RESUMO
G-protein-coupled receptors (GPCR) are now regarded as being able to acquire heterodimer conformations affecting their pharmacology, signaling and trafficking. In co-immunoprecipitation studies using differentially epitope-tagged receptors, we herein provide direct evidence for heterodimerization of human neurotensin type 1 receptor (hNTR1) and type 2 receptor (hNTR2). Using chimeric constructs, we also identified the hNTR2 transmembrane 2 (TM2) to TM4 region as crucial for the formation of the dimerization interface. At the functional level, we demonstrated that the co-expression of hNTR2 suppressed hNTR1-mediated adenylate cyclase/cAMP and phospholipase C activation. Finally, confocal microscopy revealed that whereas tagged hNTR1 expressed alone were localized to the plasma membrane, co-expression of hNTR2 caused the retention of hNTR1 in sub-cellular compartments, indicating that heterodimerization with hNTR2 interferes with the proper recruitment of hNTR1 to the plasma membrane. Overall, this study proposes a novel function of NTR2 in the regulation of NTR1 activity.
Assuntos
Receptores de Neurotensina/antagonistas & inibidores , Receptores de Neurotensina/metabolismo , Animais , Linhagem Celular , Células HeLa , Humanos , Mapeamento de Interação de Proteínas , Multimerização Proteica , Estrutura Terciária de Proteína , Ratos , Receptores de Neurotensina/genéticaRESUMO
Kisspeptin and its receptor, GPR54, play important roles in mammalian reproduction and cancer development. However, little is known about their function in nonmammalian species. In the present study, we have isolated the cDNA encoding the kisspeptin receptor, GPR54, from the bullfrog, Rana catesbeiana. The bullfrog GPR54 (bfGPR54) cDNA encodes a 379-amino acid heptahelical G protein-coupled receptor. bfGPR54 exhibits 45-46% amino acid identity with mammalian GPR54s and 70-74% identity with fish GPR54s. RT-PCR analysis showed that bfGPR54 mRNA is highly expressed in the forebrain, hypothalamus and pituitary. Upon stimulation by synthetic human kisspeptin-10 with Phe-amide residue at the C-terminus (h-Kiss-10F), bfGPR54 induces SRE-luc activity, a PKC-specific reporter, evidencing the PKC-linked signaling pathway of bfGPR54. Using a blast search, we found a gene encoding a kisspeptin-like peptide in Xenopus. The C-terminal decapeptide of Xenopus kisspeptin shows higher amino acid sequence identity to fish Kiss-10s than mammalian Kiss-10s. A synthetic Xenopus kisspeptin peptide (x-Kiss-12Y) showed a higher potency than mammalian Kiss-10s in the activation of bfGPR54. This study expands our understanding of the physiological roles and molecular evolution of kisspeptins and their receptors.
Assuntos
Rana catesbeiana , Receptores Acoplados a Proteínas G , Proteínas Supressoras de Tumor/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Feminino , Humanos , Ligantes , Masculino , Dados de Sequência Molecular , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Alinhamento de Sequência , Transdução de Sinais/fisiologia , Distribuição Tecidual , Proteínas Supressoras de Tumor/genética , Proteínas de Xenopus/genéticaRESUMO
Steroidogenic acute regulatory protein (StAR) transfers cholesterol from the outer mitochondrial membrane to the inner membrane where the cytochrome P450 side chain cleavage enzyme (P450scc) resides. This process is the rate-limiting step in steroidogenesis. StAR cDNAs have been cloned and characterized from a range of different species. To investigate the role of StAR in the amphibian system, we cloned a full-length StAR cDNA from bullfrog (Rana catesbeiana) using reverse transcription polymerase chain reaction (RT-PCR) in conjunction with rapid amplification of cDNA ends (RACE). The putative full-length bullfrog StAR (bfStAR) cDNA was 1862 base pairs (bp) in length, and the longest open reading frame (ORF) encoded a protein of 284 amino acids. Amino acid sequence comparison showed that amphibian StAR has a high degree of sequence identity, ranging from 62% to 98%, with StAR proteins of other species. Similar to other species, bfStAR contained two conserved domains, the mitochondrial targeting domain and cholesterol-binding domain, in the N-terminus and C-terminus of the protein, respectively. Northern blot analysis and RT-PCR indicated that StAR mRNA is expressed in the gonads and adrenal gland. Transfection of green monkey kidney (COS-1) cells with an expression construct for bfStAR revealed that it encoded 34 and 27kDa proteins that were recognized by antiserum raised against the human StAR-related lipid transfer (START) domain.
Assuntos
Proteínas de Anfíbios/genética , Fosfoproteínas/genética , Rana catesbeiana/genética , Sequência de Aminoácidos , Proteínas de Anfíbios/química , Proteínas de Anfíbios/metabolismo , Animais , Células COS , Chlorocebus aethiops , Clonagem Molecular , DNA Complementar/química , Dados de Sequência Molecular , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Filogenia , Rana catesbeiana/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Análise de Sequência de ProteínaRESUMO
The Glu/Asp(7.32) residue in extracellular loop 3 of the mammalian type-I gonadotropin-releasing hormone receptor (GnRHR) interacts with Arg(8) of GnRH-I, conferring preferential ligand selectivity for GnRH-I over GnRH-II. Previously, we demonstrated that the residues (Ser and Pro) flanking Glu/Asp(7.32) also play a role in the differential agonist selectivity of mammalian and non-mammalian GnRHRs. In this study, we examined the differential antagonist selectivity of wild type and mutant GnRHRs in which the Ser and Pro residues were changed. Cetrorelix, a GnRH-I antagonist, and Trptorelix-2, a GnRH-II antagonist, exhibited high selectivity for mammalian type-I and non-mammalian GnRHRs, respectively. The inhibitory activities of the antagonists were dependent on agonist concentration and subtype. Rat GnRHR in which the Ser-Glu-Pro (SEP) motif was changed to Pro-Glu-Val (PEV) or Pro-Glu-Ser (PES) had increased sensitivity to Trptorelix-2 but decreased sensitivity to Cetrorelix. Mutant bullfrog GnRHR-1 with the SEP motif had the reverse antagonist selectivity, with reduced sensitivity to Trptorelix-2 but increased sensitivity to Cetrorelix. These findings indicate that the residues flanking Glu(7.32) are important for antagonist as well as agonist selectivity.
Assuntos
Ácido Glutâmico/metabolismo , Hormônio Liberador de Gonadotropina/análogos & derivados , Antagonistas de Hormônios/metabolismo , Receptores LHRH , Sequência de Aminoácidos , Animais , Linhagem Celular , Relação Dose-Resposta a Droga , Hormônio Liberador de Gonadotropina/genética , Hormônio Liberador de Gonadotropina/metabolismo , Oligopeptídeos/metabolismo , Ratos , Receptores LHRH/antagonistas & inibidores , Receptores LHRH/genética , Receptores LHRH/metabolismoRESUMO
The SWI3-related gene product (SRG3), a component of the mouse SWI/SNF complex, has been suggested to have an alternative function. Here, we demonstrate that in the prostate transactivation of the androgen receptor (AR) is modulated by SRG3 in multiple ways. The expression of SRG3, which is developmentally regulated in the prostate, is induced by androgen through AR. SRG3 in turn enhances the transactivation of AR, providing a positive feedback regulatory loop. The SRG3 coactivation of AR transactivation is achieved through the recruitment of coactivator SRC-1, the protein level of which is upregulated by SRG3, providing another pathway of positive regulation. Interestingly, SRG3 coactivation of AR transactivation is fully functional in BRG1/BRM-deficient C33A cells and the AR/SRG3/SRC-1 complex formed in vivo contains neither BRG1 nor BRM protein, suggesting the possibility of an SRG3 function independent of the SWI/SNF complex. Importantly, the AR/SRG3/SRC-1 complex occupies androgen response elements on the endogenous SRG3 and PSA promoter in an androgen-dependent manner in mouse prostate and LNCaP cells, respectively, inducing gene expression. These results suggest that the multiple positive regulatory mechanisms of AR transactivation by SRG3 may be important for the rapid proliferation of prostate cells during prostate development and regeneration.
Assuntos
Receptores Androgênicos/metabolismo , Fatores de Transcrição/metabolismo , Ativação Transcricional , Acetiltransferases/genética , Acetiltransferases/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Histona Acetiltransferases , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Coativador 1 de Receptor Nuclear , Coativadores de Receptor Nuclear , Proteínas Oncogênicas/genética , Proteínas Oncogênicas/metabolismo , Regiões Promotoras Genéticas , Próstata/citologia , Próstata/fisiologia , Ratos , Ratos Sprague-Dawley , Receptores Androgênicos/genética , Fatores de Transcrição/genética , Técnicas do Sistema de Duplo-Híbrido , Fatores de Transcrição de p300-CBPRESUMO
The neurohypophysial nonapeptides vasopressin (VP) and oxytocin (OT) modulate a broad range of cognitive and social activities. Notably, in amphibians, vasotocin (VT), the ortholog of mammalian VP, plays a crucial role in the control of sexual behaviors. Because several neurosteroids also regulate reproduction-related behaviors, we investigated the possible effect of VT and the OT ortholog mesotocin (MT) in the control of neurosteroid production. Double immunohistochemical labeling of frog brain sections revealed the presence of VT/MT-positive fibers in close proximity of neurons expressing the steroidogenic enzymes 3beta-hydroxysteroid dehydrogenase/delta5-delta4 isomerase (3beta-HSD) and cytochrome P450 17alpha-hydroxylase/c17, 20-lyase (P450(C17)). High concentrations of VT and MT receptor mRNAs were observed in diencephalic nuclei containing the 3beta-HSD and P450(C17) neuronal populations. Exposure of frog hypothalamic explants to graded concentrations of VT or MT produced a dose-dependent increase in the formation of progesterone, 17-hydroxypregnenolone, 17-hydroxyprogesterone, and dehydroepiandrosterone. The stimulatory effect of VT and MT on neurosteroid biosynthesis was mimicked by VP and OT, as well as by a selective V1b receptor agonist, whereas V2 and OT receptor agonists had no effect. VT-induced neurosteroid production was completely suppressed by selective V1a receptor antagonists and was not affected by V2 and OT receptor antagonists. Concurrently, the effect of MT on neurosteroidogenesis was markedly attenuated by selective OT and V1a receptor antagonists but not by a V2 antagonist. The present study provides the first evidence for a regulatory effect of VT and MT on neurosteroid biosynthesis. These data suggest that neurosteroids may mediate some of the behavioral actions of VT and MT.
Assuntos
Encéfalo/efeitos dos fármacos , Ocitócicos/farmacologia , Ocitocina/análogos & derivados , Esteroides/biossíntese , Vasotocina/farmacologia , Animais , Encéfalo/metabolismo , Cromatografia Líquida de Alta Pressão/métodos , Eletroquímica/métodos , Imuno-Histoquímica/métodos , Hibridização In Situ/métodos , Masculino , Ocitocina/farmacologia , RNA Mensageiro/biossíntese , Rana esculenta , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodosRESUMO
The superfamily of G protein-coupled receptors (GPCRs) is the largest and most diverse group of membrane-spanning proteins. It plays a variety of roles in pathophysiological processes by transmitting extracellular signals to cells via heterotrimeric G proteins. Completion of the human genome project revealed the presence of approximately 168 genes encoding established nonsensory GPCRs, as well as 207 genes predicted to encode novel GPCRs for which the natural ligands remained to be identified, the so-called orphan GPCRs. Eighty-six of these orphans have now been paired to novel or previously known molecules, and 121 remain to be deorphaned. A better understanding of the GPCR structures and classification; knowledge of the receptor activation mechanism, either dependent on or independent of an agonist; increased understanding of the control of GPCR-mediated signal transduction; and development of appropriate ligand screening systems may improve the probability of discovering novel ligands for the remaining orphan GPCRs.
Assuntos
Receptores Acoplados a Proteínas G , Animais , Bioensaio , Cálcio/metabolismo , Receptores Frizzled/genética , Receptores Frizzled/metabolismo , Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Humanos , Filogenia , Conformação Proteica , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/classificação , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores dos Hormônios Gastrointestinais/genética , Receptores dos Hormônios Gastrointestinais/metabolismo , Receptores de Glutamato/genética , Receptores de Glutamato/metabolismo , Sistemas do Segundo Mensageiro/fisiologiaRESUMO
In mammals, apelin and its G protein-coupled receptor, APJ, regulate blood pressure, intake of food and water, and cardiac contractility. In this study, we report the cloning and functional characterization of APJ in the bullfrog, Rana catesbeiana. Bullfrog APJ (bfAPJ) cDNA contains an open reading frame of 1083 nucleotides encoding a protein of 360 amino acid residues. Sequence alignment reveals 75% amino acid identity with Xenopus, 63% identity with zebrafish and 40-42% identity with mammalian APJs. RT-PCR analysis and tissue binding assay reveal high expression of bfAPJ mRNA in the brain, particularly in the hypothalamus, and moderate expression in the pituitary, testis, adrenal gland and lung. Whereas [pGlu(1)]apelin-13 did not induce CRE-luc (protein kinase A-specific reporter) and SRE-luc (protein kinase C-specific reporter) activity in cells expressing bfAPJ, this apelin-13 decreased forskolin-induced CRE-luc activity and cAMP accumulation in a pertussis toxin-sensitive manner. This study indicates that bfAPJ may couple to G(i/o). [Pro(1)]apelin-13, a synthetic apelin based on the sequence of the putative apelin gene from many non-mammalian species, activates bfAPJ with 5-10-fold greater sensitivity/affinity than mammalian apelin-13. Collectively, this study expands our understanding of the physiological roles of this receptor system in non-mammalian species.
Assuntos
Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Prolina/metabolismo , Rana catesbeiana/genética , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Clonagem Molecular , DNA Complementar/genética , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Ligantes , Masculino , Dados de Sequência Molecular , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Acoplados a Proteínas G/química , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transdução de SinaisRESUMO
The basic leucine zipper transcription factor, CCAAT enhancer-binding protein-alpha (C/EBPalpha), negatively regulates cell proliferation and induces terminal differentiation of various cell types. C/EBPalpha is expressed in the prostate, but its potential role in the tissue is unknown. Herein, we show that C/EBPalpha is highly expressed at the stage of growth arrest during prostate development. Furthermore, overexpression of C/EBPalpha decreases the rate of DNA synthesis in LNCaP prostate cancer cells. Investigation of the potential cross-talk between C/EBPalpha and androgen receptor (AR) that is responsible for androgen-dependent prostate proliferation demonstrates that androgen-dependent transactivation of AR is strongly repressed by C/EBPalpha. C/EBPalpha directly binds AR in vitro and forms a complex with AR in vivo. C/EBPalpha neither prevents the nuclear translocation of AR nor disrupts the N/C-terminal interaction of AR, which are both necessary for its proper transactivation activity upon ligand binding. To modulate AR transactivation, however, C/EBPalpha does compete with AR coactivators for AR binding. Additionally, C/EBPalpha is recruited onto AR-target promoters with AR and is further able to inhibit the expression of endogenous prostate-specific antigen in prostate cancer cells. Our results suggest C/EBPalpha as a potent AR corepressor and provide insight into the role of C/EBPalpha in prostate development and cancer.
Assuntos
Antagonistas de Receptores de Andrógenos , Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Neoplasias da Próstata/metabolismo , Receptores Androgênicos/metabolismo , Proteínas Repressoras/metabolismo , Animais , Proteína alfa Estimuladora de Ligação a CCAAT/análise , Linhagem Celular Tumoral , Proliferação de Células , Regulação para Baixo , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Regiões Promotoras Genéticas , Próstata/química , Próstata/crescimento & desenvolvimento , Próstata/metabolismo , Antígeno Prostático Específico/genética , Neoplasias da Próstata/química , Neoplasias da Próstata/genética , Ratos , Receptores Androgênicos/genética , Proteínas Repressoras/análise , Ativação TranscricionalRESUMO
PURPOSE: The primary objective of this study was to examine the changes of basal cortisol and DHEA levels present in saliva and serum with age, and to determine the correlation coefficients of steroid concentrations between saliva and serum. The secondary objective was to obtain a standard diurnal rhythm of salivary cortisol and DHEA in the Korean population. MATERIALS AND METHODS: For the first objective, saliva and blood samples were collected between 10 and 11 AM from 359 volunteers ranging from 21 to 69 years old (167 men and 192 women). For the second objective, four saliva samples (post-awakening, 11 AM, 4 PM, and bedtime) were collected throughout a day from 78 volunteers (42 women and 36 men) ranging from 20 to 40 years old. Cortisol and DHEA levels were measured using a radioimmunoassay (RIA). RESULTS: The morning cortisol and DHEA levels, and the age- related steroid decline patterns were similar in both genders. Serum cortisol levels significantly decreased around forty years of age (p < 0.001, when compared with people in their 20s), and linear regression analysis with age showed a significant declining pattern (slope=-2.29, t=-4.297, p < 0.001). However, salivary cortisol levels did not change significantly with age, but showed a tendency towards decline (slope=-0.0078, t=-0.389, p=0.697). The relative cortisol ratio of serum to saliva was 3.4-4.5% and the ratio increased with age (slope=0.051, t=3.61, p < 0.001). DHEA levels also declined with age in saliva (slope=-0.007, t=-3.76, p < 0.001) and serum (slope=-0.197 t=-4.88, p < 0.001). In particular, DHEA levels in saliva and serum did not start to significantly decrease until ages in the 40s, but then decreased significantly further at ages in the 50s (p < 0.001, when compared with the 40s age group) and 60s (p < 0.001, when compared with the 50 age group). The relative DHEA ratio of serum to saliva was similar throughout the ages examined (slop=0.0016, t=0.344, p=0.73). On the other hand, cortisol and DHEA levels in saliva reflected well those in serum (r=0.59 and 0.86, respectively, p < 0.001). The highest salivary cortisol levels appeared just after awakening (about two fold higher than the 11 AM level), decreased throughout the day, and reached the lowest levels at bedtime (p < 0.001, when compared with PM cortisol levels). The highest salivary DHEA levels also appeared after awakening (about 1.5 fold higher than the 11 AM level) and decreased by 11 AM (p < 0.001). DHEA levels did not decrease further until bedtime (p=0.11, when compared with PM DHEA levels). CONCLUSION: This study showed that cortisol and DHEA levels change with age and that the negative slope of DHEA was steeper than that of cortisol in saliva and serum. As the cortisol and DHEA levels in saliva reflected those in serum, the measurement of steroid levels in saliva provide a useful and practical tool to evaluate adrenal functions, which are essential for clinical diagnosis.
Assuntos
Ritmo Circadiano , Desidroepiandrosterona/metabolismo , Hidrocortisona/metabolismo , Saliva/metabolismo , Adulto , Fatores Etários , Idoso , Análise de Variância , Desidroepiandrosterona/sangue , Feminino , Humanos , Hidrocortisona/sangue , Masculino , Pessoa de Meia-IdadeRESUMO
The effects of butyltin compounds on follicular steroidogenesis in amphibians were examined using ovarian follicles of Rana catesbeiana. Isolated follicles were cultured for 18h in the presence and absence of frog pituitary homogenate (FPH) or various steroid precursors, and the steroid levels in the follicles or culture media were measured by radioimmunoassay (RIA). Among the butyltin compounds, tributyltin (TBT) strongly inhibited the FPH-induced synthesis of pregnenolone (P(5)), progesterone (P(4)) and testosterone (T). It also inhibited the conversion of P(5)-P(4) and T to estradiol-17ß(E(2)) and it partially suppressed the conversion of androstenedione (AD) to T, but not P(4) to 17α-hydroxyprogesterone (17α-OHP(4)). A high concentration of dibutyltin (DBT) also inhibited steroidogenesis by the follicles while monobutyltin (MBT) and tetrabutylin (TeBT) had no effect. These results suggest that the initial step of steroidogenesis (P(5) synthesis) and enzymes such as 3ß-HSD, 17ß-HSD and aromatase are inhibited by TBT or DBT. However, 17α-hydroxylase was not suppressed by TBT or the other butyltin compounds.
RESUMO
Recently, we demonstrated that the mammalian type-I GnRH receptor (GnRHR) has a high preference for the phospholipase C/protein kinase C (PLC/PKC)-linked signaling pathway, whereas non-mammalian bullfrog (bf) GnRHRs couple to both adenylate cyclase/protein kinase A (AC/PKA)- and PLC/PKC-linked signaling pathways. In the pre-sent study, using AC/PKA-specific reporter (cAMP-responsive element-luciferase) and PLC/PKC-specific reporter (serum-responsive element-luciferase) systems, we attempted to identify the motif responsible for this difference. A deletion of the intracellular carboxyl-terminal tail (C tail) of bfGnRHR-1 remarkably decreased its ability to induce the AC/PKA-linked signaling pathway. Further dissection of the C tail indicated that an HFRK motif in the membrane-proximal sequence of bfGnRHR-1 C tail is a minimal requirement for the AC/PKA-linked signaling pathway as the addition of this motif to rat GnRHR or deletion of it from bfGnRHR-1 significantly affected the ability to induce the AC/PKA-linked signaling pathway. Deletion or addition of the HFRK motif, however, did not critically influence the PLC/PKC-linked signaling pathway. These results indicate that the HFRK motif in the membrane-proximal region confers the differential signal transduction pathways between mammalian and nonmammalian GnRHRs.
Assuntos
Membrana Celular/metabolismo , Receptores LHRH/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Linhagem Celular , AMP Cíclico/metabolismo , Genes Reporter , Células HeLa , Humanos , Luciferases/metabolismo , Dados de Sequência Molecular , Mutação , Plasmídeos/metabolismo , Proteína Quinase C/metabolismo , Estrutura Terciária de Proteína , Rana catesbeiana , Ratos , Receptores LHRH/metabolismo , Transdução de Sinais , Transcrição Gênica , Transfecção , Fosfolipases Tipo C/metabolismoRESUMO
CONTEXT: GnRH is known to directly regulate prostate cancer cell proliferation, but the precise mechanism of action of the peptide is still under investigation. OBJECTIVE: This study demonstrates differential effects of GnRH-I and GnRH-II on androgen-independent human prostate cancer cells. RESULTS: Both GnRH-I and GnRH-II increased the intracellular Ca(2+) concentration ([Ca(2+)](i)) either through Ca(2+) influx from external Ca(2+) source or via mobilization of Ca(2+) from internal Ca(2+) stores. Interestingly, the [Ca(2+)](i) increase was mediated by activation of the ryanodine receptor but not the inositol trisphosphate receptor. Trptorelix-1, a novel GnRH-II antagonist but not cetrorelix, a classical GnRH-I antagonist, completely inhibited the GnRH-II-induced [Ca(2+)](i) increase. Concurrently at high concentrations, trptorelix-1 and cetrorelix inhibited GnRH-I-induced [Ca(2+)](i) increase, whereas at low concentrations they exerted an agonistic action, inducing Ca(2+) influx. High concentrations of trptorelix-1 but not cetrorelix-induced prostate cancer cell death, probably through an apoptotic process. Using photoaffinity labeling with (125)I-[azidobenzoyl-D-Lys(6)]GnRH-II, we observed that an 80-kDa protein specifically bound to GnRH-II. CONCLUSIONS: This study suggests the existence of a novel GnRH-II binding protein, in addition to a conventional GnRH-I receptor, in prostate cancer cells. These data may facilitate the development of innovatory therapeutic drugs for the treatment of prostate cancer.
Assuntos
Apoptose , Hormônio Liberador de Gonadotropina/análogos & derivados , Hormônio Liberador de Gonadotropina/farmacologia , Neoplasias da Próstata/metabolismo , Receptores LHRH/fisiologia , Transdução de Sinais/fisiologia , Cálcio/metabolismo , Hormônio Liberador de Gonadotropina/antagonistas & inibidores , Humanos , Fosfatos de Inositol/metabolismo , Masculino , Marcadores de Fotoafinidade , Neoplasias da Próstata/patologia , Receptores LHRH/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Canal de Liberação de Cálcio do Receptor de Rianodina/efeitos dos fármacosRESUMO
A Glu/Asp7.32 residue in the extracellular loop 3 of the mammalian GnRH receptor (GnRHR) is known to interact with Arg8 of mammalian GnRH (mGnRH), which may confer preferential ligand selectivity for mGnRH than for chicken GnRH-II (cGnRH-II). However, some nonmammalian GnRHRs also have the Glu/Asp residue at the same position, yet respond better to cGnRH-II than mGnRH. Amino acids flanking Glu/Asp7.32 are differentially arranged such that mammalian and nonmammalian GnRHRs have an S-E/D-P motif and P-X-S/Y motif, respectively. We presumed the position of Ser7.31 or Pro7.33 of rat GnRHR as a potential determinant for ligand selectivity. Either placing Pro before Glu7.32 or placing Ser after Glu7.32 significantly decreased the sensitivity and/or efficacy for mGnRH, but slightly increased that for cGnRH-II in several mutant receptors. Among them, those with a PEV, PES, or SES motif exhibited a marked decrease in sensitivity for mGnRH such that cGnRH-II had a higher potency than mGnRH, showing a reversed preferential ligand selectivity. Chimeric mGnRHs in which positions 5, 7, and/or 8 were replaced by those of cGnRH-II revealed a greater ability to activate these mutant receptors than mGnRH, whereas they were less potent to activate wild-type rat GnRHR than mGnRH. Interestingly, a mutant bullfrog type I receptor with the SEP motif exhibited an increased sensitivity for mGnRH but a decreased sensitivity for cGnRH-II. These results indicate that the position of Pro and Ser near Glu7.32 in the extracellular loop 3 is critical for the differential ligand selectivity between mammalian and nonmammalian GnRHRs.
Assuntos
Hormônio Liberador de Gonadotropina/análogos & derivados , Hormônio Liberador de Gonadotropina/metabolismo , Receptores LHRH/química , Receptores LHRH/metabolismo , Animais , Galinhas , Primers do DNA , Ácido Glutâmico , Cinética , Ligantes , Mamíferos , Modelos Moleculares , Conformação Molecular , Mutagênese Sítio-Dirigida , Reação em Cadeia da Polimerase , Prolina , Conformação Proteica , Estrutura Secundária de Proteína , Ratos , Serina , Especificidade da Espécie , Especificidade por SubstratoRESUMO
Recently, we have identified three distinct types of bullfrog GnRH receptor (designated bfGnRHR-1, bfGnRHR-2, and bfGnRHR-3). In the present study, we have isolated three GnRHR clones in Rana dybowskii (dyGnRHR-1, dyGnRHR-2, and dyGnRHR-3). Despite high homology of dyGnRHRs with the corresponding bfGnRHRs, dyGnRHRs revealed different signaling pathways and ligand sensitivity compared with the bfGnRHR counterparts. Activation of dyGnRHRs with GnRH stimulated cAMP-mediated gene expression. However, dyGnRHR-3 but not dyGnRHR-1 and -2 induced c-fos promoter-driven gene expression. Consistently, dyGnRHR-1 and dyGnRHR-2 were not able to increase GnRH-induced inositol phosphate accumulation, whereas all bfGnRHRs and dyGnRHR-3 were, indicating that dyGnRHR-1 and dyGnRHR-2 are coupled to solely G(s), whereas all bfGnRHRs and dyGnRHR-3 are coupled to both G(s) and G(q/11). Moreover, dyGnRHR-1 and dyGnRHR-2 showed about 10-fold less sensitivity to each ligand than that of the bfGnRHR counterparts. Using type 1 chimeric and point-mutated receptors, we further elucidated that specific amino acids, Ala/Thr(201) in extracellular loop 2 and Leu/Phe(290) in transmembrane domain 6 of the type 1 receptor, are responsible for ligand sensitivity and signal transduction pathway. Particularly, substitution of Leu(290) to Phe in dyGnRHR-1 increased GnRH-induced inositol phosphate production as well as c-fos promoter-driven gene expression whereas substitution of Phe(290) to Leu in bfGnRHR-1 decreased those activities. Collectively, these results demonstrate the presence of three types of GnRHR in amphibians, and suggest species- and type-specific ligand recognition and different signaling pathways in frog GnRHRs.
Assuntos
Mutação Puntual/fisiologia , Receptores LHRH/química , Receptores LHRH/genética , Transdução de Sinais/fisiologia , Sequência de Aminoácidos , Animais , Expressão Gênica , Células HeLa , Humanos , Ligantes , Dados de Sequência Molecular , Mutagênese/fisiologia , Estrutura Terciária de Proteína , Ranidae , Receptores LHRH/metabolismo , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Especificidade da EspécieRESUMO
Gonadotropin-releasing hormone (GnRH) regulates the reproductive system through the cognate GnRH receptor (GnRHR) in vertebrates. In this study, we cloned a cDNA encoding the full-length open reading frame sequence for green monkey type-II GnRHR (gmGnRHR-2) from the genomic DNA of CV-1 cells. Transient transfection study showed that gmGnRHR-2 was able to induce both c-fos promoter- and cAMP responsive element-driven transcriptional activities, indicating that gmGnRHR-2 couples to both Gs- and Gq/11-linked signaling pathways. gmGnRHR-2 responded better to GnRH-2 ([His5, Trp7, Tyr8]GnRH) than GnRH-1 ([Tyr5, Leu7, Arg8]GnRH). Substitutions of His5, Trp7, and/or Tyr8 in GnRH-1 increased the potency to activate gmGnRHR-2, suggesting that individual His5, Trp7, and Tyr8 in GnRH-2 contributed to differential ligand sensitivity of gmGnRHR-2. Substitution of D-Ala for Gly6 in GnRH-2 increased the potency to activate the receptor, suggesting that GnRH-2 has a constrained conformation when it binds to the receptor. GnRH-induced gmGnRHR-2 activation was specifically inhibited by GnRH-2 antagonists, Trptorelix-1 and -2, but not by a GnRH-1 antagonist, Cetrorelix. In conclusion, gmGnRHR-2 revealed preferential ligand selectivity for GnRH-2 and its analogs, suggesting that gmGnRHR-2 has a functional activity that is different from mammalian type-I GnRHRs but similar to non-mammalian GnRHRs.
Assuntos
Hormônio Liberador de Gonadotropina/análogos & derivados , Hormônio Liberador de Gonadotropina/metabolismo , Receptores LHRH/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Chlorocebus aethiops , DNA Complementar/genética , Vetores Genéticos , Hormônio Liberador de Gonadotropina/química , Hormônio Liberador de Gonadotropina/farmacologia , Humanos , Ligantes , Dados de Sequência Molecular , Plasmídeos/genética , Regiões Promotoras Genéticas , Ratos , Receptores LHRH/antagonistas & inibidores , Receptores LHRH/química , Receptores LHRH/genética , Alinhamento de Sequência , Relação Estrutura-Atividade , TransfecçãoRESUMO
Cadmium (Cd) is one of the environmental pollutants affecting various tissues and organs including testis. Harmful effect of Cd in testis is known to be germ cell degeneration and impairment of testicular steroidogenesis. Animals treated with high doses of Cd (0.2 and 0.3 mg/100g BW) showed a significant decrease in serum testosterone (T) level, but a significant induction of testicular lipid peroxidation levels. TUNEL assay showed that low doses of Cd (0.13 and 0.15 mg/100g BW) exhibited typical characteristics of apoptosis while high doses of Cd caused more necrosis than apoptosis. In contrast, supplementation with ascorbic acid reduced testicular lipid peroxidation levels. Ascorbic acid supplementation restored testicular 3beta-hydroxysteroiddehydrogenase (HSD) and 17beta-HSD enzyme activities, 3beta-HSD and cytochrome P450 side chain cleavage (P450(scc)) mRNA levels and serum T concentration to normal in Cd-administered rats. Moreover, administration of ascorbic acid prevented germ cell apoptosis as demonstrated by the reduced number of TUNEL-positive cells in germinal epithelium and inhibited Cd-induced necrosis. These results indicate that ascorbic acid have protective roles in vivo on the Cd-induced overall testicular damage including impaired steroidogenesis and germ cell death possibly through scavenging the reactive oxygen species generated by Cd administration.
Assuntos
Apoptose/efeitos dos fármacos , Ácido Ascórbico/farmacologia , Cádmio/toxicidade , Hormônios Esteroides Gonadais/biossíntese , Espermatozoides/efeitos dos fármacos , Testículo/efeitos dos fármacos , 17-Hidroxiesteroide Desidrogenases/genética , 17-Hidroxiesteroide Desidrogenases/metabolismo , 3-Hidroxiesteroide Desidrogenases/genética , 3-Hidroxiesteroide Desidrogenases/metabolismo , Animais , Enzima de Clivagem da Cadeia Lateral do Colesterol/genética , Enzima de Clivagem da Cadeia Lateral do Colesterol/metabolismo , Hormônios Esteroides Gonadais/genética , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Malondialdeído/análise , Ratos , Espermatozoides/metabolismo , Testículo/citologia , Testículo/metabolismo , Testosterona/biossíntese , Testosterona/sangue , Testosterona/genéticaRESUMO
Recently, we have identified three distinct types of gonadotropin-releasing hormone receptor (GnRHR) in the bullfrog (designated bfGnRHR-1, bfGnRHR-2, and bfGnRHR-3). In the present study, we compared G protein coupling preference of mammalian and nonmammalian GnRHRs. In a transient expression system, stimulation of either bfGnRHRs or rat GnRHR by GnRH significantly increased both inositol phosphates (IP) and cAMP productions, but ratios of IP to cAMP induction levels were quite different among the receptors, indicating differential G protein coupling preference. Using cAMP-dependent protein kinase A (PKA)-specific (CRE-luc) or protein kinase C (PKC)-specific reporter (c-fos-luc) systems, we further examined G(s) and G(q/11) coupling preference of these GnRHRs. Since activities of CRE-luc and c-fos-luc were highly dependent on cell types, GnRH-induced CRE-luc or c-fos-luc activity was normalized by forskolin-induced CRE-luc or 12-O-tetradecanoylphenol-13-acetate (TPA)-induced c-fos-luc activity, respectively. This normalized result indicated that bfGnRHR-2 couples to G(s) more actively than G(q/11), while bfGnRHR-1 and -3 couple to G(s) and G(q/11) with similar strength. However, the rat GnRHR appeared to couple to G(q/11) more efficiently than G(s). This study was further confirmed by an experiment in which GnRH augmented CRE-driven luciferase activity in alphaT3-1 cells when CRE-luc was cotransfected with bfGnRHRs but not with vehicle or rat GnRHR. Collectively, these results indicate that mammalian and nonmammalian GnRHRs may induce diverse cellular and physiological responses through differential activation of PKA and PKC signaling pathways.
Assuntos
Proteínas Heterotriméricas de Ligação ao GTP/metabolismo , Receptores LHRH/metabolismo , Animais , Galinhas , AMP Cíclico/análise , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Genes Reporter , Genes fos , Células HeLa , Humanos , Fosfatos de Inositol/química , Luciferases/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Proteína Quinase C/metabolismo , Rana catesbeiana , Ratos , Receptores LHRH/química , Transdução de Sinais , Fatores de Tempo , Transcrição GênicaRESUMO
We previously demonstrated the presence of three distinct types of the gonadotropin-releasing hormone receptor (GnRHR) in a bullfrog (denoted bfGnRHR-1, bfGnRHR-2, and bfGnRHR-3). The bfGnRHRs exhibited differential tissue distribution and ligand selectivity. In the present study, we demonstrated the desensitization and internalization kinetics of these receptors in both transiently-transfected HEK293 cells and retrovirus-mediated stable cells. The time-course accumulation of the inositol phosphate in response to GnRH revealed that bfGnRHR-1 and -2 were rapidly desensitized, whereas bfGnRHR-3 was slowly desensitized. A comparison of the internalization kinetics revealed the most rapid rate and highest extent of internalization of bfGnRHR-2 among the three receptors. Interestingly, the mechanisms that underlie the receptor internalization appear to differ from each other. Internalization of bfGnRHR-1 was dependent on both dynamin and beta-arrestin, whereas those of bfGnRHR-2 and -3 were dependent on dynamin, but not on arrestin. These results, therefore, suggest that differential regulatory mechanisms for desensitization and internalization of the GnRHR are involved in diverse cellular and physiological responses to GnRH stimulation.
Assuntos
Receptores LHRH/metabolismo , Animais , Arrestinas/metabolismo , Linhagem Celular , Dinaminas/metabolismo , Humanos , Rana catesbeiana/metabolismo , Receptores LHRH/classificação , beta-ArrestinasRESUMO
Recently, we identified three types of non-mammalian gonadotropin-releasing hormone receptors (GnRHR) in the bullfrog (designated bfGnRHR-1-3), and a mammalian type-II GnRHR in green monkey cell lines (denoted gmGnRHR-2). All these receptors responded better to GnRH-II than GnRH-I, while mammalian type-I GnRHR showed greater sensitivity to GnRH-I than GnRH-II. In the present study, we designed new GnRH-II analogs and examined whether they activated or inhibited non-mammalian and mammalian type-II GnRHRs. [D-Ala6]GnRH-II, with D-Ala substituted for Gly6 in GnRH-II, increased inositol phosphate (IP) production in cells stably expressing non-mammalian GnRHRs more effectively than native GnRH-II. However, it exhibited lower activity for mammalian type-I GnRHR than GnRH-I itself. Trptorelix-1, a GnRH-II antagonist, inhibited GnRH-induced IP production in cells expressing non-mammalian GnRHRs more effectively than Cetrorelix, a GnRH-I antagonist. Trptorelix-1, however, had lower potency for mammalian type-I GnRHR than Cetrorelix. Ligand-receptor binding assays revealed that [D-Ala6]GnRH-II and Trptorelix-1 have higher affinities for non-mammalian GnRHRs but lower affinities for mammalian type-I GnRHR than GnRH-II and Cetrorelix, respectively. Moreover, [D-Ala6]GnRH-II and Trptorelix-1 had a higher affinity for gmGnRHR-2 than GnRH-II and Cetrorelix, respectively. These results indicate that [D-Ala6]GnRH-II and Trptorelix-1 are highly effective agonist and antagonist, respectively, for non-mammalian and type-II mammalian GnRHRs.