RESUMO
Exon 1 of the human luteinizing hormone receptor (LHR) gene coding region exhibits at least two forms of sequence heterogeneity between 37 and 60 bp, spanning the junction of the signal peptide and the amino terminus of the mature protein. The LHR 1 differs from the LHR 2 by the insertion of 6 bp in exon 1 but is of identical sequence in the 5' flanking region. RFLP analysis of the two haplotypes within a random population of 63 individuals revealed allele frequencies of 0. 37 and 0.63 for LHR 1 and LHR 2, respectively. 94% of the samples contained at least one LHR 2 allele, whereas only 68% contained the LHR 1 allele. No gender differences were observed, and both homozygotes and heterozygotes displayed apparently normal reproduction. Reverse-transcriptase polymerase chain-reaction analyses of LHR mRNA from testes and ovaries revealed that both haplotypes are transcribed in normal individuals, with no difference in tissue specific distribution. Thus, at least two functional polymorphic forms of exon 1 coding region of the same LHR gene are present in a random human population.
Assuntos
Éxons/genética , Frequência do Gene , Ovário/metabolismo , Receptores do LH/genética , Testículo/metabolismo , Adulto , Alelos , Sequência de Aminoácidos , Animais , Sequência de Bases , Feminino , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Dados de Sequência Molecular , Linhagem , Polimorfismo de Fragmento de Restrição , Sinais Direcionadores de Proteínas/genética , RNA Mensageiro/metabolismo , Ratos , Receptores do LH/metabolismo , Análise de Sequência de DNARESUMO
A novel human luteinizing hormone receptor (LHR) gene was isolated from a human placental genomic library. This gene (Gene II) differs from that previously isolated from a lymphocyte library (Gene I) by several base changes in the 5' flanking region and the deletion of 6 nucleotides in the coding region (+55 to +60). The sequence of the exon 1 coding region of gene II conforms to the sequence of the human ovarian LHR cDNA. Furthermore, primer extension and reporter gene analyses indicate that both the Gene II promoter and the human ovarian LHR mRNA transcriptional start sites are located within the -176 bp TATA-less 5' flanking domain. Additional upstream transcriptional start sites (> -176 bp) were identified in human testicular mRNA and the human choriocarcinoma JAR cell. Restriction enzyme analysis verifies that both LHR genes are present in the human genome, and gene dosing reveals four copies of the human LHR in contrast to a single copy in the rat genome. Chromosomal mapping localizes all copies of the human LHR to the chromosome 2p16-21 loci. These studies suggest that tissue-specific LHR promoter utilization and LHR gene expression may be correlated with gene diversity.
Assuntos
Cromossomos Humanos Par 2 , Receptores do LH/biossíntese , Receptores do LH/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Coriocarcinoma/genética , Coriocarcinoma/metabolismo , Mapeamento Cromossômico , Éxons , Feminino , Dosagem de Genes , Genes Reporter , Biblioteca Genômica , Humanos , Linfócitos/metabolismo , Masculino , Ovário/metabolismo , Placenta/metabolismo , Gravidez , Regiões Promotoras Genéticas , Ratos , Proteínas Recombinantes de Fusão/biossíntese , Mapeamento por Restrição , Testículo/metabolismo , Células Tumorais Cultivadas , Neoplasias Uterinas/genética , Neoplasias Uterinas/metabolismoRESUMO
The forskolin-induced steroidogenic block of testosterone production residing beyond pregnenolone synthesis in rat Leydig cells was localized to the level of the 17beta-hydroxysteroid dehydrogenase (17betaHSD) reaction in this study. The use of forskolin analogs that discriminate between the diterpene's inhibitory effect on the glucose transporter(s) (1,9-dideoxyforskolin) and its activation of adenylate cyclase (6-aminoethyl carbamyl forskolin) revealed that the block is related to inhibition of glucose transporter(s). 1,9-Dideoxyforskolin, but not 6-aminoethyl carbamyl forskolin, caused a significant inhibition of basal and hCG-stimulated testosterone production with accumulation of androstenedione. Glucose-deficient media produced the same metabolic block in the absence of forskolin, with a significant reduction in 17betaHSD activity and increases in the apparent Km for androstenedione. In contrast, metabolic steps before testosterone formation were not affected. Glucose-induced 17betaHSD activation was mimicked by the addition of ATP or GTP in glucose-deficient media, but not by nonhydrolyzable triphosphate analogs or NADPH. A decrease in 17betaHSD activity caused by KT-5720, a specific inhibitor of protein kinase A and the calmodulin antagonist W-7, indicates that the ATP requirement may be related to the participation of protein kinases in the activation of 17betaHSD. ATP levels derived from alternative (nonglycolytic) pathways are adequate to support basal and hormone-stimulated enzymatic activities in the metabolism of cholesterol to androstenedione. However, the integrity of the glucose transport system with subsequent ATP generation is required for activation of 17betaHSD in the final step of androgen biosynthesis. In conclusion, the conversion of androstenedione to testosterone requires the contribution of the glycolytic pathway to meet ATP requirements for 17betaHSD activity.
Assuntos
Trifosfato de Adenosina/metabolismo , Hidroxiesteroide Desidrogenases/metabolismo , Células Intersticiais do Testículo/enzimologia , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Aminoglutetimida/farmacologia , Animais , Gonadotropina Coriônica/farmacologia , Colforsina/análogos & derivados , Colforsina/farmacologia , Ativação Enzimática , Masculino , Proteínas de Transporte de Monossacarídeos/metabolismo , Ratos , Ratos Sprague-Dawley , Testosterona/biossínteseRESUMO
Studies of the regulation of androgen synthesis in steroidogenic cells have focused on both transcriptional and post-translational regulation of the proteins that catalyze these reactions: the P450c17 that catalyzes the production of DHEA or androstenedione in consecutive hydroxylase and lyase activities, and the 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) that catalyzes the conversion of androstenedione to testosterone. Our studies of the regulation of the CYP17 lyase activity at the molecular level have utilized species- and tissue-specific differences to identify target regulatory sequences. Adenovirus infection of rat CYP17 promoter/luciferase reporter gene constructs in primary cultures of rat adrenal and rat Leydig cells revealed a rat-specific domain between-1 and -108 bp that cause inhibition of both basal and cAMP-induced CYP17 transcription in the adrenal, but not the Leydig cell. In contrast, similar promoter constructs from other species exhibited substantial cAMP-induced transcriptional activity in the rat adrenal. Mutagenesis of the conserved region of the rat and human proteins reveals significant differences in the amino acid domains required for hydroxylase and lyase activities within and between the two species, consistent with their differential regulation of lyase activity. The 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) reaction requires a viable glucose transporter system for optimal activity, and a high-energy phosphate was discovered to be the requisite product of glucose metabolism in 17 beta-HSD activation. These studies have provided insight into potential mechanisms of control of androgen synthesis in the late steroidogenic pathway, at the transcriptional and post-translational levels.
Assuntos
17-Hidroxiesteroide Desidrogenases/genética , 17-Hidroxiesteroide Desidrogenases/metabolismo , Androgênios/biossíntese , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Esteroide 17-alfa-Hidroxilase/genética , Esteroide 17-alfa-Hidroxilase/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Clonagem Molecular , Sequência Conservada , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição Fushi Tarazu , Proteínas de Homeodomínio , Humanos , Dados de Sequência Molecular , Mutação , Processamento de Proteína Pós-Traducional , Ratos , Receptores Citoplasmáticos e Nucleares , Homologia de Sequência de Aminoácidos , Fator Esteroidogênico 1 , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
Isolation and structural characterization of the rat corticotropin releasing factor receptor (CRFR) gene was performed to determine the exon/intron organization of the coding region and the potential for splice variants. The CRFR gene contains 13 exons and 12 introns, and the positions of the exon/intron junctions are similar to those of other Class II G protein-coupled receptor genes including the parathyroid hormone and glucagon receptors. The promoter resides within 593 base pairs of the initiation codon and the major transcriptional start site at nucleotide -238. This domain does not possess a TATA box but contains multiple Sp1 and AP-2 sites upstream and downstream of the major transcriptional start site. Intron junctions were identified in the extracellular, transmembrane (TM), and cytoplasmic (C) domains of the CRFR, giving the potential for differential signal transduction by splice variants. CRFR cDNAs derived from rat Leydig cell mRNA included the pituitary Form A, which spans exons 1-13, and two splice variants with deletion of exon 3 or exons 7, 11, and 12. An evolutionary link between the intronless TM/C module of the glycoprotein hormone receptors and the intron-containing TM/C module of the CRFR is suggested by the common position of the luteinizing hormone receptor Form D alternate acceptor splice site and the CRFR intron 12.
Assuntos
Regiões Promotoras Genéticas , Receptores de Hormônio Liberador da Corticotropina/genética , Processamento Alternativo , Animais , Sequência de Bases , Evolução Biológica , Éxons , Variação Genética , Humanos , Íntrons , Fígado/metabolismo , Dados de Sequência Molecular , Família Multigênica , Ratos , Receptores de Hormônio Liberador da Corticotropina/biossíntese , Receptores de Hormônio Liberador da Corticotropina/química , Receptores de Glucagon/química , Receptores de Hormônios Paratireóideos/química , Deleção de Sequência , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Transcrição GênicaRESUMO
The functional importance of cysteine residues in the extracellular domain and the extracellular loops (EL1 and EL2) to hormone binding of the rat luteinizing hormone receptor (LHR) was investigated. For this purpose, cysteines in the seven-transmembrane holoreceptor (Form A) and its hormone-binding splice variant (Form B) were replaced by serine residues, and mutant receptors were expressed in COS1 and/or insect cells. Within the extracellular domain, individual replacement of all four cysteines from Exon 1 abolished hormone binding activity, and replacement of Cys-109 and Cys-134 from exons 5 and 6 caused a 75% decrease in both cell surface and total cellular solubilized LHR hormone binding activity. Mutations of Cys-257 and -258 (Exon 9), Cys-321 and -331, and Cys-417 and -492 of EL1 and EL2, respectively (Exon 11), showed no surface hormone binding activity on intact cells, but exhibited wild type levels of total hormone binding activity when recovered from detergent-solubilized cellular extracts. This finding indicated that expression of high affinity LHR binding activity at the cell surface is independent of the acquisition of the high affinity binding conformation. Other cysteine residues, including Cys-282 (exon 10), and Cys-314 (exon 11) were not essential for hormone binding activity or plasma membrane insertion. This study demonstrates that the functional hormone binding domain utilizes all cysteines N-terminal to exon 7 and localizes the binding site to this N-terminal region of the extracellular domain.
Assuntos
Gonadotropina Coriônica/metabolismo , Cisteína , Éxons , Ovário/metabolismo , Estrutura Secundária de Proteína , Receptores do LH/genética , Receptores do LH/metabolismo , Processamento Alternativo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Western Blotting , Linhagem Celular , Membrana Celular/metabolismo , Chlorocebus aethiops , Feminino , Variação Genética , Humanos , Cinética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Reação em Cadeia da Polimerase , Ratos , Receptores do LH/química , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Spodoptera , TransfecçãoRESUMO
The contribution of N-linked glycosylation to the ligand binding activity of the rat luteinizing hormone receptor (LHR) was studied in wild-type and mutant LHR expressed in mammalian (COS1) cells and overexpressed in insect (Sf9) cells. The binding affinities of the holoreceptor and its truncated splice variant (form B) lacking the transmembrane domain were equivalent in both cell lines. Tunicamycin-treated transfected Sf9 cells expressed a carbohydrate-free LH receptor that lacked hormone binding activity. Functional carbohydrate chains essential for binding activity were localized to glycosylation sites at Asn-173 and Asn-152. Glycosidase treatment of the double mutant N173Q/N152Q revealed the presence of at least one additional carbohydrate chain at Asn-269, Asn-277, or Asn-291 that does not contribute to hormone binding. Asn-77 was not glycosylated, but its mutation to Gln reduced hormone binding. LHR expressed in insect cells contained only high mannose carbohydrate chains, and those located at Asn-173 and Asn-152 were sufficient for high-affinity hormone binding. Enzymatic cleavage of glycosyl chains indicated that only the proximal N-acetylglucosamine residue, which is common to high mannose and complex carbohydrate forms, is necessary for acquisition of the high affinity conformation of the receptor. The carbohydrate chains of the LHR appear to be involved in intramolecular folding of the nascent receptor rather than in its interaction with the hormone.
Assuntos
Ovário/metabolismo , Estrutura Secundária de Proteína , Receptores do LH/química , Receptores do LH/metabolismo , Processamento Alternativo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Ligação Competitiva , Configuração de Carboidratos , Sequência de Carboidratos , Linhagem Celular , Chlorocebus aethiops , Gonadotropina Coriônica/metabolismo , Feminino , Glicosídeo Hidrolases , Glicosilação , Humanos , Rim , Cinética , Ligantes , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oligossacarídeos/química , Ratos , Receptores do LH/biossíntese , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Spodoptera , TransfecçãoRESUMO
Studies of the mechanisms controlling the expression of the rat luteinizing hormone receptor gene were pursued by characterization of the gene structure and identification of regulatory protein binding domains in the 5'-non-coding region of the gene and of 3' non-coding functional domains responsible for generation of the major mRNA forms. The coding region of the rat LHR gene contains 10 introns and 11 exons, of which the first 10 exons comprise the hormone binding extracellular domain and exon 11, the seven transmembrane/G protein coupling module. Several alternative spliced variants of the LHR were identified that conform to deletions of complete and/or partial exons. Within the 6.2 kb of the 3'-non-coding region, two functional LHR pA domains (H1) and (H2) produce two sets of major mRNA transcripts, each coding for both holoreceptor and the form B splice variant. The H1 pA domain is unique to LHR and may represent a recombinant insertion domain. The functional efficiency of each pA domain is related to the specific pA signals, distal downstream elements, and tissue-specific factors. A TATA-less promoter region is present within the 173 bp 5'flanking region of the gene, with Initiator (Inr) elements at transcriptional start sites. Transcription is dependent on the binding of the Sp1 protein at two Sp1 domains that each contribute equally to transcript initiation. Promoter activity is regulated by at least three additional DNA domains, R (-1266 to -1307 bp), C-box (-42 to -73 bp) and M1 (-24 to -42 bp) that bind multiple trans-factors in a tissue-specific manner. Basal promoter activity is enhanced by a functional M1 domain in LHR-expressing mouse Leydig tumor cells (MLTC) but not in non-expressing CHO cells. C-box binding factors either inhibit promoter activity or block inhibition through overlapping but not identical DNA binding domains that carry AP-2 and NF-1 elements. Removal of the AP-2 element within the C-box results in MLTC-specific transcriptional activation that may involve an MTLC M1/C-box interaction. In addition, competition for C-box factors by an upstream regulatory element (R) that is only inhibitory in CHO cells, indicates that both C-box binding factors compete for this upstream (R) domain in a tissue-specific manner. Competition between the inhibitory and neutral DNA binding factors within both upstream (R) and promoter domains (C-box) could provide a mechanism for the control of LH receptor gene expression in gonadal cells.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Receptores do LH/genética , Animais , Sequência de Bases , Sítios de Ligação , Proteínas de Ligação a DNA/metabolismo , Genes , Humanos , Dados de Sequência Molecular , Regiões Promotoras Genéticas , RNA Mensageiro/genética , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
Transcription of the luteinizing hormone receptor gene is dependent on Sp1-induced promoter activation from two Sp1 binding domains (Sp1(2), and Sp1(4)) within the 173-base pair promoter. Of the two Sp1 binding domains, the canonical GC box (GGGCGG) was determined by mutation to be the binding element for only the Sp1(2) domain. The Sp1 binding element within the Sp1(4) domain was identified by mutation and immunological/competition studies as the 5'-GGG GTG GGG that conforms to a Zif-268 like three zinc finger binding domain, rather than the canonical 3' Sp1(4) GC box (GGGCGG). The guanines in the third trinucleotide (GGG GTG GGG) were not required for Sp1 binding, although they increased binding affinity. Non-Sp1 protein(s) bind the 3' Sp1(4) GC box, and by themselves exhibit transcriptional activity. Tissue specific differences were localized to this non-Sp1 binding domain, which functionally substituted for the downstream activating M1 regulatory domain in non-expressing but not in expressing cells. Mutations of both non-Sp1 and M1 domains were required for inhibition of promoter activity in constructs that retained the Sp1 binding elements in non-expressing cells, indicating that together these domains may play a role in regulation of luteinizing hormone receptor gene expression.
Assuntos
Regiões Promotoras Genéticas , Receptores do LH/biossíntese , Receptores do LH/genética , Fator de Transcrição Sp1/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Ligação Competitiva , Primers do DNA , Cinética , Luciferases/biossíntese , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Reação em Cadeia da Polimerase , Ratos , Proteínas Recombinantes de Fusão/biossíntese , Sequências Reguladoras de Ácido Nucleico , Transcrição Gênica , Dedos de ZincoRESUMO
A homology model of the rat 17 alpha-hydroxylase-17,20 desmolase (CYP17) steroid binding domain was derived from the alpha/beta F supersecondary structural element of the 3 alpha/20 beta hydroxysteroid dehydrogenase (HSD) of Streptomyces hydrogenans that constitutes a major segment of the C19 steroid binding cavity. A CYP17 arginine-rich domain, including Arg346, Arg361 and Arg363, that has previously been shown to be important to CYP17 catalytic activity, is conserved in this HSD structural element between two HSD domains known to be important to C19 steroid binding. These two HSD motifs, in addition to a C-terminal domain at the apex of the steroid binding cavity, are also present in similar though not identical forms in the rat CYP17 sequence. The model was evaluated in terms of both hydroxylase/lyase activity and stability of CYP17 mutant proteins (Tyr334Phe, Phe343Ile, Arg357Ala, Arg361Ala, Asp345Ala), and further tested with mutagenesis of Glu353, Glu358, and Tyr431. Those amino acids located at folding junctions in the model steroid binding domain (Glu358, Arg361, and Tyr431) are each individually required to prevent degradation of the nascent protein, as well as for basic hydroxylase/lyase activity. Genomic analysis of the rat CYP17 gene reveals that this domain is contained in exon 6, and a correlation exists between the length of exon 6 and the boundaries of the HSD supersecondary element. These studies demonstrate that exon 6 of the rat CYP17 is essential for CYP17 activity, and may be structurally related to the NAD-linked prokaryote alpha/beta F supersecondary element.
Assuntos
Aldeído Liases/química , Simulação por Computador , Sistema Enzimático do Citocromo P-450/química , Modelos Químicos , Homologia de Sequência de Aminoácidos , Aldeído Liases/genética , Aldeído Liases/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Linhagem Celular , Sequência Conservada/genética , Cortisona Redutase/química , Cortisona Redutase/genética , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Éxons/genética , Expressão Gênica , Microssomos/enzimologia , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Pregnenos/metabolismo , Estrutura Secundária de Proteína , Ratos , Esteroide 17-alfa-Hidroxilase , Streptomyces/enzimologia , Relação Estrutura-AtividadeRESUMO
The functional importance of specific protein binding domains on transcription within the GC-rich 173-base pair promoter of the luteinizing hormone receptor gene was studied by mutagenesis and gel retardation analysis. Transcription was dependent on the presence of two Sp1 elements in the promoter domain of transfected expressing mouse Leydig tumor cells (mLTC) and nonexpressing Chinese hamster ovary cells. Mutation of two protein binding domains located downstream of the Sp1 elements (M1 and C-box) revealed tissue-specific regulation of promoter activity by each domain. Also, gel retardation studies indicated the presence of multiple trans factors that bind to the C-box and M1 domains. Removal of the AP-2 element from the C-box resulted in mLTC-specific transcriptional activation that may involve an M1/C-box interaction. In addition, competition by overlapping NF-1 and AP-2 elements was demonstrable in both the C-box and upstream R domain for separate trans factors that exhibit neutral or inhibitory functions, respectively. Competition between the inhibitory and neutral DNA binding factors within both upstream and promoter domains may be responsible for a mechanism that controls the on/off state of luteinizing hormone receptor gene expression in gonadal cells. These studies reveal a complex pattern of transcriptional regulation that may reflect targeted mechanisms for the control of luteinizing hormone receptor gene expression.
Assuntos
Regiões Promotoras Genéticas , Receptores do LH/biossíntese , Receptores do LH/genética , Sequências Reguladoras de Ácido Nucleico , Fatores de Transcrição/metabolismo , Transcrição Gênica , Animais , Sequência de Bases , Sítios de Ligação , Células CHO , Sequência Consenso , Cricetinae , DNA/isolamento & purificação , DNA/metabolismo , Dados de Sequência Molecular , Mutagênese , Oligodesoxirribonucleotídeos , Reação em Cadeia da Polimerase , Ratos , Homologia de Sequência do Ácido Nucleico , TransfecçãoRESUMO
We present 6.2 kb of the 3'-noncoding region sequence of the rat luteinizing hormone receptor (LHR) gene and identification of two functional polyadenylation (pA) domains, H1 (nt 2368-2491) and H2 (nt 5579-5768) responsible for 3'-end processing of the 2.6/2.3 kb and the 5.8 kb LHR mRNA, respectively. Two identical copies of pA elements AAUAUA in H1 and of AAUAAA in H2 account for micro-heterogeneous poly(A) addition at each of the two pA regions. Both LH holoreceptor and major splice variant form B (lacking the first 266 bp of exon 11) are identified in H1-terminated (2.6 kb and 2.3 kb) and H2-terminated (5.8 kb) mRNA transcripts. A rodent repetitive DNA LINE R domain 3' of H1 within the major 5.8 kb species and a B2 element downstream of H2 were identified. Alignment of the 3'-noncoding region of LHR with TSH, FSH and beta 2-adrenergic receptors indicate that H1 pA signal is unique to the LHR and may represent an insertion domain.
Assuntos
RNA Mensageiro/biossíntese , Receptores do LH/genética , Processamento Alternativo , Animais , Sequência de Bases , Northern Blotting , Variação Genética , Humanos , Dados de Sequência Molecular , Poli A , Ratos , Receptores Adrenérgicos beta 2/genética , Receptores do LH/biossíntese , Mapeamento por Restrição , Homologia de Sequência do Ácido NucleicoRESUMO
Site-directed mutagenesis of a domain (amino acids 343-348) within the conserved region of rat CYP17 was performed to investigate species-specific differences between rat and human/bovine delta 4-versus delta 5-lyase activity. This domain displays substantial deviations between the rat and human/bovine/pig sequences and includes Arg346, which is known to be essential for delta 4-lyase (Kitamura, M., Buczko, E., and Dufau, M. L. (1991) Mol. Endocrinol. 5, 1373-1380) and delta 5-lyase activities. Analysis of the delta 4-lyase activity of mutant rat CYP17 cDNA expressed in nonsteroidogenic COS-1 cells revealed that substitution of Phe at position 343 in the rat with Ile of the human/bovine sequence resulted in a reduction in delta 4-lyase activity to levels in the range of the delta 5-supported reaction. This Phe343-->Ile mutant CYP17 did not exhibit changes either in delta 5-supported lyase activity or in delta 4- and delta 5-hydroxylase activities. Substitution of Asn344, Ser347, and His348 in rat CYP17 with the corresponding bovine amino acids Ser, Asn, and Arg did not enhance this effect. Thus, the reduced activity of the bovine CYP17 delta 4-lyase reaction can be mimicked in part in the rat polypeptide by the substitution of Phe343 with the bovine counterpart, Ile. Unlike the bovine CYP17-catalyzed reaction, the rat Phe343-->Ile mutant exhibited a low level lyase activity (kcat) that did not discriminate between delta 4- and delta 5-substrates. These results suggest that the presence of Phe343 enhances the delta 4-supported lyase activity possibly through stabilization of a delta 4-specific interaction.
Assuntos
Aldeído Liases/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Fenilalanina , Esteroide 17-alfa-Hidroxilase/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Western Blotting , Bovinos , Linhagem Celular , Clonagem Molecular , Humanos , Cinética , Microssomos/enzimologia , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Conformação Proteica , Ratos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Esteroide 17-alfa-Hidroxilase/biossíntese , Esteroide 17-alfa-Hidroxilase/isolamento & purificação , Especificidade por Substrato , TransfecçãoRESUMO
The region of transcriptional activity in the rat luteinizing hormone receptor gene was localized to the 5'-flanking 173-base pair (bp) sequence in transfected mouse Leydig tumor cells and non-expressing Chinese hamster ovarian (CHO) and HeLa cells. Repression of activity in all cell types was induced by at least two domains located between -174 and -990 bp. An additional inhibitory region between -1237 and -2056 bp observed only in CHO and HeLa cells may contribute to the absence of receptor expression in these cells. Analysis of the 173-bp region revealed a promoter domain between -1 and -137 bp containing no TATA or CAAT boxes, but with three SP1 sites and three initiator elements located at transcriptional start sites -14, -19, and -33. Gel retardation studies showed a protein-binding DNA domain about 30 bp upstream of the transcriptional start sites and an adjacent domain containing an SP1 element that are required for maximal activity in all cell types and may play a role in basal transcription. A second promoter domain (-120 to -173 bp) with a protein-binding SP1 element had minor activity in Leydig cells but was prominent in CHO and HeLa cells. Leydig cell-specific DNA-binding protein(s) for each of the promoter domains were detected and may be of importance in transcriptional regulation. The control of gene transcription by differential activation of these promoter domains could be involved in hormone-induced regulation of luteinizing hormone receptor expression in the gonads.
Assuntos
Regiões Promotoras Genéticas , Receptores do LH/genética , Sequências Reguladoras de Ácido Nucleico , Animais , Sequência de Bases , Células CHO , Cloranfenicol O-Acetiltransferase/genética , Cricetinae , DNA , Células HeLa , Humanos , Dados de Sequência Molecular , Oligonucleotídeos , Ratos , Fatores de Transcrição/metabolismo , Transcrição GênicaRESUMO
Site directed mutagenesis of the rat ovarian luteinizing hormone (LH) receptor cDNA was performed at each of the six potential N-linked glycosylation sites to determine the effect of putative carbohydrate chains on the activity of the membrane receptor. The conversion of Asn173 to Gln resulted in the total loss of hormone binding to the surface of the transfected cell. Mutant receptors synthesized with substitutions at the remaining potential N-linked glycosylation positions of 77, 152, 269, 277 and 291 revealed no significant change in the hormone affinity. However Asn77Gln and Asn152Gln exhibited significant decreases (approximately 80%) in the number of high affinity hormone binding sites. The changes in hormone binding activity upon elimination of the potential glycosylation sites at 77, 152 and 173 indicate the presence of functional carbohydrate chains at these positions in the rat ovarian LH/hCG receptor.
Assuntos
Mutagênese Sítio-Dirigida , Receptores do LH/metabolismo , Animais , Asparagina , Sítios de Ligação , Configuração de Carboidratos , Linhagem Celular , Feminino , Vetores Genéticos , Glutamina , Glicosilação , Humanos , Ovário/química , Conformação Proteica , Ratos , Receptores do LH/química , Receptores do LH/genética , TransfecçãoRESUMO
Site-directed mutagenesis of the arginine-rich region within the putative active site of the rat testicular P45017 alpha (17 alpha-hydroxylase cytochrome P-450, the product of P450XVII gene) was performed to identify specific amino acids that contribute to either the hydroxylase or lyase activities catalyzed by this enzyme. The conversion of Arg346 to alanine differentially abolished lyase activity without affecting hydroxylase activity, resulting in an accumulation of the 17 alpha-hydroxylated intermediate, and partial lyase activity was recovered by conversion of this mutant to Lys346. Similar results were obtained with the conversion of Arg357 to alanine, although this mutant also diminished hydroxylase activity, and full lyase and hydroxylase activities were recovered with a lysine in this position. Major reductions in hydroxylase activity were apparent with the conversion of Arg363 to Ala, and this inhibition was reversed by a lysine at position 363. In contrast, differential effects were not observed with the mutants Arg361 Ala, Arg361 Lys, or Tyr334Phe. Both mutations at the Arg361 position resulted in a total loss of hydroxylase and lyase activities, and mutation at the Tyr334 position had no effect on either activity. The identification of specific amino acids that are essential for either the hydroxylase or lyase reaction indicates that the steroid substrate-protein interaction changes during the course of the two consecutive reactions and reveals the potential for separation of the two activities by chemical and biological modulators within the active site region of the P45017 alpha.
Assuntos
Aldeído Liases/genética , Sistema Enzimático do Citocromo P-450/genética , Mutagênese Sítio-Dirigida , Esteroide 17-alfa-Hidroxilase/genética , Aldeído Liases/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Northern Blotting , Linhagem Celular , Sistema Enzimático do Citocromo P-450/metabolismo , Cinética , Masculino , Dados de Sequência Molecular , RNA/genética , RNA/isolamento & purificação , Ratos , Proteínas Recombinantes/metabolismo , Esteroide 17-alfa-Hidroxilase/metabolismo , Especificidade por Substrato , Testículo/enzimologia , TransfecçãoRESUMO
The luteinizing hormone (LH)/human chorionic gonadotropin (hCG) receptor gene was isolated from rat liver genomic libraries and spanned at least 75 kilobase pairs from nucleotide -2057 of the 5'-flanking region to the 3'-noncoding end. The structural configuration of the coding region of the LH receptor gene consists of 11 exons separated by 10 introns that are all located within the putative extracellular domain prior to the first transmembrane region. The 5'-noncoding region contains several potential TATA boxes and SP1 promoter binding sites, as well as six palindromic elements, potential intron/exon splice junctions, and two extended open reading frames in frame with the initiation codon. Primer extension studies indicate the presence of multiple transcriptional initiation sites. Truncated forms of the LH/hCG receptor conform to alternative splicing patterns that are consistent either with the deletion of complete exons or alternate acceptor sites within exons. All splice site junctions correspond to known donor and acceptor consensus sequences. Exons 2-8 approximate the regions of the 14 consecutive 20 amino acid repeated motifs present in the LH, thyrotropin-stimulating hormone, and follicle-stimulating hormone receptor cDNAs, with the exception of a six to eight amino acid shift in each motif. Exons 1, 9, 10, and 11, do not conform to this repetitive intronic motif. These exons, however, contain important structural elements including the conserved cysteines (exons 1, 9, 11), the soybean lectin motif (exon 9), the seven-transmembrane domain with cytoplasmic G protein coupling elements (exon 11), and three putative N-linked glycosylation sites (exon 10), consistent with preservation of significant functional domains within single exons. Exon 10 and the beginning of exon 11 along with the lectin motif are unique to the LH receptor and may be involved in specific association with the hormonal ligand. The homologous regions with other members of the glycoprotein receptor family encoded by exons 2-8, and the common amino acid motif that contains 3 conserved cysteines immediately prior to the first transmembrane region, may be involved in common hormonal interactions and in coupling functions, respectively.
Assuntos
Receptores do LH/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Deleção Cromossômica , Clonagem Molecular , Códon , Éxons , Feminino , Biblioteca Genômica , Humanos , Íntrons , Masculino , Dados de Sequência Molecular , Fases de Leitura Aberta , Ovário/fisiologia , Plasmídeos , Ratos , Homologia de Sequência do Ácido Nucleico , Testículo/fisiologiaRESUMO
A rat testicular luteinizing hormone (LH) receptor cDNA containing a 266-base pair deletion resulting in the omission of the 1st transmembrane region and truncation of the open reading frame was isolated using a rat ovarian LH receptor cDNA probe. Comparison of this clone with a restriction fragment from the LH receptor genomic DNA revealed potential alternative splice sites following the consensus sequence TTXCAG that is present at an intron acceptor splice site and also within the next exon, accounting for the specific deletion mutation observed in this cDNA. Expression of the testicular cDNA in COS1 cells resulted in synthesis and secretion of a soluble binding protein with high affinity and specificity for LH and human chorionic gonadotropin. These studies have demonstrated that the LH receptor gene contains intron(s) within the region coding for the extracellular domain of the molecule, which determine the nature and generation of LH receptor isoforms. Expression of the soluble form of the LH receptor has indicated that the amino-terminal extracellular region plays a major role in gonadotropin binding. These features of the LH receptor are distinct from those of most other G protein-coupled receptors, which are intronless and contain their binding sites within the transmembrane region rather than the extracellular domain.
Assuntos
Íntrons , Receptores do LH/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Sondas de DNA , Enzimas de Restrição do DNA , Éxons , Feminino , Hormônio Foliculoestimulante/metabolismo , Variação Genética , Hormônio Luteinizante/metabolismo , Masculino , Dados de Sequência Molecular , Ovário/química , Splicing de RNA , Ratos , Receptores do LH/metabolismo , Solubilidade , Testículo/metabolismo , TransfecçãoRESUMO
Agonist-induced changes in expression and mRNA levels of luteinizing hormone (LH) receptors were compared during stimulation of ovarian follicular maturation and luteinization by gonadotropic hormones. Three major species of LH receptor mRNA, 5.8, 2.6 and 2.3 kb, were present throughout differentiation and changed similarly, the 5.8 kb species being consistently more abundant than the smaller forms. The increased expression of plasma-membrane LH receptors in preovulatory follicles and luteinized ovaries and their homologous down-regulation during follicular and luteal desensitization were closely correlated with the steady-state receptor mRNA levels. The reappearance of LH receptors following desensitization during the luteal stage was preceded by an increase in mRNA levels. These studies have demonstrated that the expression of LH receptors during follicular maturation, ovulation and desensitization is related to the prevailing levels of receptor mRNA in the ovary.
Assuntos
Gonadotropina Coriônica/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Gonadotropinas Equinas/farmacologia , Ovário/metabolismo , RNA Mensageiro/genética , Receptores do LH/genética , Animais , Clonagem Molecular , DNA/genética , DNA/isolamento & purificação , Sondas de DNA , Feminino , Ovário/efeitos dos fármacos , Poli A/genética , Poli A/isolamento & purificação , RNA/genética , RNA/isolamento & purificação , RNA Mensageiro/efeitos dos fármacos , Ratos , Receptores do LH/metabolismo , Maturidade SexualRESUMO
Two novel lactogen receptor cDNA clones (2.1 and 1.2 kb) were isolated from a rat ovarian cDNA library. Nucleotide sequence of the 2.1 kb clone codes for a 610 aa receptor (nonglycosylated mol. wgt. 66,000 D) with an extracellular domain, a transmembrane region and an intracellular domain, and exhibited significant overall similarity with the rat liver receptor (310 aa) and both rabbit mammary and human hepatoma receptors (616 and 622 aa). However, the ovarian lactogen receptor sequence contains a unique cytoplasmic domain of 110 aa and consensus sequences for both a tyrosine phosphorylation site and an ATP/GTP type A binding site, and thus has potential for signal transduction and mitogenic activity. The 1.2 kb clone codes for a truncated binding form of 150 aa that is identical with the ovarian long form over only the first 130 residues, and lacks the transmembrane region. Differences between long and short forms of the ovarian lactogen receptors and the truncated liver species may result from alternative splicing. The prolactin holoreceptor gene(s) has the potential for producing several receptor subtypes that differ in tissue-specific expression, size, compartmentalization and mode of signal transduction, and may subserve the divergent functions of prolactin in its several target cells.