RESUMO
We report the molecular characterization of a novel G-protein-coupled receptor, GPR48, that resembles proteins in the glycoprotein hormone receptor family. The full-length human GPR48 cDNA is comprised of 951 amino acids. The large extracellular amino terminus of 538 residues is composed of seventeen leucine-rich repeats (LRR). The genomic structure of GPR48 has several features in common with genes in the glycoprotein hormone receptor family. Analogous to these receptors, most of the LRR are encoded on single small exons, and the last exon encodes the seven transmembrane segments. The complete gene spans more than 60 kb with 18 exons and 17 introns. Northern blot analysis demonstrated high expression of GPR48 in the adult human pancreas, with moderate levels of expression in placenta, kidney, brain, and heart. Additionally, this receptor is expressed as early as 7 days post coitus in the mouse, indicating its potential involvement in development.
Assuntos
Receptores de Superfície Celular/genética , Receptores Acoplados a Proteínas G , Adulto , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Primers do DNA/genética , DNA Complementar/genética , Éxons , Etiquetas de Sequências Expressas , Feminino , Expressão Gênica , Humanos , Íntrons , Masculino , Camundongos , Dados de Sequência Molecular , Gravidez , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sequências Repetitivas de Aminoácidos , Distribuição TecidualRESUMO
The timeless (tim) gene is essential for circadian clock function in Drosophila melanogaster. A putative mouse homolog, mTimeless (mTim), has been difficult to place in the circadian clock of mammals. Here we show that mTim is essential for embryonic development, but does not have substantiated circadian function.
Assuntos
Núcleo Supraquiasmático/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica , Animais , Proteínas CLOCK , Proteínas de Ciclo Celular , Quimera , Ritmo Circadiano/genética , Genes Reporter , Heterozigoto , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Luciferases/genética , Camundongos , Camundongos Endogâmicos , Camundongos Knockout , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Filogenia , Proteínas Recombinantes/metabolismo , Spodoptera , Transativadores/genética , TransfecçãoRESUMO
We report the chromosomal localization in both mouse and human of a novel G-protein-coupled receptor, GPR48, which resembles glycoprotein hormone receptors, that may be implicated in Wilms tumor deletion syndromes such as WAGR. This receptor forms a novel sub-family of glycoprotein hormone-like GPCRs. We have mapped this receptor to human chromosome 11p14-->p13 by several approaches, including radiation hybrid and interspecific backcross mapping, and show that GPR48 is close to BDNF. This data differs from the recently published mapping of LGR4 (5q34-->q35.1) (Hsu et al., 1998). Additionally, we show that Gpr48 and Bdnf are tightly linked on mouse chromosome 2, in a region with conserved synteny to human 11p14-->p13.
Assuntos
Mapeamento Cromossômico , Cromossomos Humanos Par 11/genética , Receptores de Superfície Celular/genética , Receptores Acoplados a Proteínas G , Animais , Fator Neurotrófico Derivado do Encéfalo/genética , Cruzamentos Genéticos , Feminino , Ligação Genética/genética , Humanos , Células Híbridas , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Direct evidence is lacking to show whether the gamma-aminobutyric acid (GABA)(B) gb1-gb2 heterodimer is the signaling form of the receptor. In this study, we tested whether gb1a or gb2 subunits when coexpressed with truncated receptors or metabotropic glutamate receptor mGluR4 could form functional GABA receptors. Coexpression of the ligand binding N-terminal domain of gb1a or the C-terminal portion of gb1a composing the seven-transmembrane segments and intracellular loops with gb2 could not reconstitute functional receptors. We next examined whether mGluR4, which forms homodimers and is structurally related to GABA(B), could act as a surrogate coreceptor for gb1 or gb2. The coexpression of mGluR4 and gb1a led to the expression of gb1a monomers on cell surface membranes as determined by immunoblot analysis and flow cytometry. However, mGluR4-gb1a heterodimers were not formed, and membrane-expressed gb1a monomers were not functionally coupled to adenylyl cyclase in human embryonic kidney 293 cells or activated inwardly rectifying potassium (Kir) channels in Xenopus oocytes. Similarly, the coexpression of mGluR4 and gb2 led to nonfunctional GABA receptors. GABA-activated distal signaling events resulted only after the coexpression and heterodimerization of gb1 and gb2. Taken together with the truncated receptor studies, the data suggest that a high degree of structural specificity is required to form the functional GABA(B) receptor that is a gb1-gb2 heterodimer.
Assuntos
Canais de Potássio Corretores do Fluxo de Internalização , Receptores de GABA-B/biossíntese , Receptores de Glutamato Metabotrópico/biossíntese , Sequência de Aminoácidos , Animais , Southern Blotting , Linhagem Celular , Membrana Celular/metabolismo , Densitometria , Citometria de Fluxo , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G , Humanos , Ligantes , Camundongos , Dados de Sequência Molecular , Oócitos/metabolismo , Canais de Potássio/metabolismo , Testes de Precipitina , Receptores de GABA-B/genética , Receptores de Glutamato Metabotrópico/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Xenopus laevisRESUMO
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
G protein-coupled receptors are commonly thought to bind their cognate ligands and elicit functional responses primarily as monomeric receptors. In studying the recombinant gamma-aminobutyric acid, type B (GABAB) receptor (gb1a) and a GABAB-like orphan receptor (gb2), we observed that both receptors are functionally inactive when expressed individually in multiple heterologous systems. Characterization of the tissue distribution of each of the receptors by in situ hybridization histochemistry in rat brain revealed co-localization of gb1 and gb2 transcripts in many brain regions, suggesting the hypothesis that gb1 and gb2 may interact in vivo. In three established functional systems (inwardly rectifying K+ channel currents in Xenopus oocytes, melanophore pigment aggregation, and direct cAMP measurements in HEK-293 cells), GABA mediated a functional response in cells coexpressing gb1a and gb2 but not in cells expressing either receptor individually. This GABA activity could be blocked with the GABAB receptor antagonist CGP71872. In COS-7 cells coexpressing gb1a and gb2 receptors, co-immunoprecipitation of gb1a and gb2 receptors was demonstrated, indicating that gb1a and gb2 act as subunits in the formation of a functional GABAB receptor.
Assuntos
Canais de Potássio Corretores do Fluxo de Internalização , Receptores de GABA-B/metabolismo , Sequência de Aminoácidos , Animais , Azidas/metabolismo , Células COS , Dimerização , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G , Humanos , Hibridização In Situ , Camundongos , Dados de Sequência Molecular , Compostos Organofosforados/metabolismo , Canais de Potássio/metabolismo , Conformação Proteica , RNA Mensageiro/metabolismo , Ratos , Receptores de GABA-B/genética , Relação Estrutura-Atividade , Xenopus laevisRESUMO
Galanin is a 29- or 30-amino acid peptide with wide-ranging effects on hormone release, feeding behavior, smooth muscle contractility, and somatosensory neuronal function. Three distinct galanin receptor (GALR) subtypes, designated GALR1, 2, and 3, have been cloned from the rat. We report here the cloning of the human GALR2 and GALR3 genes, an initial characterization of their pharmacology with respect to radioligand binding and signal transduction pathways, and a profile of their expression in brain and peripheral tissues. Human GALR2 and GALR3 show, respectively, 92 and 89% amino acid sequence identity with their rat homologues. Radioligand binding studies with 125I-galanin show that recombinant human GALR2 binds with high affinity to human galanin (K(D) = 0.3 nM). Human GALR3 binds galanin with less affinity (IC50 of 12 nM for porcine galanin and 75 nM for human galanin). Human GALR2 was shown to couple to phospholipase C and elevation of intracellular calcium levels as assessed by aequorin luminescence in HEK-293 cells and by Xenopus melanophore pigment aggregation and dispersion assays, in contrast to human GALR1 and human GALR3, which signal predominantly through inhibition of adenylate cyclase. GALR2 mRNA shows a wide distribution in the brain (mammillary nuclei, dentate gyrus, cingulate gyrus, and posterior hypothalamic, supraoptic, and arcuate nuclei), and restricted peripheral tissue distribution with highest mRNA levels detected in human small intestine. In comparison, whereas GALR3 mRNA was expressed in many areas of the rat brain, there was abundant expression in the primary olfactory cortex, olfactory tubercle, the islands of Calleja, the hippocampal CA regions of Ammon's horn, and the dentate gyrus. GALR3 mRNA was highly expressed in human testis and was detectable in adrenal gland and pancreas. The genes for human GALR2 and 3 were localized to chromosomes 17q25 and 22q12.2-13.1, respectively.
Assuntos
Clonagem Molecular , Receptores de Neuropeptídeos/genética , Receptores de Neuropeptídeos/metabolismo , Sequência de Aminoácidos , Animais , Northern Blotting , Encéfalo/metabolismo , Linhagem Celular , Humanos , Isomerismo , Ligantes , Camundongos , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Ratos , Receptores de Galanina , Receptores de Neuropeptídeos/fisiologia , Ribonucleases , Transdução de Sinais/fisiologia , Suínos , Xenopus laevisRESUMO
We report the cloning of a novel human gene (GPR32) encoding a putative G-protein-coupled receptor (GPCR) of 356 amino acids and a related pseudogene psi GPR32. The deduced amino acid sequence of GPR32 shares 35-39% identity with members of the chemoattractant receptor family. psi GPR32 shares 93% nucleotide identity with GPR32. We identified a mouse EST encoding a putative GPCR (GPR33) of 309 amino acids. The deduced amino acid sequence of GPR33 shares 30-35% identity with members of the chemoattractant receptor family and 36% identity with the receptor encoded by GPR32. The human orthologue of GPR33 contains a single basepair substitution with respect to the mouse, resulting in the presence of an in-frame stop codon within the predicted second intracellular loop, demonstrating that it is a pseudogene. Through fluorescence in situ hybridization and physical mapping of YACs, both GPR32 and psi GPR32 were mapped to chromosomal 19, region q13.3, while psi GPR33 was mapped to chromosome 14q12.
Assuntos
Proteínas de Ligação ao GTP , Receptores de Superfície Celular/química , Receptores de Superfície Celular/genética , Receptores Acoplados a Proteínas G , Sequência de Aminoácidos , Animais , Sequência de Bases , Mapeamento Cromossômico , Cromossomos Humanos Par 14 , Cromossomos Humanos Par 19 , Clonagem Molecular , Códon de Terminação , Humanos , Hibridização in Situ Fluorescente , Camundongos , Dados de Sequência Molecular , PseudogenesRESUMO
We report here the molecular cloning, tissue distribution, and chromosomal localization of novel genes encoding G-protein-coupled receptors (GPCRs). A search of a mouse database of expressed sequence tags revealed an EST partially encoding a GPCR, which was used to screen a mouse genomic library to obtain the translational open reading frame (ORF). The resultant clone, GPR27, contained an intronless ORF, encoding a receptor of 379 amino acids. In an alternate strategy, human genomic DNA was subjected to polymerase chain reaction (PCR) amplification, using degenerate oligonucleotides based on GPR1. Two PCR products partially encoding GPCRs were isolated and used to screen a genomic library to obtain the translational ORF. One of the resultant clones, GPR30, contained an intronless ORF encoding a receptor of 375 amino acids. The other clone, GPR35, also contained an intronless ORF encoding a receptor of 309 amino acids. Transcripts corresponding to GPR27 and GPR30 were detected in several areas of human and rat CNS, While GPR35 expression was detected only in the rat intestine. Through fluorescence in situ hybridization analysis the gene encoding GPR30 was localized to chromosome 7p22 and GPR35 to chromosome 2q37.3.
Assuntos
Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Receptores de Superfície Celular/genética , Receptores Acoplados a Proteínas G , Proteínas de Saccharomyces cerevisiae , Sequência de Aminoácidos , Animais , Antígenos CD/genética , Cromossomos Humanos Par 2 , Cromossomos Humanos Par 7 , Clonagem Molecular , Proteínas de Ligação ao GTP/análise , Humanos , Camundongos , Dados de Sequência Molecular , Especificidade de Órgãos/genética , Ratos , Receptor da Anafilatoxina C5a , Receptores de Superfície Celular/análise , Receptores de Complemento/genética , Receptores de Estrogênio , Homologia de Sequência de AminoácidosRESUMO
In our search for novel human galanin receptor (GALR) subtypes, human genomic DNA was PCR amplified using sets of degenerate primers based on conserved sequences in human and rat GALR. The sequence of one of the subcloned PCR products revealed homology to a sequence in the 3' region of the human CD22 gene following a BLAST search of GenBank's database. A search for open reading frames (ORF) in the non-coding CD22 sequence resulted in identification of two novel putative intronless genes, GPR40 and GPR41. The recent submission of sequence overlapping the downstream CD22 sequence revealed a possible polymorphic insert containing a third intronless gene, GPR42, sharing 98% amino acid identity with GPR41, followed by a fourth intronless gene, GPR43. Thus, the GPR40, GPR41, GPR42, and GPR43 genes, respectively, occur downstream from CD22, a gene previously localized on chromosome 19q13.1. The four putative novel human genes encode new members of the GPCR family and share little homology with GALR.
Assuntos
Antígenos CD/genética , Antígenos de Diferenciação de Linfócitos B/genética , Moléculas de Adesão Celular , Cromossomos Humanos Par 19 , Proteínas de Ligação ao GTP/metabolismo , Lectinas , Família Multigênica , Receptores dos Hormônios Gastrointestinais/química , Receptores dos Hormônios Gastrointestinais/genética , Sequência de Aminoácidos , Animais , Mapeamento Cromossômico , Clonagem Molecular , Humanos , Dados de Sequência Molecular , Ratos , Receptores de Galanina , Receptores dos Hormônios Gastrointestinais/isolamento & purificação , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Lectina 2 Semelhante a Ig de Ligação ao Ácido SiálicoRESUMO
hGrb10alpha (previously named Grb-IR) is a Src-homology 2 domain-containing protein that binds with high affinity to the tyrosine-phosphorylated insulin receptor and insulin-like growth factor-1 receptor. At least two isoforms of human Grb10, (hGrb10alpha and hGrb10beta), which differ in the pleckstrin homology (PH) domain and the N-terminal sequence, have previously been identified in insulin target tissues such as human skeletal muscle and fat cells. Here we report the cloning of the third isoform of the hGrb10 family (hGrb10gamma) from human skeletal muscle and its localization to human chromosome 7. We have also determined the human chromosome localization of Grb7 to 17q21-q22 and Grb14 to chromosome 2. hGrb10gamma contains an intact PH domain and an N-terminal sequence that is present in hGrb10alpha but absent in hGrb10beta. RNase protection assays and Western blot analysis showed that hGrb10alpha and hGrb10gamma are differentially expressed in insulin target cells including skeletal muscle, liver, and adipocyte cells. hGrb10gamma is also expressed in HeLa cells and various breast cancer cell lines. The protein bound with high affinity to the insulin receptor in cells, and the interaction was dependent on the tyrosine phosphorylation of the receptor. hGrb10gamma also underwent insulin-stimulated membrane translocation and serine phosphorylation. hGrb10gamma phosphorylation was inhibited by PD98059, a specific inhibitor of mitogen-activated protein kinase kinase, and wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase. Taken together, our data suggest that hGrb10 isoforms are potential downstream signaling components of the insulin receptor tyrosine kinase and that the PH domain may play an important role in the involvement of these isoforms in signal transduction pathways initiated by insulin and other growth factors.
Assuntos
Cromossomos Humanos Par 17 , Cromossomos Humanos Par 7 , Proteínas/metabolismo , Receptor de Insulina/metabolismo , Domínios de Homologia de src , Sequência de Aminoácidos , Androstadienos/farmacologia , Mapeamento Cromossômico , Clonagem Molecular , DNA Complementar , Inibidores Enzimáticos , Flavonoides/farmacologia , Proteína Adaptadora GRB10 , Humanos , Insulina/farmacologia , Dados de Sequência Molecular , Fosforilação , Ligação Proteica , Proteínas/genética , Homologia de Sequência de Aminoácidos , Serina/metabolismo , Transdução de Sinais , WortmaninaRESUMO
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
We amplified human genomic DNA by the polymerase chain reaction (PCR) using oligonucleotides based on the primary sequence of the genes encoding the somatostatin receptors (SSTR) and the somatostatin-like receptor gene SLC-1. One resultant DNA fragment was used to screen a genomic DNA library resulting in the isolation of a gene, GPR25, encoding an additional member of the G protein-coupled receptor family (GPCR). GPR25 is intronless throughout its open reading frame (ORF) and encodes a protein of 360 amino acids. The receptor encoded by GPR25 shares highest identity to the receptor encoded by GPR15, angiotensin II type 1A receptor, and somatostatin receptor 5. Northern analysis found no transcripts expressed in liver or any of the 12 brain regions analyzed. Fluorescence in situ hybridization analysis localized GPR25 to chromosome 1q32.1.
Assuntos
Cromossomos Humanos Par 1 , Proteínas de Ligação ao GTP/genética , Receptores de Superfície Celular/genética , Receptores Acoplados a Proteínas G , Sequência de Aminoácidos , Sequência de Bases , Mapeamento Cromossômico , Primers do DNA , Proteínas de Ligação ao GTP/biossíntese , Proteínas de Ligação ao GTP/química , Biblioteca Genômica , Humanos , Hibridização in Situ Fluorescente , Dados de Sequência Molecular , Fases de Leitura Aberta , Reação em Cadeia da Polimerase , Receptores de Superfície Celular/biossíntese , Receptores de Superfície Celular/química , Receptores de Somatostatina/química , Homologia de Sequência de AminoácidosRESUMO
We have characterized a mammalian homolog of the Drosophila period gene and designated it Per2. The PER2 protein shows >40% amino acid identity to the protein of another mammalian per homolog (designated Per1) that was recently cloned and characterized. Both PER1 and PER2 proteins share several regions of homology with the Drosophila PER protein, including the protein dimerization PAS domain. Phylogenetic analysis supports the existence of a family of mammalian per genes. In the mouse, Per1 and Per2 RNA levels exhibit circadian rhythms in the SCN and eyes, sites of circadian clocks. Both Per1 and Per2 RNAs in the SCN are increased by light exposure during subjective night but not during subjective day. The results advance our knowledge of candidate clock elements in mammals.
Assuntos
Ritmo Circadiano , Regulação da Expressão Gênica/efeitos da radiação , Proteínas Nucleares/biossíntese , Proteínas Nucleares/química , Núcleo Supraquiasmático/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Ciclo Celular , DNA Complementar , Drosophila , Proteínas de Drosophila , Humanos , Luz , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Família Multigênica , Proteínas Circadianas Period , Filogenia , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Núcleo Supraquiasmático/efeitos da radiação , Fatores de TranscriçãoRESUMO
Neuropeptide Y (NPY) signals through a family of G-protein-coupled receptors present in the brain and sympathetic neurons. To further our understanding of the genetic elements involved in the regulation of NPY receptor expression, we have cloned and characterized the human gene encoding the type 2 NPY receptor (Y2 receptor, HGMW-approved symbol NPY2R).2 The transcript spans 9 kb of genomic sequence and is encoded on two exons. As in the type 1 NPY receptor (Y1 receptor) gene, the 5'-untranslated region of the Y2 receptor is interrupted by an intervening sequence ( approximately 4.5 kb). However, the Y2 receptor gene does not contain an intron analogous to that present in the coding region of the Y1 receptor. The predicted transcript size ( approximately 4.5 kb) is consistent with the size observed by Northern analysis. The 381-amino-acid sequence deduced from the open reading frame is identical to that encoded by the cDNA. The Y2 receptor gene maps to human chromosome 4q31, the same region containing the Y1 receptor locus, suggesting that these subtypes may have arisen by gene duplication despite their structural differences.
Assuntos
Cromossomos Humanos Par 4/genética , Receptores de Neuropeptídeo Y/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Bovinos , Humanos , Íntrons/genética , Dados de Sequência Molecular , Família Multigênica , Fases de Leitura Aberta , Regiões Promotoras Genéticas , Receptores de Neuropeptídeo Y/classificação , Homologia de Sequência do Ácido NucleicoRESUMO
We report the identification of a gene, named SLC-1(1), encoding a novel G protein-coupled receptor (GPCR). A customized search procedure of a database of expressed sequence tags (dbEST) retrieved a human cDNA sequence that partially encoded a GPCR. A genomic DNA fragment identical to the cDNA was obtained and used to screen a library to isolate the full-length coding region of the gene. This gene was intronless in its open reading frame, and encoded a receptor of 402 amino acids, and shared -40% amino acid identity in the transmembrane (TM) regions to the five known human somatostatin receptors. Northern blot analysis revealed that SLC-1 is expressed in human brain regions, including the forebrain and hypothalamus. Expression in the rat was highest in brain, followed by heart, kidney, and ovary. Expression of SLC-1 in COS-7 cells failed to show specific binding to radiolabelled Tyr1-somatostatin-14, naloxone, bremazocine, 1,3-di(2-tolyl)-guanidine (DTG), or haloperidol. A repeat polymorphism of the form (CA)n was discovered in the 5'-untranslated region (UTR) of the gene and SLC-1 was mapped to chromosome 22, q13.3.
Assuntos
Proteínas de Ligação ao GTP/genética , Receptores de Somatostatina/genética , Sequência de Aminoácidos , Animais , Northern Blotting , Encéfalo/metabolismo , Células COS , Mapeamento Cromossômico , Cromossomos Humanos Par 22 , Clonagem Molecular , DNA Complementar/genética , Repetições de Dinucleotídeos , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/metabolismo , Expressão Gênica , Biblioteca Genômica , Humanos , Hibridização in Situ Fluorescente , Íntrons , Ligantes , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Ratos , Receptores de Somatostatina/química , Receptores de Somatostatina/metabolismo , Somatostatina/análogos & derivados , Somatostatina/metabolismoRESUMO
Following the cloning of the dopamine receptors we continued a search of the human genome for related genes. We searched an EST data base and discovered cDNA fragments encoding novel G protein-coupled receptor genes. The available GenBank sequence of one of these EST fragments showed that it encoded a receptor with closest similarity to the D2 dopamine and adrenergic receptors. This cDNA was used to isolate the gene (GPR19), and the encoded receptor also demonstrated similarity with the neuropeptide Y receptor. The gene was mapped to chromosome 12, in region p13.2-12.3. Northern blot analysis revealed expression of GPR19 in peripheral regions, and brain regions significantly overlapping with the D2 receptor gene expression. A sequence of the rat orthologue of GPR19 was obtained and in situ hybridization analysis demonstrated a very abundant expression in rat brain.
Assuntos
Química Encefálica/genética , Cromossomos Humanos Par 12/genética , Proteínas de Ligação ao GTP/genética , Receptores Acoplados a Proteínas G , Receptores de Neurotransmissores/genética , Sequência de Aminoácidos , Animais , Mapeamento Cromossômico , Clonagem Molecular , DNA Complementar/genética , Expressão Gênica , Humanos , Hibridização in Situ Fluorescente , Dados de Sequência Molecular , Proteínas do Tecido Nervoso , Polimorfismo Conformacional de Fita Simples , Ratos , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Distribuição TecidualRESUMO
A hallmark of the mammalian brain cholecystokinin (CCK) receptor, CCK-B/gastrin (CCK-BR), is its high affinity for two structurally related peptides, CCK and gastrin. Previous radioligand binding experiments suggested that the predominant CCK receptor from Xenopus laevis brain shares high affinity for sulfated cholecystokinin octapeptide but has > or = 1000-fold lower affinity for gastrin. To determine the molecular basis for this pharmacological divergence between mammalian and lower vertebrate receptors, we isolated a cDNA encoding the X. laevis brain CCK receptor (CCK-XLR). CCK-XLR shares approximately 50% homology at the amino acid level with both the human CCK-BR and the peripheral CCK-A receptor subtypes. The recombinant X. laevis receptor has a distinct pharmacological profile of agonist and antagonist affinities and as such offers a useful tool for structure-function studies. We used CCK-XLR to map the human CCK-BR domains that confer high affinity for gastrin. A series of chimeric CCK-BR/CCK-XLR constructs was generated and pharmacologically characterized. While maintaining wild-type affinity for sulfated cholecystokinin octapeptide, receptors with increasing amino-terminal contributions from CCK-BR demonstrated a stepwise increase in gastrin affinity. Further dissection of the amino-terminal third of the human receptor, a domain that confers a > 250-fold increase in gastrin affinity, revealed the importance of interactions among at least three subdomains. Additional structural requirements for gastrin affinity mapped to a segment spanning transmembrane domains IV and V.
Assuntos
Gastrinas/metabolismo , Receptores da Colecistocinina/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Humanos , Dados de Sequência Molecular , Receptores da Colecistocinina/metabolismo , Proteínas Recombinantes de Fusão/química , Xenopus laevisRESUMO
We have cloned an orphan G protein-coupled receptor from a human pituitary cDNA library using a probe generated by PCR. The cDNA, designated H9, encodes a protein of 613 amino acids that is 45% identical at the amino acid level to the recently cloned human Mel(1a) and Mel(1b) melatonin receptors. Structural analyses of the encoded protein and its gene, along with phylogenetic analysis, further show that H9 is closely related to the G protein-coupled melatonin receptor family. Unusual features of the protein encoded by H9 include a lack of N-linked glycosylation sites and a carboxyl tail >300 amino acids long. H9 transiently expressed in COS-1 cells did not bind [125I]melatonin or [3H]melatonin. H9 mRNA is expressed in hypothalamus and pituitary, suggesting that the encoded receptor and its natural ligand are involved in neuroendocrine function.