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

AbstractWe 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

Anaerobic DNA cleavage in red light by dicopper(II) complexes on disulfide bond activation

Binuclear complexes [Cu(μ-RSSR)]2 (1) and [M2(μ-PDS)(H2O)]2 (M = Cu(II), 2; Fe(II), 3), where H2RSSR is a reduced Schiff base derived from 2-(thioethyl)salicylaldimine having a disulphide moiety and H2PDS is derived from dimerization of D-penicillamine, have been prepared, structurally characterized, and their photo-induced DNA cleavage activity studied. The crystal structure of 1 shows the complex as a discrete binuclear species with each metal in a CuN2O2 square-planar geometry (Cu...Cu, 6.420 Å). The tetradentate RSSR2- acts as a bridging ligand. The sulphur atoms in the disulphide unit do not interact with the metal ions. Complexes 1-3 do not show any DNA cleavage activity in darkness. The copper(II) complexes exhibit chemical nuclease activity in the presence of 3-mercaptopropionic acid. Cleavage of supercoiled DNA has been observed in UV-A light of 365 nm for 1 and red light of 647.1 nm for both 1 and 2 in air. Mechanistic data reveal the involvement of the disulphide unit as photosensitizer generating hydroxyl radicals (•OH) as the reactive species. Photo-induced DNA cleavage in red light seems to involve sulphide radicals in a type-I process and hydroxyl radicals. The dicopper(II) complexes show significant anaerobic photo-induced DNA cleavage activity in red light under argon following type-I pathway without involving any reactive oxygen species

Oxovanadium(IV)-based near-IR PDT agents: design to biological evaluation

An oxovanadium(IV) complex of dipyridophenazine, as a potent metal-based PDT agent, shows efficient DNA photocleavage activity at near-IR region and high photocytotoxicity in both UV-A and visible light in HeLa cells

Discounting and averaging in games across time scales

We introduce two-level discounted and mean-payoff games played by two players on a perfect-information stochastic game graph. The upper level game is a discounted or mean-payoff game and the lower level game is a (undiscounted) reachability game. Two-level games model hierarchical and sequential decision making under uncertainty across different time scales. For both discounted and mean-payoff two-level games, we show the existence of pure memoryless optimal strategies for both players and an ordered field property. We show that if there is only one player (Markov decision processes), then the values can be computed in polynomial time. It follows that whether the value of a player is equal to a given rational constant in two-level discounted or mean-payoff games can be decided in NP ∩ coNP. We also give an alternate strategy improvement algorithm to compute the value. © 2012 World Scientific Publishing Company

New paradigms in the establishment and maintenance of gradients during directed cell migration

Directional guidance of migrating cells is relatively well explored in the reductionist setting of cell culture experiments. Here spatial gradients of chemical cues as well as gradients of mechanical substrate characteristics prove sufficient to attract single cells as well as their collectives. How such gradients present and act in the context of an organism is far less clear. Here we review recent advances in understanding how guidance cues emerge and operate in the physiological context

The Hinge Region of Human Thyroid-Stimulating Hormone (TSH) Receptor Operates as a Tunable Switch between Hormone Binding and Receptor Activation

<div><p>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.</p> </div

The antibodies against the computationally designed mimic of the glycoprotein hormone receptor transmembrane domain provide insights into receptor activation and suppress the constitutively activated receptor Mutants

The exoloops of glycoprotein hormone receptors (GpHRs) transduce the signal generated by the ligand-ectodomain interactions to the transmembrane helices either through direct hormonal contact and/or by modulating the interdomain interactions between the hinge region (HinR) and the transmembrane domain (TMD). The ligand-induced conformational alterations in the HinRs and the interhelical loops of luteinizing hormone receptor/follicle stimulating hormone receptor/thyroid stimulating hormone receptor were mapped using exoloop-specific antibodies generated against a mini-TMD protein designed to mimic the native exoloop conformations that were created by joining the thyroid stimulating hormone receptor exoloops constrained through helical tethers and library-derived linkers. The antibody against the mini-TMD specifically recognized all three GpHRs and inhibited the basal and hormone-stimulated cAMP production without affecting hormone binding. Interestingly, binding of the antibody to all three receptors was abolished by prior incubation of the receptors with the respective hormones, suggesting that the exoloops are buried in the hormone-receptor complexes. The antibody also suppressed the high basal activities of gain-of-function mutations in the HinRs, exoloops, and TMDs such as those involved in precocious puberty and thyroid toxic adenomas. Using the antibody and point/deletion/chimeric receptor mutants, we demonstrate that changes in the HinR-exoloop interactions play an important role in receptor activation. Computational analysis suggests that the mini-TMD antibodies act by conformationally locking the transmembrane helices by means of restraining the exoloops and the juxta-membrane regions. Using GpHRs as a model, we describe a novel computational approach of generating soluble TMD mimics that can be used to explain the role of exoloops during receptor activation and their interplay with TMDs

Epitope mapping of MAb 311.62 by Peptide Phage Display analysis.

<p><b>A</b>. Amino acid sequences deduced from the phage clones selected after three rounds of biopanning. Three groups of sequence A, B, C were obtained respectively on analyzing the sequence and consensus obtained for each. The numbers in the parentheses denote the frequency or ratio of the number of the phage clones expressing the common peptide sequence to that of the total phage clones. <b>B</b>. Sequence alignment of the consensus sequence derived from the phage clones with the full length TSH receptor revealed sequence similarity in TSH receptor region 266–281. <b>C</b>. Multiple sequence alignment of hTSHR, hLHR and hFSHR corresponding to the region 265–281 of hTSHR. TSHR residues marked in bold were mutated to corresponding residues of LHR. D. MAb 311.62 IgG (25 µg/ml) was pre-incubated with different TSH receptor fragment protein (50 µg/ml) and then added to HEK293-hTSHR cells, and cAMP produced was determined.</p

Determination of affinity of TSHR for TSH in presence of TSHR MAbs.

<p>The membrane preparation (20 µg/ml) obtained from HEK293-hTSHR cells were incubated with either 50 µg/ml of NMIgG or TSHR MAbs. ¹²⁵I-hTSH (2×10⁵ cpm) was added to TSHR MAb –TSH complex in presence of increasing concentration of hTSH and <i>K_d</i> and <i>B_max</i> value obtained by fitting the data as mentioned in the legends of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040291#pone-0040291-g003" target="_blank">Figure 3</a>.</p>a<p>p<0.01 versus values with no IgG added. The values are expressed as the mean ± S.D. of triplicate determinations.</p>b<p>p<0.05 versus values with No IgG added.</p

Involvement of TSHR-hinge region and hormone α-subunit in hormone mediated conformational change in TSHR.

<p>Flow cytometric analysis of binding of 25 µg/ml of (<b>9A.</b>) MAb 311.62 or (<b>9B.</b>) MAb 311.82 <i>to</i> HEK293-hTSHR cells in absence of TSH (shaded solid line histogram) or pre-incubated with either 1 nM TSH (shaded dotted line histogram) or 25 nM TSH (shaded dashed line histogram). The culture supernatant of hormone α-subunit specific monoclonal antibody 2A6 was either added to HEK293-hTSHR cells previously incubated with (<b>9C.</b>) 25 nM TSH or (<b>9D.</b>) 25 nM TSH was added to HEK293-hTSHR cells previously exposed to similar concentration of 2A6 and binding of 2A6 to TSH in a TSH-TSHR complex was monitored in flow cytometry. All the flow cytometric experiments were carried out at 4°C to prevent hormone mediated receptor internalization.</p