Ligand-dependent inhibition of myoblast differentiation by overexpression of the type-1 insulin-like growth factor receptor

The insulin-like growth factors (IGFs) have paradoxical effects on skeletal myoblast differentiation. While low concentrations of IGF stimulate myoblast differentiation, high concentrations of IGF induce a progressive decrease in myoblast differentiation. The mechanism of this inhibition is unknown. Using a retroviral expression vector, we developed a subline of mouse P2 mouse myoblasts (P2-LISN) which expressed 7.5 times higher levels of type-1 IGF receptors than control (P2-LNL6) myoblasts, which were infected with a virus lacking the type-1 IGF receptor sequence. Overexpression of the type-1 IGF receptor caused the IGF dose-response curves of stimulation and progressive inhibition of differentiation to shift to the left. Additionally, at high insulin and IGF-I concentrations, complete inhibition of P2-LISN myoblast differentiation occurred. These results suggest that inhibition of differentiation at high ligand concentrations was not due to the primary involvement of other species of receptors for IGF. Type-1 IGF receptor downregulation as a mechanism for inhibition of differentiation was also ruled out since P2-LISN myoblasts constitutively expressed high levels of type-1 IGF receptors. Additionally, inhibition of differentiation at high concentrations of IGF-I was not correlated with overt stimulation of proliferation or with IGF binding protein (IGF-BP) release into the culture medium. These results indicate that the type-1 IGF receptor mediates two conflicting signal pathways in myogenic cells, differentiation-inducing and differentiation-inhibitory, which predominate at different ligand concentrations. © 1993 Wiley-Liss, Inc.     

Cleavage of the human thyrotropin receptor by ADAM10 is regulated by thyrotropin

The thyrotropin receptor (TSHR) has a unique 50 residue (317-366) ectodomain insertion that sets it apart from other glycoprotein hormone receptors (GPHRs). Other ancient members of the leucine-rich repeat G protein-coupled receptor (GPCR) (LGR) family do exhibit ectodomain insertions of variable lengths and sequences. The TSHR-specific insert is digested, apparently spontaneously, to release the ectodomain (A-subunit) leaving the balance of the ectodomain attached to the serpentine (B-subunit). Despite concerted efforts for the last 12 years by many laboratories, the enzyme involved in TSHR cleavage has not been identified and a physiologic role for this process remains unclear. Several lines of evidence had suggested that the TSHR protease is likely a member of the a disintegrin and metalloprotease (ADAM) family of metalloproteases. We show here that the expression of ADAM10 was specific to the thyroid by specially designed DNA microarrays. We also show that TSH increases TSHR cleavage in a dose-dependent manner. To prove that ADAM10 is indeed the TSHR cleavage enzyme, we investigated the effect of TSH-induced cleavage by a peptide based on a motif (TSHR residues 334-349), shared with known ADAM10 substrates. TSH increased dose dependently TSHR ectodomain cleavage in the presence of wild-type peptide but not a scrambled control peptide. Interestingly, TSH increased the abundance of non-cleaved single chain receptor, as well higher molecular forms of the A-subunit, despite their enhancement of the appearance of the fully digested A-subunit. This TSH-related increase in TSHR digested forms was further increased by wild-type peptide. We have identified for the first time ADAM10 as the TSHR cleavage enzyme and shown that TSH regulates its activation.     

Single subunit structure of the human thyrotropin receptor.

We have produced rabbit antibody against a synthetic peptide corresponding to N-terminal region of the extracellular domain of human thyrotropin receptor (hTSH-R) (N peptide, aminoacid residues 29-57). Western blot analysis revealed that N-peptide antibody recognized recombinant hTSH-R stably expressing in CHO-K1 cells as a mol. wt. about 104 kDa regardless in the presence or absence of disulfide-reducing agent. The band was not detected in untransfected CHO-K1 cells and no band was also stained by the antibody absorbed with N-peptide. In a reducing condition, the antibody also bound the rat receptor from FRTL5 cells as the same molecular size (104 kDa). These results clearly indicate that TSH-R is composed of a single subunit and that two subunit model for the TSH-R may reflect artifactual proteolytic cleavage of the receptor during membrane preparation.     

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.     

Ligand-dependent conformations and dynamics of the serotonin 5-HT(2A) receptor determine its activation and membrane-driven oligomerization properties

From computational simulations of a serotonin 2A receptor (5-HT(2A)R) model complexed with pharmacologically and structurally diverse ligands we identify different conformational states and dynamics adopted by the receptor bound to the full agonist 5-HT, the partial agonist LSD, and the inverse agonist Ketanserin. The results from the unbiased all-atom molecular dynamics (MD) simulations show that the three ligands affect differently the known GPCR activation elements including the toggle switch at W6.48, the changes in the ionic lock between E6.30 and R3.50 of the DRY motif in TM3, and the dynamics of the NPxxY motif in TM7. The computational results uncover a sequence of steps connecting these experimentally-identified elements of GPCR activation. The differences among the properties of the receptor molecule interacting with the ligands correlate with their distinct pharmacological properties. Combining these results with quantitative analysis of membrane deformation obtained with our new method (Mondal et al, Biophysical Journal 2011), we show that distinct conformational rearrangements produced by the three ligands also elicit different responses in the surrounding membrane. The differential reorganization of the receptor environment is reflected in (i)-the involvement of cholesterol in the activation of the 5-HT(2A)R, and (ii)-different extents and patterns of membrane deformations. These findings are discussed in the context of their likely functional consequences and a predicted mechanism of ligand-specific GPCR oligomerization.     

Ligand-dependent inhibition and reversal of tau filament formation

Alzheimer's disease is defined in part by the intraneuronal accumulation of filaments comprised of the microtubule associated protein tau. Because animal model studies suggest that a toxic gain of function accompanies tau aggregation in neurons, selective pharmacological inhibitors of the process may have utility in slowing neurodegeneration. Here, the properties of a candidate small molecule inhibitor of tau fibrillization, 3-(2-hydroxyethyl)-2-[2-[[3-(2-hydroxyethyl)-5-methoxy-2-benzothiazolylidene]methyl]-1-butenyl]-5-methoxybenzothiazolium (N744), were characterized in vitro using transmission electron microscopy. N744 inhibited arachidonic acid-induced aggregation of full-length, four-repeat tau protein at substoichiometric concentrations relative to total tau and with an IC(50) of approximately 300 nM. Inhibition was accompanied by a dose-dependent decrease in the number concentration of filaments, suggesting that N744 interfered with tau filament nucleation. Stoichiometric concentrations of N744 also promoted tau disaggregation when added to mature synthetic filaments. Disaggregation followed first-order kinetics and was accompanied by a steady decrease in filament number, suggesting that N744 promoted endwise loss of tau molecules with limited filament breakage. N744 at substoichiometric concentrations did not inhibit Abeta and alpha-synuclein aggregation, indicating it was tau selective under these conditions. Because of its activity in vitro, N744 may offer a pharmacological approach to the role of tau fibrillization in neurodegeneration.     

Ligand-dependent interaction of the dioxin receptor with target DNA

Wild type and nuclear transfer deficient mouse hepatoma cell lines were used to study the specific DNA binding of a dioxin inducible factor. This factor interacts with XRE only after dioxin treatment and is absent in receptor mutant containing cells even after treatment. Thus, evidence is provided to substantiate the claim that the dioxin receptor is involved in the specific DNA interaction with dioxin response enhancer elements. It is also shown that the molybdate stabilised dioxin-receptor interacts with hsp90 suggesting that, in similarity to the glucocorticoid receptor, the dioxin receptor is kept in a non-transformed state in the absence of ligand.     

Ligand-dependent switching of ubiquitin-proteasome pathways for estrogen receptor

Recent evidence indicates that the transactivation of estrogen receptor alpha (ERalpha) requires estrogen-dependent receptor ubiquitination and degradation. Here we show that estrogen-unbound (unliganded) ERalpha is also ubiquitinated and degraded through a ubiquitin-proteasome pathway. To investigate this ubiquitin-proteasome pathway, we purified the ubiquitin ligase complex for unliganded ERalpha and identified a protein complex containing the carboxyl terminus of Hsc70-interacting protein (CHIP). CHIP preferentially bound to misfolded ERalpha and ubiquitinated it to induce degradation. Ligand binding to the receptor induced the dissociation of CHIP from ERalpha. In CHIP-/- cells, the degradation of unliganded ERalpha was abrogated; however, estrogen-induced degradation was observed to the same extent as in CHIP+/+ cells. Our findings suggest that ERalpha is regulated by two independent ubiquitin-proteasome pathways, which are switched by ligand binding to ERalpha. One pathway is necessary for the transactivation of the receptor and the other is involved in the quality control of the receptor.     

Graves' IgG recognizes linear epitopes in the human thyrotropin receptor.

Twenty-nine peptides covering the full extracellular domain of the human thyrotropin receptor have been synthesized and tested for reactivity with Graves' patients' and normal sera in ELISA. Two peptides, amino acids 331-350 and the second extracellular loop of the transmembrane segment, bound IgG-s from 5 and 4 of 10 Graves' disease patients' sera, respectively. Neither of these two peptides showed enhanced binding to normal IgG. There were no apparent differences between the Graves' disease and normal group with respect to the other 27 peptides. We conclude that peptide 331-350 and the second extracellular loop carry important linear epitopes which may contribute to the disease process in selected Graves' patients.     

Ligand-dependent and -independent degradation of the human aryl hydrocarbon receptor (hAHR) in cell culture models

Studies have shown that zebrafish and rodent aryl hydrocarbon receptors (AHRs) are degraded following ligand exposure and that reductions in AHR protein can impact growth and development in vivo. The current study was designed to evaluate the degradation of the AHR in seven human cell lines that were derived from various carcinomas or from normal tissue. Consistent with studies in other species, the results show that the human AHR (hAHR) is degraded in a ligand dependent manner following exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin for up to 16h. However, the hAHRs expressed in the various cell lines show differences in the time course and magnitude of degradation. The ligand dependent degradation is completely blocked by treatment with the proteasome inhibitor, MG-132. Ligand-independent degradation of the hAHR following exposure to geldanamycin (GA) is also observed in the different cell lines, although the magnitude of hAHR degradation is also is variable. These findings are significant since they indicate that ligand-dependent and independent degradation of the AHR is a conserved aspect of this signal transduction cascade from fish to human. In addition, the study identifies several cell lines that may provide novel models to further assess the regulation of AHR-mediated signaling and degradation of the human AHR.     

Monitoring receptor oligomerization using time-resolved fluorescence resonance energy transfer and bioluminescence resonance energy transfer. The human delta -opioid receptor displays constitutive oligomerization at the cell surface, which is not regulated by receptor occupancy

Oligomerization of the human delta-opioid receptor and its regulation by ligand occupancy were explored following expression in HEK293 cells using each of co-immunoprecipitation of differentially epitope-tagged forms of the receptor, bioluminescence resonance energy transfer and time-resolved fluorescence resonance energy transfer. All of the approaches identified constitutively formed receptor oligomers, and the time-resolved fluorescence studies confirmed the presence of such homo-oligomers at the cell surface. Neither the agonist ligand [d-Ala(2),d-Leu(5)]enkephalin nor the inverse agonist ligand ICI174864 were able to modulate the oligomerization status of this receptor. Interactions between co-expressed delta-opioid receptors and beta(2)-adrenoreceptors were observed in co-immunoprecipitation studies. Such hetero-oligomers could also be detected using bioluminescence resonance energy transfer although the signal obtained was substantially smaller than for homo-oligomers of either receptor type. Signal corresponding to the delta-opioid receptor-beta(2)-adrenoreceptor hetero-oligomer was increased in the presence of agonist for either receptor. However, substantial levels of this hetero-oligomer were not detected at the cell surface using time-resolved fluorescence resonance energy transfer. These studies demonstrate that, following transient transfection of HEK293 cells, constitutively formed oligomers of the human delta-opioid receptor can be detected by a variety of approaches. However, these are not regulated by ligand occupancy. They also indicate that time-resolved fluorescence resonance energy transfer represents a means to detect such oligomers at the cell surface in populations of intact cells.     

Biological activities of rabbit antibodies against synthetic human thyrotropin receptor peptides representing thyrotropin binding regions.

Recently, we have shown that the thyrotropin (TSH) binding regions of human thyrotropin receptor (TSHR) reside in two areas within residues 12-44 and 308-344. Serial antisera were raised against four overlapping synthetic peptides representing these two regions of TSHR (peptides 12-30, 24-44, 308-328, and 324-344) and were investigated for their ability to stimulate or block the cultured porcine thyroid cells. In addition, serum concentrations of triiodothyronine (T3) and thyroxine (T4) in serial sera obtained from each rabbit were examined. It was shown that residues of 12-30 and 324-344 of TSHR, respectively, are the site (at least a part of the site) where stimulating (TSAb) and blocking type (TSBAb) immunoglobulins are directed.     

Functional analysis of the cytoplasmic domains of the human thyrotropin receptor by site-directed mutagenesis

The thyrotropin (TSH) receptor belongs to a family of guanine nucleotide protein-coupled receptors with seven transmembrane-spanning regions joined regulatory together by extracellular and intracellular loops. The cytoplasmic domain comprises three cytoplasmic loops and a cytoplasmic tail that are likely to be important in coupling of the receptor to the guanine nucleotide proteins. To address the question of which portions of the cytoplasmic domain of the TSH receptor are important in this process, we have altered groups of amino acids in the region of the TSH receptor by site-directed mutagenesis. Because of the low affinity of TSH binding to the TSH receptor mutated in the amino terminus of the second cytoplasmic loop and the amino terminus of the cytoplasmic tail, definitive conclusions cannot be made regarding the roles of these regions in signal transduction. However, our data indicate that the first cytoplasmic loop (residues 441-450), the carboxyl-terminal region of the second cytoplasmic loop (residues 528-537), and the carboxyl-terminal (but not the amino-terminal) region of the third cytoplasmic loop (residues 617-625) are important in the ability of the TSH receptor to mediate an increase in intracellular cAMP production. Furthermore, two-thirds of the carboxyl-terminal end of the cytoplasmic tail (residues 709-764; corresponding to the region not conserved between the TSH and lutropin/chorionic gonadotropin receptors) can be removed without functional impairment of the TSH receptor.     

Oligomerization of the human thyrotropin receptor: fluorescent protein-tagged hTSHR reveals post-translational complexes

To examine thyrotropin (TSH) receptor homophilic interactions we fused the human TSH receptor (hTSHR) carboxyl terminus to green fluorescent protein (GFP) and the corresponding chimeric cDNA was expressed in Chinese hamster ovary cells. Fluorescent TSH receptors on the plasma membrane were functional as assessed by TSH-induced cAMP synthesis. The binding of TSH, as well as TSHR autoantibodies, induced time- and dose-dependent receptor capping. Fluorescence resonance energy transfer between receptors differentially tagged with GFP variants (RFP and YFP) provided evidence for the close proximity of individual receptor molecules. This was consistent with previous studies demonstrating the presence of TSHR dimers and oligomers in thyroid tissue. Co-immunoprecipitation of GFP-tagged and Myc-tagged receptor complexes was performed using doubly transfected cells with Myc antibody. Western blotting of the immunoprecipitated complex revealed the absence of noncleaved TSH holoreceptors. This further suggested that cleavage of the holoreceptor into its two-subunit structure, comprising disulfide-linked TSHR-alpha and TSHR-beta subunits, was required for the formation of TSHR dimers and higher order complexes.     

Ligand-dependent linkage of the ATP site to inhibition gate closure in the KATP channel

Major advances have been made on the inhibition gate and ATP site of the K(ir)6.2 subunit of the K(ATP) channel, but little is known about conformational coupling between the two. ATP site mutations dramatically disrupt ATP-dependent gating without effect on ligand-independent gating, observed as interconversions between active burst and inactive interburst conformations in the absence of ATP. This suggests that linkage between site and gate is conditionally dependent on ATP occupancy. We studied all substitutions at position 334 of the ATP site in K(ir)6.2deltaC26 that express in Xenopus oocytes. All substitutions disrupted ATP-dependent gating by 10-fold or more. Only positive-charged arginine or lysine at 334, however, slowed ligand-independent gating from the burst, and this was in some but not all patches. Moreover, the polycationic peptide protamine reversed the slowed gating from the burst of 334R mutant channels, and speeded the slow gating from the burst of wild-type SUR1/K(ir)6.2 in the absence of ATP. Our results support a two-step ligand-dependent linkage mechanism for K(ir)6.2 channels in which ATP-occupied sites function to electrostatically dissociate COOH-terminal domains from the membrane, then as in all K(ir) channels, free COOH-terminal domains and inner M2 helices transit to a lower energy state for gate closure.