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CCR5 is a G-protein-coupled receptor activated by the chemokines RANTES (regulated on activation normal T cell expressed and secreted), macrophage inflammatory protein 1alpha and 1beta, and monocyte chemotactic protein 2 and is the main co-receptor for the macrophage-tropic human immunodeficiency virus strains. We have identified a sequence motif (TXP) in the second transmembrane helix of chemokine receptors and investigated its role by theoretical and experimental approaches. Molecular dynamics simulations of model alpha-helices in a nonpolar environment were used to show that a TXP motif strongly bends these helices, due to the coordinated action of the proline, which kinks the helix, and of the threonine, which further accentuates this structural deformation. Site-directed mutagenesis of the corresponding Pro and Thr residues in CCR5 allowed us to probe the consequences of these structural findings in the context of the whole receptor. The P84A mutation leads to a decreased binding affinity for chemokines and nearly abolishes the functional response of the receptor. In contrast, mutation of Thr-82(2.56) into Val, Ala, Cys, or Ser does not affect chemokine binding. However, the functional response was found to depend strongly on the nature of the substituted side chain. The rank order of impairment of receptor activation is P84A > T82V > T82A > T82C > T82S. This ranking of impairment parallels the bending of the alpha-helix observed in the molecular simulation study.

CCR5 is a functional receptor for MIP-1alpha, MIP-1beta, RANTES (regulated on activation normal T cell expressed), MCP-2, and MCP-4 and constitutes the main coreceptor for macrophage tropic human and simian immunodeficiency viruses. By using CCR5-CCR2b chimeras, we have shown previously that the second extracellular loop of CCR5 is the major determinant for chemokine binding specificity, whereas the amino-terminal domain plays a major role for human immunodeficiency virus type 1 (HIV-1) and simian immunodeficiency virus coreceptor function. In the present work, by using a panel of truncation and alanine-scanning mutants, we investigated the role of specific residues in the CCR5 amino-terminal domain for chemokine binding, functional response to chemokines, HIV-1 gp120 binding, and coreceptor function. Truncation of the amino-terminal domain resulted in a progressive decrease of the binding affinity for chemokines, which correlated with a similar drop in functional responsiveness. Mutants lacking residues 2-13 exhibited fairly weak responses to high concentrations (500 nM) of RANTES or MIP-1beta. Truncated mutants also exhibited a reduction in the binding affinity for R5 Env proteins and coreceptor activity. Deletion of 4 or 12 residues resulted in a 50 or 80% decrease in coreceptor function, respectively. Alanine-scanning mutagenesis identified several charged and aromatic residues (Asp-2, Tyr-3, Tyr-10, Asp-11, and Glu-18) that played an important role in both chemokine and Env high affinity binding. The overlapping binding site of chemokines and gp120 on the CCR5 amino terminus, as well as the involvement of these residues in the epitopes of monoclonal antibodies, suggests that these regions are particularly exposed at the receptor surface.

CCR5 is the major coreceptor for macrophage-tropic human immunodeficiency virus type I (HIV-1). For most G-protein-coupled receptors that have been tested so far, the disulfide bonds linking together the extracellular loops (ECL) are required for maintaining the structural integrity necessary for ligand binding and receptor activation. A natural mutation affecting Cys20, which is thought to form a disulfide bond with Cys269, has been described in various human populations, although the consequences of this mutation for CCR5 function are not known. Using site-directed mutagenesis, we mutated the four extracellular cysteines of CCR5 singly or in combination to investigate their role in maintaining the structural conformation of the receptor, its ligand binding and signal transduction properties, and its ability to function as a viral coreceptor. Alanine substitution of any single Cys residue reduced surface expression levels by 40-70%. However, mutation of Cys101 or Cys178, predicted to link ECL1 and ECL2 of the receptor, abolished recognition of CCR5 by a panel of conformation sensitive anti-CCR5 antibodies. The effects of the mutations on receptor expression and conformation were partially temperature-sensitive, with partial restoration of receptor expression and conformation achieved by incubating cells at 32 degrees C. All cysteine mutants were unable to bind detectable levels of MIP-1beta, and did not respond functionally to CCR5 agonists. Surprisingly, all cysteine mutants did support infection by R5 strains of HIV, though at reduced levels. These results indicate that both disulfide bonds of CCR5 are necessary for maintaining the structural integrity of the receptor necessary for ligand binding and signaling. Env binding and the mechanisms of HIV entry appear much less sensitive to alterations of CCR5 conformation.

p24 is a thyroid protein (Mr 24,000) identified by two‐dimensional gel electrophoresis on the basis that its synthesis and phosphorylation are up‐regulated by thyrotropin and cyclic AMP agonists. p24 cDNA was cloned from a lambda gt11 cDNA library using a polyclonal antibody raised against the protein recovered from a Western blot spot. The encoded polypeptide (189 residues) displays a putative target‐site for phosphorylation by cyclic AMP‐dependent protein kinase and belongs to the superfamily of proteins binding Ca2+ through ‘EF hand’ domains. It presents four such domains of which two agree closely with the consensus. The ability of p24 to bind Ca2+ has been directly confirmed on Western blots. p24 was detected in many tissues including the salivary glands, the lung and the brain. The ubiquitous nature of p24, together with its regulatory and sequence characteristics suggest that it constitutes an important target common to the cyclic AMP and Ca2+‐phosphatidylinositol cascades.

The structure of thyroglobulin mRNA was analyzed in an inbred herd of Afrikander cattle with hereditary goitre. Northern transfer of RNA from affected animals revealed both a shorter (approximately 7100 bases) and a normal‐sized (approximately 8200 bases) thyroglobulin mRNA when hybridized to bovine thyroglobulin cDNA clones. S1 nuclease mapping experiments established that 1100 bases are deleted in the 5′ region of the smaller mRNA. Electron microscopy of RNA from animals with goitre hybridized to a bovine genomic DNA clone showed that the region deleted corresponds to exon 9 of the thyroglobulin gene. Southern blot analysis of the exon 9 region revealed differences between affected and control animals with the enzymes PstI and TaqI. Although they could reflect a linkage disequilibrium between the mutation and restriction fragment length polymorphism, it is noteworthy that these differences map in the region of the exon 9/intron 9 junction. Our results show that a genetic lesion in the thyroglobulin gene causes aberrant splicing of the pre‐mRNA, and suggest that the responsible mutation is at the exon 9/intron 9 junction.

A lambda gt11 cDNA library was constructed from a normal human thyroid and screened with a rabbit anti‐porcine thyroperoxidase antibody. A series of thyroperoxidase (TPO) clones were obtained which allowed determination of the complete primary structure of the protein. The library was also screened with serum from a patient with Hashimoto's thyroiditis, an autoimmune disease characterized by the presence in the serum of high titers of autoantibodies directed against the ‘microsomal antigen’ (McAg). Comparison of the cDNA sequences from TPO clones and McAg clones provides definite proof that the McAg is TPO. A short segment of TPO was characterized as bearing a major epitope involved in autoimmunity. The primary structure of TPO was 42% homologous to myeloperoxidase (MPO). It contains, in addition, a C‐terminal extension with a membrane anchor region contiguous to two domains encoded by modules belonging to the EGF and C4b gene families. The existence in TPO of still another domain presenting a significant homology with a putative heme‐binding region of cytochrome C oxidase polypeptide I raises the possibility that a mitochondrial gene module has contributed a piece to the evolution of a typical nuclear mosaic gene.

The availability of rat thyroglobulin cDNA clones was exploited to study the regulation of thyroglobulin gene transcription by thyrotropin (TSH). Groups of rats were subjected to treatments leading to reduction or increase in the rat serum TSH (rTSH) levels. Thyroid gland nuclei were isolated, incubated in vitro in the presence of 32P-labeled uridine triphosphate, and thyroglobulin transcripts were quantitated by hybridization to immobilized rat thyroglobulin cDNA clones. Transcription of the thyroglobulin gene was found to be very active in thyroid nuclei from control animals. It represented about 10% of total RNA polymerase II activity. Chronic hyperstimulation of the thyroid glands with endogenous rTSH was achieved in rats treated with the goitrogen propylthiouracil. No significant increase of thyroglobulin gene transcription could be measured in thyroid nuclei from these animals. On the contrary, a dramatic decrease in thyroglobulin gene transcription was observed in those animals in which endogenous rTSH levels had been suppressed by hypophysectomy or by the administration of triiodothyronine. Injection of exogenous bovine TSH in such animals readily restored transcriptional activity of the gene. Our results identify transcription as an important regulatory step involved in TSH action. They suggest that normal TSH levels induce close to maximal expression of the thyroglobulin gene but that continuous presence of TSH is required in order to maintain the gene in an activated state.

Treatment of ox and dog thyroid slices in vitro with either thyrotropin or dibutyryl cyclic AMP elicited a variety of changes in polyribosome distribution. The most marked and consistent responses were decreases in both free and membrane-bound monoribosomes with a concomitant increase in the specific peak of thyroglobulin-synthesizing polyribosomes. On some occasions there was a shift towards heavier aggregates in the free polyribosomes. The increase in the amount of thyroglobulin-synthesizing polyribosomes was not accompanied by a shift in its location on the gradients. These changes were apparent within 30 min of thyrotropin addition and within 60 min of the addition of dibutyryl cyclic AMP. It is suggested that the major initial effect on translation of both thyrotropin and dibutyryl cyclic AMP is to stimulate the recruitment of pre-existing free monoribosomes on to pre-existing unloaded or under-loaded thyroglobulin mRNA molecules.

The aim of this study was to define, at the molecular level, epitopes of thyroglobulin recognized by heterologous antibodies and autoantisera.One hundred thousand clones from a λgt11 human thyroid cDNA library were screened using a rabbit antibody to human thyroglobulin at a 1:2000 dilution. Twenty clones were plaque-purified to homogeneity, and characterization and sequencing of their cDNA inserts showed that they represented four distinct regions of the thyroglobulin molecule, one of them being the 22 carboxyl-terminal amino acids. Rescreening of the library with the same rabbit antibody to human thyroglobulin absorbed with peptides encoded by the carboxyl-terminal clone, led to the definition of six further epitope-bearing fragments of thyroglobulin. The ten regions that we have identified were recognized by ten further rabbit antibodies to human thyroglobulin, showing that they are representative of the repertoire of heterologous epitopes.In contrast, none of the ten heteroepitope-bearing fragments was recognized by sera from ten patients with autoimmune thyroid disease with various titres of thyroglobulin antibodies. Screening of 2 × 106clones from the library using a pool of ten autoantisera (individual sera diluted to 1:1000), and of 1 × 106clones using a single autoantiserum of very high antithyroglobulin titre (diluted 1:400) resulted in no thyroglobulin clones being isolated. The significance of these results to the immune process is discussed.Journal of Endocrinology(1989) 122,169–176

Transcription of thyroglobulin (Tg) gene is under the positive control of thyrotropin (TSH). The mechanism of this control has been further investigated. Rats were treated with triiodothyronine (T 3) to decrease their endogenous TSH production. Following the intravenous injection of bovine TSH, a 3-fold stimulation of Tg gene transcription could be detected in isolated nuclei as early as 1 h after treatment. The TSH effect was also observed in tissue fragments incubated in vitro under conditions where a concomitant stimulation of cAMP accumulation was detected. Forskolin, a universal activator of adenylate cyclase, was able to mimic TSH action on Tg gene transcription. We conclude that TSH controls transcription of Tg gene directly via its known interaction with receptors on thyrocytes and that cAMP is a physiological mediator of this effect.

The chemokine receptor CCR5 binds macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, and regulated on activation, normal T-cell expressed and secreted (RANTES), and constitutes the major co-receptor allowing infection of CD4(+) T lymphocytes, macrophages, and microglial cells by macrophage-tropic strains of human and simian immunodeficiency virus. CCR5 is most closely related to CCR2b, another chemokine receptor that responds to monocyte chemoattractant protein (MCP)-1, MCP-2, MCP-3, and MCP-4. We have investigated by mutagenesis the regions of CCR5 and CCR2b involved in the specificity of binding and functional response to their respective ligands. We demonstrate that the key region of CCR5 involved in its specific interaction with MIP-1alpha, MIP-1beta, and RANTES, and its subsequent activation, lies within the second extracellular loop (and possibly the adjacent transmembrane segments). Conversely, the NH2-terminal domain of CCR2b is responsible for the high affinity binding of MCP-1, but is not sufficient to confer activation of the intracellular cascades. Extracellular loops of the receptor, among which the second loop plays a prominent role, are necessary to achieve efficient signaling of the receptor. These data complement our previous mapping of CCR5 domains functionally involved in the fusion process with the human immunodeficiency virus envelope, and will help in the development of agents able to interfere with the early steps of viral infection.

The goiters in a breed of hypothyroid goats contain only minute amounts of thyroblobulin-related antigens (0.01% of normal value). We have analyzed these goiters for the presence of mRNA coding for thyroglobulin. Using DNA complementary to beef 33S thyroglobulin mRNA as a probe, we found that the mRNA sequence is present in the goat goiter but at a concentration 1/10-1/40 that of normal goat thyroid. Hybrids of cDNA with either goiter or normal thyroid RNA exhibited identical sharp melting curves which suggests that the same RNA sequence is responsible for hybridization in both tissues. Normal goat thyroid contains a population of large membrane-bound polysomes engaged in throglobulin synthesis. In contrast, such polysomes are absent in the goiter. In regard to subcellular distribution, the relative amount of the thyroglobulin mRNA sequences from the goiter in nuclear RNA was 42% of normal, in cytoplasmic RNA was 7% of normal, and in the membrane fraction was only 1-2% of normal. Our results suggest that the lack of thyroglobulin in these goiters is due to a defect in thyroglobulin mRNA which leads to aberrant processing and/or transport of it from its site of synthesis to the endoplasmic reticulum.

In a cell-free protein-synthesizing system from a rabbit reticulocyte lysate, total RNA extracted from cultured rat pituitary tumor (GH3) cells directed, in a dose-related manner, the synthesis of proteins that were precipitated by antisera specific to rat growth hormone (somatotropin) and rat prolactin. A marked decrease in growth hormone secretion and growth hormone mRNA activity was observed when cells were grown in a medium deficient in thyroid hormone. Addition of triiodothyronine in physiologic amounts both prevented and completely reversed this effect within 48 hr. Thyroid hormone had no effect on prolactin secretion or prolactin mRNA activity. These data suggest that thyroid hormone may stimulate synthesis of growth hormone through induction of transcriptional activity. The possibility of an additional effect at the posttranscriptional level has not been excluded. Although thyroid hormone is believed to have a general effect on a variety of metabolic processes, some effects, at the molecular level, may be quite selective, as indicated by the observed changes in growth hormone but not prolactin mRNA activity. The GH3 cell model is useful in the study of triiodothyronine action because of independence from secondary hormonal effects caused by hypothyroidism and because simultaneous measurement of prolactin mRNA activity serves as a unique internal control.

The hereditary goitre of Afrikander cattle is an autosomal recessive disease characterized in homozygotes by the production of abnormal thyroglobulin (Tg) and the coexistence in the thyroid of normal-sized 8.4-kilobase (kb) Tg mRNA with a misspliced 7.3-kb message having lost exon 9. We have cloned and sequenced the cDNA segment corresponding to the abnormal exon 8-exon 10 junction and the relevant genomic DNA region. The mutation responsible for the disease is a cytosine to thymine transition creating a stop codon at position 697 in exon 9. The original reading frame is maintained in the 7.3-kb mRNA, which, as it lacks the mutated exon, is translatable into a potentially functional protein. This puzzling phenotype in which a mutated exon is apparently removed selectively from transcripts by alternative splicing leads us to suggest that the 7.3-kb transcript could be present in normal animals. Using a sensitive oligonucleotide hybridization assay, we have demonstrated that a 7.3-kb mRNA lacking exon 9 does exist in normal thyroids as a minor mRNA species. As it is fully translatable, the 7.3-kb mRNA is expected to be more stable than the normal-sized 8.4-kb message. This probably accounts for the higher proportion of 7.3-kb transcript found in the goitre.