Sabine Costagliola

Cloning of two human thyroid cDNAs encoding new members of the NADPH oxidase family

Two cDNAs encoding NADPH oxidases and constituting the thyroid H2O2 generating system have been cloned. The strategy of cloning was based on the functional similarities between H2O2 generation in leukocytes and the thyroid, according to the hypothesis that one of the components of the thyroid system would belong to the gp91Phox/Mox1 gene family and display sequence similarities with gp91Phox. Screening at low stringency with a gp91Phox probe of cDNA libraries from thyroid cells in primary culture yielded two distinct human cDNA clones harboring open reading frames of 1551 (ThOX1) and 1548 amino acids (ThOX2), respectively. The encoded polypeptides display 83% sequence similarity and are clearly related to gp91Phox (53 and 47% similarity). The theoretical molecular mass of 177 kDa is close to the apparent molecular mass of 180 kDa of the native corresponding porcine flavoprotein and the protein(s) detected by Western blot in dog and human thyroid. ThOX1 and ThOX2 display sequence similarities of 53% and 61%, respectively, with a predicted protein ofCaenorhabditis elegans over their entire length. They show along their first 500 amino acids a similarity of 43% with thyroperoxidase. The corresponding genes of ThOX1 and ThOX2 are closely linked on chromosome 15q15.3. The dog mRNA expression is thyroid-specific and up-regulated by agents activating the cAMP pathway as is the synthesis of the polypeptides they are coding for. In human thyroid the positive regulation by cAMP is less pronounced. The proteins ThOX1 and ThOX2 accumulate at the apical membrane of thyrocytes and are co-localized with thyroperoxidase.

Glycoprotein hormone receptors: link between receptor homodimerization and negative cooperativity.

The monomeric model of rhodopsin‐like G protein‐coupled receptors (GPCRs) has progressively yielded the floor to the concept of GPCRs being oligo(di)mers, but the functional correlates of dimerization remain unclear. In this report, dimers of glycoprotein hormone receptors were demonstrated in living cells, with a combination of biophysical (bioluminescence resonance energy transfer and homogenous time resolved fluorescence/fluorescence resonance energy transfer), functional and biochemical approaches. Thyrotropin (TSHr) and lutropin (LH/CGr) receptors form homo‐ and heterodimers, via interactions involving primarily their heptahelical domains. The large hormone‐binding ectodomains were dispensable for dimerization but modulated protomer interaction. Dimerization was not affected by agonist binding. Observed functional complementation indicates that TSHr dimers may function as a single functional unit. Finally, heterologous binding‐competition studies, performed with heterodimers between TSHr and LH/CG–TSHr chimeras, demonstrated the unsuspected existence of strong negative cooperativity of hormone binding. Tracer desorption experiments indicated an allosteric behavior in TSHr and, to a lesser extent, in LH/CGr and FSHr homodimers. This study is the first report of homodimerization associated with negative cooperativity in rhodopsin‐like GPCRs. As such, it may warrant revisitation of allosterism in the whole GPCR family.

Tyrosine sulfation is required for agonist recognition by glycoprotein hormone receptors

The glycoprotein hormone receptors (thyrotrophin receptor, TSHr; luteinizing hormone/chorionic gonadotrophin receptor, LH/CGr; follicle‐stimulating hormone receptor, FSHr) constitute a subfamily of rhodopsin‐like G protein‐coupled receptors (GPCRs) with a long N‐terminal extracellular extension responsible for high‐affinity hormone binding. These ectodomains contain two cysteine clusters flanking nine leucine‐rich repeats (LRR), a motif found in several protein families involved in protein–protein interactions. Similar to the situation described recently in CCR5, we demonstrate here that the TSHr, as it is present at the cell surface, is sulfated on tyrosines in a motif located downstream of the C‐terminal cysteine cluster. Sulfation of one of the two tyrosines in the motif is mandatory for high‐affinity binding of TSH and activation of the receptor. Site‐directed mutagenesis experiments indicate that the motif, which is conserved in all members of the glycoprotein hormone receptor family, seems to play a similar role in the LH/CG and FSH receptors.

Glycoprotein hormone receptors: determinants in leucine-rich repeats responsible for ligand specificity

Glycoprotein hormone receptors [thyrotropin (TSHr), luteinizing hormone/chorionic gonadotropin (LH/CGr), follicle stimulating hormone (FSHr)] are rhodopsin‐like G protein‐coupled receptors with a large extracellular N‐terminal portion responsible for hormone recognition and binding. In structural models, this ectodomain is composed of two cysteine clusters flanking nine leucine‐rich repeats (LRRs). The LRRs form a succession of β‐strands and α‐helices organized into a horseshoe‐shaped structure. It has been proposed that glycoprotein hormones interact with residues of the β‐strands making the concave surface of the horseshoe. Gain‐of‐function homology scanning of the β‐strands of glycoprotein hormone receptors allowed identification of the critical residues responsible for the specificity towards human chorionic gonadotropin (hCG). Substitution of eight or two residues of the LH/CGr into the TSHr or FSHr, respectively, resulted in constructs displaying almost the same affinity and sensitivity for hCG as wild‐type LH/CGr. Molecular dynamics simulations and additional site‐directed mutagenesis provided a structural rationale for the evolution of binding specificity in this duplicated gene family.

Generation of functional thyroid from embryonic stem cells

The primary function of the thyroid gland is to metabolize iodide by synthesizing thyroid hormones, which are critical regulators of growth, development and metabolism in almost all tissues. So far, research on thyroid morphogenesis has been missing an efficient stem-cell model system that allows for the in vitro recapitulation of the molecular and morphogenic events regulating thyroid follicular-cell differentiation and subsequent assembly into functional thyroid follicles. Here we report that a transient overexpression of the transcription factors NKX2-1 and PAX8 is sufficient to direct mouse embryonic stem-cell differentiation into thyroid follicular cells that organize into three-dimensional follicular structures when treated with thyrotropin. These in vitro-derived follicles showed appreciable iodide organification activity. Importantly, when grafted in vivo into athyroid mice, these follicles rescued thyroid hormone plasma levels and promoted subsequent symptomatic recovery. Thus, mouse embryonic stem cells can be induced to differentiate into thyroid follicular cells in vitro and generate functional thyroid tissue.

Constitutive activation of the TSH receptor by spontaneous mutations affecting the N-terminal extracellular domain.

Activating mutations of the TSH receptor gene have been found in toxic adenomas and hereditary toxic thyroid hyperplasia. Up to now, all mutations have been located in the serpentine portion of the receptor. We now describe two additional mutations affecting Ser‐281 (Ser‐281‐Thr and Ser‐281‐Asn) in the ectodomain of the receptor. After transfection in COS cells, both mutants displayed increased constitutive activity for cAMP generation despite expression at a lower level than the wild type. The mutants were responsive to TSH. The present results are compatible with a model in which the activity of the unliganded receptor is kept at a low level by an inhibitory interaction between the N‐terminal domain and the serpentine portion of the receptor.

Allosteric modulation of binding properties between units of chemokine receptor homo- and hetero-oligomers.

We have demonstrated previously that the chemokine receptors CCR2 and CCR5 form homo- and heterodimers and that dimers can only bind a single chemokine molecule with high affinity. We provide here evidence from bioluminescence resonance energy transfer experiments that stimulation by chemokines does not influence the CCR2/CCR5 heterodimerization status. In addition, we show that the rate of radioligand dissociation from one unit of the heterodimer in “infinite” tracer dilution conditions is strongly increased in the presence of an unlabeled chemokine ligand of the other unit. These results demonstrate unambiguously that the interaction between heterodimer units is of allosteric nature. Agonists, but also some monoclonal antibodies, could promote such negative binding cooperativity, indicating that this phenomenon does not require the full conformational change associated with receptor activation. Finally, we show that G protein coupling is required for high-affinity binding of macrophage inflammatory protein-1β (CCL4) to CCR5 and that the dissociation from G proteins, after incubation with Gpp(NH)p, promotes the release of prebound radiolabeled chemokines with kinetics similar to those measured after the addition of an excess of unlabeled chemokines. These observations suggest that the association with G proteins probably participates in the negative cooperativity observed between receptor monomers. We propose that negative cooperativity within homo- and heterodimers of chemokine receptors and probably other G protein-coupled receptors will probably have major implications in their pharmacology in vivo and in the physiopathology of the diseases with which they are associated.

An activation switch in the rhodopsin family of G protein coupled receptors: The thyrotropin receptor

We aimed at understanding molecular events involved in the activation of a member of the G protein-coupled receptor family, the thyrotropin receptor. We have focused on the transmembrane region and in particular on a network of polar interactions between highly conserved residues. Using molecular dynamics simulations and site-directed mutagenesis techniques we have identified residue Asn-7.49, of the NPxxY motif of TM 7, as a molecular switch in the mechanism of thyrotropin receptor (TSHr) activation. Asn-7.49 appears to adopt two different conformations in the inactive and active states. These two states are characterized by specific interactions between this Asn and polar residues in the transmembrane domain. The inactive gauche+ conformation is maintained by interactions with residues Thr-6.43 and Asp-6.44. Mutation of these residues into Ala increases the constitutive activity of the receptor by factors of ∼14 and ∼10 relative to wild type TSHr, respectively. Upon receptor activation Asn-7.49 adopts the trans conformation to interact with Asp-2.50 and a putatively charged residue that remains to be identified. In addition, the conserved Leu-2.46 of the (N/S)LxxxD motif also plays a significant role in restraining the receptor in the inactive state because the L2.46A mutation increases constitutive activity by a factor of ∼13 relative to wild type TSHr. As residues Leu-2.46, Asp-2.50, and Asn-7.49 are strongly conserved, this molecular mechanism of TSHr activation can be extended to other members of the rhodopsin-like family of G protein-coupled receptors.

Identification and characterization of an endogenous chemotactic ligand specific for FPRL2

Chemotaxis of dendritic cells (DCs) and monocytes is a key step in the initiation of an adequate immune response. Formyl peptide receptor (FPR) and FPR-like receptor (FPRL)1, two G protein–coupled receptors belonging to the FPR family, play an essential role in host defense mechanisms against bacterial infection and in the regulation of inflammatory reactions. FPRL2, the third member of this structural family of chemoattractant receptors, is characterized by its specific expression on monocytes and DCs. Here, we present the isolation from a spleen extract and the functional characterization of F2L, a novel chemoattractant peptide acting specifically through FPRL2. F2L is an acetylated amino-terminal peptide derived from the cleavage of the human heme-binding protein, an intracellular tetrapyrolle-binding protein. The peptide binds and activates FPRL2 in the low nanomolar range, which triggers intracellular calcium release, inhibition of cAMP accumulation, and phosphorylation of extracellular signal–regulated kinase 1/2 mitogen-activated protein kinases through the Gi class of heterotrimeric G proteins. When tested on monocytes and monocyte-derived DCs, F2L promotes calcium mobilization and chemotaxis. Therefore, F2L appears as a new natural chemoattractant peptide for DCs and monocytes, and the first potent and specific agonist of FPRL2.

G protein-coupled receptors: mutations and endocrine diseases

Over the past 20 years, naturally occurring mutations that affect G protein-coupled receptors (GPCRs) have been identified, mainly in patients with endocrine diseases. The study of loss-of-function or gain-of-function mutations has contributed to our understanding of the pathophysiology of several diseases with classic hypophenotypes or hyperphenotypes of the target endocrine organs, respectively. Simultaneously, study of the mutant receptors ex vivo was instrumental in delineating the relationships between the structure and function of these important physiological and pharmacological molecules. Now that access to the crystallographic structure of a few GPCRs is available, the mechanics of these receptors can be studied at the atomic level. Progress in the fields of cell biology, molecular pharmacology and proteomics has also widened our view of GPCR functions. Initially considered simply as guanine nucleotide exchange factors capable of activating G protein-dependent regulatory cascades, GPCRs are now known to display several additional characteristics, each susceptible to alterations by disease-causing mutations. These characteristics include functionally important basal activity of the receptor; differential activation of various G proteins; differential activation of G protein-dependent and independent effects (biased agonism); interaction with proteins that modify receptor function; dimerization-dependent effects; and interaction with allosteric modulators. This Review attempts to illustrate how natural mutations of GPCR could contribute to our understanding of these novel facets of GPCR biology.