RESUMO
Joubert syndrome (JS) is rare recessive disorders characterized by the combination of hypoplasia/aplasia of the cerebellar vermis, thickened and elongated superior cerebellar peduncles, and a deep interpeduncular fossa which is defined by neuroimaging and is termed the 'molar tooth sign'. JS is genetically highly heterogeneous, with at least 29 disease genes being involved. To further understand the genetic causes of JS, we performed whole-exome sequencing in 24 newly recruited JS families. Together with six previously reported families, we identified causative mutations in 25 out of 30 (24 + 6) families (83.3%). We identified eight mutated genes in 27 (21 + 6) Japanese families, TMEM67 (7/27, 25.9%) and CEP290 (6/27, 22.2%) were the most commonly mutated. Interestingly, 9 of 12 CEP290 disease alleles were c.6012-12T>A (75.0%), an allele that has not been reported in non-Japanese populations. Therefore c.6012-12T>A is a common allele in the Japanese population. Importantly, one Japanese and one Omani families carried compound biallelic mutations in two distinct genes (TMEM67/RPGRIP1L and TMEM138/BBS1, respectively). BBS1 is the causative gene in Bardet-Biedl syndrome. These concomitant mutations led to severe and/or complex clinical features in the patients, suggesting combined effects of different mutant genes.
Assuntos
Anormalidades Múltiplas/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Antígenos de Neoplasias/genética , Cerebelo/anormalidades , Anormalidades do Olho/genética , Doenças Renais Císticas/genética , Proteínas de Membrana/genética , Proteínas Associadas aos Microtúbulos/genética , Proteínas de Neoplasias/genética , Retina/anormalidades , Anormalidades Múltiplas/diagnóstico por imagem , Anormalidades Múltiplas/epidemiologia , Anormalidades Múltiplas/fisiopatologia , Alelos , Proteínas de Ciclo Celular , Cerebelo/diagnóstico por imagem , Cerebelo/fisiopatologia , Proteínas do Citoesqueleto , Anormalidades do Olho/diagnóstico por imagem , Anormalidades do Olho/epidemiologia , Anormalidades do Olho/fisiopatologia , Feminino , Heterogeneidade Genética , Predisposição Genética para Doença , Humanos , Japão/epidemiologia , Doenças Renais Císticas/diagnóstico por imagem , Doenças Renais Císticas/epidemiologia , Doenças Renais Císticas/fisiopatologia , Masculino , Mutação , Omã/epidemiologia , Linhagem , Retina/diagnóstico por imagem , Retina/fisiopatologiaRESUMO
We have recently identified RFamide-related peptide (RFRP) gene that would encode three peptides (i.e., RFRP-1, -2, and -3) in human and bovine, and demonstrated that synthetic RFRP-1 and -3 act as specific agonists for a G protein-coupled receptor OT7T022. However, molecular characteristics and tissue distribution of endogenous RFRPs have not been determined yet. In this study, we prepared a monoclonal antibody for the C-terminal portion of rat RFRP-1. As this antibody could recognize a consensus sequence among the C-terminal portions of rat, human, and bovine RFRP-1, we purified endogenous RFRP-1 from bovine hypothalamus on the basis of immunoreactivity to the antibody. The purified bovine endogenous RFRP-1 was found to have 35-amino-acid length that corresponds to 37-amino-acid length in human and rat. We subsequently constructed a sandwich enzyme immunoassay using the monoclonal antibody and a polyclonal antibody for the N-terminal portion of rat RFRP-1, and analyzed the tissue distribution of endogenous RFRP-1 in rats. Significant levels of RFRP-1 were detected only in the central nervous system, and the highest concentration of RFRP-1 was detected in the hypothalamus. RFRP-1-positive nerve cells were detected in the rat hypothalamus by immunohistochemical analyses using the monoclonal antibody. In culture, RFRP-1 lowered cAMP production in Chinese hamster ovary cells expressing OT7T022 and it was abolished by pre-treatment with pertussis toxin, suggesting that OT7T022 couples G(i)/G(o) in the signal transduction pathway.
Assuntos
Hipotálamo/metabolismo , Neuropeptídeos/metabolismo , Receptores Acoplados a Proteínas G , Proteínas de Saccharomyces cerevisiae , Sequência de Aminoácidos , Animais , Encéfalo/metabolismo , Células CHO , Bovinos , Cromatografia em Gel , Cricetinae , Técnicas Imunoenzimáticas , Imuno-Histoquímica , Dados de Sequência Molecular , Neuropeptídeos/análise , Neuropeptídeos/isolamento & purificação , Ratos , Receptores de Superfície Celular/metabolismo , Alinhamento de SequênciaRESUMO
We analyzed the tissue distribution of apelin mRNA in rats by a quantitative reverse transcription-polymerase chain reaction and that of immunoreactive apelin (ir-apelin) by an enzyme immunoassay (EIA) using a monoclonal antibody. The expression levels of apelin mRNA and ir-apelin seemed to be consistent among tissues: they were highly expressed in the lung and mammary gland. By the combination of gel filtration and EIA, we found that the molecular forms of apelin differ among respective tissues: apelin molecules with sizes close to apelin-36 (long forms) were major components in the lung, testis, and uterus, but both long and short (whose sizes were close to [Assuntos
Proteínas de Transporte/análise
, Receptores Acoplados a Proteínas G
, Sequência de Aminoácidos
, Animais
, Apelina
, Receptores de Apelina
, Proteínas de Transporte/síntese química
, Proteínas de Transporte/metabolismo
, Cromatografia em Gel
, Feminino
, Técnicas Imunoenzimáticas
, Peptídeos e Proteínas de Sinalização Intercelular
, Pulmão/metabolismo
, Masculino
, Glândulas Mamárias Animais/metabolismo
, Dados de Sequência Molecular
, Peso Molecular
, RNA Mensageiro/análise
, Ratos
, Ratos Wistar
, Receptores de Dopamina D2
, Reação em Cadeia da Polimerase Via Transcriptase Reversa
, Testículo/metabolismo
, Útero/metabolismo
RESUMO
Only a few RFamide peptides have been identified in mammals, although they have been abundantly found in invertebrates. Here we report the identification of a human gene that encodes at least three RFamide-related peptides, hRFRP-1-3. Cells transfected with a seven-transmembrane-domain receptor, OT7T022, specifically respond to synthetic hRFRP-1 and hRFRP-3 but not to hRFRP-2. RFRP and OT7T022 mRNAs are expressed in particular regions of the rat hypothalamus, and intracerebroventricular administration of hRFRP-1 increases prolactin secretion in rats. Our results indicate that a variety of RFamide-related peptides may exist and function in mammals.
Assuntos
Neuropeptídeos/isolamento & purificação , Receptores de Neuropeptídeos/isolamento & purificação , Sequência de Aminoácidos , Animais , Bovinos , Clonagem Molecular , Humanos , Camundongos , Dados de Sequência Molecular , Neuropeptídeos/química , Neuropeptídeos/genética , Ratos , Receptores de Neuropeptídeos/genética , Homologia de Sequência de Aminoácidos , Especificidade da EspécieRESUMO
We validated the effect of prolactin-releasing peptide (PrRP) on prolactin (PRL) secretion from rat anterior pituitary cells in in vitro culture. We found that culture conditions considerably influenced the response of the anterior pituitary cells to PrRP. Longer culture term (4 d) was required to obtain better responses of the anterior pituitary cells to PrRP in comparison to thyrotropin-releasing hormone (TRH). Under the culture conditions employed here, PrRP was comparable to TRH in the potency promoting PRL secretion, and the action of PrRP was very specific for PRL secretion. The susceptibility of the anterior pituitary cells to PrRP varied in female rats depending on the process of reproduction: the cells prepared from lactating rats were the most sensitive to PrRP compared with those from random-cycle and pregnant rats. Because the expression levels of PrRP receptor mRNA in the pituitary varied during the reproductive process, we speculated that the susceptibility of the anterior pituitary cells would reflect cellular changes including the expression level of PrRP receptors. In addition, treatment with estrogen in vivo enhanced the susceptibility of the cultured anterior pituitary cells in male rats. Our results indicate that the susceptibility of the rat anterior pituitary cells to PrRP is regulated by physiological mechanisms.
Assuntos
Hormônios Hipotalâmicos/farmacologia , Neuropeptídeos/farmacologia , Adeno-Hipófise/efeitos dos fármacos , Prolactina/metabolismo , Animais , Células Cultivadas , Hormônio Liberador da Corticotropina/farmacologia , Feminino , Expressão Gênica , Hormônio Liberador de Gonadotropina/farmacologia , Hormônio Liberador de Hormônio do Crescimento/farmacologia , Cinética , Lactação , Masculino , Adeno-Hipófise/química , Adeno-Hipófise/metabolismo , Gravidez , Hormônio Liberador de Prolactina , RNA Mensageiro/análise , Ratos , Ratos Endogâmicos F344 , Receptores de Neuropeptídeos/genética , Receptores de Neuropeptídeos/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Hormônio Liberador de Tireotropina/farmacologia , Fatores de TempoRESUMO
Neuromedin U is a bioactive peptide isolated originally from the porcine spinal cord. We recently identified neuromedin U as the cognate ligand for the orphan G protein-coupled receptor FM-3. In this study, we isolated cDNA coding for a novel G protein-coupled receptor, TGR-1, which was highly homologous with FM-3. We found that neuromedin U specifically and clearly elevated the extracellular acidification rates, arachidonic acid metabolite release, and intracellular Ca(2+) mobilization in Chinese hamster ovary cells expressing TGR-1. Radiolabeled neuromedin U specifically bound with high affinity to membrane fractions prepared from these cells. These results show that TGR-1, like FM-3, is a specific and functional receptor for neuromedin U. We analyzed TGR-1 mRNA tissue distribution in rats using quantitative reverse transcription-polymerase chain reaction and found it to considerably differ from that of FM-3 mRNA. TGR-1 mRNA was primarily expressed in the uterus, suggesting that TGR-1 mediates the contractile activity of neuromedin U in this tissue. The identification of specific and functional receptor subtypes for neuromedin U will facilitate the study of their physiological roles and the search for their specific agonists and antagonists.
Assuntos
Proteínas de Membrana , Neuropeptídeos/metabolismo , Receptores de Neurotransmissores/genética , Receptores de Neurotransmissores/metabolismo , Transdução de Sinais , Sequência de Aminoácidos , Animais , Clonagem Molecular , Cricetinae , Proteínas de Ligação ao GTP/metabolismo , Humanos , Dados de Sequência Molecular , Ratos , Alinhamento de SequênciaRESUMO
Neuromedin U is a bioactive peptide first isolated from porcine spinal cord. In this paper, we demonstrate that neuromedin U is the cognate ligand for the orphan G protein-coupled receptor, FM-3, isolated originally as a homologue of neurotensin and growth hormone secretogogue receptors. Neuromedin U induced specific and evident elevation of extracellular acidification rates, arachidonic acid metabolite release, and intracellular Ca(2+) mobilization in Chinese hamster ovary cells expressing human FM-3. In addition, radiolabeled neuromedin U specifically bound to membrane fractions prepared from these cells with high affinity. We subsequently analyzed the tissue distribution of neuromedin U and FM-3 mRNAs in rats using quantitative reverse transcription-polymerase chain reaction. Neuromedin U mRNA was highly expressed in the gastrointestinal tract, and the highest expression was detected in the pituitary gland. On the other hand, FM-3 mRNA was highly expressed in the small intestine and lung, suggesting that neuromedin U plays important roles in these tissues. The identification of a specific and functional receptor for neuromedin U will facilitate studies on their physiological roles and the search for receptor agonists and antagonists.
Assuntos
Proteínas de Membrana , Neuropeptídeos/genética , Neuropeptídeos/metabolismo , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Receptores de Neurotransmissores , Sequência de Aminoácidos , Animais , Ácido Araquidônico/metabolismo , Sequência de Bases , Encéfalo/metabolismo , Células CHO , Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Cricetinae , Feminino , Humanos , Concentração de Íons de Hidrogênio , Ligantes , Masculino , Dados de Sequência Molecular , Neuropeptídeos/farmacologia , Especificidade de Órgãos , RNA Mensageiro/genética , Ratos , Receptores de Superfície Celular/química , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transcrição Gênica , TransfecçãoRESUMO
We have recently identified apelin as the endogenous ligand for human APJ. In rats, the highest expression of APJ mRNA was detected in the lung, suggesting that APJ and its ligand play an important role in the pulmonary system. When apelin-36 and its pyroglutamylated C-terminal peptide, [Assuntos
Encéfalo/metabolismo
, Proteínas de Transporte/metabolismo
, Receptores de Dopamina D2/genética
, Receptores de Dopamina D2/metabolismo
, Receptores Acoplados a Proteínas G
, Sequência de Aminoácidos
, Animais
, Apelina
, Receptores de Apelina
, Células CHO
, Bovinos
, Membrana Celular/metabolismo
, Cricetinae
, Feminino
, Humanos
, Peptídeos e Proteínas de Sinalização Intercelular
, Cinética
, Ligantes
, Masculino
, Dados de Sequência Molecular
, Especificidade de Órgãos
, Gravidez
, RNA Mensageiro/metabolismo
, Ratos
, Receptores de Dopamina D2/química
, Proteínas Recombinantes/química
, Proteínas Recombinantes/metabolismo
, Reação em Cadeia da Polimerase Via Transcriptase Reversa
, Alinhamento de Sequência
, Homologia de Sequência de Aminoácidos
, Transfecção
, Fatores de Virulência de Bordetella/farmacologia
RESUMO
By using a strategy that we have developed to search for the ligands of orphan seven-transmembrane-domain receptors [S. Hinuma et al., Nature 393 (1998) 272-276], we have recently identified a natural ligand, apelin, for the orphan 7TMR, APJ [K. Tatemoto et al., Biochem. Biophys. Res. Commun. 251 (1998) 471-476]. In this paper, we isolated rat and mouse apelin cDNAs, and analyzed the tissue distribution of apelin mRNA in rats. Although apelin mRNA was widely detected in a variety of tissues, the highest expression of apelin mRNA was detected in the mammary gland of pregnant rats. In the mammary gland, biologically active apelin and its mRNA considerably increased during pregnancy and lactation, and reached a maximal level around parturition. Moreover, a large amount of apelin (14-93 pmol/ml) was found to be secreted in the bovine colostrum, and it was still detectable even in commercial bovine milk. Since apelin partially suppressed cytokine production by mouse spleen cells in response to T cell receptor/CD3 cross-linking, the oral intake of apelin in the colostrum and milk might modulate immune responses in neonates.
Assuntos
Proteínas de Transporte/metabolismo , Colostro/metabolismo , Receptores Acoplados a Proteínas G , Adipocinas , Sequência de Aminoácidos , Animais , Apelina , Receptores de Apelina , Células CHO , Proteínas de Transporte/síntese química , Proteínas de Transporte/genética , Bovinos , Colforsina , Cricetinae , AMP Cíclico/metabolismo , DNA Complementar/isolamento & purificação , Feminino , Peptídeos e Proteínas de Sinalização Intercelular , Lactação/metabolismo , Ligantes , Masculino , Glândulas Mamárias Animais/metabolismo , Camundongos , Leite/química , Dados de Sequência Molecular , Gravidez/metabolismo , RNA Mensageiro/análise , Ratos , Ratos Wistar , Receptores de Dopamina D2/química , Receptores de Dopamina D2/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Prolactin-releasing peptide (PrRP) is a novel bioactive peptide, originally isolated from bovine hypothalamus by utilizing an orphan seven-transmembrane-domain receptor expressed in the human pituitary gland. In this paper, we analyzed the tissue distribution of rat and human PrRP and their receptor mRNAs by quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Northern blotting. In RT-PCR analysis, rat PrRP receptor mRNA was detected in the central nervous system, and the highest expression was detected in the pituitary gland. In addition, in situ hybridization revealed that rat PrRP receptor mRNA was highly expressed in the anterior lobe of the pituitary. On the other hand, rat PrRP mRNA was most abundantly expressed in the medulla oblongata, while significant levels of expression were widely detected in other tissues. In Northern blot analyses, human PrRP receptor mRNA was detected only in the pituitary gland among tissues examined. Human PrRP mRNA was detected in the medulla oblongata and in the pancreas. In contrast to the pattern of mRNA expression, the highest content of bioactive PrRP was found in the hypothalamus rather than the medulla oblongata in the rat brain, indicating that PrRP mRNA does not always parallel with mature PrRP in tissue distribution. The wide distribution of PrRP and its receptor suggests that they have various functions not only in the pituitary gland but also in the other tissues.
Assuntos
Hormônios Hipotalâmicos/biossíntese , Neuropeptídeos/biossíntese , Receptores de Neuropeptídeos/biossíntese , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA Complementar , Humanos , Hormônios Hipotalâmicos/genética , Dados de Sequência Molecular , Neuropeptídeos/genética , Hormônio Liberador de Prolactina , Ratos , Receptores de Neuropeptídeos/genética , Distribuição TecidualRESUMO
We established a sensitive and specific two-site enzyme immunoassay (EIA) for prolactin-releasing peptide (PrRP) using two region-specific monoclonal antibodies. We investigated the tissue distribution and the plasma concentration of immunoreactive (ir-) PrRP in rats using this assay. Ir-PrRP was widely distributed in the central nervous system and pituitary gland. The highest concentration of ir-PrRP was found in the hypothalamus. In peripheral tissues, appreciable levels of ir-PrRP were found only in the adrenal gland. The mean plasma concentration of ir-PrRP was 0.13 +/- 0.01 fmol/ml (mean +/- SEM). In reverse-phase and gel-filtration high performance liquid chromatography, hypothalamic ir-PrRP eluted at a position identical to that of PrRP31 and PrRP20. On the other hand, ir-PrRP from the adrenal gland and plasma eluted only at the position of synthetic PrRP31, indicating that molecular forms of ir-PrRP in vivo differed among tissues.
Assuntos
Hormônios Hipotalâmicos/sangue , Hormônios Hipotalâmicos/metabolismo , Neuropeptídeos/sangue , Neuropeptídeos/metabolismo , Glândulas Suprarrenais/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Especificidade de Anticorpos , Sistema Nervoso Central/metabolismo , Cromatografia Líquida de Alta Pressão , Feminino , Hormônios Hipotalâmicos/imunologia , Técnicas Imunoenzimáticas , Masculino , Neuropeptídeos/imunologia , Fragmentos de Peptídeos/sangue , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/metabolismo , Hipófise/metabolismo , Hormônio Liberador de Prolactina , Isoformas de Proteínas/sangue , Isoformas de Proteínas/imunologia , Isoformas de Proteínas/metabolismo , Ratos , Ratos Wistar , Sensibilidade e EspecificidadeRESUMO
In the search for an endogenous ligand of the orphan G protein-coupled receptor APJ, the presence of the ligand in various tissue extracts was examined by measuring the increase in extracellular acidification rate of the cells expressing the APJ receptor as a specific signal induced by the interaction of the receptor and ligand. By monitoring this activity, we isolated an APJ receptor ligand, designated apelin, from bovine stomach extracts. The structures of bovine and human apelin preproproteins were deduced from the sequences of the corresponding cDNAs. The preproproteins consisted of 77 amino acid residues, and the apelin sequence was encoded in the C-terminal regions. Synthetic peptides derived from the C-terminal amino acid sequence of bovine preproapelin were capable of specifically promoting the acidification rate in the cells expressing the APJ receptor in a range from 10(-7) to 10(-10) M, indicating that apelin is an endogenous ligand for the APJ receptor.
Assuntos
Proteínas de Transporte/metabolismo , Mucosa Gástrica/metabolismo , Receptores de Dopamina D2/metabolismo , Receptores Acoplados a Proteínas G , Sequência de Aminoácidos , Tonsila do Cerebelo/metabolismo , Animais , Apelina , Receptores de Apelina , Sequência de Bases , Células CHO , Proteínas de Transporte/química , Proteínas de Transporte/genética , Bovinos , Clonagem Molecular , Cricetinae , Proteínas de Ligação ao GTP/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Ligantes , Pulmão/metabolismo , Dados de Sequência Molecular , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Reação em Cadeia da Polimerase , Precursores de Proteínas/química , Precursores de Proteínas/metabolismo , Receptores de Dopamina D2/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Extratos de Tecidos/química , Extratos de Tecidos/metabolismo , TransfecçãoRESUMO
Hypothalamic peptide hormones regulate the secretion of most of the anterior pituitary hormones, that is, growth hormone, follicle-stimulating hormone, luteinizing hormone, thyroid-stimulating hormone and adrenocorticotropin. These peptides do not regulate the secretion of prolactin, at least in a specific manner, however. The peptides act through specific receptors, which are referred to as seven-transmembrane-domain receptors or G-protein-coupled receptors. Although prolactin is important in pregnancy and lactation in mammals, and is involved in the development of the mammary glands and the promotion of milk synthesis, a specific prolactin-releasing hormone has remained unknown. Here we identify a potent candidate for such a hormone. We first proposed that there may still be unknown peptide hormone factors that control pituitary function through seven-transmembrane-domain receptors. We isolated the complementary DNA encoding an 'orphan' receptor (that is, one for which the ligand is unknown). This receptor, hGR3, is specifically expressed in the human pituitary. We then searched for the hGR3 ligand in the hypothalamus and identified a new peptide, which shares no sequence similarity with known peptides and proteins, as an endogenous ligand. We show that this ligand is a potent prolactin-releasing factor for rat anterior pituitary cells; we have therefore named this peptide prolactin-releasing peptide.
Assuntos
Hormônios Hipotalâmicos/fisiologia , Neuropeptídeos/fisiologia , Prolactina/metabolismo , Receptores Acoplados a Proteínas G , Sequência de Aminoácidos , Animais , Ácido Araquidônico/metabolismo , Células CHO , Bovinos , Cricetinae , Feminino , Humanos , Hormônios Hipotalâmicos/genética , Hormônios Hipotalâmicos/isolamento & purificação , Lactação , Ligantes , Dados de Sequência Molecular , Neuropeptídeos/genética , Neuropeptídeos/isolamento & purificação , Adeno-Hipófise/metabolismo , Hormônio Liberador de Prolactina , Precursores de Proteínas/genética , Precursores de Proteínas/isolamento & purificação , Ratos , Ratos Endogâmicos F344 , Receptores de Superfície Celular/metabolismo , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Células Tumorais CultivadasRESUMO
Recombinant glycosaminoglycan-modified urinary thrombomodulin (GAG-UTM) expressed in mouse C-127 cells has potent antithrombotic activity available as an anticoagulant. GAG-UTM, a glycoprotein with sialic acid, was investigated regarding the influence of the terminal sialic acid on its pharmacokinetics upon rapid intravenous injection in rat. Asialo GAG-UTM desialated by neuraminidase was cleared rapidly from plasma. Sialyzed GAG-UTM, a sialyzed asialo GAG-UTM with alpha-2, 6-sialyltransferase, containing sialic acid similarly to native sialo GAG-UTM, had only a short half-life in plasma, suggesting that the binding site of sialic acid on galactose was not only sialyzed with alpha-2, 6-sialyltransferase but also with 2, 3-sialyltransferase. Asialo GAG-UTM with oxidized terminal galactose, however, had a long half-life. These results suggest that terminal sialic acid may be important to the pharmacokinetics of GAG-UTM; therefore, an analysis of asialo GAG-UTM became significant for quality control. In order to analyze sialo- and asialo-types in the early stage of purification, we investigated separation and analysis methods for both types and found a suitable sample of each: RCA-120-Agarose column for separation and ELISA using anti-thrombomodulin antibody and RCA lectin for analysis.
Assuntos
Anticoagulantes/farmacologia , Anticoagulantes/farmacocinética , Glicoproteínas/química , Ácidos Siálicos/farmacologia , Ácidos Siálicos/farmacocinética , Trombomodulina/química , Animais , Anticoagulantes/química , Ensaio de Imunoadsorção Enzimática , Humanos , Injeções Intravenosas , Masculino , Camundongos , Controle de Qualidade , Ratos , Ratos Wistar , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacocinética , Proteínas Recombinantes/farmacologia , Ácidos Siálicos/químicaRESUMO
A cDNA encoding a pituitary adenylate cyclase-activating polypeptide (PACAP) receptor was cloned from a bovine brain cDNA library using a synthetic oligonucleotide probe corresponding to the partial N-terminal amino acid sequence of the PACAP receptor purified from the bovine brain. The cloned cDNA encoded a polypeptide of 513 amino acid residues with seven putative transmembrane domains. The deduced amino acid sequence exactly matched the N-terminal amino acid sequence of the purified PACAP receptor. It also shared an apparent similarity with the vasoactive intestinal peptide (VIP), secretin, growth hormone releasing hormone, calcitonin, and glucagon receptors, suggesting that the PACAP receptor is a member of the secretin receptor subfamily of the guanine nucleotide-binding regulatory protein-coupled receptor family. Northern blot analysis showed that the size of the major mRNA band which hybridized with the cDNA was about 7 kb in the bovine cerebral-cortex and hippocampus. An expression vector containing the cloned cDNA for the PACAP receptor was introduced into Chinese hamster ovary (CHO) cells. The affinity of PACAP receptors expressed on the transfected CHO cells was quite similar to that of natural PACAP receptors on the bovine brain membranes. Competitive binding experiments showed that PACAP38 displaced the binding of 125I-labeled PACAP27 to the receptors on the CHO cells more efficiently than PACAP27, while VIP was less effective. In addition, both of PACAP27 and PACAP38 elevated the levels of cAMP and inositol phosphates in the transformed CHO cells. These results indicate that the PACAP receptors encoded by the cloned cDNA are identical to the purified PACAP receptors, and that they can stimulate dual signaling cascades.
Assuntos
DNA Complementar/metabolismo , Neuropeptídeos/metabolismo , Hipófise/metabolismo , Receptores do Hormônio Hipofisário/biossíntese , Sequência de Aminoácidos , Animais , Sequência de Bases , Células CHO , Bovinos , Clonagem Molecular , Cricetinae , AMP Cíclico/metabolismo , Biblioteca Gênica , Fosfatos de Inositol/metabolismo , Cinética , Dados de Sequência Molecular , Sondas de Oligonucleotídeos , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase , Receptores do Hormônio Hipofisário/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismo , Mapeamento por Restrição , TransfecçãoRESUMO
A functional cDNA clone for a human pituitary adenylate cyclase activating polypeptide (PACAP) receptor was isolated from a human pituitary cDNA library. The cDNA encoded a polypeptide consisting of 525 amino acids with putative seven hydrophobic domains. Chinese hamster ovary (CHO)-K1 cells transfected with the cDNA specifically bound PACAP and mediated PACAP-triggered intracellular accumulation of cAMP, indicating that this cDNA encoded a functional human PACAP Type I receptor. This receptor was structurally related to the vasoactive intestinal peptide (VIP), secretin, calcitonin and parathyroid hormone receptors and is much more homologous to a rat PACAP receptor. Northern blot analysis revealed that PACAP receptor mRNAs were expressed mainly in the brain and widely distributed in the central nervous system.
Assuntos
Hipófise/metabolismo , Receptores do Hormônio Hipofisário/biossíntese , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Southern Blotting , Células CHO , Membrana Celular/metabolismo , Clonagem Molecular , Cricetinae , AMP Cíclico/metabolismo , DNA Complementar/metabolismo , Expressão Gênica , Biblioteca Gênica , Humanos , Cinética , Dados de Sequência Molecular , Neuropeptídeos/metabolismo , Neurotransmissores/metabolismo , Sondas de Oligonucleotídeos , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase , Conformação Proteica , Ratos , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase , Receptores do Hormônio Hipofisário/genética , Receptores do Hormônio Hipofisário/metabolismo , Homologia de Sequência de Aminoácidos , Transfecção , Peptídeo Intestinal Vasoativo/farmacologiaRESUMO
Sexual cell fusion is an initial step of macrocyst formation in Dictyostelium discoideum and requires environmental conditions such as darkness, plenty of water and the presence of calcium ions. We have been analyzing the mechanism of sexual cell fusion between HM1 and NC4, heterothallic strains in D. discoideum. Cells of these strains have been shown to be fusion competent when cultured in a liquid medium in darkness, but not so when cultured on agar plates or in a liquid medium in the light. Two cell-surface proteins, gp70 and gp138, have been identified as target molecules for fusion-blocking antibodies and therefore as relevant to sexual cell fusion. In the present study, gp70 was shown to be present in HM1 cells cultured in the light, and fusion incompetent. Intact HM1 cells cultured in the light were unable to absorb the fusion-blocking activity of antibodies against membrane components of fusion-competent HM1 cells, whose activity had been shown to be absorbed by gp70, but they did so after separation of proteins in the SDS-PAGE. In addition, fusion-competent HM1 cells were found to lose their fusion competence by subsequent cultivation in the light. This loss of competence was cycloheximide sensitive, indicating that de novo synthesis of proteins was necessary for this inhibition. From these results, we presume that light induces a protein that hinders the interaction of gp70 in HM1 cells with its receptor on the NC4 cell surface and thereby inhibits the sexual process between these strains.
Assuntos
Dictyostelium/fisiologia , Fertilização/fisiologia , Células Germinativas/fisiologia , Luz , Proteínas de Membrana/fisiologia , Animais , Fusão Celular/fisiologia , Células Cultivadas , Eletroforese em Gel de Poliacrilamida , Fertilização/efeitos dos fármacos , Células Germinativas/química , Células Germinativas/metabolismo , Células Germinativas/ultraestrutura , Immunoblotting , Proteínas de Membrana/análise , Receptores de Superfície Celular/efeitos dos fármacos , Receptores de Superfície Celular/metabolismoRESUMO
Sexual cell fusion occurs between HM1 and NC4, heterothallic strains in Dictyostelium discoideum. A membrane component of HM1 cells with a molecular mass of 70 kDa (70K protein) has been shown to be implicated in cell fusion (Urushihara et al. (1988) Cell Differ. Dev. 25, 81-88). In the present study, 70K protein was partially purified using affinity Sepharose on which membrane proteins of NC4 cells were immobilized. Through this process, involvement of Ca2+ in the interaction of 70K protein with its receptor was suggested. Lectin staining of partially purified 70K protein indicated it to be a glycoprotein containing D-mannose and/or D-glucose residues.
Assuntos
Cálcio/fisiologia , Dictyostelium/fisiologia , Proteínas Fúngicas/isolamento & purificação , Glicoproteínas de Membrana/isolamento & purificação , Fusão Celular , Cromatografia de Afinidade , Glicoproteínas de Membrana/fisiologia , ReproduçãoRESUMO
The molecular mechanism of sexual cell fusion in Dictyostelium discoideum was studied using the heterothallic strains HM1 and NC4. Monovalent antibodies (Fab) prepared from rabbit antiserum against a crude membrane preparation of fusion-competent HM1 cells inhibited fusion between HM1 and NC4 cells. Six out of 43 antigenic proteins were found in fusion-competent HM1 cells but not in fusion-incompetent cells. Among them, only one protein with a molecular mass of 70 kDa was able to neutralize the fusion-inhibiting activity of Fab, suggesting its possible participation in sexual cell fusion.