RESUMO
Treatment-resistant major depressive disorder remains inadequately treated with currently available antidepressants. Opioid receptors (ORs) are involved in the pathophysiology of depression yet remain an untapped therapeutic intervention. The µ-δ OR heteromer represents a unique signaling complex with distinct properties compared with µ- and δ-OR homomers; however, its role in depression has not been characterized. As there are no ligands exclusively targeting the µ-δ heteromer, we devised a strategy to selectively antagonize the function of the µ-δOR complex using a specific interfering peptide derived from the δOR distal carboxyl tail, a sequence implicated in µ-δOR heteromerization. In vitro studies using a minigene expressing this peptide demonstrated a loss of the unique pharmacological and trafficking properties of δ-agonists at the µ-δ heteromer, with no effect on µ- or δ-OR homomers, and a dissociation of the µ-δOR complex. Intra-accumbens administration of the TAT-conjugated interfering peptide abolished the antidepressant-like and anxiolytic-like actions of the δ-agonist UFP-512 (H-Dmt-Tic-NH-CH(CH2-COOH)-Bid) measured in the forced swim test, novelty-induced hypophagia and elevated plus maze paradigms in rats. UFP-512's antidepressant-like and anxiolytic-like actions were abolished by pretreatment with either µOR or δOR antagonists. Overall, these findings demonstrate that the µ-δ heteromer may be a potential suitable therapeutic target for treatment-resistant depression and anxiety disorders.
Assuntos
Ansiedade/tratamento farmacológico , Depressão/tratamento farmacológico , Núcleo Accumbens/efeitos dos fármacos , Receptores Opioides delta/metabolismo , Receptores Opioides mu/metabolismo , Animais , Ansiolíticos/farmacologia , Antidepressivos , Ansiedade/fisiopatologia , Benzimidazóis/farmacologia , Membrana Celular/metabolismo , Depressão/fisiopatologia , Células HEK293 , Humanos , Masculino , Antagonistas de Entorpecentes/farmacologia , Núcleo Accumbens/fisiopatologia , Oligopeptídeos/farmacologia , Ratos Sprague-Dawley , Receptores Opioides delta/agonistas , Receptores Opioides delta/antagonistas & inibidores , Receptores Opioides delta/genética , Receptores Opioides mu/genética , Estresse PsicológicoRESUMO
Globoid cell leukodystrophy (GLD) is characterized histopathologically by apoptosis of oligodendrocytes, progressive demyelination, and the existence of large, multinuclear (globoid) cells derived from perivascular microglia. The glycosphingolipid, psychosine (d-galactosyl-beta-1,1' sphingosine), accumulates to micromolar levels in GLD patients who lack the degradative enzyme galactosyl ceramidase. Here we document that an orphan G protein-coupled receptor, T cell death-associated gene 8, is a specific psychosine receptor. Treatment of cultured cells expressing this receptor with psychosine or structurally related glycosphingolipids results in the formation of globoid, multinuclear cells. Our discovery of a molecular target for psychosine suggests a mechanism for the globoid cell histology characteristic of GLD, provides a tool with which to explore the disjunction of mitosis and cytokinesis in cell cultures, and provides a platform for developing a medicinal chemistry for psychosine.
Assuntos
Divisão Celular/fisiologia , Metabolismo dos Lipídeos , Oligodendroglia/fisiologia , Psicosina/metabolismo , Receptores de Superfície Celular/metabolismo , Receptores Acoplados a Proteínas G , Cálcio/metabolismo , Linhagem Celular , Separação Celular , AMP Cíclico/metabolismo , Citometria de Fluxo , Proteínas de Ligação ao GTP/metabolismo , Genes Reporter/genética , Humanos , Immunoblotting , Leucodistrofia de Células Globoides/patologia , Leucodistrofia de Células Globoides/fisiopatologia , Microscopia Confocal , Estrutura Molecular , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
The first intracellular loop of the G protein-coupled receptors (GPCRs) is probably the domain that has been studied least. According to the limited data available, mutations of this region can increase, decrease or not affect receptor-G protein coupling, depending on the receptor. Melanocyte-stimulating hormone (MSH) receptors with a Ser69Leu mutation of the first intracellular loop phenotypically confer tobacco color to the coat of mice, and have constitutive activity and enhanced agonist stimulation of adenylyl cyclase. Since the human D1 dopamine receptor (D1DR) has a serine at the equivalent position, we were interested to see if this serine is involved in receptor-G protein coupling in a similar fashion. Our site-directed mutagenesis study showed that the replacement of this serine by leucine (Ser56Leu) in D1DR did not affect the ability of the receptors to bind ligand or couple to G protein.
Assuntos
Adenilil Ciclases/metabolismo , Mutação , Receptores de Dopamina D1/genética , Receptores de Dopamina D1/metabolismo , Adenilil Ciclases/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Células COS/efeitos dos fármacos , Dopamina/metabolismo , Dopamina/farmacologia , Humanos , Leucina , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Receptores do Hormônio Hipofisário/genética , Receptores do Hormônio Hipofisário/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , SerinaRESUMO
A search of the expressed sequence tag (EST) database retrieved a human cDNA sequence which partially encoded a novel G protein-coupled receptor (GPCR) GPR26. A human genomic DNA fragment encoding a partial open reading frame (ORF) and a rat cDNA encoding the full length ORF of GPR26 were obtained by library screening. The rat GPR26 cDNA encoded a protein of 317 amino acids, most similar (albeit distantly related) to the serotonin 5-HT(5A) and gastrin releasing hormone BB2 receptors. GPR26 mRNA expression analysis revealed signals in the striatum, pons, cerebellum and cortex. HEK293 and Rh7777 cells transfected with GPR26 cDNA displayed high basal cAMP levels, slow growth rate of clonal populations and derangements of normal cell shape. We also used a sequence reported only in the patent literature encoding GPR57 (a.k.a. HNHCI32) to PCR amplify a DNA fragment which was used to screen a human genomic library. This resulted in the cloning of a genomic fragment containing a pseudogene, psiGPR57, with a 99.6% nucleotide identity to GPR57. Based on shared sequence identities, the receptor encoded by GPR57 was predicted to belong to a novel subfamily of GPCRs together with GPR58 (a.k.a. phBL5, reported only in the patent literature), putative neurotransmitter receptor (PNR) and a 5-HT(4) pseudogene. Analysis of this subfamily revealed greatest identities (approximately 56%) between the receptors encoded by GPR57 and GPR58, each with shared identities of approximately 40% with PNR. Furthermore, psiGPR57, GPR58, PNR and the 5-HT(4) pseudogene were mapped in a cluster localized to chromosome 6q22-24. PNR and GPR58 were expressed in COS cells, however no specific binding was observed for various serotonin receptor-specific ligands.
Assuntos
Encéfalo/metabolismo , Receptores de Superfície Celular/genética , Receptores Acoplados a Proteínas G , 1-Metil-3-Isobutilxantina , Sequência de Aminoácidos , Animais , Sequência de Bases , Células COS , Linhagem Celular , Clonagem Molecular , DNA Complementar/isolamento & purificação , Humanos , Hibridização in Situ Fluorescente , Ligantes , Dados de Sequência Molecular , Fases de Leitura Aberta , Pseudogenes , RNA Mensageiro/metabolismo , Ratos , Receptores de Superfície Celular/química , Receptores de Superfície Celular/metabolismo , TransfecçãoRESUMO
Nearly all molecules known to signal cells via G proteins have been assigned a cloned G-protein-coupled-receptor (GPCR) gene. This has been the result of a decade-long genetic search that has also identified some receptors for which ligands are unknown; these receptors are described as orphans (oGPCRs). More than 80 of these novel receptor systems have been identified and the emphasis has shifted to searching for novel signalling molecules. Thus, multiple neurotransmitter systems have eluded pharmacological detection by conventional means and the tremendous physiological implications and potential for these novel systems as targets for drug discovery remains unexploited. The discovery of all the GPCR genes in the genome and the identification of the unsolved receptor-transmitter systems, by determining the endogenous ligands, represents one of the most important tasks in modern pharmacology.
Assuntos
Proteínas de Ligação ao GTP/fisiologia , Receptores de Superfície Celular/fisiologia , Animais , Humanos , Ligantes , Receptores de Superfície Celular/efeitos dos fármacos , Receptores de Superfície Celular/genéticaRESUMO
The majority of genes encoding G protein-coupled receptors were isolated by methods based on sequence similarities found throughout this family. Experimental techniques have exploited these similarities (including low-stringency hybridization, polymerase chain reaction and electronic database searching) to identify genes encoding many pharmacologically recognized receptors and their subtypes. Homology-based searches have revealed receptors for which the endogenous ligands were unknown and these were named orphan receptors. Many orphan receptors are expressed in the brain, suggesting the existence of unidentified neurotransmitters. Methods used to identify ligands for these orphan receptors resulted in the identification of novel ligands and succeeded in pairing previously identified ligands with their receptors. Similar successful strategies are required to characterize the physiological and pathological importance of the remaining orphan receptors to facilitate the discovery of novel drugs for these systems.
Assuntos
Sistema Nervoso Central/efeitos dos fármacos , Receptores de Superfície Celular/efeitos dos fármacos , Animais , Sistema Nervoso Central/metabolismo , Humanos , Receptores de Angiotensina/efeitos dos fármacos , Receptores de Angiotensina/metabolismo , Receptores de Canabinoides , Receptores de Superfície Celular/metabolismo , Receptores de Droga/efeitos dos fármacos , Receptores de Droga/metabolismo , Receptores de Galanina , Receptores de Neuropeptídeos/efeitos dos fármacos , Receptores de Neuropeptídeos/metabolismo , Receptores de Neuropeptídeo Y/efeitos dos fármacos , Receptores de Neuropeptídeo Y/metabolismo , Receptores de Somatostatina/efeitos dos fármacos , Receptores de Somatostatina/metabolismoRESUMO
TRH (thyroliberin) is a tripeptide (pGlu-His-ProNH2) that signals via G protein-coupled receptors. Until recently, only a single receptor for TRH was known (TRH-R1), but two groups identified a second receptor, TRH-R2. We independently discovered TRH-R2. Using an extensive set of TRH analogs, we found no differences in TRH-R1 and TRH-R2 binding or in acute stimulation of signaling. TRH-R2 was more rapidly internalized upon binding TRH and exhibited a greater level of TRH-induced down-regulation than TRH-R1. During prolonged exposure to TRH, cells expressing TRH-R2 exhibited a lower level of gene induction than cells expressing TRH-R1. TRH-R2 receptor mRNA was present in very discrete nuclei and regions of rat brain. A major mRNA transcript for TRH-R2 was seen in the cerebral cortex, pons, thalamus, hypothalamus, and midbrain with faint bands found in the striatum and pituitary. The extensive distribution of TRH-R2 in the brain suggests that it mediates many of the known functions of TRH that are not transduced by TRH-R1. The variations in agonist-induced internalization and down-regulation/desensitization, and anatomic distribution of TRH-R2 compared with TRH-R1, suggest important functional differences between the two receptors.
Assuntos
Encéfalo/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Receptores Acoplados a Proteínas G , Receptores do Hormônio Liberador da Tireotropina/metabolismo , Sequência de Aminoácidos , Animais , Células COS , Clonagem Molecular , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , DNA Complementar/metabolismo , Relação Dose-Resposta a Droga , Regulação para Baixo , Hibridização In Situ , Proteínas de Membrana/química , Dados de Sequência Molecular , Hipófise/metabolismo , Ligação Proteica , Ratos , Receptores do Hormônio Liberador da Tireotropina/química , Receptores do Hormônio Liberador da Tireotropina/genética , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Hormônio Liberador de Tireotropina/metabolismo , Hormônio Liberador de Tireotropina/farmacologia , Fatores de Tempo , Transcrição GênicaRESUMO
Dopamine D2 receptor agonists are commonly used in the control of PRL-secreting adenomas, and the sensitivity of dopamine agonists during long term therapy is exquisite. However, the molecular mechanisms responsible for the maintenance of this cellular sensitivity to dopamine agonists remain poorly understood. In the present study, we examined the agonist-induced regulation of the human D2L receptor expressed to a specific activity of approximately 1 pmol receptor/mg protein in Sf9 insect cells. Treatment of D2L receptor-expressing cells with dopamine for up to 3 h resulted in no detectable change in the ligand-binding properties of the receptor and a approximately 120-fold reduction in the potency, but not the efficacy, of D2L receptors to mediate dopamine inhibition of forskolin-stimulated adenylyl cyclase activity. This resistance of the D2L receptor to agonist-induced desensitization was accompanied by a approximately 28% translocation of intracellular D2L receptors to the cell surface, as quantified by cellular fractionation and radioligand binding and visualized by whole cell immunocytochemical staining and confocal microscopy. Immunoblot analysis of the P2 membrane fraction revealed that surface D2L receptors comprised monomers and dimers. Treatment of D2L receptor-expressing cells with the protein synthesis inhibitor cycloheximide significantly reduced the basal expression level of receptors, but did not block the agonist-induced up-regulation of receptors. Longer periods of dopamine exposure for 24 h brought about a small increase in surface receptor density. However, when these studies were conducted in the presence of cycloheximide, receptor density was marginally reduced, suggesting that receptor synthesis accounts for the maintenance of cellular receptor density under these conditions. We conclude that the resistance of the D2L receptor-coupled adenylyl cyclase system to agonist-induced desensitization is attributed to the up-regulation of surface receptors after the translocation of existing intracellular receptors and de novo receptor synthesis.
Assuntos
Receptores de Superfície Celular/fisiologia , Receptores de Dopamina D2/efeitos dos fármacos , Receptores de Dopamina D2/fisiologia , Regulação para Cima/fisiologia , Animais , Fracionamento Celular , Linhagem Celular , Membrana Celular/química , Membrana Celular/efeitos dos fármacos , Colforsina/farmacologia , Cicloeximida/farmacologia , Dopamina/farmacologia , Relação Dose-Resposta a Droga , Eletroforese em Gel de Poliacrilamida , Feminino , Regulação da Expressão Gênica , Humanos , Immunoblotting , Imuno-Histoquímica , Mariposas , Ovário/química , Ovário/citologia , Ligação Proteica , Inibidores da Síntese de Proteínas/farmacologia , RNA Mensageiro/análise , RNA Mensageiro/química , RNA Mensageiro/genética , Receptores de Superfície Celular/análise , Receptores de Superfície Celular/genética , Receptores de Dopamina D2/genética , Spodoptera , Regulação para Cima/efeitos dos fármacosRESUMO
Selectively outbred Dahl salt-sensitive (DS) and salt-resistant (DR) rats were compared with the inbred Dahl salt-sensitive (SS/Jr) and salt-resistant (SR/Jr) rats developed from the original Brookhaven stocks by J.P. Rapp. The animals were evaluated for genotype at the renin locus. The inbred strains are uniformly homozygous for their respective alleles, s in SS/Jr and r in SR/Jr. DR rats were also uniformly homozygous for the r renin allele. In DS rats, however, three renin alleles were segregating. In addition to the s and r alleles, a third allele, designated the z allele, was found. The gene frequencies in DS rats were s = 0.690, r = 0.284, and z = 0.026. Continued use of DS and DR rats in most experimental work is inappropriate because of genetic heterogeneity in the DS stock.
Assuntos
Ratos/genética , Renina/genética , Alelos , Animais , Pressão Sanguínea/genética , Southern Blotting , HeterozigotoRESUMO
The gene encoding the human D4 dopamine receptor has evolved by gaining at least five internal repeats which are located within exons 3 and 4, and in the intervening intron 3 sequence. The amino acid sequence in the cytoplasmic loop of the receptor, involved in G protein coupling, has been altered by these gene changes.
Assuntos
Receptores de Dopamina D2 , Receptores Dopaminérgicos/genética , Sequências Repetitivas de Ácido Nucleico/genética , Sequência de Aminoácidos , Sequência de Bases , Éxons/genética , Humanos , Íntrons/genética , Dados de Sequência Molecular , Conformação Proteica , Receptores Dopaminérgicos/química , Receptores de Dopamina D4 , Homologia de Sequência do Ácido NucleicoRESUMO
Molecular cloning studies have now identified five structurally homologous genes encoding the biosynthesis of the human dopamine receptors, DRD1, DRD2, DRD3, DRD4, and DRD5. Two of these dopamine receptors (DRD1 and DRD5) are encoded by intronless genes. To ascertain whether there are other intronless genes that share identity with the gene (DRD5) encoding the DRD5 receptor, we used a cloning method based on the polymerase chain reaction (PCR). Human genomic DNA was amplified by PCR with oligodeoxyribonucleotides (oligos) based on the DRD5 nucleotide (nt) sequence. Amplification of nt sequences between these oligos allowed the isolation of two independent intronless genes that share identity with DRD5. The full-length clones have also been isolated by screening human genomic libraries. The deduced amino acid sequences for these genes, PG-1 and PG-2, share 91% and 92% identity to DRD5, respectively. However, each of the genes contains differences in the coding regions that would render these genes incapable of encoding functional receptors. Thus, the human genome contains at least two DRD5 pseudogenes, consistent with in situ human chromosomal hybridization analysis which reveals the presence of two pseudogenes.
Assuntos
Família Multigênica/genética , Pseudogenes/genética , Receptores de Dopamina D1 , Receptores Dopaminérgicos/genética , Sequência de Aminoácidos , Sequência de Bases , Southern Blotting , Cromossomos Humanos Par 1 , Cromossomos Humanos Par 2 , Cromossomos Humanos Par 4 , Clonagem Molecular , Códon/genética , Humanos , Íntrons/genética , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Oligodesoxirribonucleotídeos/genética , Reação em Cadeia da Polimerase , Receptores de Dopamina D5 , Homologia de Sequência do Ácido NucleicoRESUMO
We report the discovery of four novel human putative G-protein-coupled receptor (GPCR) genes. Gene GPR20 was isolated by amplifying genomic DNA with oligos based on the opioid and somatostatin related receptor genes and subsequent screening of a genomic library. Also, using our customized search procedure of a database of expressed sequence tags (dbEST), cDNA sequences that partially encoded novel GPCRs were identified. These cDNA fragments were obtained and used to screen a genomic library to isolate the full-length coding region of the genes. This resulted in the isolation of genes GPR21, GPR22 and GPR23. The four encoded receptors share significant identity to each other and to other members of the receptor family. Northern blot analysis revealed expression of GPR20 and GPR22 in several human brain regions while GPR20 expression was detected also in liver. Fluorescence in situ hybridization (FISH) was used to map GPR20 to chromosome 8q, region 24.3-24.2, GPR21 to chromosome 9, region q33, GPR22 to chromosome 7, region q22-q31.1, and GPR23 to chromosome X, region q13-q21.1.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Receptores de Superfície Celular/genética , Sequência de Aminoácidos , Northern Blotting , Mapeamento Cromossômico , Cromossomos Humanos Par 7 , Cromossomos Humanos Par 8 , Cromossomos Humanos Par 9 , Clonagem Molecular , Primers do DNA , Humanos , Hibridização in Situ Fluorescente , Dados de Sequência Molecular , Receptores de Superfície Celular/química , Receptores de Superfície Celular/metabolismo , Análise de Sequência , Homologia de Sequência de Aminoácidos , Cromossomo XRESUMO
The human dopamine D5 receptor (DRD5) gene family consists of the DRD5-encoding gene (DRD5) and the pseudogenes psi DRD5-1 and psi DRD5-2. Analysis of the 5' UTR of DRD5 and homologous regions in the pseudogenes revealed that the nucleotide identity (approx. 95%) extended for 1.9 kb and terminated at a monomeric Alu sequence in each of the pseudogenes. The presence of Alu sequences in the pseudogenes, at this point of divergence with DRD5, suggests that Alu sequences were involved in the evolution of the DRD5 family. This report is the first to describe a possible mechanism involved in the duplication of genes in the G-protein-coupled receptor (GPCR) family. The pseudogenes continue to share identity (approx. 98%) beyond this 5' UTR point of divergence with DRD5 for at least another 6 kb. Analysis of the 3' UTR of DRD5 and homologous regions in the pseudogenes revealed that the identity (approx. 95%) extends at least 14 kb, and the identity between the pseudogenes (approx. 98%) extends for at least 18 kb. Thus, the duplication unit that produced the first pseudogene was at least 16 kb, whereas the second pseudogene was at least 28 kb. We have also located two DRD5 pseudogenes in gorilla demonstrating that these closely related pseudogenes were present in a common ancestor of human and gorilla.
Assuntos
Gorilla gorilla/genética , Família Multigênica/genética , Pseudogenes/genética , Receptores de Dopamina D1 , Receptores Dopaminérgicos/genética , Animais , Sequência de Bases , Clonagem Molecular , Humanos , Dados de Sequência Molecular , Filogenia , Receptores de Dopamina D5 , Sequências Reguladoras de Ácido Nucleico , Mapeamento por Restrição , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
An examination of mRNA expression of the serotonin genes encoding both psi 5HT1D alpha and the related 5HT1D alpha, demonstrated that the pseudogene is transcribed, and has a tissue distribution similar to the 5HT1D alpha receptor-encoding gene. This psi 5HT1D alpha transcript is capable of being translated into a polypeptide of only 28 amino acids in length, and the psi 5HT1D alpha pseudogene most likely arose from a gene duplication or transposition event.
Assuntos
Pseudogenes/genética , RNA Mensageiro/análise , Receptores de Serotonina/genética , Transcrição Gênica , Sequência de Aminoácidos , Sequência de Bases , Humanos , Dados de Sequência Molecular , Família Multigênica/genética , Especificidade de Órgãos , Biossíntese de Proteínas , Receptor 5-HT1D de Serotonina , Receptores de Serotonina/química , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência do Ácido NucleicoRESUMO
Molecular cloning studies have now identified six HTR genes encoding the biosynthesis of the structurally homologous human serotonin (5-hydroxytryptamine; 5-HT) receptors, namely 5-HTR1A, 5-HTR1B, 5-HTR1C, 5-HTR1D, 5-HTR2 and 5-HTRS31. Several of these receptors are encoded by intronless genes, and we now report the cloning of another intronless serotonergic HTR gene. This gene was cloned by a method using the polymerase chain reaction. The nucleotide sequence of this gene is most closely homologous to the 5-HTR1D gene; however, several stop codons, frame shifts and deletions are present in the coding region suggesting that this is a pseudogene which could not encode a functional receptor. Sequence analysis also revealed that the coding sequence of this pseudogene is disrupted by insertion of a 283-bp Alu repeat sequence.
Assuntos
Pseudogenes , Receptores de Serotonina/genética , Sequências Repetitivas de Ácido Nucleico , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Southern Blotting , Clonagem Molecular , Humanos , Dados de Sequência Molecular , Pan troglodytes , Reação em Cadeia da Polimerase , Ratos , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Especificidade da EspécieRESUMO
We report the cloning of a gene, intronless in its coding region, which we have named APJ. This gene was cloned using the polymerase chain reaction (PCR), with a set of primers designed on the basis of the conservation that members of G protein-coupled receptors (GPCR) have in their transmembrane (TM) regions. The putative receptor protein, APJ, shares closest identity to the angiotensin receptor (AT1) ranging from 40 to 50% in the hydrophobic TM regions of these receptors. The transcripts for this gene were detected in many regions of the brain. PCR analysis of somatic cell lines found APJ-related sequences to be only present on chromosome 11, and high-resolution mapping by fluorescence in situ hybridization (FISH) sublocalized APJ on band q12.
Assuntos
Cromossomos Humanos Par 11 , Receptores de Angiotensina/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Células Cultivadas , Mapeamento Cromossômico , Clonagem Molecular , DNA , Humanos , Hibridização in Situ Fluorescente , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Especificidade da EspécieRESUMO
We report the identification, cloning and tissue distributions of ten novel human genes encoding G protein-coupled receptors (GPCRs) GPR78, GPR80, GPR81, GPR82, GPR93, GPR94, GPR95, GPR101, GPR102, GPR103 and a pseudogene, psi GPR79. Each novel orphan GPCR (oGPCR) gene was discovered using customized searches of the GenBank high-throughput genomic sequences database with previously known GPCR-encoding sequences. The expressed genes can now be used in assays to determine endogenous and pharmacological ligands. GPR78 shared highest identity with the oGPCR gene GPR26 (56% identity in the transmembrane (TM) regions). psi GPR79 shared highest sequence identity with the P2Y(2) gene and contained a frame-shift truncating the encoded receptor in TM5, demonstrating a pseudogene. GPR80 shared highest identity with the P2Y(1) gene (45% in the TM regions), while GPR81, GPR82 and GPR93 shared TM identities with the oGPCR genes HM74 (70%), GPR17 (30%) and P2Y(5) (40%), respectively. Two other novel GPCR genes, GPR94 and GPR95, encoded a subfamily with the genes encoding the UDP-glucose and P2Y(12) receptors (sharing >50% identities in the TM regions). GPR101 demonstrated only distant identities with other GPCR genes and GPR102 shared identities with GPR57, GPR58 and PNR (35-42% in the TM regions). GPR103 shared identities with the neuropeptide FF 2, neuropeptide Y2 and galanin GalR1 receptors (34-38% in the TM regions). Northern analyses revealed GPR78 mRNA expression in the pituitary and placenta and GPR81 expression in the pituitary. A search of the GenBank databases with the GPR82 sequence retrieved an identical sequence in an expressed sequence tag (EST) partially encoding GPR82 from human colonic tissue. The GPR93 sequence retrieved an identical, human EST sequence from human primary tonsil B-cells and an EST partially encoding mouse GPR93 from small intestinal tissue. GPR94 was expressed in the frontal cortex, caudate putamen and thalamus of brain while GPR95 was expressed in the human prostate and rat stomach and fetal tissues. GPR101 revealed mRNA transcripts in caudate putamen and hypothalamus. GPR103 mRNA signals were detected in the cortex, pituitary, thalamus, hypothalamus, basal forebrain, midbrain and pons.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Receptores de Superfície Celular/genética , Sequência de Aminoácidos , Mapeamento Cromossômico , Clonagem Molecular , DNA/química , DNA/genética , DNA Complementar/química , DNA Complementar/genética , Chaperona BiP do Retículo Endoplasmático , Feminino , Expressão Gênica , Humanos , Masculino , Dados de Sequência Molecular , Pseudogenes/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Superfície Celular/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Distribuição TecidualRESUMO
The serotonin (5-hydroxytryptamine (5-HT)) 1B and 1D receptor subtypes share a high amino acid sequence identity and have similar ligand binding properties. In this study, we demonstrate that both receptor subtypes exist as monomers and homodimers when expressed alone and as monomers and heterodimers when co-expressed. Gene expression studies have shown that there are brain regions where the 5-HT1B and 5-HT1D receptors are co-localized and where heterodimerization may occur physiologically. This is the first direct visualization of the physical association between G protein-coupled receptors of different subtypes.
Assuntos
Receptores de Serotonina/química , Receptores de Serotonina/metabolismo , Animais , Benzamidas/metabolismo , Ligação Competitiva , Membrana Celular/metabolismo , Células Cultivadas , Dimerização , Expressão Gênica , Immunoblotting , Ketanserina/metabolismo , Peso Molecular , Piridinas/metabolismo , Receptor 5-HT1B de Serotonina , Receptor 5-HT1D de Serotonina , Receptores de Serotonina/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Serotonina/análogos & derivados , Serotonina/metabolismo , Antagonistas da Serotonina/metabolismo , Agonistas do Receptor de Serotonina/metabolismoRESUMO
In order to determine whether dimers of dopamine D2 receptors can occur in mammalian brain, rat and human brain striatal membranes were photolabelled with two radioactive photoaffinity compounds selective for dopamine D2 receptors, [125I]azidophenethylspiperone and [125I]-4-azido-5-iodonemonapride. It was found that [125I]azidophenethylspiperone only labelled the D2 monomer, while [125I]-4-azido-5-iodonemonapride labelled both D2 monomers and dimers, despite the fact that very high concentrations (6 nM) of both radiocompounds were used. In addition, human cloned D2 receptors were probed with a D2-specific antibody, revealing multiple bands indicating the existence of trimers, tetramers and pentamers of D2 receptors. The different D2-binding patterns of the spiperone and benzamide congeners may explain the different densities of dopamine D2 receptors found with these two radioligands in human brain positron tomography in health and disease.
Assuntos
Núcleo Caudado/metabolismo , Corpo Estriado/metabolismo , Receptores de Dopamina D2/metabolismo , Animais , Dimerização , Humanos , Pessoa de Meia-Idade , Ratos , Proteínas Recombinantes/metabolismo , SpodopteraRESUMO
An approach based on the polymerase chain reaction (PCR) was used to isolate additional members of the G-linked receptor family from a rat striatal lambda gtII cDNA library. Priming with one degenerate probe corresponding to highly conserved consensus sequences in the third transmembrane (TM) domain of 15 G-linked receptors and sequences in the phage vector resulted in one clone (G-13) encoding a dopamine D2 receptor variant with a 29 amino acid insert in the third cytoplasmic loop. In addition, the amino acid sequence encoded by clone G-36 contained conserved sequences characteristic of the G-linked class of receptors and displayed sequence homology in TM domains with the beta 2-adrenergic receptor (48%). Two conserved serine residues in TM5 postulated to be part of a ligand binding site in the adrenergic receptor, suggests that G-36 encodes a catecholaminergic receptor. Northern blot analysis confirmed the expression of G-36 in rat brain, but not in kidney, heart and lung. Several strong hybridizing bands to G-36 were obtained in both human and rat genomic DNA. The general PCR strategy employed here should prove to be extremely useful for the isolation of other members of the G-linked receptor family.