insulin receptor; insulin in the glucagon receptor; glucagon in the glucagon receptor; glucagon in the insulin receptor; and gastrin in gastrin binding protein and its receptor. Most of these homologies are in regions or sequences known to contribute to receptor binding of the respective hormone. These results suggest that the Dwyer hypothesis for receptor evolution may be generalizable beyond self-aggregating to complementary peptides. The evolution of receptors may have been driven by small molecule complementarity augmented by modular evolutionary processes that left a "molecular paleontology" that is still evident in the genome today. This "paleontology" may allow identification of peptide receptor sites.     

Role of the beta 93-100 determinant loop sequence in receptor binding and biological activity of human luteinizing hormone and chorionic gonadotropin

The intercysteine loop sequence (93-100) in the beta-subunit has been postulated to be important for receptor binding and specificity in the glycoprotein hormones, LH and human CG (hCG). To demonstrate this directly, and to characterize the structural features essential for activity, we prepared a series of synthetic peptides and analogs incorporating this determinant loop region. Peptides were assayed for inhibition of labeled hCG binding to ovarian membrane receptors and stimulation of testosterone production in Leydig cells. Peptides with the native (93-100) sequence from hCG and hLH inhibited hCG binding half maximally at 2.18 and 2.62 x 10(-4) M, respectively, while the sequence from FSH was inactive. Isosteric substitution of Ala for Cys resulted in an inactive peptide, indicating that the (93-100) disulfide bridge is essential for activity. Optimal binding activity requires at least one net positive charge among the side chains, as shown by loss of activity in hybrid analogs with neutral or negative charges conferred by progressive replacement of Arg by Asp at 94 and 95 or by introduction of Asp at 96 and 97. Despite binding to receptors, the native sequence did not promote testosterone production at doses up to 10(-2) M. This contrasts with a second receptor binding sequence, beta (38-57) that activates testosterone production. There are differences between the (93-100) and (38-57) loop sequences in their chemical and physical properties, biological activity and antigenicity. While the cumulative evidence suggests that they associate with counterpart sites in alpha-subunit to form a topographical binding domain in the whole hormone, our results suggest that each sequence may contribute in different ways to activation of postreceptor events.     

Topographic antigenic determinants recognized by monoclonal antibodies on human choriogonadotropin beta-subunit

We describe a first attempt to study the antibody-combining sites recognized by monoclonal antibodies raised against the beta-subunit of human choriogonadotropin (hCG). Two groups of antibodies were first defined by their ability to recognize only the free beta-subunit or the free and combined subunit. Antibodies FBT-11 and FBT-11-L bind only to hCG beta-subunit but not to hCG, whereas antibodies FBT-10 and D1E8 bind to both the beta-subunit and the hormone. In both cases, the antigenic determinants were localized to the core of the protein (residues 1-112), indicating the weak immunogenicity of the specific carboxyl-terminal extension of hCG-beta. Nine synthetic peptides spanning different regions of hCG-beta and lutropin-beta were assessed for their capacity to inhibit antibody binding. A synthetic peptide inclusive of the NH2-terminal region (residues 1-7) of the hCG beta-subunit was found to inhibit binding to the radiolabeled subunit of a monoclonal antibody specific for free hCG-beta (FBT-11). Further delineation of the antigenic site recognized by this antibody provided evidence for the involvement of fragment 82-92. Moreover, monoclonal antibody FBT-11 inhibited the recombination of hCG-beta to hCG-alpha, indicating that its antigenic determinant might be located nearby or in the hCG-beta portion interacting with the alpha-subunit. Binding of monoclonal antibody FBT-10, corresponding to the second antigenic determinant, was weakly inhibited by fragment 82-105 and did not impair the recombination of the hCG beta-subunit to the hCG alpha-subunit. Its combining site appeared to be located in a region of the intact native choriogonadotropin present at the surface of the hormone-receptor complex.     

Tumor-associated antigen human chorionic gonadotropin beta contains numerous antigenic determinants recognized by in vitro-induced CD8+ and CD4+ T lymphocytes

The beta subunit of human chorionic gonadotropin (hCG beta) is markedly overexpressed by neoplastic cells of differing histological origin including those present in colon, breast, prostate and bladder tumors. We have previously shown that some patients with hCG beta-producing urothelial tumors have circulating T cells that proliferate in response to hCG beta. To make a comprehensive study of hCG beta as a potential target for cancer immunotherapy, we investigated whether hCG beta peptides could induce CD4+ or CD8+ T-cell responses in vitro. By stimulating peripheral blood mononuclear cells (PBMCs) from three donors with mixtures of overlapping 16-mer synthetic peptides analogous to portions of either the hCG beta 20-71 or the hCG beta 102-129 region, we established six CD4+ T-cell lines that proliferated specifically in response to five distinct determinants located within these two hCG beta regions. Three antigenic determinants (hCG beta 52-67, 106-121 and 114-125) were presented by HLA-DR molecules, while the two other antigenic determinants (hCG beta 48-63 and 56-67) were presented by HLA-DQ molecules. Interestingly, one T-cell line specific for peptide hCG beta 106-121 recognized hCG beta peptides comprising, at position 117, either an alanine or an aspartic acid residue, with the latter residue being present within the protein expressed by some tumor cells. In addition, three other hCG beta-derived peptides that exhibited HLA-A*0201 binding ability were able to stimulate CD8+ cytotoxic T cells from two HLA-A*0201 donors. These three immunogenic peptides corresponded to regions hCG beta 40-48, hCG beta 44-52 and hCG beta 75-84. Our results indicate that the tumor-associated antigen hCG beta possesses numerous antigenic determinants liable to stimulate CD4+ and CD8+ T lymphocytes, and might thus be an effective target antigen for the immunotherapy of hCG beta-producing tumors.     

Ligand-induced receptor dimerization may be critical for signal transduction by choriogonadotropin.

A mechanism of signal transduction by human choriogonadotropin (hCG) has been proposed. Competitive inhibition of the binding of hCG to its receptor by the serine protease inhibitors led to the identification of local structural homology of an extracellular region of the receptor with the reactive site loop of chymotrypsin inhibitor. Synthetic peptides from the extracellular domain of luteinizing hormone-choriogonadotropin (LH/CG) receptor, rationally designed on the basis of this homology, were found to affect hormone-receptor binding and bioactivity. A receptor peptide incorporating one complete structural unit of the leucine-rich repeats motif of the extracellular domain of the receptor significantly increased hCG-receptor binding in a dose-dependent manner. However, the testosterone production in a Leydig cell bioassay was inhibited in the presence of this peptide. The agonistic effect on the hCG-receptor binding and the antagonistic effect on the testosterone production of a receptor peptide suggests the possibility of more than one quasi-equivalent receptor-binding site on the hormone. Hormone-induced receptor oligomerization may therefore be involved in the mechanism of signal transduction by hCG.     

Enhancing the affinity of SEB-binding peptides by repeating their sequence

The utilization of peptide ligands in biosensors and bioassays is dependent on achieving high affinity of these peptides toward their targets. In a previous report, we identified 12-mer peptides that could selectively bind to Staphylococcal enterotoxin B (SEB) using a phage-display library. In this study, we explore for new modification approaches to enhance the affinity of two different SEB-binding peptides. In order to identify the binding regions of selected peptides, the charged residues and the ones, critical for the structure of peptide, were replaced with alanine. However, a specific binding region could not be suggested as all mutant peptides have lost their affinities toward SEB completely. The modifications for the affinity enhancement were done by repeating the 12-mer peptide sequences. A 10-fold increase was observed in the binding affinity of one of the two-repeated peptides, while this modification did not affect the affinity of the other tested peptide. The peptide, with enhanced affinity, was further modified as three repeats; however the affinity of the peptide decreased. The structural basis of the affinity difference between modified peptides was examined by molecular dynamics simulation. The results showed that the conformational differences hold the key for affinity of peptides modified by repeating the sequence. This high affinity peptide with increased affinity is a promising molecular recognition agent to be used in the detection of SEB to be utilized in biosensing systems.     

Receptor epitope usage by an interleukin-5 mimetic peptide

The cyclic peptide AF17121 is a library-derived antagonist for human interleukin-5 (IL5) receptor alpha (IL5Ralpha) and inhibits IL5 activity. Our previous results have demonstrated that the sixth arginine residue of the peptide is crucial for the inhibitory effect and that several acidic residues in the N- and C-terminal regions also make a contribution, although to a lesser extent (Ruchala, P., Varadi, G., Ishino, T., Scibek, J., Bhattacharya, M., Urbina, C., Van Ryk, D., Uings, I., and Chaiken, I. (2004) Biopolymers 73, 556-568). However, the recognition mechanism of the receptor has remained unresolved. In this study, AF17121 was fused to thioredoxin by recombinant DNA techniques and examined for IL5Ralpha interaction using a surface plasmon resonance biosensor method. Kinetic analysis revealed that the dissociation rate of the peptide.receptor complex is comparable with that of the cytokine.receptor complex. The fusion peptide competed with IL5 for both biological function and interaction with IL5Ralpha, indicating that the binding sites on the receptor are shared by AF17121 and IL5. To define the epitope residues for AF17121, we defined its binding footprint on IL5Ralpha by alanine substitution of Asp(55), Asp(56), Glu(58), Lys(186), Arg(188), and Arg(297) of the receptor. Marked effects on the interaction were observed in all three fibronectin type III domains of IL5Ralpha, in particular Asp(55), Arg(188), and Arg(297) in the D1, D2, and D3 domains, respectively. This footprint represents a significant subset of that for IL5 binding. The fact that AF17121 mimics the receptor binding capability of IL5 but antagonizes biological function evokes several models for how IL5 induces activation of the multisubunit receptor system.     

Binding of Biotinylated Thrombin Receptor Peptide to Cloned Human Thrombin Receptor Overexpressed in Baby Hamster Kidney Cells

Abstract

Baby hamster kidney (BHK) cells transfected with an expression vector for the human thrombin receptor, and then treated with basic fibroblast growth factor, were found to express specific and saturable binding sites for biotinylated thrombin receptor peptide (SFLLRNPNDKYEPF). Analysis of the binding to live BHK cells yielded an equilibrium dissociation constant (Kd) of 3.0±0.3 μmol/l and a maximal binding capacity (Bmax) of 31.0±0.5 nmol/mg of protein. In competitive binding experiments, the thrombin receptor agonist peptide (SFLLRN), which is a strong inducer of human platelet aggregation, was the most potent competitor. In contrast, position 1 to 2 inverted peptides such as FSLLRNPNDKYEPF and FSLLRNP, which fail to induce for the platelet aggregation, were less potent. This simple and convenient binding assay system using the biotinylated thrombin receptor peptide as a labeled ligand and the cloned thrombin receptor overexpressed in BHK cells may be useful for exploring specific antagonists of the receptor.     

Alpha 1B-adrenoceptor interacts with multiple sites of transglutaminase II: characteristics of the interaction in binding and activation

We previously reported that a novel GTP binding protein (G alpha h) is tissue type transglutaminase (TGII) and transmits the alpha 1B-adrenoceptor (AR) signal to phospholipase C (PLC) through its GTPase function. We have also shown that PLC-delta 1 is the effector in TGII-mediated signaling. In this study, interaction sites on TGII for the alpha 1B-AR were identified using a peptide approach and site-directed mutagenesis, including in vivo reconstitution of TGIIs with the alpha 1B-AR and PLC-delta 1. To identify the interaction sites, 11 synthetic peptides covering approximately 132 amino acid residues of the C-terminal domain of TGII were tested. The studies with the peptides revealed that three peptides, L547-I561, R564-D581, and Q633-E646, disrupted formation of an alpha 1-agonist-alpha 1B-AR-TGII complex and blocked alpha 1B-AR-mediated TGase inhibition in a dose-dependent manner, indicating that these peptide regions are involved in recognition and activation of TGII by the alpha 1B-AR. These three regions were further evaluated with full-length TGIIs by constructing and coexpressing each site-directed mutant with the alpha 1B-AR and PLC-delta 1 in COS-1 cells. Supporting the findings with these peptides, these TGII mutants lost 56-82% the receptor binding ability and reduced by 29-68% the level of alpha 1B-AR-mediated IP3 production via PLC-delta 1 as compared to those with wild-type TGII. The results also revealed that the regions of R564-D581 and Q633-E646 were the high-affinity binding sites of TGII for the receptor and critical for the activation of TGII by the receptor. Taken together, the studies demonstrate that multiple regions of TGII interact with the alpha 1B-AR and that the alpha 1B-AR stimulates PLC-delta 1 via TGII.