RESUMO
Here we describe the results of an interlaboratory test for RT-PCR-based BCR/ABL analysis. The test was organized in two parts. The number of participating laboratories in the first and second part was 27 and 20, respectively. In the first part samples containing various concentrations of plasmids with the ela2, b2a2 or b3a2 BCR/ABL transcripts were analyzed by PCR. In the second part of the test, cell samples containing various concentrations of BCR/ABL-positive cells were analyzed by RT-PCR. Overall PCR sensitivity was sufficient in approximately 90% of the tests, but a significant number of false positive results were obtained. There were significant differences in sensitivity in the cell-based analysis between the various participants. The results are discussed, and proposals are made regarding the choice of primers, controls, conditions for RNA extraction and reverse transcription.
Assuntos
Proteínas de Fusão bcr-abl , Leucemia Mielogênica Crônica BCR-ABL Positiva/diagnóstico , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Sequência de Bases , Biomarcadores Tumorais , Primers do DNA , Proteínas de Fusão bcr-abl/normas , Humanos , Controle de QualidadeRESUMO
Uncoupling protein-3 (UCP3) is considered as an important regulator of energy expenditure and thermogenesis in humans. To get insight into the mechanisms regulating its expression we have cloned and characterized about 5 kb of the 5'-flanking region of the human UCP3 (hUCP3) gene. 5'-RACE analysis suggested a single transcription initiation site 187 bp upstream from the translational start site. The promoter region contains both TATA and CAAT boxes as well as consensus motifs for PPRE, TRE, CRE and muscle-specific factors like MyoD and MEF2 sites. Functional characterization of a 3 kb hUCP3 promoter fragment in multiple cell lines using a CAT-ELISA identified a cis-acting negative regulatory element between -2983 and -982 while the region between -982 and -284 showed greatly increased basal promoter activity suggesting the presence of a strong enhancer element. Promoter activity was particularly enhanced in the murine skeletal muscle cell line C2C12 reflecting the tissue-selective expression pattern of UCP3.
Assuntos
Proteínas de Transporte/genética , Regiões Promotoras Genéticas/fisiologia , Desacopladores , Regiões 5' não Traduzidas , Animais , Sequência de Bases , Células Cultivadas , Cloranfenicol O-Acetiltransferase/genética , Humanos , Canais Iônicos , Proteínas Mitocondriais , Dados de Sequência Molecular , Especificidade de Órgãos , RNA Mensageiro/análise , Proteína Desacopladora 3RESUMO
As a member of the uncoupling protein family, UCP2 is ubiquitously expressed in rodents and humans, implicating a major role in thermogenesis. To analyze promoter function and regulatory motifs involved in the transcriptional regulation of UCP2 gene expression, 3.3 kb of 5'-flanking region of the human UCP2 (hUCP2) gene have been cloned. Sequence analysis showed that the promoter region of hUCP2 lacks a classical TATA or CAAT box, however, appeared GC-rich resulting in the presence of several Sp-1 motifs and Ap-1/-2 binding sites near the transcription initiation site. Functional characterization of human UCP2 promoter-CAT fusion constructs in transient expression assays showed that minimal promoter activity was observed within 65 bp upstream of the transcriptional start site (+1). 75 bp further upstream (from nt -141 to -66) a strong cis-acting regulatory element (or enhancer) was identified, which significantly enhanced basal promoter activity. The regulation of human UCP2 gene expression involves complex interactions among positive and negative regulatory elements distributed over a minimum of 3.3 kb of the promoter region.
Assuntos
Proteínas de Membrana Transportadoras , Proteínas Mitocondriais , Regiões Promotoras Genéticas , Proteínas/genética , Desacopladores , Animais , Sequência de Bases , Linhagem Celular , Clonagem Molecular , DNA/genética , Primers do DNA/genética , Regulação da Expressão Gênica , Genes Reguladores , Humanos , Canais Iônicos , Dados de Sequência Molecular , Proteínas Recombinantes de Fusão/genética , Transcrição Gênica , Proteína Desacopladora 2RESUMO
Uncoupling proteins (UCPs) are mitochondrial membrane transporters which are involved in dissipating the proton electrochemical gradient thereby releasing stored energy as heat. This implies a major role of UCPs in energy metabolism and thermogenesis which when deregulated are key risk factors for the development of obesity and other eating disorders. Recent studies have shown that the sympathetic nervous system, via norepinephrine (beta-adrenoceptors) and cAMP, as well as thyroid hormones and PPAR gamma ligands seem to be major regulators of UCP expression. From the three different UCPs identified so far by gene cloning UCP1 is expressed exclusively in brown adipocytes while UCP2 is widely expressed. The third analogue, UCP3, is expressed predominantly in human skeletal muscle and was found to exist in a long and a short form. At the amino acid level UCP2 has about 59% homology to UCP1 while UCP3 is 73% identical to UCP2. Both UCP2 and UCP3 were mapped in close proximity (75-150 kb) to regions of human chromosome 11 (11q13) that have been linked to obesity and hyper-insulinaemia. Furthermore, there is strong evidence that UCP2, by virtue of its ubiquitous expression, may be important for determining basal metabolic rate. Based on the published full-length cDNA sequence we have deduced the genomic structure of the human UCP2 (hUCP2) gene by PCR and direct sequence analysis. The hUCP2 gene spans over 8.4 kb distributed on 8 exons. The localization of the exon/intron boundaries within the coding region matches precisely the one found in the human UCP1 gene and is almost conserved in the recently discovered UCP3 gene as well. However, the size of each of the introns in the hUCP2 gene differs from its UCP1 and UCP3 counterparts. It varies from 81 bp (intron 5) to about 3 kb (intron 2). The high degree of homology at the nucleotide level and the conservation of the exon/intron boundaries among the three UCP genes suggests that they may have evolved from a common ancestor or are the result from gene duplication events. Mutational analysis of the hUCP2 gene in a cohort of 25 children of caucasian origin (aged 7-13) characterized by low BMR values revealed a point mutation in exon 4 (C to T transition at position 164 of the corresponding cDNA resulting in the substitution of an alanine residue by a valine at codon 55) and an insertion polymorphism in exon 8. The insertion polymorphism consists of a 45 bp repeat located 150 bp downstream of the stop codon in the 3'-UTR. The allele frequencies were 0.61 and 0.39 for the alanine and valine encoded alleles, respectively, and 0.71 versus 0.29 for the insertion polymorphism. Expression studies of the wildtype and mutant forms of UCP2 should clarify the functional consequences these mutations may have on energy metabolism and body weight regulation. In addition, mapping of the promoter region and the identification of putative promoter regulatory sequences should give insight into the transcriptional regulation of UCP2 expression--in particular by anyone of the above mentioned factors--in vitro and in vivo.
Assuntos
Proteínas de Membrana Transportadoras , Proteínas Mitocondriais , Mutação , Obesidade/genética , Proteínas/genética , Adolescente , Sequência de Aminoácidos , Sequência de Bases , Proteínas de Transporte/genética , Estudos de Casos e Controles , Criança , Mapeamento Cromossômico , Cromossomos Humanos Par 11/genética , Clonagem Molecular , Análise Mutacional de DNA , Primers do DNA/genética , Éxons , Genótipo , Humanos , Íntrons , Canais Iônicos , Proteínas de Membrana/genética , Dados de Sequência Molecular , Mutação Puntual , Polimorfismo Genético , Desacopladores/metabolismo , Proteína Desacopladora 1 , Proteína Desacopladora 2 , Proteína Desacopladora 3RESUMO
Uncoupling proteins (UCPs) are mitochondrial membrane transporters which are involved in dissipating the proton electrochemical gradient thereby releasing stored energy as heat. This implies a major role of UCPs in energy metabolism and thermogenesis which when deregulated are key risk factors for the development of obesity and other eating disorders. From the three different human UCPs identified so far by gene cloning both UCP2 and UCP3 were mapped in close proximity (75-150 kb) to regions of human chromosome 11 (11q13) that have been linked to obesity and hyperinsulinaemia. At the amino acid level hUCP2 has about 55% identity to hUCP1 while hUCP3 is 71% identical to hUCP2. In this study we have deduced the genomic structure of the human UCP2 gene by PCR and direct sequence analysis. The hUCP2 gene spans over 8.7 kb distributed on 8 exons. The localization of the exon/intron boundaries within the coding region matches precisely that of the hUCP1 gene and is almost conserved in the recently discovered hUCP3 gene as well. The high degree of homology at the nucleotide level and the conservation of the exon /intron boundaries among the three UCP genes suggests that they may have evolved from a common ancestor or are the result from gene duplication events. Mutational analysis of the hUCP2 gene in a cohort of 172 children (aged 7 - 13) of Caucasian origin revealed a polymorphism in exon 4 (C to T transition at position 164 of the cDNA resulting in the substitution of an alanine by a valine at codon 55) and an insertion polymorphism in exon 8. The insertion polymorphism consists of a 45 bp repeat located 150 bp downstream of the stop codon in the 3'-UTR. The allele frequencies were 0.63 and 0.37 for the alanine and valine encoded alleles, respectively, and 0.71 versus 0.29 for the insertion polymorphism. The allele frequencies of both polymorphisms were not significantly elevated in a subgroup of 25 children characterized by low Resting Metabolic Rates (RMR). So far a direct correlation of the observed genotype with (RMR) and Body Mass Index (BMI) was not evident. Expression studies of the wild type and mutant forms of UCP2 should clarify the functional consequences these polymorphisms may have on energy metabolism and body weight regulation.
Assuntos
Proteínas de Membrana Transportadoras , Proteínas Mitocondriais , Mutação , Proteínas/genética , Regiões 3' não Traduzidas/genética , Adolescente , Sequência de Aminoácidos , Sequência de Bases , Proteínas de Transporte/genética , Criança , Análise Mutacional de DNA , Éxons/genética , Frequência do Gene , Genótipo , Humanos , Íntrons/genética , Canais Iônicos , Proteínas de Membrana/genética , Dados de Sequência Molecular , Obesidade , Fenótipo , Polimorfismo Genético/genética , Proteínas/química , Homologia de Sequência de Aminoácidos , Regiões Terminadoras Genéticas/genética , Proteína Desacopladora 1 , Proteína Desacopladora 2 , Proteína Desacopladora 3 , População Branca/genéticaRESUMO
OBJECTIVE: The missense mutation (64Trp to 64Arg) in the beta 3-adrenergic-receptor has previously been described to confer a genetic predisposition to the development of obesity. DESIGN: To test the hypothesis we evaluated allele frequencies in children, adolescents and young adults who belonged to different weight groups that were delineated with percentiles for the body mass index (BMI; kg/m2). SUBJECTS: 99 underweight probands (BMI < or = 15th percentile). 80 normal weight probands (BMI: 5th-85th percentile). 238 obese children and adolescents (BMI > or = 97th percentile). 84 patients with anorexia nervosa (AN). MEASUREMENTS: The cohorts were screened by polymerase chain reaction with subsequent restriction fragment length polymorphism (PCR-RFLP) analysis. Data were statistically analysed for association. In addition to these case control studies, the transmission disequilibrium test (TDT) was applied to 80 families of obese probands and to 52 families of patients with AN. RESULTS: Both the tests for association and linkage were negative. The Trp64Arg allele frequencies in the three weight groups (obesity: 0.071; normal weight: 0.081; underweight: 0.056) and the AN patients (0.054) were similar. Extremely obese individuals showed no excess of the Trp64Arg allele. No homozygotes for the Trp64Arg allele were detected. CONCLUSION: Heterozygosity for the Trp64Arg allele is not of major importance in regulation of body weight in individuals younger than 35 y. Additionally, the extreme obese subgroup is not enriched for the polymorphism.
Assuntos
Alelos , Anorexia Nervosa/genética , Peso Corporal , Obesidade/genética , Receptores Adrenérgicos beta/genética , Adolescente , Adulto , Arginina/genética , Índice de Massa Corporal , Criança , Estudos de Coortes , Feminino , Frequência do Gene , Ligação Genética , Humanos , Masculino , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Triptofano/genéticaRESUMO
Several lines of evidence implicate a role for the serotonergic system in body weight regulation and eating disorders. The magnitude and duration of postsynaptic responses to serotonin (5-HT) is directed by the transport into and release from the presynaptic neuron. Recently, a common polymorphism of a repetitive element in the region of the serotonin transporter (5-HTT) gene-linked polymorphic region (5-HTTLPR) was identified that results in a system of two common alleles. The activity of the 5-HTT, as measured in in vitro assays and in human lymphoblastoid cell lines, is dependent on the respective genotype. We thus hypothesized that this polymorphism is relevant for weight regulation in general and is possibly involved in the etiology of anorexia nervosa (AN). Allele frequencies and genotypes were determined in a total of 385 unrelated obese children, adolescents and adults, 112 underweight subjects and 96 patients with AN. Furthermore, both parents of 98 obese children and adolescents and of 55 patients with AN, respectively, were genotyped, thus allowing to test for both association and linkage. The comparison of allele frequencies between obese and underweight probands provided no evidence for a major role of the 5-HTTLPR in weight regulation. Patients with AN had allele frequencies not significantly different to those observed for obese and underweight individuals.
Assuntos
Alelos , Anorexia Nervosa/genética , Peso Corporal/genética , Proteínas de Transporte/genética , Glicoproteínas de Membrana/genética , Proteínas de Membrana Transportadoras , Proteínas do Tecido Nervoso , Polimorfismo Genético , Adolescente , Criança , Estudos de Coortes , Comportamento Alimentar , Feminino , Frequência do Gene , Genótipo , Homozigoto , Humanos , Masculino , Obesidade/genética , Proteínas da Membrana Plasmática de Transporte de SerotoninaRESUMO
Serotonin is a neurotransmitter involved in a large number of psychophysiological processes including the regulation of mood, arousal, aggression, sleep, learning, nociceptions, nerve growth and importantly, appetitive functions. Alterations of 5-HT receptor activity have been shown to occur in many psychiatric diseases including depression, anxiety, eating disorders, schizophrenia etc. Hence, genetic variation in genes coding for serotonin receptor proteins might well be involved in the genetic predisposition to these diseases and therefore are of great pharmacogenetic relevance. Knockout mice deficient of a functional 5-HT2C receptor have implicated a potential role of this receptor subtype in the serotonergic control of appetite. A Cys23Ser mutation in the human 5-HT2C receptor gene discovered recently prompted us to investigate this mutation with regard to the development of human obesity. We have evaluated this mutation in 241 obese children and adolescents (mean BMI > or = 97th percentile), 80 normal weight children (BMI 5th-85th percentile) and 92 underweight probands (BMI < or = 15th percentile) for a possible association with obesity. The frequencies of the mutant allele in all three weight groups (obese subjects: 0.1597; normal weight: 0.168; underweight: 0.1575) were very similar. Association as well as linkage studies were negative. Therefore it is unlikely that this receptor mutation plays a direct role in the development of human obesity.
Assuntos
Peso Corporal/fisiologia , Mutação/genética , Obesidade/genética , Receptores de Serotonina/genética , Adolescente , Adulto , Alelos , Criança , Feminino , Humanos , MasculinoRESUMO
Transcripts of a gene, GIRK4, that encodes for a 419-amino-acid protein and shows high structural similarity to other subfamily members of G-protein-activated inwardly rectifying K+ channels (GIRK) have been identified in the human hippocampus. When expressed in Xenopus oocytes, GIRK4 yielded functional GIRK channels with activity that was enhanced by the stimulation of coexpressed serotonin 1A receptors. GIRK4 potentiated basal and agonist-induced currents mediated by other GIRK channels, possibly because of channel heteromerization. Despite the structural similarity to a putative rat KATP channel, no ATP sensitivity or KATP-typical pharmacology was observed for GIRK4 alone or GIRK4 transfected in conjunction with other GIRK channels in COS-7 cells. In rat brain, GIRK4 is expressed together with three other subfamily members, GIRK1-3, most likely in identical hippocampal neurons. Thus, heteromerization or an unknown molecular interaction may cause the physiological diversity observed within this class of K+ channels.
Assuntos
Hipocampo/química , Proteínas do Tecido Nervoso/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização , Canais de Potássio/metabolismo , Potássio/metabolismo , Trifosfato de Adenosina/farmacologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Química Encefálica , Linhagem Celular Transformada , Chlorocebus aethiops , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G , Proteínas de Ligação ao GTP/metabolismo , Genes , Humanos , Substâncias Macromoleculares , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/isolamento & purificação , Canais de Potássio/genética , Canais de Potássio/isolamento & purificação , Ratos , Receptores de Serotonina/metabolismo , Receptores 5-HT1 de Serotonina , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Serotonina/farmacologia , Especificidade da Espécie , TransfecçãoRESUMO
The basophilic leucaemia cell line RBL-2H3 exhibits a robust inwardly rectifying potassium current, IKIR, which is likely to be modulated by G proteins. We examined the physiological and molecular properties of this KIR conductance to define the nature of the underlying channel species. The macroscopic conductance revealed characteristics typical of classical K+ inward rectifiers of the IRK type. Channel gating was rapid, first order (tau approximately 1 ms at -100 mV) and steeply voltage dependent. Both activation potential and slope conductance were dependent on extracellular K+ concentration ([K+]o) and inward rectification persisted in the absence of internal Mg2+. The current was susceptible to a concentration- and voltage-dependent block by extracellular Na+, Cs+ and Ba2+. Initial IKIR whole-cell amplitudes as well as current rundown were dependent on the presence of 1 mM internal ATP. Perfusion of intracellular guanosine 5'-Q-(3-thiotriphosphate) (GTP[gamma S]) suppressed IKIR with an average half-time of decline of approximately 400 s. It was demonstrated that the dominant IRK-type 25 pS conductance channel was indeed suppressed by 100 microM preloaded GTP[gamma S]. Reverse transcriptase-polymerase chain reactions (RT-PCR) with RBL cell poly(A)+ RNA identified a full length K+ inward rectifier with 94% base pair homology to the recently cloned mouse IRK1 channel. It is concluded that RBL cells express a classical voltage-dependent IRK-type K+ inward rectifier RBL-IRK1 which is negatively controlled by G proteins.
Assuntos
Mastócitos/fisiologia , Canais de Potássio/fisiologia , Trifosfato de Adenosina/farmacologia , Sequência de Aminoácidos , Animais , Bário/farmacologia , Sequência de Bases , Césio/farmacologia , Ácido Edético/farmacologia , Condutividade Elétrica , Guanosina 5'-O-(3-Tiotrifosfato)/farmacologia , Ativação do Canal Iônico/fisiologia , Cinética , Leucemia Basofílica Aguda , Magnésio/farmacologia , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Potássio/farmacologia , Canais de Potássio/química , Canais de Potássio/genética , Ratos , Sódio/farmacologia , Células Tumorais CultivadasRESUMO
Substantial pharmacological evidence in vitro and in vivo has suggested the existence of subtypes of the kappa opioid receptor. Quantitative radioligand binding techniques resolved the presence of two high affinity binding sites for the kappa 1 ligand [3H]U69,593 in mouse brain membranes, termed kappa 1a and kappa 1b, respectively. Whereas the kappa 1a site has high affinity for fedotozine and oxymorphindole and low affinity for bremazocine and alpha-neoendorphin, site kappa 1b has high affinity for bremazocine and alpha-neoendorphin and low affinity for fedotozine and oxymorphindole. CI-977 and U69,593 bind equally well at both sites. To determine the relationship between these kappa 1 receptor subtypes and the recently cloned mouse kappa 1 receptor (KOR), we examined [3H]U69,593 binding to the KOR in stably transfected cells (KORCHN-8). Competition of [3H]U69,593 binding to the KOR by bremazocine, alpha-neoendorphin, fedotozine and oxymorphindole resolved a single class of binding sites at which these agents had binding affinities similar to that of the kappa 1b site present in mouse brain. These results suggest that the cloned KOR corresponds to the kappa 1 site in mouse brain defined as kappa 1b.
Assuntos
Receptores Opioides kappa/efeitos dos fármacos , Animais , Ligação Competitiva/fisiologia , Clonagem Molecular , Masculino , Camundongos , Camundongos Endogâmicos ICR , Ensaio Radioligante , Receptores Opioides kappa/análiseRESUMO
To investigate a possible genetic basis for reported differences in beta-2 receptor expression in atopic subjects, DNA from 42 atopic children (22 asthmatics and 22 with allergic rhinitis) and 30 non-atopic subjects was Southern blotted and Ban-1 restriction fragment polymorphisms (RFLPS) were studied using a 2.6 kb probe of the human beta-2 receptor gene. Two alleles 3.1 kb and 2.9 kb were identified. Homozygotes and heterozygotes for the two alleles were found with equal frequency in the atopic patients who had asthma and in those who had allergic rhinitis only. The gene frequencies for the upper and lower alleles were 0.45 and 0.55 respectively. Our studies do not provide evidence for an association between a particular polymorphic form of the human beta-2 receptor gene and atopy.
Assuntos
Hipersensibilidade Imediata/genética , Polimorfismo de Fragmento de Restrição , Receptores Adrenérgicos beta 2/genética , Adulto , Alelos , Asma/genética , Southern Blotting , Criança , Análise Mutacional de DNA , Desoxirribonucleases de Sítio Específico do Tipo II , Frequência do Gene , Predisposição Genética para Doença , Genótipo , Humanos , Hipersensibilidade Imediata/metabolismo , Rinite Alérgica Perene/genética , Rinite Alérgica Sazonal/genéticaRESUMO
alpha-Thrombin stimulates G-protein-coupled effectors leading to secretion and aggregation in human platelets, and to a mitogenic response in CCL39 hamster fibroblasts. alpha-Thrombin receptors can be activated by synthetic peptides corresponding to the receptor sequence starting with serine-42, at the proposed cleavage site. We have previously determined that the agonist domain of receptor-activating peptides resides within the five N-terminal residues [Vouret-Craviari, Van Obberghen-Schilling, Rasmussen, Pavirani, Lecocq and Pouysségur (1992) Mol. Biol. Cell. 3, 95-102], although the 7-residue peptide (SFFLRNP) corresponding to the hamster alpha-thrombin receptor was 10 times more potent than the 5-residue peptide for activation of human platelets. In the present study we have analysed the role of individual amino acids in receptor activation by using a series of modified hexa- or hepta-peptides derived from the human alpha-thrombin-receptor sequence. Cellular events examined here include phospholipase C activation, adenylyl cyclase inhibition and DNA synthesis stimulation in non-transformed CCL39 fibroblasts and a tumorigenic variant of that line (A71 cells). Modification of the peptide sequence had similar functional consequence for each of the assays described, indicating that either a unique receptor or pharmacologically indistinguishable receptor subtypes activate distinct G-protein signalling pathways. Furthermore, we found that: (1) the N-terminal serine can be replaced by small or intermediately sized amino acids (+/- hydroxyl groups) without loss of activity. However, its replacement by an aromatic side-chain or omission of the N-terminal amino group severely reduces activity. (2) An aromatic side-chain on the penultimate N-terminal residue appears to play a critical role since phenylalanine in this position can be substituted by tyrosine without complete loss of activity whereas an alanine in its place is not tolerated. (3) Deletion of the first, second or third N-terminal residue leads to a loss of activity, suggesting that a defined spacing of more than one structural component may be important for ligand-receptor interaction. Finally, we did not observe an antagonistic effect of the inactive peptides on phospholipase C activation or DNA synthesis induced by alpha-thrombin (1 nM) or SFLLRNP (3 microM).
Assuntos
Fosfatos de Inositol/metabolismo , Oligopeptídeos/farmacologia , Receptores de Superfície Celular/efeitos dos fármacos , Trombina/farmacologia , Fosfolipases Tipo C/metabolismo , 1-Metil-3-Isobutilxantina/farmacologia , Inibidores de Adenilil Ciclases , Sequência de Aminoácidos , Animais , Linhagem Celular , Colforsina/farmacologia , Cricetinae , Cricetulus , Replicação do DNA/efeitos dos fármacos , Ativação Enzimática , Fator 2 de Crescimento de Fibroblastos/farmacologia , Humanos , Cinética , Pulmão , Dados de Sequência Molecular , Oligopeptídeos/síntese química , Receptores de Superfície Celular/metabolismo , Receptores de Trombina , Proteínas Recombinantes/farmacologia , Relação Estrutura-Atividade , Trombina/metabolismoRESUMO
Current methods for comparative analyses of protein sequences are 1D-alignments of amino acid sequences based on the maximization of amino acid identity (homology) and the prediction of secondary structure elements. This method has a major drawback once the amino acid identity drops below 20-25%, since maximization of a homology score does not take into account any structural information. A new technique called Hydrophobic Cluster Analysis (HCA) has been developed by Lemesle-Varloot et al. (Biochimie 72, 555-574), 1990). This consists of comparing several sequences simultaneously and combining homology detection with secondary structure analysis. HCA is primarily based on the detection and comparison of structural segments constituting the hydrophobic core of globular protein domains, with or without transmembrane domains. We have applied HCA to the analysis of different families of G-protein coupled receptors, such as catecholamine receptors as well as peptide hormone receptors. Utilizing HCA the thrombin receptor, a new and as yet unique member of the family of G-protein coupled receptors, can be clearly classified as being closely related to the family of neuropeptide receptors rather than to the catecholamine receptors for which the shape of the hydrophobic clusters and the length of their third cytoplasmic loop are very different. Furthermore, the potential of HCA to predict relationships between new putative and already characterized members of this family of receptors will be presented.
Assuntos
Proteínas de Ligação ao GTP/química , Receptores de Superfície Celular/química , Homologia de Sequência de Aminoácidos , Sequência de Aminoácidos , Animais , Análise por Conglomerados , Humanos , Dados de Sequência Molecular , Receptores Adrenérgicos/química , Receptores de Catecolaminas , Receptores de Superfície Celular/fisiologia , Receptores Muscarínicos/química , Receptores de Trombina , Alinhamento de Sequência , Solubilidade , ÁguaRESUMO
Peptide mapping can be used to elucidate further the structural similarities of the benzodiazepine binding proteins in different vertebrate species. Crude synaptic membrane preparations were photoaffinity-labeled with [3H]flunitrazepam and subsequently degraded with various concentrations of trypsin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by fluorography allowed a comparison of the molecular weights of photolabeled peptides in different species. Tryptic degradation led to a common peptide of 40K in all species investigated, a finding indicating that the benzodiazepine binding proteins are structurally homologous in higher bony fishes and tetrapods.
Assuntos
Fragmentos de Peptídeos/metabolismo , Receptores de GABA-A/metabolismo , Membranas Sinápticas/metabolismo , Tripsina/metabolismo , Marcadores de Afinidade , Jacarés e Crocodilos , Animais , Bovinos , Patos , Eletroforese em Gel de Poliacrilamida , Peixes , Flunitrazepam/metabolismo , Cobaias , Iguanas , Peso Molecular , Fotoquímica , Ranidae , Homologia de Sequência do Ácido Nucleico , Serpentes , Especificidade da Espécie , SuínosRESUMO
The presence of hormones, neurotransmitters, their receptors and biosynthetic and degradative enzymes is clearly not only associated with the present and the recent past but with the past several hundred million years. Evidence is mounting which indicates substantial conservation of protein structure and function of these receptors and enzymes over these tremendous periods of time. These findings indicate that the evolution and development of the nervous system was not dependent upon the formation of new or better transmitter substances, receptor proteins, transducers and effector proteins but involved better utilization of these highly developed elements in creating advanced and refined circuitry. This is not a new concept; it is one that is now substantiated by increasingly sophisticated studies. In a 1953 article discussing chemical aspects of evolution (Danielli, 1953) Danielli quotes Medawar, "... endocrine evolution is not an evolution of hormones but an evolution of the uses to which they are put; an evolution not, to put it crudely, of chemical formulae but of reactivities, reaction patterns and tissue competences." To also quote Danielli, "In terms of comparative biochemistry, one must ask to what extent the evolution of these reactivities, reaction patterns and competences is conditional upon the evolution of methods of synthesis of new proteins, etc., and to what extent the proteins, etc., are always within the synthetic competence of an organism. In the latter case evolution is the history of changing uses of molecules, and not of changing synthetic abilities." (Danielli, 1953). Figure 4 outlines a phylogenetic tree together with an indication of where evidence exists for both the enzymes that determine the biosynthesis and metabolism of the cholinergic and adrenergic transmitters and their specific cholinergic and adrenergic receptors. This figure illustrates a number of important points. For example, the evidence appears to show that the transmitters and their associated enzymes existed for a substantial period before their respective receptor proteins. While the transmitters and enzymes appear to exist in single cellular organisms, there is no solid evidence for the presence of adrenergic or cholinergic receptors until multicellular organisms where the receptors appear to be clearly associated with specific cellular and neuronal communication (Fig. 4). One can only speculate as to the possible role for acetylcholine and the catecholamine in single cell organisms.(ABSTRACT TRUNCATED AT 400 WORDS)
Assuntos
Evolução Biológica , Neurônios/fisiologia , Receptores Adrenérgicos/fisiologia , Receptores Colinérgicos/fisiologia , Receptores de Neurotransmissores/fisiologia , Animais , Nucleotídeos de Guanina/fisiologia , Humanos , Neurônios/metabolismo , Receptores Adrenérgicos/metabolismo , Receptores Colinérgicos/metabolismo , Receptores de Neurotransmissores/metabolismo , Homologia de Sequência do Ácido NucleicoRESUMO
Two cDNA clones, lambda RHM-MF and lambda RHB-DAR, encoding the muscarinic cholinergic receptor and the beta-adrenergic receptor, respectively, have been isolated from a rat heart cDNA library. The cDNA clones were characterized by restriction mapping and automated DNA sequence analysis utilizing fluorescent dye primers. The rat heart muscarinic receptor consists of 466 amino acids and has a calculated molecular weight of 51,543. The rat heart beta-adrenergic receptor consists of 418 amino acids and has a calculated molecular weight of 46,890. The two cardiac receptors have substantial amino acid homology (27.2% identity, 50.6% with favored substitutions). The rat cardiac beta receptor has 88.0% homology (92.5% with favored substitutions) with the human brain beta receptor and the rat cardiac muscarinic receptor has 94.6% homology (97.6% with favored substitutions) with the porcine cardiac muscarinic receptor. The muscarinic cholinergic and beta-adrenergic receptors appear to be as conserved as hemoglobin and cytochrome c but less conserved than histones and are clearly members of a multigene family. These data support our hypothesis, based upon biochemical and immunological evidence, that suggests considerable structural homology and evolutionary conservation between adrenergic and muscarinic cholinergic receptors. To our knowledge, this is the first report utilizing automated DNA sequence analysis to determine the structure of a gene.
Assuntos
Receptores Adrenérgicos beta/genética , Receptores Muscarínicos/genética , Sequência de Aminoácidos , Animais , Automação , Sequência de Bases , Dados de Sequência Molecular , Família Multigênica , Miocárdio/análise , Ratos , SolubilidadeRESUMO
Two cDNA clones, lambda-CLFV-108 and lambda-CLFV-119, encoding for the beta-adrenergic receptor, have been isolated from a human brain stem cDNA library. One human genomic clone, LCV-517 (20 kb), was characterized by restriction mapping and partial sequencing. The human brain beta-receptor consists of 413 amino acids with a calculated Mr of 46480. The gene contains three potential glucocorticoid receptor-binding sites. The beta-receptor expressed in human brain was homology with rodent (88%) and avian (52%) beta-receptors and with porcine muscarinic cholinergic receptors (31%), supporting our proposal [(1984) Proc. Natl. Acad. Sci. USA 81, 272 276] that adrenergic and muscarinic cholinergic receptors are structurally related. This represents the first cloning of a neurotransmitter receptor gene from human brain.