RESUMO
The gene encoding the mouse somatostatin receptor subtype 5 has been isolated from a genomic library and the mRNA start point mapped to position -95 relative to the translational start codon. The promoter region is devoid of TATA and CAAT boxes but contains putative binding sites for AP-1, AP-2 and SP1 and response elements for glucocorticoids (GRE) and phorbol esters (TRE). The encoded receptor protein with a predicted molecular weight of 42.5 kDa is comprised of 385 amino acids and thus contains 22 and 21 amino acids more than rat and human counterparts. The extra amino acids are caused by another translational initiation codon located further upstream. In the region of overlap the mouse somatostatin receptor subtype 5 displays 96.7% sequence identity to the rat and 81.7% to the human homologue. Application of somatostatin-14 and -28 to human embryonic kidney cells expressing the recombinant receptor resulted in the inhibition of forskolin-stimulated adenylyl cyclase with comparable EC50 values. Consistent with the observed sequence relationship, the mouse somatostatin receptor subtype 5 displays a pharmacological profile that resembles the rat homologue more closely than the human counterpart. mRNA for the mouse somatostatin type 5 receptor has been detected in pituitary, kidney, spleen and ovary and, to a lesser extent, in brain, stomach, intestine and thymus but was not observed in heart, pancreas and liver.
Assuntos
Biblioteca Genômica , Receptores de Somatostatina/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Código Genético , Humanos , Camundongos , Dados de Sequência Molecular , Ratos , Receptores de Somatostatina/efeitos dos fármacos , Receptores de Somatostatina/metabolismo , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Especificidade da EspécieRESUMO
Rat insulinoma 1046-38 cells represent a model system to study beta-cell function. The mRNAs for sst1 and sst2, two of the five somatostatin receptors, were detected by reverse transcription polymerase chain reaction amplification in these cells. Displacement binding analysis suggested that sstl represents the major somatostatin receptor subtype. The sstl selective compound CH-275 did not inhibit adenylyl cyclases while compounds that activated sst2 did. In contrast, CH-275 caused a marked inhibition of voltage-operated Ca2+ channels while the sst2 specific analog octreotide elicited a less pronounced effect suggesting that in rat insulinoma 1046-38 cells sst1 preferably mediates the inhibition of Ca2+ channels.
Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Receptores de Somatostatina/efeitos dos fármacos , Somatostatina/análogos & derivados , Animais , Insulinoma/metabolismo , Insulinoma/patologia , Potenciais da Membrana/efeitos dos fármacos , Ratos , Somatostatina/farmacologia , Células Tumorais CultivadasRESUMO
Endocytosis of somatostatin receptors could regulate cellular responses to the two natural peptides, somatostatin-14 and somatostatin-28, and to synthetic ligands used in the clinical diagnosis and symptomatic therapy of neuroendocrine tumours. The five cloned SSTRs with or without epitope tags at their carboxyl-termini were expressed in rat insulinoma 1046-38 cells. Application of the two natural peptides or octreotide, at 37 degrees C but not at 4 degrees C, to cells transfected with somatostatin receptor subtype 2 or 3 cDNA resulted in a significant decrease of cell surface binding-sites for 125I-Tyr11-somatostatin-14. In contrast, cells transfected with subtype 5 cDNA only responded to stimulation with octreotide or somatostatin-28. Cells transfected with subtype 1 cDNA responded to somatostatin-14 and 28, while cells expressing subtype 4 cDNA showed no response. Confocal microscopy revealed that 6 min after stimulation with somatostatin-14 at 37 degrees C, tagged somatostatin receptor subtypes 1, 2 and 3 were internalized into vesicles. Internalization was not observed at 4 degrees C in the presence of 0.4 M sucrose and 80 microM phenylarsine oxide and hence proceeded via endocytosis through clathrin-coated pits and vesicles. After 20 min the internalized receptors appeared in perinuclear vesicles and after 120 min they reappeared at the plasma membrane. This recycling was not sensitive to cycloheximide and, hence, not dependent on de novo protein synthesis. Recovery of cell surface receptors was, however, inhibited by brefeldin A, monensin and bafilomycin A1, indicating that receptor recycling proceeded through vesicular traffic of acidified compartments. The data are consistent with the assumption that the observed agonist and subtype specific internalization of somatostatin receptors in a neuroendocrine cell line may be important for tumour diagnosis and therapy and, thus, suggest a manifold control in cellular signalling.
Assuntos
Endocitose/efeitos dos fármacos , Insulinoma/ultraestrutura , Receptores de Somatostatina/agonistas , Receptores de Somatostatina/metabolismo , Adenilil Ciclases/metabolismo , Animais , Sítios de Ligação , Clonagem Molecular , DNA Complementar/genética , DNA Complementar/metabolismo , Epitopos/metabolismo , Insulinoma/metabolismo , Microscopia Confocal , Ratos , Receptores de Somatostatina/classificação , Sensibilidade e Especificidade , Transdução de Sinais/fisiologia , Somatostatina/metabolismo , Somatostatina/farmacologia , Somatostatina-28 , TransfecçãoRESUMO
The present study describes the expression pattern of somatostatin receptor genes during the development of rat cerebellum. Characterization of somatostatin receptors was carried out by binding studies using receptor subtype-selective ligands in the germinative epithelium and granule cell layer of the cerebellum from postnatal day 4 (P4) to P21 and in granule cell cultures. Quantitative reverse transcription-PCR carried out for the five receptor subtype mRNAs in cerebellar extracts showed that sst1 mRNAs are predominant at the end of gestation. A transient high expression of the sst2 gene was observed from P7 to P14. In parallel, high levels of binding sites sensitive to sst2 ligands were detected in the granule cell germinative epithelium and in granule cell cultures. sst3 mRNAs rapidly increased from P14 and became the predominant form at P21, but respective binding sites were not detected. Whereas sst4 mRNA levels were generally low, those of sst5 were nearly undetectable. Reverse transcription-PCR carried out in granule cell cultures revealed the relative abundance of sst mRNAs as follows: sst2 > sst1 > sst3 = sst4. sst5 mRNA was undetectable. The results show the expression of four somatostatin receptor genes, but only three receptors (sst1, sst4, and mainly sst2) were detected as binding sites during cerebellar development.
Assuntos
Cerebelo/embriologia , Neurônios/química , Receptores de Somatostatina/genética , Animais , Autorradiografia , Ligação Competitiva/fisiologia , Células Cultivadas/fisiologia , Cerebelo/química , Cerebelo/citologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Antagonistas de Hormônios/metabolismo , Antagonistas de Hormônios/farmacologia , Hormônios/metabolismo , Hormônios/farmacologia , Humanos , Radioisótopos do Iodo , Rim/citologia , Masculino , Neurônios/citologia , Neurônios/fisiologia , Octreotida/metabolismo , Octreotida/farmacologia , Oligopeptídeos/metabolismo , Oligopeptídeos/farmacologia , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/farmacologia , Peptídeos Cíclicos/metabolismo , Peptídeos Cíclicos/farmacologia , Reação em Cadeia da Polimerase , Gravidez , RNA Mensageiro/análise , Ratos , Ratos Wistar , Receptores de Somatostatina/metabolismo , Somatostatina/análogos & derivados , Somatostatina/farmacologiaRESUMO
Endocytosis of the five rat somatostatin receptor subtypes (SSTR1-5) was investigated in transfected HEK cells by biochemical ligand binding assays and confocal microscopic analysis. Phenylarsine oxide-sensitive internalization of SSTR1-3 is dependent on SST-14 or SST-28, whereas only the octacosapeptide triggers this reaction with SSTR5. SSTR4 is not internalized with either SST. Internalized SSTR3 is cycled back to the plasma membrane while endocytosed rho-Ala1-SST-14 remains inside the cell. Delineation of sequence motifs responsible for internalization of SSTR3 revealed multiple serines and a threonine (Ser-341, Ser-346, Ser-351, and Thr-357) within the carboxy-terminal tail of which Ser-351 and Thr-357 were the most effective ones. Chimeras in which various segments of the carboxyl terminus of SSTR4 were replaced by the corresponding regions of SSTR3 were internalized as long as they contain the Ser/Thr motif. However, this internalization reaction was suppressed when the chimeras were extended by the carboxyl terminus of SSTR4 (residues 320-384), suggesting the presence of a negative control element in that region. Step-wise truncation of the carboxyl terminus of wild-type SSTR4 revealed a motif of three amino acid residues Glu-Thr-Thr (SSTR4-330-332) that is responsible for preventing internalization and may be important in regulating endocytosis of this receptor subtype.
Assuntos
Endocitose , Receptores de Somatostatina/metabolismo , Somatostatina/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Linhagem Celular , Clonagem Molecular , Ligantes , Microscopia Confocal , Dados de Sequência Molecular , Ratos , Receptores de Somatostatina/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , TransfecçãoRESUMO
We report the isolation and characterisation of cDNAs encoding three different, human members of the cysteine-rich secretory protein (CRISP) family. The novel CRISP-1 exists in five cDNA subtypes differing by the presence or absence of a stretch coding for a C-terminal cysteine-rich domain so far found in all members of the family, and by the length of their 3'-untranslated region. CRISP-2 cDNA corresponds to the previously described TPX1 form, with so far unreported 5'-untranslated sequence heterogeneities while CRISP-3 cDNA codes for a new, unique protein. Northern blot analysis of various human organs indicates that CRISP-1 transcripts are epididymis-specific whereas CRISP-2/TPX1 transcripts are detected mainly in the testis and also in the epididymis. CRISP-3 transcripts are more widely distributed and found predominantly in the salivary gland, pancreas and prostate, and in less abundance in the epididymis, ovary, thymus and colon. A protein reacting with an anti-mouse CRISP-1 antibody was isolated from human epididymal extracts and N-terminal sequencing revealed that it corresponded to the CRISP-1 cDNA we have isolated. In contrast to findings on its rat counterpart epididymal protein DE/acidic epididymal glycoprotein (AEG), no significant association of CRISP-1 with human spermatozoa was observed.
Assuntos
Epididimo/metabolismo , Glicoproteínas/biossíntese , Glicoproteínas/química , Glicoproteínas de Membrana , Proteínas e Peptídeos Salivares/biossíntese , Proteínas e Peptídeos Salivares/química , Proteínas de Plasma Seminal , Testículo/metabolismo , Idoso , Sequência de Aminoácidos , Animais , Sequência de Bases , Moléculas de Adesão Celular , DNA Complementar , Ensaio de Imunoadsorção Enzimática , Biblioteca Gênica , Glicoproteínas/análise , Humanos , Masculino , Proteínas de Membrana , Camundongos , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Especificidade de Órgãos , Reação em Cadeia da Polimerase , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , Ratos , Proteínas Recombinantes/análise , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas e Peptídeos Salivares/análise , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Espermatozoides/metabolismo , Transcrição GênicaRESUMO
In the rat, the secretory glycoprotein DE/AEG is one of the main constituents of the epididymal fluid. We have recently reported the cloning of the cDNA for the related cysteine-rich secretory protein-1 (CRISP-1) from murine epididymis (Haendler et al., 1993; Endocrinology 133:192-198). The protein has now been isolated from the same organ and its N-terminal amino acid sequence has been determined. CRISP-1 exhibited an isoelectric point of approximately 6.8. High levels of CRISP-1 antigen were detected in the corpus and cauda of the epididymis, vas deferens, seminal vesicle, prostate, and in the salivary gland by immunohistochemistry. A quantitative analysis of the cauda epididymal fluid by sandwich ELISA revealed that CRISP-1 represented approximately 15% of the total protein. For heterologous expression, the CRISP-1 coding sequence was introduced into the pMPSV/CMV vector before transfection of baby hamster kidney (BHK) cells and selection with puromycin and neomycin. Expression in insect cells was achieved by co-transfection of Sf9 cells with a transfer vector and baculovirus DNA. Recombinant CRISP-1 was isolated in quantities sufficient for structural analysis. Ethyl maleimide treatment showed that all 16 cysteines were engaged in disulfide bonds. Proteolytic digestion demonstrated that the six cysteines localized in the N-terminal moiety formed three bonds with each other, suggesting the existence of two discrete domains in the protein.