Novel information on the epitope of an inverse agonist monoclonal antibody provides insight into the structure of the TSH receptor

The TSH receptor (TSHR) comprises an extracellular leucine-rich domain (LRD) linked by a hinge region to the transmembrane domain (TMD). Insight into the orientation of these components to each other is required for understanding how ligands activate the receptor. We previously identified residue E251 at the LRD-hinge junction as contributing to coupling TSH binding with receptor activation. However, a single residue cannot stabilize the LRD-hinge unit. Therefore, based on the LRD crystal structure we selected for study four other potential LRD-hinge interface charged residues. Alanine substitutions of individual residues K244, E247, K250 and R255 (as well as previously known E251A) did not affect TSH binding or function. However, the cumulative mutation of these residues in varying permutations, primarily K250A and R255A when associated with E251A, partially uncoupled TSH binding and function. These data suggest that these three residues, spatially very close to each other at the LRD base, interact with the hinge region. Unexpectedly and most important, monoclonal antibody CS-17, a TSHR inverse agonist whose epitope straddles the LRD-hinge, was found to interact with residues K244 and E247 at the base of the convex LRD surface. These observations, together with the functional data, exclude residues K244 and E247 from the TSHR LRD-hinge interface. Further, for CS-17 accessibility to K244 and E247, the concave surface of the TSHR LRD must be tilted forwards towards the hinge region and plasma membrane. Overall, these data provide insight into the mechanism by which ligands either activate the TSHR or suppress its constitutive activity.     

The cysteine bonding in TSH receptor and it function in autoantibodies recognition

The majority of epitopes for TSH receptor (TSHR) stimulating autoantibodies are clustered around the Nterminal region of the TSH receptor. The characteristic feature of this region is the presence of four cysteine residues. It was proposed that cysteines in positions 29 and 41 in the receptor are connected by disulfide bonds and they are the target for receptor stimulating antibodies. The present study was aimed to check this possibility. The synthetic peptides: peptide corresponding to the part of TSHR containing the above 29-41 cysteine bond, the peptide similar to this peptide but without disulfide bond and the control peptide, containing sequence absent in the receptor were used for rabbit immunization. The thyroid status of all immunized rabbits was the same. Rabbits immunized with peptides related to TSHR generated antisera reactive with TSHR in immunoenzymatic assay. To check specificity of this reaction the influence of the peptides and the antisera on TSH binding to the receptor in competitive assay (TRAK) and their influence on adenylate cyclase activity were studied. It was found that neither synthetic peptides nor antiserum from any rabbit influenced TSH binding to the receptor in TRAK. In contrast low, but significant adenylate cyclase stimulating activity was noticed for antisera from two of six rabbit immunized by peptide containing the disulfide bond. We concluded that such a bond between cysteine residues 29 and 41 are present in TSHR in the site of stimulating antibodies epitope.     

The hinge region of human thyroid-stimulating hormone (TSH) receptor operates as a tunable switch between hormone binding and receptor activation

The mechanism by which the hinge regions of glycoprotein hormone receptors couple hormone binding to activation of downstream effecters is not clearly understood. In the present study, agonistic (311.62) and antagonistic (311.87) monoclonal antibodies (MAbs) directed against the TSH receptor extracellular domain were used to elucidate role of the hinge region in receptor activation. MAb 311.62 which identifies the LRR/Cb-2 junction (aa 265-275), increased the affinity of TSHR for the hormone while concomitantly decreasing its efficacy, whereas MAb 311.87 recognizing LRR 7-9 (aa 201-259) acted as a non-competitive inhibitor of Thyroid stimulating hormone (TSH) binding. Binding of MAbs was sensitive to the conformational changes caused by the activating and inactivating mutations and exhibited differential effects on hormone binding and response of these mutants. By studying the effects of these MAbs on truncation and chimeric mutants of thyroid stimulating hormone receptor (TSHR), this study confirms the tethered inverse agonistic role played by the hinge region and maps the interactions between TSHR hinge region and exoloops responsible for maintenance of the receptor in its basal state. Mechanistic studies on the antibody-receptor interactions suggest that MAb 311.87 is an allosteric insurmountable antagonist and inhibits initiation of the hormone induced conformational changes in the hinge region, whereas MAb 311.62 acts as a partial agonist that recognizes a conformational epitope critical for coupling of hormone binding to receptor activation. The hinge region, probably in close proximity with the α-subunit in the hormone-receptor complex, acts as a tunable switch between hormone binding and receptor activation.     

The thyrotropin receptor hinge region is not simply a scaffold for the leucine-rich domain but contributes to ligand binding and signal transduction

The glycoprotein hormone receptor hinge region connects the leucine-rich and transmembrane domains. The prevalent concept is that the hinge does not play a significant role in ligand binding and signal transduction. Portions of the hinge are redundant and can be deleted by mutagenesis or are absent in certain species. A minimal hinge will be more amenable to future investigation of its structure and function. We, therefore, combined and progressively extended previous deletions (Delta) in the TSH receptor (TSHR) hinge region (residues 277-418). TSHRDelta287-366, Delta287-371, Delta287-376, and Delta287-384 progressively lost their response to TSH stimulation of cAMP generation in intact cells, consistent with a progressive loss of TSH binding. The longest deletion (TSHRDelta287-384), reducing the hinge region from 141 to 43 amino acids, totally lost both functions. Surprisingly, however, with deletions extending from residues 371-384, constitutive (ligand-independent) activity increased severalfold, reversing the suppressive (inverse agonist) effect of the TSHR extracellular domain. TSHR-activating point mutations I486F and I568T in the first and second extracellular loops (especially the former) had reduced activity on a background of TSHRDelta287-371. In summary, our data support the concept that the TSHR hinge contributes significantly to ligand binding affinity and signal transduction. Residues within the hinge, particularly between positions 371-384, appear involved in ectodomain inverse agonist activity. In addition, the hinge is necessary for functionality of activating mutations in the first and second extracellular loops. Rather than being an inert linker between the leucine-rich and transmembrane domains, the TSHR hinge is a signaling-specificity domain.     

Extended hormone binding site of the human thyroid stimulating hormone receptor: distinctive acidic residues in the hinge region are involved in bovine thyroid stimulating hormone binding and receptor activation

The human thyroid stimulating hormone receptor (hTSHR) belongs to the glycoprotein hormone receptors that bind the hormones at their large extracellular domain. The extracellular hinge region of the TSHR connects the N-terminal leucine-rich repeat domain with the membrane-spanning serpentine domain. From previous studies we reasoned that apart from hormone binding at the leucine-rich repeat domain, additional multiple hormone contacts might exist at the hinge region of the TSHR by complementary charge-charge recognition. Here we investigated highly conserved charged residues in the hinge region of the TSHR by site-directed mutagenesis to identify amino acids interacting with bovine TSH (bTSH). Indeed, the residues Glu-297, Glu-303, and Asp-382 in the TSHR hinge region are essential for bTSH binding and partially for signal transduction. Side chain substitutions showed that the negative charge of Glu-297 and Asp-382 is necessary for recognition of bTSH by the hTSHR. Multiple combinations of alanine mutants of the identified positions revealed an increased negative effect on hormone binding. An assembled model suggests that the deciphered acidic residues form negatively charged patches at the hinge region resulting in an extended binding mode for bTSH on the hTSHR. Our data indicate that certain positively charged residues of bTSH might be involved in interaction with the identified negatively charged amino acids of the hTSHR hinge region. We demonstrate that the hinge region represents an extracellular intermediate connector for both hormone binding and signal transduction of the hTSHR.     

Relationship between thyrotropin receptor hinge region proteolytic posttranslational modification and receptor physiological function

The glycoprotein hormone receptor hinge region is the least conserved component and the most variable in size; the TSH receptor (TSHR) being the longest (152 amino acids; residues 261-412). The TSHR is also unique among the glycoprotein hormone receptor in undergoing in vivo intramolecular cleavage into disulfide-linked A- and B-subunits with removal of an intervening 'C-peptide' region. Experimentally, hinge region amino acids 317-366 (50 residues) can be deleted without alteration in receptor function. However, in vivo, more than 50 amino acids are deleted during TSHR intramolecular cleavage; furthermore, the boundaries of this deleted region are ragged and poorly defined. Studies to determine the extent to which hinge region deletions can be tolerated without affecting receptor function ('minimal hinge') are lacking. Using as a template the functionally normal TSHR with residues 317-366 deleted, progressive downstream extension of deletions revealed residue 371 to be the limit compatible with normal TSH binding and coupling with cAMP signal transduction. Based on the foregoing downstream limit, upstream deletion from residue 307 (307-371 deletion) was also tolerated without functional alteration, as was deletion of residues 303-366. Addressing a related issue regarding the functional role of the TSHR hinge region, we observed that downstream hinge residues 377-384 contribute to coupling ligand binding with cAMP signal transduction. In summary, we report the first evaluation of TSHR function in relation to proteolytic posttranslational hinge region modifications. Deletion of TSHR hinge amino acids 303-366 (64 residues) or 307-371 (65 residues) are the maximum hinge region deletions compatible with normal TSHR function.     

Generation and epitope analysis of thyroid stimulating hormone-specific monoclonal antibodies for enzyme immunoassays

By using an improved hybridoma technique with a semisolid medium of methylcellulose for initial cloning, numerous high affinity monoclonal antibodies against human thyroid stimulating hormone (TSH) were generated. These antibodies were characterized with respect to their subunit and epitope specificity. Epitope analysis of antibodies specific to the beta-subunit of TSH was performed by a sandwich pairing procedure. Based on the results of this analysis, it was concluded that there are four distinct TSH-specific epitopes on the beta-subunit of TSH; these are designated a, b, c, and ab. The five antibodies binding to epitopes a, b, and c are not mutually exclusive. However, the antibody binding to epitope ab prevents further binding of other antibodies to epitope a or b, but not to epitope c. This epitope analysis enabled us to combine three high affinity monoclonal antibodies, each of which reacts with epitopes a, b, and c, respectively, in a typical sandwich enzyme immunoassay. One was immobilized on polystyrene beads and the other two were conjugated with horseradish peroxidase and served as the second antibodies. This enzyme immunoassay can be performed within 90 min and with a minimum sensitivity of 0.2-0.3 microIU/ml.     

The Presence of Thyroid-Stimulation Blocking Antibody Prevents High Bone Turnover in Untreated Premenopausal Patients with Graves’ Disease

Osteoporosis-related fractures are one of the complications of Graves’ disease. This study hypothesized that the different actions of thyroid-stimulating hormone receptor (TSHR) antibodies, both stimulating and blocking activities in Graves’ disease patients might oppositely impact bone turnover. Newly diagnosed premenopausal Graves’ disease patients were enrolled (n = 93) and divided into two groups: patients with TSHR antibodies with thyroid-stimulating activity (stimulating activity group, n = 83) and patients with TSHR antibodies with thyroid-stimulating activity combined with blocking activity (blocking activity group, n = 10). From the stimulating activity group, patients who had matched values for free T4 and TSH binding inhibitor immunoglobulin (TBII) to the blocking activity group were further classified as stimulating activity-matched control (n = 11). Bone turnover markers BS-ALP, Osteocalcin, and C-telopeptide were significantly lower in the blocking activity group than in the stimulating activity or stimulating activity-matched control groups. The TBII level showed positive correlations with BS-ALP and osteocalcin levels in the stimulating activity group, while it had a negative correlation with the osteocalcin level in the blocking activity group. In conclusion, the activation of TSHR antibody-activated TSH signaling contributes to high bone turnover, independent of the actions of thyroid hormone, and thyroid-stimulation blocking antibody has protective effects against bone metabolism in Graves’ disease.     

Delineation of the discontinuous-conformational epitope of a monoclonal antibody displaying full in vitro and in vivo thyrotropin activity

An experimental murine model of Graves' disease was used to produce monoclonal antibodies (mAbs) with thyroid stimulating activity. Two of these, IRI-SAb2 and IRI-SAb3, showed particularly high potency (in the low nanomolar range) and efficacy. IRI-SAb2 behaved as a full agonist of the human TSH receptor (TSHr), even when tested in physiological salt concentrations. Both IRI-SAb2 and IRI-SAb3 were displaced from the TSHr by autoantibodies from patients with Graves' disease or harboring thyroid-blocking antibodies, but not from control subjects or patients with Hashimoto thyroiditis. The epitopes of IRI-SAb2 and IRI-SAb3 were precisely mapped, at the amino acid level, to the amino-terminal portion of the concave portion of the horseshoe structure of TSHr ectodomain. They overlap closely with each other and, surprisingly, with the epitope of a mAb with blocking activity. When injected iv in mice, both mAbs caused biological and histological signs of hyperthyroidism. Unexpectedly, they also triggered an inflammatory response in the thyroid glands. Delineation of the conformational epitopes of these stimulating antibodies opens the way to the identification of the molecular mechanisms implicated in the activation of the TSHr.     

A full biological response to autoantibodies in Graves' disease requires a disulfide-bonded loop in the thyrotropin receptor N terminus homologous to a laminin epidermal growth factor-like domain

We observed amino acid homology between the cysteine-rich N terminus of the thyrotropin receptor (TSHR) ectodomain and epidermal growth factor-like repeats in the laminin gamma1 chain. Thyroid-stimulating autoantibodies (TSAb), the cause of Graves' disease, interact with this region of the TSHR in a manner critically dependent on antigen conformation. We studied the role of the cluster of four cysteine (Cys) residues in this region of the TSHR on the functional response to TSAb in Graves' patients' sera. As a benchmark we also studied TSH binding and action. Removal in various permutations of the four cysteines at TSHR positions 24, 29, 31, and 41 (signal peptide residues are 1-21) revealed Cys(41) to be the key residue for receptor expression. Forced pairing of Cys(41) with any one of the three upstream Cys residues was necessary for trafficking to the cell surface of a TSHR with high affinity TSH binding similar to the wild-type receptor. However, for a full biological response to TSAb, forced pairing of Cys(41) with Cys(29) or with Cys(31), but not with Cys(24), retained functional activity comparable with the wild-type TSHR. These data suggest that an N-terminal disulfide-bonded loop between Cys(41) and Cys(29) or its close neighbor Cys(31) comprises, in part, the highly conformational epitope for TSAb at the critical N terminus of the TSHR. Amino acid homology, as well as cysteine pairing similar to the laminin gamma1 chain epidermal growth factor-like repeat 11, suggests conformational similarity between the two molecules and raises the possibility of molecular mimicry in the pathogenesis of Graves' disease.     

Immunoglobulins from Graves' disease patients interact with different sites on TSH receptor/LH-CG receptor chimeras than either TSH or immunoglobulins from idiopathic myxedema patients

To examine the identity of binding sites for thyrotropin (TSH) and thyroid stimulating antibodies (TSAbs) associated with Graves' disease, we constructed eight human TSH receptor/rat LH-CG receptor chimeras. Substitution of amino acid residues 8-165 of the TSH receptor with the corresponding LH-CG receptor segment (Mc1 + 2) results in a chimera which retains high affinity TSH binding and the cAMP response to TSH but loses both the cAMP response to Graves' IgG and Graves' IgG inhibition of TSH binding. Two of three IgGs from idiopathic myxedema patients which contain thyroid stimulation blocking antibodies (TSBAbs) still, however, react with this chimera. Chimeras which substitute residues 90-165 (Mc2) and 261-370 (Mc4) retain the ability to interact with TSH, Graves' IgG, and idiopathic myxedema IgG. The data thus suggest that residues 8-165 contain an epitope specific for TSAbs and that TSH receptor determinants important for the activities of TSAbs and TSH are not identical. Further, binding sites for TSBAbs in idiopathic myxedema may be different from receptor binding sites for both Graves' IgG TSAb as well as TSH and may be different in individual patients.     

A Tyrosine Residue on the TSH Receptor Stabilizes Multimer Formation

Background

The thyrotropin stimulating hormone receptor (TSHR) is a G protein coupled receptor (GPCR) with a large ectodomain. The ligand, TSH, acting via this receptor regulates thyroid growth and thyroid hormone production and secretion. The TSH receptor (TSHR) undergoes complex post –translational modifications including intramolecular cleavage and receptor multimerization. Since monomeric and multimeric receptors coexist in cells, understanding the functional role of just the TSHR multimers is difficult. Therefore, to help understand the physiological significance of receptor multimerization, it will be necessary to abrogate multimer formation, which requires identifying the ectodomain and endodomain interaction sites on the TSHR. Here, we have examined the contribution of the ectodomain to constitutive multimerization of the TSHR and determined the possible residue(s) that may be involved in this interaction.

Methodology/Principal Findings

We studied ectodomain multimer formation by expressing the extracellular domain of the TSHR linked to a glycophosphotidyl (GPI) anchor in both stable and transient expression systems. Using co-immunoprecipitation and FRET of tagged receptors, we established that the TSH receptor ectodomain was capable of multimerization even when totally devoid of the transmembrane domain. Further, we studied the effect of two residues that likely made critical contact points in this interaction. We showed that a conserved tyrosine residue (Y116) on the convex surface of the LRR3 was a critical residue in ectodomain multimer formation since mutation of this residue to serine totally abrogated ectodomain multimers. This abrogation was not seen with the mutation of cysteine 176 on the inner side of the LRR5, demonstrating that inter-receptor disulfide bonding was not involved in ectodomain multimer formation. Additionally, the Y116 mutation in the intact wild type receptor enhanced receptor degradation.

Conclusions/Significance

These data establish the TSH receptor ectodomain as one site of multimerization, independent of the transmembrane region, and that this interaction was primarily via a conserved tyrosine residue in LRR3.     

Localization of epitopes of herpes simplex virus type 1 glycoprotein D.

We previously defined eight groups of monoclonal antibodies which react with distinct epitopes of herpes simplex virus glycoprotein D (gD). One of these, group VII antibody, was shown to react with a type-common continuous epitope within residues 11 to 19 of the mature glycoprotein (residues 36 to 44 of the predicted sequence of gD). In the current investigation, we have localized the sites of binding of two additional antibody groups which recognize continuous epitopes of gD. The use of truncated forms of gD as well as computer predictions of secondary structure and hydrophilicity were instrumental in locating these epitopes and choosing synthetic peptides to mimic their reactivity. Group II antibodies, which are type common, react with an epitope within residues 268 to 287 of the mature glycoprotein (residues 293 to 312 of the predicted sequence). Group V antibodies, which are gD-1 specific, react with an epitope within residues 340 to 356 of the mature protein (residues 365 to 381 of the predicted sequence). Four additional groups of monoclonal antibodies appear to react with discontinuous epitopes of gD-1, since the reactivity of these antibodies was lost when the glycoprotein was denatured by reduction and alkylation. Truncated forms of gD were used to localize these four epitopes to the first 260 amino acids of the mature protein. Competition experiments were used to assess the relative positions of binding of various pairs of monoclonal antibodies. In several cases, when one antibody was bound, there was no interference with the binding of an antibody from another group, indicating that the epitopes were distinct. However, in other cases, there was competition, indicating that these epitopes might share some common amino acids.     

Human apolipoprotein E. Determination of the heparin binding sites of apolipoprotein E3

The interaction of human apolipoprotein (apo-) E3 with heparin was examined using heparin-Sepharose as a model system. The approach taken to determine the region of apo-E that is responsible for binding to heparin was to identify apo-E monoclonal antibodies that inhibited heparin binding, to determine the epitopes of the inhibiting antibodies, and finally to examine the heparin binding of fragments containing the inhibiting antibody epitopes. Three antibodies, designated 1D7, 6C5, and 3H1, were found to inhibit binding, suggesting that multiple heparin binding sites were present on apo-E. The epitopes of the inhibiting antibodies were determined by immunoblot analysis of synthetic or proteolytic fragments of apo-E. Measurement of the heparin binding activity of fragments containing epitopes of the inhibiting antibodies demonstrated that apo-E3 contains two heparin binding sites. The first site is located in the vicinity of residues 142-147 and coincides with the 1D7 epitope. The second binding site is contained in the carboxyl-terminal region of apo-E and is inhibited by 3H1, the epitope of which is located between residues 243 and 272. The epitope of the third inhibiting antibody, 6C5, is located at the amino terminus of apo-E; however, this antibody inhibits the second heparin binding site located in the carboxyl-terminal region. A head-to-tail association of apo-E, in which the 6C5 epitope and the second heparin binding site would be in close proximity, is proposed to account for this observation. In the lipid-free state both heparin binding sites on apo-E are expressed; however, when apo-E is complexed to phospholipid or on the surface of a lipoprotein particle, only the first binding site (residues 142-147) is expressed.     

Regulation of the phosphatidylinositol 3-kinase,Akt/protein kinase B,FRAP/mammalian target of rapamycin,and ribosomal S6 kinase 1 signaling pathways by thyroid-stimulating hormone (TSH) and stimulating type TSH receptor antibodies in the thyroid gland

Thyroid-stimulating hormone (TSH) regulates the growth and differentiation of thyrocytes by activating the TSH receptor (TSHR). This study investigated the roles of the phosphatidylinositol 3-kinase (PI3K), PDK1, FRAP/mammalian target of rapamycin, and ribosomal S6 kinase 1 (S6K1) signaling mechanism by which TSH and the stimulating type TSHR antibodies regulate thyrocyte proliferation and the follicle activities in vitro and in vivo. The TSHR immunoprecipitates exhibited PI3K activity, which was higher in the cells treated with either TSH or 8-bromo-cAMP. TSH and cAMP increased the tyrosine phosphorylation of TSHR and the association between TSHR and the p85alpha regulatory subunit of PI3K. TSH induced a redistribution of PDK1 from the cytoplasm to the plasma membrane in the cells in a PI3K- and protein kinase A-dependent manner. TSH induced the PDK1-dependent phosphorylation of S6K1 but did not induce Akt/protein kinase B phosphorylation. The TSH-induced S6K1 phosphorylation was inhibited by a dominant negative p85alpha regulatory subunit or by the PI3K inhibitors wortmannin and LY294002. Rapamycin inhibited the phosphorylation of S6K1 in the cells treated with either TSH or 8-bromo-cAMP. The stimulating type TSHR antibodies from patients with Graves disease also induced S6K1 activation, whereas the blocking type TSHR antibodies from patients with primary myxedema inhibited TSH- but not the insulin-induced phosphorylation of S6K1. In addition, rapamycin treatment in vivo inhibited the TSH-stimulated thyroid follicle hyperplasia and follicle activity. These findings suggest an interaction between TSHR and PI3K, which is stimulated by TSH and cAMP and might involve the downstream S6K1 but not Akt/protein kinase B. This pathway may play a role in the TSH/stimulating type TSH receptor antibody-mediated thyrocyte proliferation in vitro and in the response to TSH in vivo.     

Antigenic determinants on chicken riboflavin carrier protein. A study with monoclonal antibodies

Monoclonal antibodies raised against chicken egg white riboflavin carrier protein were classified into seven categories each recognizing a distinct epitope. Of these, six were directed against conformation dependent epitopes and one to a sequential epitope. The roles of lysine residues and the post-translationally attached phosphate and oligosaccharide moieties in the antigenicity of riboflavin carrier protein recognized by the monoclonal antibodies were investigated. The binding region of three monoclonal antibodies could be located within the 87–219 amino acid sequence of the protein and one antibody among these recognized a sequence of 182–204 amino acid residues. All the monoclonal antibodies were able to recognize riboflavin carrier proteins present in the sera of pregnant rats, cows and humans indicating that the epitopes to which they are directed are conserved through evolution from chicken to the human.     

Fine epitope mapping of anti-epidermal growth factor receptor antibodies through random mutagenesis and yeast surface display

Fine epitope mapping of therapeutically relevant monoclonal antibodies (mAbs) specific for the epidermal growth factor receptor (EGFR) was accomplished through random mutagenesis and yeast surface display. Using this method, we have identified key residues energetically important for the binding of EGFR to the mAbs 806, 225, and 13A9. A yeast-displayed library of single point mutants of an EGFR ectodomain fragment (residues 273-621) was constructed by random mutagenesis and was screened for reduced binding to EGFR mAbs. If an EGFR mutant showed loss of binding to a mAb, this suggested that the mutated residue was potentially a contact residue. The mAb 806 binding epitope was localized to one face of a loop comprised of EGFR residues Cys287-Cys302, which is constrained by a disulfide bond and two salt bridges. The mAb 806 epitope as identified here is not fully accessible in the autoinhibited EGFR monomer conformation, which is consistent with the hypothesis that mAb 806 binds to a transitional form of EGFR as it changes from an autoinhibited to extended monomer. The amino acids Lys465 and Ile467 were identified as energetic hot spot residues for mAb 225 binding to EGFR. These residues are adjacent to the EGFR ligand-binding site, which is consistent with the ability of mAb 225 to block binding of epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha) ligands. Ser468 and Glu472 were identified as energetically important for mAb 13A9 binding to EGFR, and the location of this epitope suggests that mAb 13A9 mediates observed TGF-alpha blocking effects through conformational perturbation of EGFR domain III. Combinatorial library screening of yeast-displayed mutagenic proteins is a novel method to identify discontinuous and heat-denaturable mAb binding epitopes with residue-level resolution.     

Localization of conserved and nonconserved epitopes within the Rous sarcoma virus-encoded src protein.

We investigated the location of binding sites of pp60src-specific monoclonal antibodies by immunoprecipitating a panel of structurally altered src proteins. Two families of antibodies which recognized epitopes mapping to either amino acid residues 28 to 38 or 92 to 128 were identified. The highly conserved nature of the epitope defined by residues 92 to 128 suggests that it may represent an important functional region of the cellular src protein.     

Insights into differential modulation of receptor function by hinge region using novel agonistic lutropin receptor and inverse agonistic thyrotropin receptor antibodies

We report two antibodies, scFv 13B1 and MAb PD1.37, against the hinge regions of LHR and TSHR, respectively, which have similar epitopes but different effects on receptor function. While neither of them affected hormone binding, with marginal effects on hormone response, scFv 13B1 stimulated LHR in a dose-dependent manner, whereas MAb PD1.37 acted as an inverse agonist of TSHR. Moreover, PD1.37 could decrease the basal activity of hinge region CAMs, but had varied effects on those present in ECLs, whereas 13B1 was refractory to any CAMs in LHR. Using truncation mutants and peptide phage display, we compared the differential roles of the hinge region cysteine box-2/3 as well as the exoloops in the activation of these two homologus receptors.