Unraveling the intrafamilial correlations and heritability of tumor types in MEN1: a Groupe d'étude des Tumeurs Endocrines study.

BACKGROUND: MEN1, which is secondary to the mutation of the MEN1 gene, is a rare autosomal-dominant disease that predisposes mutation carriers to endocrine tumors. Most studies demonstrated the absence of direct genotype-phenotype correlations. The existence of a higher risk of death in the Groupe d'étude des Tumeurs Endocrines-cohort associated with a mutation in the JunD interacting domain suggests heterogeneity across families in disease expressivity. This study aims to assess the existence of modifying genetic factors by estimating the intrafamilial correlations and heritability of the six main tumor types in MEN1. METHODS: The study included 797 patients from 265 kindred and studied seven phenotypic criteria: parathyroid and pancreatic neuroendocrine tumors (NETs) and pituitary, adrenal, bronchial, and thymic (thNET) tumors and the presence of metastasis. Intrafamilial correlations and heritability estimates were calculated from family tree data using specific validated statistical analysis software. RESULTS: Intrafamilial correlations were significant and decreased along parental degrees distance for pituitary, adrenal and thNETs. The heritability of these three tumor types was consistently strong and significant with 64% (s.e.m.=0.13; P<0.001) for pituitary tumor, 65% (s.e.m.=0.21; P<0.001) for adrenal tumors, and 97% (s.e.m.=0.41; P=0.006) for thNETs. CONCLUSION: The present study shows the existence of modifying genetic factors for thymus, adrenal, and pituitary MEN1 tumor types. The identification of at-risk subgroups of individuals within cohorts is the first step toward personalization of care. Next generation sequencing on this subset of tumors will help identify the molecular basis of MEN1 variable genetic expressivity.

Systematic screening for PRKAR1A gene rearrangement in Carney complex: identification and functional characterization of a new in-frame deletion.

BACKGROUND: Point mutations of the PRKAR1A gene are a genetic cause of Carney complex (CNC) and primary pigmented nodular adrenocortical disease (PPNAD), but in 30% of the patients no mutation is detected. OBJECTIVE: Set up a routine-based technique for systematic detection of large deletions or duplications of this gene and functionally characterize these mutations. METHODS: Multiplex ligation-dependent probe amplification (MLPA) of the 12 exons of the PRKAR1A gene was validated and used to detect large rearrangements in 13 typical CNC and 39 confirmed or putative PPNAD without any mutations of the gene. An in-frame deletion was characterized by western blot and bioluminescence resonant energy transfer technique for its interaction with the catalytic subunit. RESULTS: MLPA allowed identification of exons 3-6 deletion in three patients of a family with typical CNC. The truncated protein is expressed, but rapidly degraded, and does not interact with the protein kinase A catalytic subunit. CONCLUSIONS: MLPA is a powerful technique that may be used following the lack of mutations detected by direct sequencing in patients with bona fide CNC or PPNAD. We report here one such new deletion, as an example. However, these gene defects are not a frequent cause of CNC or PPNAD.

Acrodysostosis.

Acrodysostosis refers to a group of rare skeletal dysplasias that share in common characteristic clinical and radiological features including brachydactyly, facial dysostosis, and nasal hypoplasia. In the past, the term acrodysostosis has been used to describe patients with heterogeneous phenotypes, including, in some cases, patients that today would be given alternative diagnoses. The recent finding that mutations impairing the cAMP binding to PRKAR1A are associated with "typical" acrodysostosis and hormonal resistance initiates the era where this group of disorders can be categorized on a genetic basis. In this review, we will first discuss the clinical, radiologic, and metabolic features of acrodysostosis, emphasizing evidence that several forms of the disease are likely to exist. Second, we will describe recent results explaining the pathogenesis of acrodysostosis with hormonal resistance (ADOHR). Finally, we will discuss the similarities and differences observed comparing patients with ADOHR and other diseases resulting from defects in the PTHR1 signaling pathway, in particular, pseudohypoparathyroidism type 1a and pseudopseudohypoparathyroidism.

Acrodysostosis syndromes.

Acrodysostosis (ADO) refers to a heterogeneous group of rare skeletal dysplasia that share characteristic features including severe brachydactyly, facial dysostosis and nasal hypoplasia. The literature describing acrodysostosis cases has been confusing because some reported patients may have had other phenotypically related diseases presenting with Albright Hereditary Osteodystrophy (AHO) such as pseudohypoparathyroidism type 1a (PHP1a) or pseudopseudohypoparathyroidism (PPHP). A question has been whether patients display or not abnormal mineral metabolism associated with resistance to PTH and/or resistance to other hormones that bind G-protein coupled receptors (GPCR) linked to Gsα, as observed in PHP1a. The recent identification in patients affected with acrodysostosis of defects in two genes, PRKAR1A and PDE4D, both important players in the GPCR-Gsα-cAMP-PKA signaling, has helped clarify some issues regarding the heterogeneity of acrodysostosis, in particular the presence of hormonal resistance. Two different genetic and phenotypic syndromes are now identified, both with a similar bone dysplasia: ADOHR, due to PRKAR1A defects, and ADOP4 (our denomination), due to PDE4D defects. The existence of GPCR-hormone resistance is typical of the ADOHR syndrome. We review here the PRKAR1A and PDE4D gene defects and phenotypes identified in acrodysostosis syndromes, and discuss them in view of phenotypically related diseases caused by defects in the same signaling pathway.

Inhibition by transmembrane peptides of chimeric insulin receptors.

Receptor tyrosine kinases play essential roles in cell proliferation and differentiation. We have recently shown that peptides corresponding to the transmembrane domains of the epidermal growth factor (EGF) and ErbB2 receptors inhibit their corresponding receptor activation in cancer cell lines. We extend this observation to cells transfected with chimeric insulin receptors where the transmembrane domain has been replaced by that of the EGF receptor or a mutated Erb2 domain. Peptides corresponding to the transmembrane domains of the EGF receptor and ErbB2 are able to inhibit specifically the autophosphorylation of insulin receptors with the corresponding domain. This inhibitory effect is correlated with the propensity of the different transmembrane domains to self-associate in a genetic reporter assay. Thus, our data strengthen the notion that transmembrane domains are involved in erbB receptor activation, and that these receptors can be modulated by inhibiting protein-protein interactions within the membrane.

A functional enhanced green fluorescent protein (EGFP)-tagged angiotensin II at(1a) receptor recruits the endogenous Galphaq/11 protein to the membrane and induces its specific internalization independently of receptor-g protein coupling in HEK-293 cells.

The angiotensin II (Ang II) AT(1A) receptor was tagged at its C terminus with the enhanced green fluorescent protein (EGFP), and the corresponding chimeric cDNA was expressed in HEK-293 cells. This tagged receptor presents wild-type pharmacological and signaling properties and can be immunodetected by Western blotting and immunoprecipitation using EGFP antibodies. Therefore, this EGFP-tagged AT(1A) receptor is the perfect tool for analyzing in parallel the subcellular distributions of the receptor and its interacting G protein and their trafficking using confocal microscopy. Morphological observation of both the fluorescent receptor and its cognate Galphaq/11 protein, identified by indirect immunofluorescence, and the development of a specific software for digital image analysis together allow examination and quantification of the cellular distribution of these proteins before and after the binding of different agonist or antagonist ligands. These observations result in several conclusions: 1) Expression of increasing amounts of the AT(1A) receptor at the cell surface is associated with a progressive recruitment of the cytosolic Galphaq/11 protein at the membrane; 2) Internalization of the EGFP-tagged AT(1A) induced by peptide ligands but not nonpeptide ligands is accompanied by a Galphaq/11 protein intracellular translocation, which presents a similar kinetic pattern but occurs predominantly in a different compartment; and 3) This Galphaq/11 protein cellular translocation is dependent on receptor internalization process, but not G protein coupling and signal transduction mechanisms, as assessed by pharmacological data using agonists and antagonists and the characterization of AT(1A) receptor mutants (D(74)N and Delta329) for which the coupling and internalization functions are modified.

The expression of thyrotropin receptor in the brain.

The regulation of the thyroid gland by TSH is mediated by a heterotrimeric G protein-coupled receptor. Nonthyroid effects of TSH have been reported, and expression of its receptor has been described in adipocytes and lymphocytes. We have previously reported the existence of specific and saturable binding sites of TSH and specific TSH effects in primary cultured rat brain astroglial cells. We now report expression of the TSH receptor gene in these cells; the coding sequence of the corresponding complementary DNA is identical to that previously established in thyroid. Using specific antisense RNA probe, expression of this gene was detected in some isolated or clustered glial fibrillary acidic protein-positive primary cultured cells by in situ hybridization. With this technique, we further detected TSH receptor messenger RNA (mRNA) expression in rat brain cryoslices in both neuronal cells and astrocytes. Its presence predominated in neuron-rich areas (pyriform and postcingulate cortex, hippocampus, and hypothalamic nuclei) and was mostly colocalized with neuron-specific enolase. In astrocytes, this mRNA was detected in the ependymal cell layer and the subependymal zone, and several isolated cells were also found in the brain parenchyma. We also detected TSH receptor mRNA and protein in primary cultured human astrocytes. The protein was detected as well in both rat and human brain cryoslices. Together, these findings clearly demonstrate the expression of the TSH receptor gene in the brain in both neuronal cells and astrocytes.

Systematic identification of mutations that constitutively activate the angiotensin II type 1A receptor by screening a randomly mutated cDNA library with an original pharmacological bioassay.

The constitutive activation of G-protein-coupled receptors is a major new approach to investigating their physiopathology and pharmacology. A large number of spontaneous and site-directed mutations resulting in constitutive activity have been identified, but systematic mapping of the amino acids involved for a given receptor would be extremely useful for complete elucidation of the molecular mechanisms underlying its activation. We carried out such mapping for the angiotensin II type 1A (AT(1A)) receptor by screening a randomly mutated cDNA library after expressing the mutated clones in eukaryotic cells. To test the AT(1A) mutants generated, we developed an original, specific, and highly sensitive assay based on the properties of CGP42112A. This classical AT(2) agonist is a weak partial agonist of the wild-type AT(1A) receptor and becomes a full agonist for constitutively active AT(1A) mutants, as shown experimentally and in allostery-based theoretical models. Activation of the mutated receptors by CGP42112A was monitored by using the bioluminescent protein aequorin, a very sensitive and specific sensor of intracellular calcium mobilization. The screening of 4,800 clones, providing an exhaustive coverage of all of the mutations generated, led to the identification of 16 mutations in sequences encoding the transmembrane domains that were responsible for high sensitivity to CGP42112A. The constitutive activity was confirmed by agonist-independent production of inositol phosphates, which showed that at least half of the clones had significantly increased basal activity. These data demonstrate that this new type of approach is very efficient for the systematic identification of constitutively active mutants of G-protein-coupled receptors.

[Synthesis and local and distant actions of vasoactive peptides]. / Synthèse et actions locales et à distance des peptides vasoactifs.

In this short review, we compare the structure, biosynthetis and processing pathways, physiological actions, receptor systems and signaling pathways of 3 vasoactive peptides: vasopressin, angiotensin II and endothelins. This comparison shows clearly that all these peptides are synthesized as preproproteins and are processed by proteolytic enzymes. They all act via different types of membrane-bound G protein coupled receptors. Their actions on cardiovascular target tissues are either of an endocrine nature (vasopressin) or a paracrine nature (endothelins) or both (angiotensin II).

Substitution of the insulin receptor transmembrane domain with that of glycophorin A inhibits insulin action.

To study the role of transmembrane (TM) domains interactions in the activation of the insulin receptor, we have replaced the insulin receptor TM domain with that of glycophorin A (GpA), an erythrocyte protein that spontaneously forms detergent-resistant dimers through TM-TM interactions. Insulin receptor cDNA sequences with the TM domain replaced by that of GpA were constructed and stably transfected in CHO cells. Insulin binding to cells and solubilized receptors was not modified. Electrophoresis after partial reduction of disulfide bonds revealed an altered structure for the soluble chimeric receptors, seen as an altered mobility apparently due to increased interactions between the beta subunits of the receptor. Insulin signaling was markedly decreased for cells transfected with chimeric receptors compared with cells transfected with normal receptors. A decrease in insulin-induced receptor kinase activity was observed for solubilized chimeric receptors. In conclusion, substitution by the native GpA TM domain of the insulin receptor results in structurally modified chimeric receptors that are unable to transmit the insulin signal properly. It is hypothesized that this substitution may impose structural constraints that prevent the proper changes in conformation necessary for activation of the receptor kinase. Other mutants modifying the structure or the membrane orientation of the glycophorin A TM domain are required to better understand these constraints.

Gene and cDNA cloning and characterization of the mouse V3/V1b pituitary vasopressin receptor.

The gene of the mouse V3/V1b receptor was identified by homology cloning. One of the genomic clones contained the entire coding sequence. The cDNA presented high identity with rat (92%) and human (84%) sequences. Southern blot analysis indicated the existence of a single gene. Tissue distribution was studied by RT-PCR. The major site of expression was the pituitary. A faint signal was also present in hypothalamus, brain, adrenal, pancreas and colon. The mouse corticotroph cell line, AtT20, did not express the transcript. In order to confirm the identity of the sequence, the V3/V1b receptor cDNA was cloned and stably expressed in CHO-AA8 Tet-Off cells under the control of tetracycline. When transfected cells were treated with arginine vasopressin (AVP), inositol phosphate production increased in a dose-dependent manner, indicating that the V3/V1b receptor couples to phospholipase C. Moreover, AVP did not stimulate cAMP production. Binding studies with [3H]AVP indicated that the affinity of the mouse V3/V1b receptor (Kd=0.5 nM) is similar to that reported for rat and human receptors. The rank order of potency established in competition binding experiments with different analogues was representative of a V3/V1b profile, distinct from V1a and V2. However, significant differences were found between human and mouse receptors tested in parallel. Thus the pharmacology of V3/V1b receptors can not be transposed among different species.

Sheep 5HT2A receptors: partial cloning of the coding sequence and mRNA localization by in situ hybridization in the ewe hypothalamus.

UNLABELLED: Serotonin and serotonin receptors of class II (5HT2-R) are thought to be involved in the neural mechanisms which regulate the LH release associated with photoperiodic changes in sheep. A specific premammillary hypothalamic area displaying a significant binding of 3H-ketanserin, a potent 5HT2-R antagonist, was previously identified. The aim of the present study was to ascertain by in situ hybridization (ISH) that 5HT2-R mRNA-containing cells were also present in this specific hypothalamic area. Total RNA was prepared from sheep pars tuberalis/median eminence, and a cDNA fragment of 546 bp was amplified by reverse transcriptase polymerase chain reaction (RT-PCR) using degenerated primers deduced from the human and rat 5HT2A-R sequences. After cloning and sequencing, the sheep nucleotide sequence had the highest homology (85.1-92.3%) with the other known mammalian 5HT2-R or 5HT2A-R sequences. Homology with other 5HT-R subtypes or other monoamine receptors was much lower, 60% at maximum. After ISH using sense and antisense 35S-riboprobes, specific labelling was found in different parts of the hypothalamus, especially in the mammillary bodies where the binding was higher. Within the hypothalamus, the density of labelled cells, mainly neurons, varied considerably. It was maximal in the mammillary bodies and also in a restricted ventral region of the premammillary hypothalamus located from about 500/700 micrometer to 1200/1400 micrometer in front of the mammillary recess, where 3H-ketanserin binding was previously reported. IN CONCLUSION: (1) the structural study of the sequence indicated that the new cloned cDNA corresponds to the sheep 5HT2-R class and, probably, to the 5HT2A-R subtype and (2) the ISH studies revealed that a restricted area of the premammillary hypothalamus shows a large number of 5HT2-R mRNA-containing neurons.

Several interesting phenotypes of the AT1 receptor produced by site-directed mutagenesis.

The angiotensin II (AngII) AT1 receptor is a seven-transmembrane domain receptor coupled to a Gq/11 protein and phospholipase C, but also to other G proteins and to several tyrosine kinase pathways. These signaling pathways transduce inside the cells the classical actions of AngII (vasoconstriction, aldosterone secretion, etc.), but also the mitogenic action of this vasoactive peptide. In the past 5 yr, site-directed mutagenesis has elucidated the molecular determinants of the AngII and nonpeptidic analogue-binding sites together with those of G protein interaction. In addition, these studies have demonstrated that modifications of the specific interactions between transmembrane domains are responsible for the activation of the receptor. Therefore, several mutations of these domains are able to block the receptor in active or inactive states. Finally, these mutagenesis studies identify two interesting phenotypes of the AT1 receptor. (1) A carboxy-terminal truncation of the AT1 receptor produces a mutant that is unable to be internalized and desensitized and therefore is functionally hyper-reactive. (2) A replacement of the distal part of the third intracellular loop of the AT1 receptor by the homologous segment of the beta2-adrenergic receptor produces a mutant coupled to both Gq and Gs proteins, which is unable to transduce the mitogenic action of AngII.

[Molecular structure and function of angiotensin ii receptors]. / Structure et fonctions moléculaires des récepteurs de l'angiotensine II.

Angiotensin II (Ang II) receptors are 7 transmembrane domain receptors corresponding to 2 pharmacologically and molecularly distinct receptors, called AT1 and AT2, the primary structures of which have been established by molecular cloning. Most if not all the physiological actions of Ang II are mediated by the AT1 receptor, which is coupled to a Gq protein activating a phospholipase C (PLC), which in turn mobilizes the intracellular calcium stores and activates protein kinases C. Many site directed mutagenesis works have allowed to identify short extracellular sequences responsible for the Ang II binding, whereas non-peptidic AT1-specific antagonists bind to a different transmembranar site. Structural modifications are responsible for the change of the receptor from an inactive to an active state. At the basal state, the receptor is mostly in an inactive state; agonists present a better affinity for the active state, displacing the equilibrium to this state; at the opposite, the inverse agonists present a better affinity for the inactive state. Antagonists present a similar affinity for both states of the receptor. Several mutations of polar residues of the transmembrane domains block the receptor either in an inactive state (D74D, S115A, Y292F) or in a constitutively active state (N111A and N295A). After activation, the receptor is coupled to different intracellular proteins, the first of them being the G proteins of the Gq/11 family. The sequences of the receptor involved in this coupling correspond to the 2nd, the 3rd intracellular loops and the proximal segment of the carboxyterminal domain. Other sequences interact with other proteins, such as the 319YIPP332 sequence of the carboxyterminus, which interacts with the Jak2 tyrosine kinase. After the binding of a peptidic ligands, the ligand-receptor complex is internalized independently for the G protein coupling. Again, site directed mutagenesis experiments have localized a sequence of the carboxyterminus (329STLSTKMSTLS338) involved in the internalization. This serine and threonine-rich sequence plays also a role in the desensitization of the AT1 receptor, consecutively to its phosphorylation. The AT2 receptor is only 34% identical to the AT1 receptor and its functions are far less understood. Its physiological functions (apoptosis and antiproliferative actions) and its signaling pathways (activation of Gi proteins and tyrosine phosphatases) are still a matter of debate.

[Angiotensin II receptors: classification, structure, and signal transduction]. / Récepteurs de l'angiotensine II: classification, structure et transduction du signal.

Angiotensin II (AngII), a circulating vasoactive peptide, interacts with specific membrane-bound receptors on the target tissues (vessels, kidneys and adrenal gland). Using new pharmacological tools and molecular cloning, these receptors have been classified in two types, called AT1 et AT2, whereas two subtypes, called AT1A et AT1B, have been identified for the rodent AT1 receptors, but not in humans. All these receptors present a seven hydrophobic transmembrane domain structure, which is classical for G protein coupled receptors. The interspecies molecular homology of these AngII receptors is high (> 90 per cent identity) within the same type of receptor, but is rather low (approximately 35 per cent identity) between the two types of receptors. The AT1 receptors are responsible for most of the AngII physiological actions and are coupled to a Gq protein, which activates a phospholipase C producing second messengers which activate protein kinases C and mobilize calcium intracellular stores. More recently, a strong interaction of this receptor has been demonstrated with the signalling pathways of the tyrosine kinases. The molecular mechanisms and the physiological importance of these interactions remain to be elucidated. The intracellular signalling (Gi coupling and tyrosine phosphatase activation) and the physiological actions (cellular differentiation, apoptosis) of the AT2 receptors are more controversial.

Detection and quantification of the A3243G mutation of mitochondrial DNA by ligation detection reaction.

The A3243G mutation of mitochondrial DNA is associated to the MELAS syndrome and to transmitted forms of diabetes mellitus. This mutation exists in a heteroplasmic state and can be present at a minor and hardly detectable level. The aim was to design a method which could be applied to large series of samples and could provide rapid, sensitive and quantitative detection of this mutation in the wild-type mitochondrial DNA background. The ability of ligation detection reaction (LDR) to satisfy these objectives was evaluated. Ligation detection reaction was performed on a model template composed of mixtures of various proportions of plasmids bearing the wild-type or mutant mitochondrial DNA sequence. Radiolabelled or fluorescent primers and the wild-type and mutant LDR products were separated by electrophoresis on conventional denaturating gel or on an Applied Biosystem 373. The ratios of mutant/wild-type products were consistent with the initial ratios of the plasmids in the template. The sensitivity and accuracy of the fluorescence and isotopic detection methods were similar. The detection limit of mutant DNA was 10% of total mitochondrial DNA. The percentage of mutant DNA in DNA samples extracted from leukocytes of 19 patients having the mutation at different levels, was evaluated by fluorescent or isotopic LDR.

Identification of the rat adapter Grb14 as an inhibitor of insulin actions.

We cloned by interaction with the beta-subunit of the insulin receptor the rat variant of the human adapter Grb14 (rGrb14). rGrb14 is specifically expressed in rat insulin-sensitive tissues and in the brain. The binding of rGrb14 to insulin receptors is insulin-dependent in vivo in Chinese hamster ovary (CHO) cells overexpressing both proteins and importantly, in rat liver expressing physiological levels of proteins. However, rGrb14 is not a substrate of the tyrosine kinase of the receptor. In the two-hybrid system, two domains of rGrb14 can mediate the interaction with insulin receptors: the Src homology 2 (SH2) domain and a region between the PH and SH2 domains that we named PIR (for phosphorylated insulin receptor-interacting region). In vitro interaction assays using deletion mutants of rGrb14 show that the PIR, but not the SH2 domain, is able to coprecipitate insulin receptors, suggesting that the PIR is the major binding domain of rGrb14. The interaction between rGrb14 and the insulin receptors is almost abolished by mutating tyrosine residue Tyr1150 or Tyr1151 of the receptor. The overexpression of rGrb14 in CHO-IR cells decreases insulin stimulation of both DNA and glycogen synthesis. These effects are accompanied by a decrease in insulin-stimulated tyrosine phosphorylation of IRS-1, but insulin receptor autophosphorylation is unaltered. These findings suggest that rGrb14 could be a new downstream signaling component of the insulin-mediated pathways.

Functional interactions of L-162,313 with angiotensin II receptor subtypes and mutants.

A nonpeptide ligand, L-162,313 (5,7-dimethyl-2-ethyl-3-[[4-[2(n-butyloxycarbonylsulfonamido)-5-is obutyl-3-thienyl]phenyl]methyl]imidazo[4,5,6]pyridine) was characterized on the angiotensin II receptors. This compound displaced [125I][Sar1]angiotensin II from rat angiotensin AT1A, AT1B or AT2 receptor individually expressed in COS-7 cells (Ki = 207 nM, 226 nM and 276 nM, respectively). In monkey kidney cells expressing angiotensin AT1A or AT1B receptors, it stimulated inositol phosphate accumulation, but the maximal response was 34.9 and 23.3%, respectively, of those of angiotensin II. Furthermore, an antagonist effect of L-162.313 was observed in response to angiotensin II. Single-point substitutions in the second and third transmembrane domains of the rat angiotensin AT1A receptor, which impaired the binding of losartan (2-n-butyl-4-chloro-5-hydroxymethyl-1[(1H-tetrazol-5-yl)biphenyl-4 -yl)methyl]imidazole), also affected the binding of L-162,313. Losartan and L-162,313 require some common structural determinants for non-peptide recognition on the angiotensin AT1 receptor. Furthermore, some of these substitutions, which impaired the inositol phosphate accumulation in response to angiotensin II, also impaired the response to L-162,313. Angiotensin II and L-162,313 require common critical residues for angiotensin AT1 receptor activation.