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1.
Horm Metab Res ; 37(6): 369-74, 2005 Jun.
Article in English | MEDLINE | ID: mdl-16001329

ABSTRACT

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant disease predisposed by heterozygous germline mutations in the MEN1 tumor suppressor gene. Biallelic loss of MEN1 resulting from small mutation and/or loss of heterozygosity occurs in a large tissue spectrum of MEN1 tumors or non-hereditary tumors. Mouse models of MEN1 underexpression or overexpression have also supported the tumor-suppressor effect of the MEN1 gene. Menin, the 610-amino-acid protein encoded by MEN1, is expressed ubiquitously and found predominantly in the nucleus. Sequence analyses do not reveal motifs of known function other than two nuclear localization sequences. Menin has been found to partner in vitro with a variety of proteins that comprise transcription factors, DNA processing factors, DNA repair proteins, and cytoskeletal proteins. The diverse functions of menin interactors suggest roles for menin in multiple biological pathways. Inactivation of menin switches its JunD partner from a downstream action of growth suppression to growth promotion. This is a plausible mechanism for menin tumorigenesis.


Subject(s)
Gene Expression Regulation, Neoplastic , Multiple Endocrine Neoplasia Type 1/metabolism , Proto-Oncogene Proteins/metabolism , Animals , Cell Nucleus/metabolism , Cytoskeletal Proteins/metabolism , DNA/metabolism , Gene Expression Regulation, Neoplastic/genetics , Humans , Multiple Endocrine Neoplasia Type 1/genetics , Protein Binding , Proto-Oncogene Proteins/genetics , Transcription Factors/metabolism , Transcription, Genetic
2.
Genesis ; 30(4): 259-63, 2001 Aug.
Article in English | MEDLINE | ID: mdl-11536432

ABSTRACT

In an effort to create a conventional knockout mouse model for multiple endocrine neoplasia type 1 (MEN1), we targeted disruption of the mouse Men1 gene through homologous recombination in ES cells. Men1 exons 2-4 were replaced by a PGK-neomycin cassette inserted in the opposite direction of Men1 transcription (Men1(MSK/+)). Unexpectedly, the Men1 conventional knockout was lethal in heterozygous, chimeric animals. Analysis of embryos revealed late gestational lethality with some embryos showing omphalocele. This was a very surprising phenotype, given that humans and mice that are heterozygotes for loss of function mutations in MEN1 are phenotypically normal except for a risk of endocrine tumors. Northern analysis of Men1(MSK/+) embryonic stem cell RNA revealed the presence of an abundant, novel transcript of 2.1 kb, in addition to the expected wild-type transcripts of 2.7 kb and 3.1 kb. RT-PCR analysis identified this aberrant transcript as arising from the antisense strand of the PGK promoter. We hypothesize that this transcript is producing either a toxic effect at the RNA level, or a dominant negative effect through the production of an amino-terminal truncated protein product. This example serves as a cautionary reminder that mouse knockouts using PGK-neo may sometimes display phenotypes that reflect more than just the loss of function of the targeted gene.


Subject(s)
Embryo Loss/genetics , Genes, Lethal/genetics , Heterozygote , Mutagenesis, Insertional/genetics , Neoplasm Proteins/genetics , Proto-Oncogene Proteins , Transcription, Genetic/genetics , Animals , Blotting, Western , Chimera/genetics , Embryo, Mammalian/metabolism , Exons/genetics , Gene Deletion , Gene Targeting/methods , Genes, Dominant/genetics , Genes, Reporter/genetics , Hernia, Umbilical/genetics , Mice , Mice, Knockout , Neomycin/biosynthesis , Phenotype , Precipitin Tests , Promoter Regions, Genetic/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism
3.
Oncogene ; 20(36): 4917-25, 2001 Aug 16.
Article in English | MEDLINE | ID: mdl-11526476

ABSTRACT

Multiple endocrine neoplasia type 1 is an autosomal dominant tumor syndrome. Manifestations include neoplasms of the parathyroid glands, enteropancreatic neuroendocrine cells, and the anterior pituitary gland. The MEN1 tumor suppressor gene encodes menin, a 610 amino acid nuclear protein without sequence homology to other proteins. To elucidate menin function, we used immunoprecipitation to identify interacting proteins. The NF-kappaB proteins p50, p52 and p65 were found to interact specifically and directly with menin in vitro and in vivo. The region of NF-kappaB proteins sufficient for binding to menin is the N-terminus. Furthermore, amino acids 305-381 of menin are essential for this binding. Menin represses p65-mediated transcriptional activation on NF-kappaB sites in a dose-dependent and specific manner. Also, PMA (phorbol 12-myristate 13-acetate)-stimulated NF-kappaB activation is suppressed by menin. These observations suggest that menin's ability to interact with NF-kappaB proteins and its modulation of NF-kappaB transactivation contribute to menin's tumor suppressor function.


Subject(s)
Genes, Tumor Suppressor , NF-kappa B/antagonists & inhibitors , NF-kappa B/metabolism , Neoplasm Proteins/physiology , Proto-Oncogene Proteins , Animals , COS Cells , Cell Line , Glutathione Transferase/chemistry , HeLa Cells , Humans , NF-kappa B/chemistry , Neoplasm Proteins/chemistry , Neoplasm Proteins/immunology , Precipitin Tests , Protein Structure, Tertiary , Tetradecanoylphorbol Acetate/pharmacology , Transcriptional Activation
4.
J Biol Chem ; 276(34): 32145-51, 2001 Aug 24.
Article in English | MEDLINE | ID: mdl-11399760

ABSTRACT

The G protein-coupled Ca(2+) receptor (CaR) possesses an approximately 600-residue extracellular domain involved in ligand binding and receptor activation. Based on an alignment of the amino acid sequence of the CaR with that of bacterial periplasmic-binding proteins, the first approximately 530 residues of the extracellular domain are believed to form a domain resembling a bilobed Venus's flytrap (VFT). Four insertions in the CaR sequence that do not align with those of bacterial periplasmic-binding proteins correspond to four loops within lobe I of the VFT. We constructed a series of deletion mutants of these four loops and tested their ability to form fully processed CaR as well as their ability to be activated by Ca(2+). As many as 21 residues (365) of loop III could be deleted without impairing receptor expression or activation. Deletion of portions of either loops I (50) or IV (438) did not impair receptor expression but significantly reduced Ca(2+) activation. Deletion of the entire loop II (117) abolished receptor expression and function, but the replacement of even a single residue within this deletion mutant led to expression of a monomeric form of the receptor showing increased Ca(2+) sensitivity but reduced maximal activation. Our results reveal that certain residues within loops I and IV are dispensable in formation of the VFT domain but are critical for Ca(2+) activation of the receptor. In contrast, the residues in loop II are critical for maintaining the inactive state of the CaR. We discuss these results in light of the recently defined crystal structure of the homologous domain of the type 1 metabotropic glutamate receptor.


Subject(s)
Calcium-Binding Proteins/physiology , Mutagenesis, Site-Directed , Sequence Deletion , Amino Acid Sequence , Calcium-Binding Proteins/chemistry , Calcium-Binding Proteins/genetics , Cell Line , Humans , Molecular Sequence Data
5.
Trends Endocrinol Metab ; 12(4): 173-8, 2001.
Article in English | MEDLINE | ID: mdl-11295574

ABSTRACT

Multiple endocrine neoplasia type 1 (MEN1) provides a prime example of how a rare disease can advance our understanding of basic cell biology, neoplasia and common endocrine tumors. MEN1 is expressed mainly as parathyroid, enteropancreatic neuroendocrine, anterior pituitary and foregut carcinoid tumors. It is an autosomal dominant disease caused by mutation of the MEN1 gene. Since its identification, the MEN1 gene has been implicated in many common endocrine and non-endocrine tumors. This is a brief overview of recent scientific advances relating to MEN1, including newly recognized clinical features that are now better characterized by genetic analysis, insights into the function of the MEN1-encoded protein menin, and refined recommendations for mutation testing and tumor screening, which highlight our increasing understanding of this complex syndrome.


Subject(s)
Multiple Endocrine Neoplasia Type 1 , Proto-Oncogene Proteins , Adrenal Cortex Neoplasms/genetics , Angiofibroma/genetics , Humans , Leiomyoma/genetics , Multiple Endocrine Neoplasia Type 1/genetics , Mutation , Neoplasm Proteins/genetics , Neoplasm Proteins/physiology , Pheochromocytoma/genetics , Skin Neoplasms/genetics , Thyroid Neoplasms/genetics
6.
J Biol Chem ; 276(10): 6901-4, 2001 Mar 09.
Article in English | MEDLINE | ID: mdl-11238442

ABSTRACT

The extracellular N-terminal domain of the human Ca(2+) receptor (hCaR) consists of a Venus's-flytrap (VFT) domain and a cysteine-rich (Cys-rich) domain. We have shown earlier that the Cys-rich domain is critical for signal transmission from the VFT domain to the seven-transmembrane domain. The VFT domain contains 10 cysteines: two of them (Cys(129) and Cys(131)) were identified as involved in intermolecular disulfide bonds necessary for homodimerization, and six others (Cys(60)-Cys(101), Cys(358)-Cys(395), and Cys(437)-Cys(449)) are predicted to form three intramolecular disulfide bonds. The Cys-rich domain contains nine cysteines, the involvement of which in disulfide bond formation has not been defined. In this work, we asked whether the remaining cysteines in the hCaR VFT, namely Cys(236) and Cys(482), form disulfide bond(s) with cysteines in the Cys-rich domain. We constructed mutant hCaRs with a unique tobacco etch virus (TEV) protease recognition site inserted between the VFT domain and the Cys-rich domain. These mutant hCaRs remain fully functional compared with the wild type hCaR. After TEV protease digestion of the mutant hCaR proteins, dimers of the VFT were identified on Western blot under nonreducing conditions. We concluded that there is no disulfide bond between the VFT and the Cys-rich domains in the hCaR.


Subject(s)
Calcium-Binding Proteins/chemistry , Cysteine/chemistry , Amino Acid Sequence , Binding Sites , Cell Line , Dimerization , Disulfides , Dose-Response Relationship, Drug , Endopeptidases/metabolism , Glutamic Acid/chemistry , Humans , Immunoblotting , Molecular Sequence Data , Mutagenesis, Site-Directed , Protein Conformation , Protein Sorting Signals , Protein Structure, Tertiary , Transfection , Valine/chemistry
7.
Gene ; 263(1-2): 31-8, 2001 Jan 24.
Article in English | MEDLINE | ID: mdl-11223240

ABSTRACT

Multiple endocrine neoplasia type 1 (MEN1) is a familial cancer syndrome characterized by tumors of the parathyroid, entero-pancreatic neuroendocrine and pituitary tissues and caused by inactivating mutations in the MEN1 gene. Menin, the 610-amino acid nuclear protein encoded by MEN1, binds to the transcription factor JunD and can repress JunD-induced transcription. We report here the identification of a MEN1 ortholog in Drosophila melanogaster, Menin1, that encodes a 763 amino acid protein sharing 46% identity with human menin. Additionally, 69% of the missense mutations and in-frame deletions reported in MEN1 patients appear in amino acid residues that are identical in the Drosophila and human protein, suggesting the importance of the conserved regions. Drosophila Menin1 gene transcripts use alternative polyadenylation sites resulting in 4.3 and 5-kb messages. The 4.3-kb transcript appears to be largely maternal, while the 5-kb transcript appears mainly zygotic. The binding of Drosophila menin to human JunD or Drosophila Jun could not be demonstrated by the yeast two-hybrid analysis. The identification of the MEN1 ortholog from Drosophila melanogaster will provide an opportunity to utilize Drosophila genetics to enhance our understanding of the function of human menin.


Subject(s)
Drosophila melanogaster/genetics , Neoplasm Proteins/genetics , Proto-Oncogene Proteins , Amino Acid Sequence , Animals , Blotting, Northern , DNA, Complementary/chemistry , DNA, Complementary/genetics , Drosophila melanogaster/embryology , Drosophila melanogaster/growth & development , Embryo, Nonmammalian/metabolism , Embryonic Development , Exons , Female , Gene Expression Regulation, Developmental , Genes, Insect/genetics , Glutathione Transferase/genetics , Glutathione Transferase/metabolism , Humans , In Situ Hybridization , Introns , Male , Mice , Molecular Sequence Data , Proto-Oncogene Proteins c-jun/genetics , Proto-Oncogene Proteins c-jun/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Saccharomyces cerevisiae/genetics , Sequence Alignment , Sequence Analysis, DNA , Sequence Homology , Transcription, Genetic , Two-Hybrid System Techniques , Zebrafish
8.
Proc Natl Acad Sci U S A ; 98(3): 1118-23, 2001 Jan 30.
Article in English | MEDLINE | ID: mdl-11158604

ABSTRACT

Multiple endocrine neoplasia type 1 (MEN1) is an autosomal dominant cancer syndrome, characterized primarily by multiple tumors in the parathyroid glands, endocrine pancreas, and anterior pituitary. Other tumors, including gastrinoma, carcinoid, adrenal cortical tumors, angiofibroma, collagenoma, and lipoma, also occur in some patients. Individuals with MEN1 almost always have loss-of-function mutations in the MEN1 gene on chromosome 11, and endocrine tumors arising in these patients usually show somatic loss of the remaining wild-type allele. To examine the role of MEN1 in tumor formation, a mouse model was generated through homologous recombination of the mouse homolog Men1. Homozygous mice die in utero at embryonic days 11.5-12.5, whereas heterozygous mice develop features remarkably similar to those of the human disorder. As early as 9 months, pancreatic islets show a range of lesions from hyperplasia to insulin-producing islet cell tumors, and parathyroid adenomas are also frequently observed. Larger, more numerous tumors involving pancreatic islets, parathyroids, thyroid, adrenal cortex, and pituitary are seen by 16 months. All of the tumors tested to date show loss of the wild-type Men1 allele, further supporting its role as a tumor suppressor gene.


Subject(s)
Multiple Endocrine Neoplasia Type 1/genetics , Neoplasm Proteins/genetics , Proto-Oncogene Proteins , Adenoma/genetics , Adenoma/pathology , Animals , Chromosome Mapping , Chromosomes, Artificial, Bacterial , Chromosomes, Human, Pair 11 , Crosses, Genetic , Disease Models, Animal , Exons , Female , Fetal Death , Genes, Lethal , Genes, Tumor Suppressor , Homozygote , Humans , Hyperparathyroidism/genetics , Hyperparathyroidism/pathology , Islets of Langerhans/pathology , Male , Mice , Mice, Inbred Strains , Mice, Transgenic , Multiple Endocrine Neoplasia Type 1/pathology , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/pathology , Parathyroid Neoplasms/genetics , Parathyroid Neoplasms/pathology , Phosphoglycerate Kinase/genetics , Pregnancy , Recombination, Genetic
9.
Cytotechnology ; 35(2): 127-35, 2001 Mar.
Article in English | MEDLINE | ID: mdl-19003289

ABSTRACT

A process was developed for producing human menin from transformed Drosophila Schneider 2 cells. Protein expression was achieved after inducing the metallothionein promoter by adding copper sulfate to cells growing in suspension in a stirred-tank reactor. Experiments in shake flasks showed that the production of menin was improved when the induction was conducted late in the exponential phase of cell growth at a concentration of 1-2 x 10(7) cells ml(-1), with a copper concentration of 0.2 mM for no more than 24 h. This observation was confirmed by experiments in bench-scale fermentors. Subsequently, a pilot-scale fermentation yielded 1 mg l(-1) culture of purified menin.

10.
J Clin Endocrinol Metab ; 85(11): 4131-7, 2000 Nov.
Article in English | MEDLINE | ID: mdl-11095444

ABSTRACT

Extracellular calcium levels are able to influence the secretion of gastrin by gastrinomas and possibly affect the growth pattern. The molecular mechanisms of these functions are not known. The purpose of the present study was to investigate the presence of the calcium-sensing receptor (CaR) in 10 gastrinomas and determine the extent of expression in the tumors. The amounts of CaR messenger ribonucleic acid in eight tumors were determined by quantitative RT-PCR. Protein expression was analyzed by Western blot and immunohistochemistry using a monoclonal antibody (ADD). CaR messenger ribonucleic acid was detected in all gastrinomas with levels ranging from 0.04-3.16 times the amount of beta-actin transcripts. The Western blot showed a major immunoreactive band at 250 kDa and a minor at 140 kDa, corresponding to the receptor dimer and monomer, respectively. Immunohistochemistry demonstrated variable membranous staining in all gastrinomas and normal pancreatic islets. No staining was observed in the normal liver, lymph node, or exocrine pancreas. We conclude that the CaR is present in all gastrinomas, with expression varying by 80-fold. It probably contributes to the calcium-stimulated gastrin release by gastrinomas. Whether the density of the CaR is a determining factor of the magnitude of this gastrin release or plays a role in regulating the growth pattern of the gastrinoma, as it does in other cells, remains unclear at present.


Subject(s)
Gastrinoma/genetics , Pancreatic Neoplasms/genetics , Receptors, Cell Surface/genetics , Zollinger-Ellison Syndrome/genetics , Adult , Blotting, Western , Calcium/metabolism , Female , Gastrinoma/pathology , Gastrinoma/surgery , Humans , Islets of Langerhans/pathology , Male , Middle Aged , Multiple Endocrine Neoplasia Type 1/genetics , Neoplasm Metastasis , Pancreatic Neoplasms/pathology , Pancreatic Neoplasms/surgery , Polymerase Chain Reaction , RNA, Messenger/analysis , Receptors, Calcium-Sensing , Receptors, Cell Surface/analysis , Transcription, Genetic , Zollinger-Ellison Syndrome/pathology , Zollinger-Ellison Syndrome/surgery
11.
Endocrinology ; 141(11): 4156-63, 2000 Nov.
Article in English | MEDLINE | ID: mdl-11089548

ABSTRACT

Naturally occurring mutations identified in subjects with autosomal dominant hypocalcemia (ADH) and the calcimimetic compound, R-568, have both been reported to increase Ca2+ sensitivity of the Ca2+ receptor (CaR). To gain insight into their mechanism of action, we studied interactions between four different ADH mutations located in the amino-terminal extracellular domain (ECD) and R-568. We found that R-568 increased the sensitivity of three of the ADH mutant receptors, but the Leu125Pro mutant appeared to be maximally left-shifted in that neither R-568 addition nor combining other ADH mutations with Leu125Pro gave increases in sensitivity comparable to those seen with the three other ADH mutations studied. We also made use of truncation and deletion mutants of the CaR and CaR/metabotropic glutamate receptor type 1 (mGluR1) chimeras to study both the site of action of R-568 and the effect of the Leu125Pro activating mutation. R-568 was effective in receptor constructs containing the seven transmembrane domain (7TM) of the CaR, but not in those containing the mGluR1 7TM. R-568, moreover, imparted Ca2+ responsiveness to CaR constructs lacking all or part of the CaR ECD. The Leu125Pro mutation in contrast conferred no or minimal increase in Ca2+ responsiveness to CaR constructs lacking part of the CaR ECD but showed a striking increase in basal activity in the context of chimeras containing an mGluR1 7TM. Our results localize the site of action of NPS-568 specifically to the CaR 7TM. Our results with the Leu125Pro mutant, furthermore, suggest that the mGluR1 7TM domain may be more permissive for activation than the 7TM domain of the CaR.


Subject(s)
Aniline Compounds/pharmacology , Calcium-Binding Proteins/drug effects , Calcium-Binding Proteins/genetics , Calcium/agonists , Mutagenesis , Receptors, Metabotropic Glutamate/drug effects , Receptors, Metabotropic Glutamate/genetics , Amino Acid Sequence , Calcium/pharmacology , Gene Deletion , Humans , Hydrolysis , Immunoblotting , Molecular Sequence Data , Phenethylamines , Phosphatidylinositols/metabolism , Propylamines , Receptors, Metabotropic Glutamate/metabolism , Recombinant Fusion Proteins/metabolism , Transfection
12.
Oncogene ; 19(41): 4706-12, 2000 Sep 28.
Article in English | MEDLINE | ID: mdl-11032020

ABSTRACT

Menin, the product of the MEN1 tumor suppressor gene, binds to the AP1 transcription factor JunD and represses JunD transcriptional activity. The effects of human or mouse JunD missense mutations upon menin interaction were studied by random and alanine scanning mutagenesis of the menin binding region of JunD (amino acids 1-70). JunD mutant proteins were tested for menin binding in a reverse yeast two-hybrid assay, and for transcriptional regulation by menin in AP1-reporter assays. Random mutagenesis identified two different mutations that disrupted menin interaction at mouse JunD amino acid 42 (G42E and G42R). Mutation G42A generated by alanine scanning did not affect menin binding, likely reflecting the conserved nature of this amino acid substitution. Furthermore, by size exclusion chromatography menin co-migrated with wild type JunD but not with the JunD mutant tested (G42E). Alanine scanning mutagenesis of residues 30-55 revealed two different amino acids, P41 and P44, of mouse JunD that were critical for interaction with menin. Mouse JunD missense mutants P41A, G42R, G42E and P44A failed to bind menin and also escaped menin's control over their transcriptional activity. At lower amounts of transfected menin, the transcriptional effect of menin on the mutants P41A, G42R and G42E was changed from repression to activation, similar to that with c-jun. In conclusion, a small N-terminal region of JunD mediates a key difference between JunD and c-jun, and a component of this difference is dependent on JunD binding to menin.


Subject(s)
Amino Acid Substitution , Mutation, Missense , Neoplasm Proteins/metabolism , Protein Isoforms/genetics , Proto-Oncogene Proteins c-jun/genetics , Proto-Oncogene Proteins , Amino Acid Sequence , Animals , Cell Line , Chromatography, Gel , Humans , Kidney , Mice , Molecular Sequence Data , Mutagenesis, Site-Directed , Protein Binding , Protein Isoforms/metabolism , Protein Structure, Tertiary , Proto-Oncogene Proteins c-jun/metabolism , Rats , Sequence Alignment , Sequence Homology, Amino Acid , Structure-Activity Relationship , Transcriptional Activation , Transfection , Two-Hybrid System Techniques
13.
Endocrine ; 13(1): 63-70, 2000 Aug.
Article in English | MEDLINE | ID: mdl-11051048

ABSTRACT

We studied the activity of mutants involving the aminoterminal extracellular, seven-transmembrane (7TM) and carboxy-terminal tail domains of the human Ca2+ receptor to gain insight into the functional interactions between these domains during receptor activation. Missense mutations of highly conserved residues, D190 and E297, in the extracellular domain (ECD), and a mutation within part of the proximal carboxyterminal tail, A877-880E, resulted in receptors with severely reduced response to Ca2+ despite adequate cell surface expression. Coexpression of either D190A or E297K mutants with A877-880E led to significant reconstitution of function. No such reconstitution occurred when D190A or E297K mutants were coexpressed with a truncation mutant possessing an intact amino-terminal extracellular and first transmembrane domain, despite evidence for heterodimerization and cell surface expression of the respective mutant receptors. In addition, no reconstitution of function was observed when D190A was coexpressed with a deletion Ca2+ receptor mutant lacking only a cysteine-rich region located in the ECD of the Ca2+ receptor (Ca-//-Ca). Moreover, coexpression of this Ca-//-Ca with A877-880E did not recover function. The results show that Ca2+ receptor extracellular and 7TM domains are discrete entities that can communicate within the context of a heterodimer composed of complementary mutant receptors. Two intact 7TM domains and two intact cysteine-rich regions appear to be required for such communication to occur. The results are discussed in the context of a speculative model of receptor structure and function.


Subject(s)
Calcium-Binding Proteins/chemistry , Calcium-Binding Proteins/physiology , Amino Acid Sequence , Biotinylation , Calcium/pharmacology , Calcium-Binding Proteins/genetics , Cell Line , Cell Membrane/metabolism , Dimerization , Gene Expression , Humans , Hydrolysis , Immunosorbent Techniques , Molecular Sequence Data , Mutagenesis, Site-Directed , Phosphatidylinositols/metabolism , Structure-Activity Relationship , Transfection
14.
Horm Res ; 53 Suppl 3: 17-22, 2000.
Article in English | MEDLINE | ID: mdl-10971097

ABSTRACT

G proteins couple receptors for many hormones to effectors that regulate second messenger metabolism. Several endocrine disorders have been shown to be caused by either loss- or gain-of-function mutations in G proteins or G protein-coupled receptors. In pseudohypoparathyroidism type Ia (PHP Ia), there are generalized hormone resistance (parathyroid hormone [PTH], thyroid-stimulating hormone, gonadotropins) and associated abnormal physical features, Albright hereditary osteodystrophy. Subjects with PHP Ib are normal in appearance and show renal resistance to PTH. In McCune-Albright syndrome (MAS), subjects show autonomous endocrine hyperfunction associated with fibrous dysplasia of bone and skin hyperpigmentation. Germline loss-of-function mutations have been identified in the G(s)-alpha gene in PHP Ia, and recent evidence suggests that the G(s)-alpha gene is paternally imprinted in a tissue-specific manner. Abnormal imprinting of the G(s)-alpha gene may be the cause of PHP Ib. MAS, in contrast, is caused by gain-of-function missense mutations of the G(s)-alpha gene.


Subject(s)
GTP-Binding Proteins/physiology , Signal Transduction/physiology , Animals , GTP-Binding Proteins/genetics , Humans , Pseudohypoparathyroidism/genetics , Pseudohypoparathyroidism/physiopathology , Signal Transduction/genetics
15.
Mamm Genome ; 11(6): 448-54, 2000 Jun.
Article in English | MEDLINE | ID: mdl-10818209

ABSTRACT

Mutations in the MEN1 gene lead to an autosomal dominant disorder, multiple endocrine neoplasia type 1 (MEN1), which is characterized by tumors of the parathyroid, entero-pancreatic neuroendocrine, and pituitary tissues. The protein encoded by MEN1, 610-amino acid menin, resides primarily in the nucleus and binds to the transcription factor JunD, resulting in the repression of JunD-induced transcription. We report here a detailed characterization of the zebrafish men1 gene and its full-length (2551 nt) transcript, encoding a 617-amino acid protein with 67% identity and 80% similarity to human menin. Of the 81 missense mutations and in-frame deletions reported in MEN1 patients, 72 occur in residues that are identical in zebrafish, suggesting the importance of the conserved regions. The zebrafish men1 gene maps 61 cM from the top of linkage group 7 (LG7), a region that appears to show conserved synteny to the MEN1 loci at human 11q13. A 2.7-kb men1 message is detected at all stages of zebrafish development analyzed, from one-cell embryos to adult fish. Whole-mount in situ hybridization showed ubiquitous distribution of men1 message in zebrafish embryos at cleavage, blastula, gastrula, and early segmentation stages, with relatively abundant expression in blood cell progenitors (24 h post fertilization) and mesenchymal tissues (48 h post fertilization) at later stages. Zebrafish menin binds both human and mouse JunD, and represses JunD-induced transcription, indicating that the JunD-binding ability of menin is evolutionarily conserved.


Subject(s)
DNA, Complementary/genetics , Neoplasm Proteins/genetics , Proto-Oncogene Proteins , Zebrafish/genetics , Amino Acid Sequence , Animals , Base Sequence , Blotting, Northern , Chromosome Mapping , DNA/chemistry , DNA/genetics , DNA, Complementary/chemistry , DNA, Complementary/isolation & purification , Embryo, Nonmammalian/metabolism , Embryonic Development , Exons , Gene Expression , Gene Expression Regulation, Developmental , Humans , In Situ Hybridization , Introns , Mice , Molecular Sequence Data , Neoplasm Proteins/metabolism , Protein Binding , Proto-Oncogene Proteins c-jun/genetics , Proto-Oncogene Proteins c-jun/metabolism , RNA/genetics , RNA/metabolism , Rats , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sequence Alignment , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Transcriptional Activation , Zebrafish/embryology
16.
J Biol Chem ; 275(21): 16382-9, 2000 May 26.
Article in English | MEDLINE | ID: mdl-10747888

ABSTRACT

The 612-residue extracellular domain of the human Ca(2+) receptor (hCaR) has been speculated to consist of a Venus's-flytrap domain (VFT) and a cysteine-rich domain. We studied the function of the hCaR Cys-rich domain by using mutagenesis and chimera approaches. A chimeric hCaR with the sequence from residues 540-601 replaced by the corresponding sequence from the Fugu CaR remained fully functional. Another chimeric hCaR with the same region of sequence replaced by the corresponding sequence from metabotropic glutamate receptor subtype 1 (mGluR1) still was activated by extracellular Ca(2+) ([Ca(2+)](o)), but its function was severely compromised. Chimeric receptors with the hCaR VFT and mGluR1 seven-transmembrane domain plus C-tail domain retained good response to [Ca(2+)](o) whether the Cys-rich domain was from hCaR or from mGluR1. Mutant hCaR with the Cys-rich domain deleted failed to respond to [Ca(2+)](o), although it was expressed at the cell surface and capable of dimerization. Our results indicate that the hCaR Cys-rich domain plays a critical role in signal transmission from VFT to seven-transmembrane domain. This domain tolerates a significant degree of amino acid substitution and may not be directly involved in the binding of [Ca(2+)](o).


Subject(s)
Receptors, Cell Surface/genetics , Recombinant Fusion Proteins/genetics , Amino Acid Sequence , Calcium/pharmacology , Calcium Signaling , Cell Line , Cysteine/chemistry , Humans , Immunoblotting , Membrane Proteins/genetics , Membrane Proteins/metabolism , Molecular Sequence Data , Mutagenesis, Site-Directed , Phosphatidylinositols/metabolism , Protein Binding , Receptors, Calcium-Sensing , Receptors, Cell Surface/chemistry , Receptors, Metabotropic Glutamate/genetics , Recombinant Fusion Proteins/metabolism , Signal Transduction , Transfection
17.
Genes Chromosomes Cancer ; 28(1): 58-65, 2000 May.
Article in English | MEDLINE | ID: mdl-10738303

ABSTRACT

Neuroendocrine tumors of the lung consist of a spectrum of neoplasms, including typical carcinoids, atypical carcinoids, large-cell neuroendocrine carcinomas (LCNEC), and small-cell lung carcinomas (SCLC). We previously reported frequent inactivation of the gene responsible for multiple endocrine neoplasia type 1 (MEN1) in both typical and atypical carcinoid tumors. In the present study, we extend the analysis of human NE lung tumors to include 9 primary SCLCs, 36 SCLC cell lines, and 13 primary LCNECs for MEN1 gene inactivation. In SCLC, loss of heterozygosity (LOH) at the MEN1 gene on chromosome band 11q13 was detected in one primary tumor and two cell lines. The coding sequence and splice junctions of the MEN1 gene were screened for mutations in all 44 tumors and cell lines, and no mutations were detected. Northern blot analysis of 13 SCLC cell lines showed the MEN1 transcript to be present and of normal size. In LCNECs, a somatic frameshift in the MEN1 gene (1226delC) was found in one of 13 tumors, representing the first mutation observed outside the spectrum of neoplasms associated with MEN1. Interestingly, neither a deletion nor a mutation was detected in the other allele, and wild-type mRNA sequence was expressed in the tumor, suggesting that the MEN1 gene was not inactivated by a conventional two-hit mechanism. The data support the hypothesis that SCLC and lung carcinoids develop via distinct molecular pathways; however, further investigation is necessary to determine the significance of the MEN1 gene mutation observed in a single case of LCNEC. Published 2000 Wiley-Liss, Inc.


Subject(s)
Carcinoma, Neuroendocrine/genetics , DNA, Neoplasm/analysis , Lung Neoplasms/genetics , Neoplasm Proteins/genetics , Proto-Oncogene Proteins , Carcinoma, Large Cell/genetics , Carcinoma, Small Cell/genetics , Chromosomes, Human, Pair 11/genetics , DNA Mutational Analysis/methods , Humans , Loss of Heterozygosity/genetics , Multiple Endocrine Neoplasia Type 1/genetics , Tumor Cells, Cultured
18.
J Bone Miner Res ; 15(1): 120-8, 2000 Jan.
Article in English | MEDLINE | ID: mdl-10646121

ABSTRACT

Activating missense mutations of the GNAS1 gene, encoding the alpha subunit of the stimulatory G protein (Gs), have been identified in patients with the McCune-Albright syndrome (MAS; characterized by polyostotic fibrous dysplasia, café au lait skin pigmentation, and endocrine disorders). Because fibrous dysplasia (FD) of bone also commonly occurs outside of the context of typical MAS, we asked whether the same mutations could be identified routinely in non-MAS FD lesions. We analyzed a series of 8 randomly obtained, consecutive cases of non-MAS FD and identified R201 mutations in the GNAS1 gene in all of them by sequencing cDNA generated by amplification of genomic DNA using a standard primer set and by using a novel, highly sensitive method that uses a protein nucleic acid (PNA) primer to block amplification of the normal allele. Histologic findings were not distinguishable from those observed in MAS-related FD and included subtle changes in cell shape and collagen texture putatively ascribed to excess endogenous cyclic adenosine monophosphate (cAMP). Osteomalacic changes (unmineralized osteoid) were prominent in lesional FD bone. In an in vivo transplantation assay, stromal cells isolated from FD failed to recapitulate a normal ossicle; instead, they generated a miniature replica of fibrous dysplasia. These data provide evidence that occurrence of GNAS1 mutations, previously noted in individual cases of FD, is a common and perhaps constant finding in non-MAS FD. These findings support the view that FD, MAS, and nonskeletal isolated endocrine lesions associated with GNAS1 mutations represent a spectrum of phenotypic expressions (likely reflecting different patterns of somatic mosaicism) of the same basic disorder. We conclude that mechanisms underlying the development of the FD lesions, and hopefully mechanism-targeted therapeutic approaches to be developed, must also be the same in MAS and non-MAS FD.


Subject(s)
Fibrous Dysplasia of Bone/genetics , GTP-Binding Protein alpha Subunits, Gs/genetics , Mutation , Osteomalacia/pathology , Stromal Cells/pathology , Adolescent , Adult , Amino Acid Sequence , Animals , Base Sequence , Cell Transplantation , Child , DNA , Female , Fibrous Dysplasia of Bone/pathology , Humans , Male , Mice , Polymerase Chain Reaction
19.
J Clin Endocrinol Metab ; 85(1): 116-23, 2000 Jan.
Article in English | MEDLINE | ID: mdl-10634374

ABSTRACT

Multiple endocrine neoplasia type 1 (MEN1) gene mutations are reported in some gastrinomas occurring in patients without MEN1 as well as in some other pancreatic endocrine tumors (PETs). In some inherited syndromes phenotype-genotype correlations exist for disease severity, location, or other manifestations. The purpose of the present study was to correlate mutations of the MEN1 gene in a large cohort of patients with sporadic gastrinomas to disease activity, tumor location, extent, and growth pattern. DNA was extracted from frozen gastrinomas from 51 patients and screened by dideoxyfinger-printing (ddF) for abnormalities in the 9 coding exons and adjacent splice junctions of the MEN1 gene. Tumor DNA exhibiting abnormal ddF patterns was sequenced for mutations. The findings were correlated with clinical manifestations of the disease, primary tumor site, disease extent, and tumor growth postoperatively. Tumor growth was determined by serial imaging studies. Sixteen different MEN1 gene mutations in the 51 sporadic gastrinomas (31%) were identified (11 truncating, 4 missense, and 1 in-frame deletion). Nine of the 16 mutations were located in exon 2 compared to 7 of 16 in the remaining 8 coding exons (P = 0.005 on a per nucleotide basis). Primary pancreatic or lymph node gastrinomas with a mutation had only exon 2 mutations, whereas duodenal tumors uncommonly harbored exon 2 mutations (P = 0.011). Similarly, small primary tumors (<1 cm) more frequently contained a nonexon 2 mutation (P = 0.02). There was no difference between patients with or without a mutation with respect to clinical characteristics, primary tumor site, disease extent, or proportion of patients disease free after surgery. Postoperative tumor growth tended to be more aggressive in patients with a mutation (P = 0.09). No correlation in the rate of disease-free status or postoperative tumor growth in patients with active disease to the location of the mutation was seen. These results demonstrate that the MEN1 gene is mutated in 31% of sporadic gastrinomas, and mutations are clustered between amino acids 66-166, which differs from patients with familial MEN1, in whom mutations occur throughout the gene. The presence of an MEN1 gene mutation does not correlate with clinical characteristics of patients with gastrinomas, gastrinoma extent, or growth pattern; however, the location of the mutation differed with gastrinoma location. These data suggest that mutations in the MEN1 gene are important in a proportion of sporadic gastrinomas, but the presence or absence of these mutations will not identify the clinically important subgroups with different growth patterns.


Subject(s)
Gastrinoma/genetics , Multiple Endocrine Neoplasia Type 1/genetics , Mutation/genetics , Pancreatic Neoplasms/genetics , Base Sequence , DNA, Neoplasm/analysis , DNA, Neoplasm/genetics , Exons/genetics , Female , Genotype , Humans , Male , Middle Aged , Molecular Sequence Data , Phenotype , Polymerase Chain Reaction , Zollinger-Ellison Syndrome/genetics
20.
Surgery ; 126(6): 1145-50; discussion 1150-1, 1999 Dec.
Article in English | MEDLINE | ID: mdl-10598200

ABSTRACT

BACKGROUND: Rapid intraoperative parathyroid hormone (RI-PTH) assay is used to guide adequacy of resection during operation for hyperparathyroidism. We compared the RI-PTH assay (15 minutes) with a standard PTH assay, determined whether the PTH half-life varied between patients, and constructed a kinetic analysis of the RI-PTH data. METHODS: Forty-five patients with hyperparathyroidism had blood sampled at baseline and at times after parathyroid resection. Intact PTH was determined using RI-PTH and a standard assay. Values were fitted to an exponential decay curve using the baseline and the follow-up time points. PTH half-life and the new postexcision baseline value were calculated from the decay curve. RESULTS: The RI-PTH assay and the standard PTH assay correlated well. Average PTH half-life was 1.68 +/- 0.94 minutes (0.42 to 3.81 minutes). A kinetic analysis yielded a formula for the generation of a PTH decay curve. Using a 50% reduction in RI-PTH at 5 minutes as the criterion for adequate resection, 2 patients were incorrectly classified as not being cured. These patients were correctly classified using the kinetic analysis. CONCLUSIONS: PTH half-life can vary substantially. A kinetic analysis may be more accurate in assessing adequacy of resection. This method allows the surgeon to interpret RI-PTH data independent of the timing of samples.


Subject(s)
Hyperparathyroidism/blood , Hyperparathyroidism/surgery , Parathyroid Hormone/blood , Parathyroidectomy , Adult , Algorithms , Chemistry, Clinical/methods , Chemistry, Clinical/standards , False Negative Reactions , False Positive Reactions , Female , Humans , Immunoassay , Intraoperative Period , Kinetics , Luminescent Measurements , Male , Middle Aged , Reference Values , Time Factors
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