RESUMEN
Homozygous and compound heterozygous mutations in SLC34A3, the gene encoding the sodium-dependent co-transporter NaPi-IIc, cause hereditary hypophosphatemic rickets with hypercalciuria (HHRH), a disorder characterized by renal phosphate-wasting resulting in hypophosphatemia, elevated 1,25(OH)(2) vitamin D levels, hypercalciuria, rickets/osteomalacia, and frequently kidney stones or nephrocalcinosis. Similar albeit less severe biochemical changes are also observed in heterozygous carriers, which are furthermore indistinguishable from those encountered in idiopathic hypercalciuria (IH). We now searched for SLC34A3 mutations (exons and introns) in two previously not reported HHRH kindreds, which resulted in the identification of three novel mutations. The affected members of kindred A were compound heterozygous for two different mutations, c.1046_47del and the intronic mutation c.560+23_561-42del, while the index case in kindred B was homozygous for the nonsense SLC34A3 mutation c.1764C>G (p.Y588X). The patient in kindred C was diagnosed with IH because of bilateral medullary nephrocalcinosis, suppressed PTH levels, and hypercalciuria; she was found to have a novel heterozygous c.1571_1880del mutation. The HHRH patients in kindred A were treated for up to 7years with oral phosphate, which led to reversal of hypophosphatemia, hypercalciuria, and prevention or healing of the mild bone abnormalities. PTH levels were normal throughout the observation period, while 1,25(OH)(2) vitamin D levels remained elevated and may thus be helpful for assessing treatment efficacy and patient compliance in HHRH.
Asunto(s)
Raquitismo Hipofosfatémico Familiar/complicaciones , Raquitismo Hipofosfatémico Familiar/genética , Hipercalciuria/complicaciones , Hipercalciuria/genética , Mutación/genética , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIc/genética , Niño , Preescolar , Análisis Mutacional de ADN , Familia , Femenino , Estudios de Seguimiento , Humanos , Masculino , Linaje , Factores de TiempoRESUMEN
Hereditary hypophosphatemic rickets with hypercalciuria (HHRH) is caused by mutations in SLC34A3, the gene encoding the renal sodium-phosphate co-transporter NaPi-IIc. Despite increased urinary calcium excretion, HHRH is typically not associated with kidney stones prior to treatment. However, here we describe two sisters, who displayed nephrolithiasis or nephrocalcinosis upon presentation. The index patient, II-4, presented with short stature, bone pain, and knee X-rays suggestive of mild rickets at age 8.5 years. Laboratory evaluation showed hypophosphatemia, elevated 1,25(OH) (2) vitamin D levels, and hypercalciuria, later also developing vitamin D deficiency. Her sister, II-6, had a low normal serum phosphorous level, biochemically vitamin D deficiency and no evidence for osteomalacia, but had undergone left nephro-ureterectomy at age 17 because of ureteral stricture secondary to renal calculi. Nucleotide sequence analysis of DNA from II-4 and II-6 revealed a homozygous missense mutation c.586G>A (p.G196R) in SLC34A3/NaPi-IIc. Ultrasonographic examinations prior to treatment showed grade I nephrocalcinosis for II-4, while II-6 had grade I-II nephrocalcinosis in her remaining kidney. Four siblings and the mother were heterozygous carriers of the mutation, but showed no biochemical abnormalities. With oral phosphate supplements, hypophosphatemia and hypercalciuria improved in both homozygous individuals. Renal calcifications that are presumably due to increased urinary calcium excretion can be the presenting finding in homozygous carriers of G196R in SLC34A3/NaPi-IIc, and some or all laboratory features of HHRH may be masked by vitamin D deficiency.
Asunto(s)
Calcinosis/metabolismo , Hipercalciuria/metabolismo , Hipofosfatemia/metabolismo , Enfermedades Renales/metabolismo , Raquitismo/metabolismo , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIc/metabolismo , Adolescente , Adulto , Calcinosis/complicaciones , Calcinosis/diagnóstico por imagen , Calcinosis/genética , Niño , Susceptibilidad a Enfermedades , Femenino , Humanos , Hipercalciuria/complicaciones , Hipercalciuria/diagnóstico por imagen , Hipercalciuria/genética , Hipofosfatemia/complicaciones , Hipofosfatemia/diagnóstico por imagen , Hipofosfatemia/genética , Enfermedades Renales/complicaciones , Enfermedades Renales/diagnóstico por imagen , Enfermedades Renales/genética , Masculino , Persona de Mediana Edad , Mutación/genética , Linaje , Raquitismo/complicaciones , Raquitismo/diagnóstico por imagen , Raquitismo/genética , Proteínas Cotransportadoras de Sodio-Fosfato de Tipo IIc/genética , UltrasonografíaRESUMEN
This report describes a 60-yr-old white male presenting with decompensated liver cirrhosis. He had a history of Addison's disease for 36 yr, primary hypothyroidism for 5 yr, and moderate alcohol consumption. His laboratory studies and a liver biopsy supported the diagnosis of autoimmune hepatitis. Furthermore, he was found to be heterozygous for the piZ allele of the alpha1-antitrypsin gene with normal serum alpha1-antitrypsin levels and absence of pulmonary affection. Mucosal biopsies revealed moderately severe atrophic gastritis; however, signs of pernicious anemia were missing. An association of autoimmune hepatitis with endocrine disorders and atrophic gastritis has been described. Long term hydrocortisone therapy for his adrenal insufficiency may have prevented a faster course of the liver disease, whereas the heterozygous alpha1-antitrypsin deficiency and moderate alcohol consumption constituted additional risk factors ultimately leading to the development of cirrhosis.
Asunto(s)
Enfermedad de Addison/complicaciones , Insuficiencia Pancreática Exocrina/complicaciones , Gastritis Atrófica/complicaciones , Hepatitis Autoinmune/complicaciones , Heterocigoto , Cirrosis Hepática/etiología , Tiroiditis/complicaciones , Deficiencia de alfa 1-Antitripsina/complicaciones , Enfermedad de Addison/patología , Atrofia/patología , Insuficiencia Pancreática Exocrina/patología , Gastritis Atrófica/patología , Hepatitis Autoinmune/patología , Humanos , Cirrosis Hepática/patología , Masculino , Persona de Mediana Edad , Tiroiditis/patología , Deficiencia de alfa 1-Antitripsina/patologíaRESUMEN
The 23-year-old Caucasian male propositus presented with symptomatic hypercalcemia, hypophosphatemia and normocalciuria for 2 months. His 29-year-old brother had undergone an operation for recurrent parathyroid adenoma at age 26 and 28. No other member of the family was affected. His father and mother were second-degree relatives. Laboratory studies showed primary hyperparathyroidism (pHPT), while the remaining endocrine studies and genetic testing for multiple endocrine neoplasia 1 and 2A were normal. Technetium-cardiolite scintigraphy and ultrasound scans revealed a parathyroid mass at the left lower neck. Apart from bilateral hearing loss due to gentamicin treatment as a pre-term child, the patient was in of good health. Signs or symptoms of other endocrinopathies were absent. The patient was referred for parathyroidectomy with subsequent autotransplantation of the remaining glands into his sternocleidomastoid muscle. Histological examination revealed an adenoma with oncocytic differentiation, similar to that seen in his brother. The disease may follow a recessive mode of inheritance or may be due to a dominant germ-cell mutation in one of the parents. The presented case may ultimately help in elucidating the molecular genetic basis of this rare form of pHPT.
Asunto(s)
Adenoma/genética , Consanguinidad , Neoplasias de las Paratiroides/genética , Adenoma/diagnóstico , Adenoma/cirugía , Adulto , Calcio/orina , Humanos , Hipercalcemia , Hiperparatiroidismo/genética , Hiperparatiroidismo/patología , Hipofosfatemia , Masculino , Neoplasia Endocrina Múltiple Tipo 1 , Neoplasia Endocrina Múltiple Tipo 2a , Glándulas Paratiroides/patología , Neoplasias de las Paratiroides/diagnóstico , Neoplasias de las Paratiroides/cirugía , Paratiroidectomía , Linaje , Tecnecio Tc 99m Sestamibi , UltrasonografíaRESUMEN
The wingless- and int-related proteins (Wnts) have an important role during embryonic development and limb patterning. To investigate their function during chondrocyte differentiation, we used NIH3T3 cells producing seven members of the Wnt family and secreted frizzled-related protein (sFRP-2) for co-culture experiments with the rat chondrogenic cell line pColl(II)-EGFP-5. Pilot experiments showed a negative effect of Wnt-7a on the proliferation of three rodent chondrogenic cell lines, RCJ3.1(C5.18), CFK-2, and C1. To establish a reporter system for chondrogenic differentiation we then produced a stably transfected chondrogenic cell line based on RCJ3.1(C5.18) for further experiments, which expresses green fluorescence protein (EGFP) under the collagen type II promoter (pColl(II)-EGFP-5). This cell line permits convenient observation of green fluorescence as a marker for differentiation in life cultures. The colony size of this cell line in agarose suspension cultures was reduced to 20-40% of control, when exposed to Wnt-1, 3a, 4, 7a, and 7b for 14 days. Similarly, reporter gene expression and the synthesis of cartilage-specific proteoglycans were inhibited by this group of Wnts. In contrast, pColl(II)-EGFP-5 cells exposed to Wnt-5a and Wnt-11 reached 140% of control, and reporter gene expression and proteoglycan synthesis were stimulated. The effects of Wnt-7a and Wnt-5a were additive in pColl(II)-EGFP-5 cells and some but not all Wnt effects were antagonized by the inhibition of proteoglycan sulfation with chlorate, by sFRP-2, which may modulate Wnt receptor binding, or by inhibitors of protein kinase C. These results suggest two functional Wnt subclasses that differentially regulate proliferation and chondrogenic differentiation in vitro which may have implications for cartilage differentiation in vivo. Since some, but not all Wnt effects were sensitive to inhibitors of proteoglycan synthesis or protein kinase C, multiple modes of signal transduction may be involved.
Asunto(s)
Condrocitos/efectos de los fármacos , Glicoproteínas/farmacología , Proteínas Proto-Oncogénicas/farmacología , Proteínas de Pez Cebra , Células 3T3 , Animales , Antígenos CD/farmacología , Diferenciación Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Línea Celular , Cloratos/farmacología , Condrocitos/citología , Técnicas de Cocultivo , Colágeno/genética , Expresión Génica , Genes Reporteros , Integrina alfa3 , Integrinas , Ratones , Plásmidos , Proteínas Proto-Oncogénicas/biosíntesis , Proteínas Proto-Oncogénicas/genética , Ratas , Cráneo , Transfección , Proteínas Wnt , Proteína Wnt1RESUMEN
Core binding factor A1 (CBFA1/RUNX2) is a runt-like transcription factor essential for osteoblast differentiation. Haplotype insufficiency causes cleidocranial dysplasia (CCD), a syndrome featuring supernumerary tooth buds, delayed tooth eruption, patent fontanels, Wormian bones, short stature, dysplasia of the clavicles, growth retardation and hypoplasia of the distal phalanges. We identified novel CBFAI/RUNX2 mutations after PCR and direct sequencing of patient leukocyte DNA. In family 1 mother and son are affected by CCD. Both carry the missense mutation R190W (CGG > TGG). This nucleotide change introduced a BsmI restriction site, which was used to independently confirm the mutation. It was absent in healthy members of the family. Family 2, in which father and daughter are affected by CCD, shows a deletion of nucleotide C821. This deletion causes a frameshift mutation with premature stop after the insertion of 18 aberrant amino acids. Healthy family members did not have this mutation. The clavicular dysplasia was more pronounced with the R19OW mutation, while the bone density was markedly reduced in individuals with either mutation, suggesting a previously underemphasized increased risk for osteoporosis in CCD.
Asunto(s)
Displasia Cleidocraneal/genética , Proteínas de Unión al ADN/genética , Mutación Missense/genética , Proteínas Proto-Oncogénicas , Factores de Transcripción/genética , Adolescente , Adulto , Densidad Ósea/genética , Subunidad alfa 2 del Factor de Unión al Sitio Principal , Densitometría , Exones/genética , Femenino , Humanos , Masculino , Persona de Mediana Edad , Osteoporosis/genética , Linaje , Factores de RiesgoRESUMEN
The clonal rat calvaria cell line RCJ3.1C5.18 (RCJ) undergoes chondrogenic differentiation after long-term culture post confluence. To allow flexible genetic manipulation, a tetracycline-regulated gene expression system was established in this cell line. Treatment with tetracycline in operational doses does not affect the differentiation of RCJ cells with respect to the markers tested. After stable transfection with pUHD15.1 containing the tetracycline transactivator (tTA) in the presence of pTK-hyg for hygromycin selection, 28 clones were isolated and characterized for alcian blue staining of cartilage-specific proteoglycans and for collagen type II expression. Clone R-tTA-24 was selected on the basis of phenotype and displayed tetracycline-dependent down-regulation of luciferase activity (tet-OFF system) by two orders of magnitude (57-149-fold) after stable transfection with the reporter gene pBI-EGFP/luc. The novel, chondrogenic cell line R-tTA-24 may be stably transfected with various genes of interest for tetracycline-regulated gene expression using neomycin selection and may be a valuable tool to study the process of chondrogenic differentiation in vitro.
Asunto(s)
Condrogénesis , Regulación de la Expresión Génica/efectos de los fármacos , Cráneo/citología , Tetraciclina/farmacología , Azul Alcián , Animales , Diferenciación Celular , Línea Celular , Células Clonales , Colorantes , Genes Reporteros , Ingeniería Genética , Luciferasas/genética , Ingeniería de Proteínas , Ratas , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Represoras/genética , Transactivadores/genética , TransfecciónRESUMEN
Genomic and cDNA clones encoding portions of a putative catfish parathyroid hormone (PTH) 2 receptor (PTH2R) led to the isolation of a cDNA encoding a full-length zebrafish PTH2R (zPTH2R). The zPTH2R shared 63 and 60% amino acid sequence identity with human and rat PTH2Rs, respectively, 47-52% identity with mammalian and frog PTH/PTHrP receptors (PTH1R), and less than 37% with other members of this family of G protein-coupled receptors. COS-7 cells expressing zPTH2R(43), a 5' splice variant that lacked 17 amino acids in the amino-terminal extracellular domain, showed cAMP accumulation when challenged with [Tyr(34)]hPTH(1-34)-amide (hPTH) (EC(50), 1.64 +/- 0. 95 nM) and [Ile(5),Trp(23),Tyr(36)]hPTHrP-(1-36)-amide ([Ile(5), Trp(23)]hPTHrP) (EC(50), 46.8 +/- 12.1 nM) but not when stimulated with [Tyr(36)]hPTHrP-(1-36)-amide (hPTHrP), [Trp(23), Tyr(36)]hPTHrP-(1-36)-amide ([Trp(23)]hPTHrP), or [Ala(29),Glu(30), Ala(34),Glu(35),Tyr(36)]fugufish PTHrP-(1-36)amide (fuguPTHrP). FuguPTHrP also failed to activate the human PTH2R but had similar efficiency and efficacy as hPTH and hPTHrP when tested with cells expressing the human PTH1R. Agonist-dependent activation of zPTH2R was less efficient than that of zPTH2R(43), and both receptor variants showed no cAMP accumulation when stimulated with either secretin, growth hormone-releasing hormone, or calcitonin. The zPTH2R thus has ligand specificity similar to that of the human homolog, which raises the possibility that a PTH-like molecule exists in zebrafish, species which lack parathyroid glands.
Asunto(s)
Evolución Biológica , Proteínas de Unión al GTP/metabolismo , Hormona Paratiroidea/metabolismo , Proteínas/metabolismo , Receptores de Hormona Paratiroidea/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Southern Blotting , Clonación Molecular , Sondas de ADN , ADN Complementario , Humanos , Ictaluridae , Datos de Secuencia Molecular , Hormona Paratiroidea/genética , Proteína Relacionada con la Hormona Paratiroidea , Proteínas/genética , Empalme del ARN , Ratas , Receptor de Hormona Paratiroídea Tipo 2 , Homología de Secuencia de Aminoácido , Pez CebraRESUMEN
Cell-cell adhesion, as mediated by the cadherin-catenin system, is a prerequisite for normal cell function and the preservation of tissue integrity. With recent progress in our understanding, beta-catenin as a component of a complex signal transduction pathway may serve as a common switch in central processes that regulate cellular differentiation and growth. The function of the cadherin-catenin system in cell adhesion as well as in intracellular signaling, appears to be subjected to multifactorial control by a variety of different mechanisms, and data on a hormonal control of these signaling pathways, even though scarce to date, suggest an important regulatory influence in many cellular systems. Loss of E-cadherin-catenin function was described in many tumors along with an increased invasiveness and a decreased prognosis of many carcinomas, including tumors of endocrine glands and their target systems, and a causal role of this loss-of-function in the multifactorial process of tumorigenesis was recently proven in genetic mouse models. Modification of E-caderin-catenin function in endocrine and nonendocrine tumors may involve germline and somatic gene mutations, epigenetic mechanisms such as gene silencing due to promotor-hypermethylation, and posttranscriptional events, likely to be involved in many endocrine tissues and their target organs. Such events may converge on nuclear activation of oncogenes such as c-myc by the beta-catenin/TCF4 complex. The expression and functional status of the components of the cadherin-catenin system may serve as prognostic markers for endocrine and nonendocrine tumors. The frequent involvement of functional dysregulation in many tumors raises hopes that better definition of the regulation of all components of the cadherin-catenin system and their response to extracellular modulators may eventually lead to new therapeutic approaches for these tumors and help to prevent, more specifically, growth, invasion, and metastasis of these carcinomas.
Asunto(s)
Cadherinas/fisiología , Proteínas del Citoesqueleto/fisiología , Glándulas Endocrinas/crecimiento & desarrollo , Transactivadores , Animales , Cadherinas/química , Cadherinas/genética , Adhesión Celular , Diferenciación Celular , Proteínas del Citoesqueleto/química , Proteínas del Citoesqueleto/genética , Neoplasias de las Glándulas Endocrinas/genética , Neoplasias de las Glándulas Endocrinas/patología , Glándulas Endocrinas/patología , Humanos , Ratones , Mutación , Pronóstico , Transducción de Señal , Relación Estructura-Actividad , beta CateninaRESUMEN
Complementary DNAs encoding two nonallelic PTH/PTH-related peptide (PTHrP) receptor (PPR) isoforms, xPPR-A and xPPR-B, were isolated from a kidney complementary DNA library of the tetraploid African clawed frog Xenopus laevis. Both isoforms differ in their coding region by 19 amino acids, and lack the region corresponding to the mammalian exon E2. When expressed in mammalian COS-7 cells, both receptor isoforms bound radiolabeled PTH-(1-34) and PTHrP-(1-36) analogs with comparable affinity, and both unlabeled peptides equivalently stimulated the accumulation of cAMP. xPPR-A also mediated inositol phosphate turnover in COS cells and stimulated channel-mediated current changes in voltage clamp experiments after injection into oocytes. Using ribonuclease protection analysis, significant xPPR-A messenger RNA expression was first detected in neurula stage embryos, which subsequently increased approximately 30-fold during tadpole development. Expression reached a maximum at the metamorphotic climax, when isoform B also became detectable at significant levels, and subsequently declined in postmetamorphotic froglets. In the adult frog, xPPR-A was prominently expressed in lung, brain, small bowel, and skin, whereas isoform B was highest in lung, heart, and brain. Using an xPPR-A antisense riboprobe for in situ hybridization, expression appeared during metamorphosis at all sites of chondrogenesis, specifically in the maturing zone of the amphibian growth plate. xPPR-A expression was also seen in a subpopulation of mononuclear cells, possibly representing osteoblasts that line perichondral bone and diaphyseal bone trabeculae. Our findings suggest that xPPRs serve a prominent role in amphibian skeletal development and possibly other functions during embryonal and early larval development.
Asunto(s)
Envejecimiento/metabolismo , Receptores de Hormona Paratiroidea/metabolismo , Xenopus laevis/metabolismo , Alelos , Secuencia de Aminoácidos , Animales , Células COS , Embrión no Mamífero/fisiología , Hibridación in Situ , Isomerismo , Datos de Secuencia Molecular , Receptor de Hormona Paratiroídea Tipo 1 , Receptores de Hormona Paratiroidea/genética , Receptores de Hormona Paratiroidea/fisiología , Homología de Secuencia de Aminoácido , Xenopus laevis/embriología , Xenopus laevis/crecimiento & desarrolloRESUMEN
The parathyroid hormone (PTH)-2 receptor displays strong ligand selectivity in that it responds fully to PTH but not at all to PTH-related peptide (PTHrP). In contrast, the PTH-1 receptor (PTH/PTHrP receptor) responds fully to both ligands. Previously it was shown that two divergent residues in PTH and PTHrP account for PTH-2 receptor selectivity; position 23 (Trp in PTH and Phe in PTHrP) determines binding selectivity and position 5 (Ile in PTH and His in PTHrP) determines signaling selectivity. To identify sites in the PTH-2 receptor involved in discriminating between His5 and Ile5, we constructed PTH-2 receptor/PTH-1 receptor chimeras, expressed them in COS-7 cells, and tested for cAMP responsiveness to [Trp23] PTHrP-(1-36), and to the nondiscriminating peptide [Ile5, Trp23]PTHrP-(1-36) (the Phe23 --> Trp modification enabled high affinity binding of each ligand to the PTH-2 receptor). The chimeras revealed that the membrane-spanning/loop region of the receptor determined His5/Ile5 signaling selectivity. Subsequent analysis of smaller cassette substitutions and then individual point mutations led to the identification of two single residues that function as major determinants of residue 5 signaling selectivity. These residues, Ile244 at the extracellular end of transmembrane helix 3, and Tyr318 at the COOH-terminal portion of extracellular loop 2, are replaced by Leu and Ile in the PTH-1 receptor, respectively. The results thus indicate a functional interaction between two residues in the core region of the PTH-2 receptor and residue 5 of the ligand.
Asunto(s)
Hormona Paratiroidea/metabolismo , Fragmentos de Péptidos/metabolismo , Proteínas/metabolismo , Receptores de Hormona Paratiroidea/metabolismo , Transducción de Señal , Secuencia de Aminoácidos , Animales , Células COS , Membrana Celular/metabolismo , AMP Cíclico/metabolismo , Histidina/metabolismo , Humanos , Isoleucina/metabolismo , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Proteína Relacionada con la Hormona Paratiroidea , Fragmentos de Péptidos/química , Proteínas/química , Receptor de Hormona Paratiroídea Tipo 2 , Receptores de Hormona Paratiroidea/química , Receptores de Hormona Paratiroidea/genética , Homología de Secuencia de AminoácidoRESUMEN
The Vitamin D receptor (VDR), a member of the nuclear receptor superfamily, mediates the effects of 1,25-dihydroxyvitamin D3 on mineral ion homeostasis. Although the mammalian and avian VDRs have been extensively studied, little is known about the VDR in lower vertebrate species. To address this, we have isolated the Xenopus laevis VDR (xVDR) complementary DNA. Overall, the xVDR shares 79%, 73%, 73%, and 75% identity at the amino acid level with the chicken, mouse, rat, and human VDRs, respectively. The amino acid residues and subdomains important for DNA binding, hormone binding, dimerization, and transactivation are mostly conserved among all VDR species. The xVDR polypeptide can heterodimerize with the mouse retinoid X receptor alpha, bind to the rat osteocalcin vitamin D response element (VDRE), and induce vitamin D-dependent transactivation in transfected mammalian cells. Northern analysis reveals two xVDR messenger RNA species of 2.2 kb and 1.8 kb in stage 60 Xenopus tissues. In the adult, xVDR expression is detected in many tissues including kidney, intestine, skin, and bone. During Xenopus development, xVDR messenger RNA first appears at developmental stage 13 (pre-neurulation), increasing to maximum at stages 57-61 (metamorphosis). Our data demonstrate that, in Xenopus, VDR expression is developmentally regulated and that the vitamin D endocrine system is highly conserved during evolution.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Intestino Delgado/metabolismo , Receptores de Calcitriol/biosíntesis , Envejecimiento , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Huesos/metabolismo , Pollos , Clonación Molecular , Dimerización , Embrión no Mamífero/fisiología , Femenino , Humanos , Riñón/metabolismo , Ratones , Datos de Secuencia Molecular , Especificidad de Órganos , Reacción en Cadena de la Polimerasa , Ratas , Receptores de Calcitriol/química , Receptores de Calcitriol/metabolismo , Receptores de Ácido Retinoico/química , Receptores de Ácido Retinoico/metabolismo , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Receptor alfa de Ácido Retinoico , Homología de Secuencia de Aminoácido , Piel/metabolismo , Especificidad de la Especie , Xenopus laevisRESUMEN
Calcitonin (CT) and parathyroid hormone (PTH), whose receptors belong to the same family of G protein-coupled receptors, share no amino acid sequence homology and selectively activate either CT or PTH receptors. We now show, however, that reciprocal hybrid ligands (CT/PTH and PTH/CT), which do not activate the "wild-type" receptors, activate PTH/CT and CT/PTH receptor chimeras, respectively. Our findings indicate that PTH and CT share a similar architecture with at least two functional, receptor-specific domains. These domains are sufficiently independent to permit synthetic hybrid ligands to efficiently activate appropriate receptor chimeras. Therefore, both ligands follow, despite their very different primary sequences, a common pattern of ligand-receptor interaction.
Asunto(s)
Calcitonina/metabolismo , Hormona Paratiroidea/metabolismo , Receptores de Calcitonina/metabolismo , Receptores de Hormona Paratiroidea/metabolismo , Animales , Células COS , Ratas , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
Pseudohypoparathyroidism type Ib (PHP-Ib) is thought to be caused by a PTH/PTH-related peptide (PTHrP) receptor defect. To search for receptor mutations in genomic DNA from 17 PHP-Ib patients, three recently isolated human genomic DNA clones were further characterized by restriction enzyme mapping and nucleotide sequencing across intron/exon borders. Regions including all 14 coding exons and their splice junctions were amplified by polymerase chain reaction, and the products were analyzed by either temperature gradient gel electrophoresis or direct nucleotide sequencing. Silent polymorphisms were identified in exons G (1 of 17), M4 (1 of 17), and M7 (15 of 17). Two base changes were found in introns, 1 at the splice-donor site of the intron between exons E2 and E3 (1 of 17) and the other between exons G and M1 (2 of 17). Total ribonucleic acid from COS-7 cells expressing minigenes with or without the base change between exons E2 and E3 showed no difference by either Northern blot analysis or reverse transcriptase-polymerase chain reaction. Radioligand binding was indistinguishable for both transiently expressed constructs. A missense mutation (E546 to K546) in the receptor's cytoplasmic tail (3 of 17) was also found in 1 of 60 healthy individuals, and PTH/PTHrP receptors with this mutation were functionally indistinguishable from wild-type receptors. PHP-Ib thus appears to be rarely, if ever, caused by mutations in the coding exons of the PTH/PTHrP receptor gene.
Asunto(s)
Exones , Genes , Mutación , Proteínas/genética , Seudohipoparatiroidismo/genética , Receptores de Hormona Paratiroidea/genética , Adolescente , Adulto , Secuencia de Aminoácidos , Secuencia de Bases , Niño , Preescolar , Femenino , Humanos , Masculino , Sondas Moleculares/genética , Datos de Secuencia Molecular , Proteína Relacionada con la Hormona Paratiroidea , Polimorfismo Genético , Seudohipoparatiroidismo/clasificaciónRESUMEN
The pulsatile but not the continuous application of parathyroid hormone (PTH) increase bone mass in vivo. To study the effects of intermittent hormonal administration on bone-derived cells in vitro, we established a perifusion system using the human osteosarcoma cell line SaOS-2. Cells were grown in suspension culture attached to collagen beads and were then loaded into a 3 ml syringe for perifusion experiments. The application of PTH(1-34) resulted in a dose-dependent increase of cAMP release by SaOS-2 cells into the effluent medium. Cyclic AMP accumulation was rapidly desensitized by approx. 80% after 30 min of continuous exposure to PTH(1-34) (10(-7) M), while cells remained responsive to forskolin. The recovery of PTH responsiveness required at least 2 h of hormone-free perifusion. Desensitization in the experimental setting was dose-dependent (EC50 = 1 x 10(-10) M PTH(1-34)). Neither 8Br-cAMP (2 x 10(-4) M) nor PMA(1 x 10(-7) M) had an effect on the PTH(1-34)-induced desensitization of the adenylate cyclase. Radioreceptor assays showed that [125I]-[Tyr36]hPTHrP(1-36)amide binding to SaOS-2 cells was decreased by 60-70% by PTH(1-34) (1 x 10(-6) M), bPTH(1-84) (1.8 x 10(-6) M) and bPTH(3-34) (2 x 10(-6) M), whereas 8Br-cAMP (2 x 10(-4) M) had no effect on radioligand binding. PMA (1 x 10(-7) M) appeared to slightly increase [125I]PTHrP binding. This observation is consistent with a small (3-fold) increase in PTH-induced cAMP release as a result of PMA pre-treatment. Receptor internalization was dose-dependent EC50 = 3 x 10(-7) M PTH(1-34)). The maximal effect occurred after 10-30 min and was largely reversible within 2 h. Monensin (3 x 10(-5) M) inhibited the recovery from receptor internalization. We conclude that a perifusion system using SaOS-2 cells is a suitable model to study the effect of discontinuous application of PTH on cAMP release. A rapid, homologous desensitization of PTH(1-34) stimulated cAMP accumulation has been observed that does not appear to involve protein kinase A or C.
Asunto(s)
Adenilil Ciclasas/metabolismo , Osteosarcoma/enzimología , Hormona Paratiroidea/farmacología , Receptores de Hormona Paratiroidea/fisiología , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , AMP Cíclico/análisis , Humanos , Perfusión/métodos , Receptores de Hormona Paratiroidea/efectos de los fármacos , Acetato de Tetradecanoilforbol/farmacología , Células Tumorales CultivadasRESUMEN
Mycoplasmataceae are known to express various proteins that are similar to those present in mammals. We report a strain of Mycoplasma hyorhinis isolated from opossum kidney cells with specific, high-affinity binding sites for human angiotensin II (Kd = 5.1 +/- 1.9 nM). In contrast, two strains of M. hominis revealed no specific binding. These binding sites resembled mammalian angiotensin II receptors by their high affinity and by their sensitivity to dithiothreitol. However, they are different from mammalian angiotensin II receptors in that they bind angiotensin I with high affinity (Kd = 1.6 +/- 0.29 nM) but not angiotensin III (Kd approximately 330,000 nM). [125I]-angiotensin II binding was not inhibited by angiotensin receptor subtype antagonists DuP 753 and CGP 42112A but it was sensitive to bacitracin and aprotinin. Positions Asp1, Ile5, His6 and Pro7 were essential for binding to M. hyorhinis as deletion of these residues led to a more than 10,000-fold decrease in affinity.