RESUMO
The modulation of glucagon receptor (GR) expression and biological response was investigated in human embryonic kidney cell (HEK-293) clones permanently expressing the GR with different densities. The GR mRNA expression level in these clones was upregulated by cellular cAMP accumulation and presented a good correlation with both the protein expression level and the maximum number of glucagon binding sites. However, the determination of glucagon-induced cAMP accumulation in these cell lines revealed that the enhancement of receptor expression did not lead to a proportional increase in cAMP formation. Under these conditions, the maximum cAMP production induced by NaF and forskolin was not significantly different among selected clones, regardless of the receptor expression level. High receptor-expressing clones showed the greatest susceptibility for agonist-induced desensitization compared with clones with lower GR expression levels. The results of the present study suggest that the GR can recruit non-GR-specific desensitization mechanism(s). Furthermore, the partial inhibition or alteration of the overall cAMP synthesis pathway at the receptor level may be a necessary adaptive step for a cell in response to a massive increase in membrane receptor expression level.
Assuntos
AMP Cíclico/metabolismo , Rim/citologia , Receptores de Glucagon/genética , 8-Bromo Monofosfato de Adenosina Cíclica/farmacologia , Animais , Linhagem Celular , Colforsina/farmacologia , Dactinomicina/farmacologia , Inibidores Enzimáticos/farmacologia , Proteínas de Ligação ao GTP/metabolismo , Expressão Gênica/efeitos dos fármacos , Expressão Gênica/fisiologia , Glucagon/farmacologia , Humanos , Isoproterenol/farmacologia , Inibidores da Síntese de Proteínas/farmacologia , RNA Mensageiro/análise , Ratos , Simpatomiméticos/farmacologia , TransfecçãoRESUMO
We prepared a stable cell line expressing the glucagon receptor to characterize the effect of G(s)-coupled receptor stimulation on extracellular signal-regulated protein kinase 1/2 (ERK1/2) activity. Glucagon treatment of the cell line caused a dose-dependent increase in cAMP concentration, activation of cAMP-dependent protein kinase (PKA), and transient release of intracellular calcium. Glucagon treatment also caused rapid dose-dependent phosphorylation and activation of mitogen-activated protein kinase kinase/ERK kinase (MEK1/2) and ERK1/2. Inhibition of either PKA or MEK1/2 blocked ERK1/2 activation by glucagon. However, no significant activation of several upstream activators of MEK, including Ras, Rap1, and Raf, was observed in response to glucagon treatment. In addition, chelation of intracellular calcium reduced glucagon-mediated ERK1/2 activation. In transient transfection experiments, glucagon receptor mutants that bound glucagon but failed to increase intracellular cAMP and calcium concentrations showed no glucagon-stimulated ERK1/2 phosphorylation. We conclude that glucagon-induced MEK1/2 and ERK1/2 activation is mediated by PKA and that an increase in intracellular calcium concentration is required for maximal ERK activation.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Receptores de Glucagon/metabolismo , Animais , Cálcio/metabolismo , Linhagem Celular , Ativação Enzimática/efeitos dos fármacos , Proteínas de Ligação ao GTP/metabolismo , Glucagon/farmacologia , Humanos , Proteína Quinase 3 Ativada por Mitógeno , Ratos , Receptores de Glucagon/genética , Transdução de Sinais , TransfecçãoRESUMO
These studies were performed to establish whether functional receptors for calcitonin gene-related peptide (CGRP) are present on human dendritic cells (DCs) and to investigate potential immunomodulatory effects of CGRP on DCs other than Langerhans cells. Reverse transcriptase-PCR revealed expression of mRNA for a type 1 CGRP receptor by mature and immature blood-derived DCs. Sequence analysis confirmed the identity of the type 1 CGRP receptor (CGRP-R1). Addition of CGRP (10-7 M) to mature and immature DCs resulted in mobilization of intracellular calcium. Treatment of immature DCs with CGRP (10-7 M), before and after maturation in monocyte-conditioned medium, resulted in decreased cell surface expression of HLA-DR MHC class II and the costimulatory molecule, CD86. Treatment of immature DCs with CGRP (10-7 M) also resulted in decreased expression of CD86, but expression of HLA-DR was unchanged. When CGRP-treated mature DCs were used to stimulate allogeneic T cells, proliferative responses were dampened (approximately 50%), especially at low DC:T cell ratios (1:360). This effect was not observed with CGRP-treated, immature DCs. In contrast, CGRP-treated mature or immature DCs were no less efficient than untreated DCs in driving syngeneic T cell-proliferative responses to staphylococcal enterotoxin B. We conclude that mature and immature DCs express type 1 CGRP receptors and that signaling through these receptors may dampen mature DC-driven T cell proliferation most likely via down-regulation of CD86 and HLA-DR.
Assuntos
Antígenos CD/biossíntese , Peptídeo Relacionado com Gene de Calcitonina/farmacologia , Células Dendríticas/imunologia , Antígenos HLA-DR/biossíntese , Imunossupressores/farmacologia , Ativação Linfocitária/imunologia , Glicoproteínas de Membrana/biossíntese , Receptores de Peptídeo Relacionado com o Gene de Calcitonina/fisiologia , Linfócitos T/imunologia , Antígenos CD/farmacologia , Antígeno B7-2 , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Cálcio/metabolismo , Diferenciação Celular/imunologia , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Enterotoxinas/farmacologia , Humanos , Isoantígenos/imunologia , Teste de Cultura Mista de Linfócitos , Glicoproteínas de Membrana/farmacologia , RNA Mensageiro/biossíntese , Staphylococcus aureus/imunologia , Superantígenos/farmacologia , Linfócitos T/microbiologiaRESUMO
G protein-coupled receptors (GPCRs) are seven-transmembrane (TM) helical proteins that bind extracellular molecules and transduce signals by coupling to heterotrimeric G proteins in the cytoplasm. The human D4 dopamine receptor is a particularly interesting GPCR because the polypeptide loop linking TM helices 5 and 6 (loop i3) may contain from 2 to 10 similar direct hexadecapeptide repeats. The precise role of loop i3 in D4 receptor function is not known. To clarify the role of loop i3 in G protein coupling, we constructed synthetic genes for the three main D4 receptor variants. D4-2, D4-4, and D4-7 receptors contain 2, 4, and 7 imperfect hexadecapeptide repeats in loop i3, respectively. We expressed and characterized the synthetic genes and found no significant effect of the D4 receptor polymorphisms on antagonist or agonist binding. We developed a cell-based assay where activated D4 receptors coupled to a Pertussis toxin-sensitive pathway to increase intracellular calcium concentration. Studies using receptor mutants showed that the regions of loop i3 near TM helices 5 and 6 were required for G protein coupling. The hexadecapeptide repeats were not required for G protein-mediated calcium flux. Cell membranes containing expressed D4 receptors and receptor mutants were reconstituted with purified recombinant G protein alpha subunits. The results show that each D4 receptor variant is capable of coupling to several G(i)alpha subtypes. Furthermore, there is no evidence of any quantitative difference in G protein coupling related to the number of hexadecapeptide repeats in loop i3. Thus, loop i3 is required for D4 receptors to activate G proteins. However, the polymorphic region of the loop does not appear to affect the specificity or efficiency of G(i)alpha coupling.
Assuntos
Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genética , Variação Genética , Engenharia de Proteínas/métodos , Receptores de Dopamina D2/genética , Éster Metílico do Ácido 3-Piridinacarboxílico, 1,4-Di-Hidro-2,6-Dimetil-5-Nitro-4-(2-(Trifluormetil)fenil)/farmacologia , Sequência de Aminoácidos , Animais , Células COS , Cálcio/antagonistas & inibidores , Cálcio/metabolismo , Linhagem Celular , Citoplasma/química , Citoplasma/metabolismo , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/biossíntese , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/química , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/metabolismo , Expressão Gênica , Genes Sintéticos , Humanos , Camundongos , Dados de Sequência Molecular , Fragmentos de Peptídeos/biossíntese , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/genética , Ligação Proteica/genética , Estrutura Secundária de Proteína , Quimpirol/farmacologia , Receptores de Dopamina D2/biossíntese , Receptores de Dopamina D2/química , Receptores de Dopamina D2/metabolismo , Receptores de Dopamina D4 , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/síntese química , Proteínas Recombinantes/síntese química , Proteínas Recombinantes/metabolismo , Espiperona/metabolismoRESUMO
The glucagon receptor is a member of a distinct class of G protein-coupled receptors (GPCRs) sharing little amino acid sequence homology with the larger rhodopsin-like GPCR family. To identify the components of the glucagon receptor necessary for G-protein coupling, we replaced sequentially all or part of each intracellular loop (i1, i2, and i3) and the C-terminal tail of the glucagon receptor with the 11 amino acids comprising the first intracellular loop of the D4 dopamine receptor. When expressed in transiently transfected COS-1 cells, the mutant receptors fell into two different groups with respect to hormone-mediated signaling. The first group included the loop i1 mutants, which bound glucagon and signaled normally. The second group comprised the loop i2 and i3 chimeras, which caused no detectable adenylyl cyclase activation in COS-1 cells. However, when expressed in HEK 293T cells, the loop i2 or i3 chimeras caused very small glucagon-mediated increases in cAMP levels and intracellular calcium concentrations, with EC50 values nearly 100-fold higher than those measured for wild-type receptor. Replacement of both loops i2 and i3 simultaneously was required to completely abolish G protein signaling as measured by both cAMP accumulation and calcium flux assays. These results show that the i2 and i3 loops play a role in glucagon receptor signaling, consistent with recent models for the mechanism of activation of G proteins by rhodopsin-like GPCRs.
Assuntos
Cálcio/metabolismo , AMP Cíclico/metabolismo , Receptores de Glucagon/química , Receptores de Glucagon/metabolismo , Adenilil Ciclases/metabolismo , Sequência de Aminoácidos , Animais , Células COS , Glucagon/metabolismo , Glicosídeo Hidrolases/metabolismo , Dados de Sequência Molecular , Conformação Proteica , Estrutura Secundária de Proteína , Ratos , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Relação Estrutura-Atividade , TransfecçãoRESUMO
Polyclonal antibodies were prepared against synthetic peptides corresponding to four different extramembrane segments of the rat glucagon receptor. The antibodies bound specifically to native glucagon receptor as judged by immunofluorescence microscopy of cultured cells expressing a synthetic gene for the receptor. Antibodies to peptides designated PR-15 and DK-12 were directed against amino acid residues 103-117 and 126-137, respectively, of the extracellular N-terminal tail. Antibody to peptide KD-14 was directed against residues 206-219 of the first extracellular loop, and antibody to peptide ST-18, against the intracellular C-terminal tail, residues 468-485. The DK-12 and KD-14 antibodies, but not the PR-15 and ST-18 antibodies, could effectively block binding of 125I-labeled glucagon to its receptor in liver membranes. Incubation of these antibodies with rat liver membranes resulted in both a decrease in the maximal hormonal binding capacity and an apparent decrease in glucagon affinity for its receptor. These effects were abolished in the presence of excess specific peptide antigen. In addition, DK-12 and KD-14 antibodies, but not PR-15 and ST-18 antibodies, interfered with glucagon-induced adenylyl cyclase activation in rat liver membranes and behaved as functional glucagon antagonists. These results demonstrate that DK-12 and KD-14 antibodies are pharmacologically active glucagon antagonists and strongly suggest that residues 126-137 of the N-terminal tail and residues 206-219 of the first extracellular loop contain determinants of ligand binding and may comprise the primary ligand-binding site on the glucagon receptor.
Assuntos
Glucagon/metabolismo , Receptores de Glucagon/química , Adenilil Ciclases/metabolismo , Sequência de Aminoácidos , Animais , Reações Antígeno-Anticorpo , Ligação Competitiva , Membrana Celular/metabolismo , Células Cultivadas , Chlorocebus aethiops , Ativação Enzimática , Mapeamento de Epitopos , Epitopos/química , Espaço Extracelular , Fígado/metabolismo , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/imunologia , Ligação Proteica , Ratos , Receptores de Glucagon/imunologia , Receptores de Glucagon/metabolismo , Transdução de Sinais , TransfecçãoRESUMO
Glucagon receptor mutants were characterized with the aim of elucidating minimal structural requirements for proper biosynthesis, ligand binding, and adenylyl cyclase coupling. One N-terminal deletion mutant and five truncation mutants with progressively shorter C termini were expressed in transiently transfected monkey kidney (COS-1) cells. Each truncation mutant was designed so that the truncated C-terminal tail would remain on the cytoplasmic surface of the receptor. In order to characterize the cellular location of the expressed receptor mutants, a highly specific, high affinity antipeptide antibody was prepared against the extracellular, N-terminal tail of the receptor. Immunoblot analysis and immunofluorescence microscopy showed that the presence of all seven putative transmembrane segments, but not not an intact N-terminal tail, was required for cell surface expression of the receptor. Membranes from cells expressing receptor mutants lacking a large portion of the N-terminal tail or any of the seven putative transmembrane segments failed to bind glucagon. Membranes from cells expressing the C-terminal tail truncation mutants, which retained all seven transmembrane segments, bound glucagon with affinities similar to that of the native receptor and activated cellular adenylyl cyclase in response to glucagon. These results indicate that all seven helices are necessary for the proper folding and processing of the glucagon receptor. Glycosylation is not required for the receptor to reach the cell surface, and it may not be required for ligand binding. However, the N-terminal extracellular portion of the receptor is required for ligand binding. Most of the distal C-terminal tail is not necessary for ligand binding, and the absence of the tail may increase slightly the receptor binding affinity for glucagon. The C-terminal tail is also not necessary for adenylyl cyclase coupling and therefore does not play a direct role in G protein (GS) activation by the glucagon receptor.
Assuntos
Glucagon/metabolismo , Estrutura Secundária de Proteína , Receptores de Glucagon/química , Receptores de Glucagon/metabolismo , Deleção de Sequência , Sequência de Aminoácidos , Animais , Ligação Competitiva , Linhagem Celular , Membrana Celular/metabolismo , Chlorocebus aethiops , Imunofluorescência , Genes Sintéticos , Cinética , Dados de Sequência Molecular , Mutagênese , Mutagênese Insercional , Ratos , Receptores de Glucagon/biossíntese , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Mapeamento por Restrição , TransfecçãoRESUMO
Various first messengers linked to phospholipase C, including acetylcholine and interleukin 1, regulate the production both of the secreted form of the amyloid protein precursor (APP) and of amyloid beta-protein. We have now identified intracellular signals which are responsible for mediating these effects. We show that activation of phospholipase C may affect APP processing by either of two pathways, one involving an increase in protein kinase C and the other an increase in cytoplasmic calcium levels. The effects of calcium on APP processing appear to be independent of protein kinase C activation. The observed effects of calcium on APP processing may be of therapeutic utility.
Assuntos
Peptídeos beta-Amiloides/biossíntese , Precursor de Proteína beta-Amiloide/metabolismo , Cálcio/metabolismo , Proteína Quinase C/metabolismo , Processamento de Proteína Pós-Traducional , Peptídeos beta-Amiloides/isolamento & purificação , Precursor de Proteína beta-Amiloide/isolamento & purificação , Animais , Células CHO , ATPases Transportadoras de Cálcio/antagonistas & inibidores , Carbacol/farmacologia , Linhagem Celular , Cricetinae , Eletroforese em Gel de Poliacrilamida , Glioma , Humanos , Immunoblotting , Interleucina-1/farmacologia , Modelos Biológicos , Peso Molecular , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Receptores Muscarínicos/biossíntese , Receptores Muscarínicos/metabolismo , Terpenos/farmacologia , Tapsigargina , Transfecção , Células Tumorais CultivadasRESUMO
Rhodopsin mutants responsible for autosomal dominant retinitis pigmentosa (ADRP) were prepared by site-directed mutagenesis and characterized. The aim was to evaluate ADRP mutations that occur at three locations on the cytoplasmic surface of rhodopsin: Thr-58 near the cytoplasmic border of helix A, the tetrapeptide Leu-68 to Pro-71 in the first cytoplasmic loop, and Arg-135 at the cytoplasmic border of helix C. It was hypothesized that amino acid changes at these sites would result in mutant rhodopsins with normal spectral properties but defects in their ability to interact with the rod outer segment G protein, transducin. A set of 12 mutant opsin genes was prepared. Four of the mutants were known to cause ADRP: Thr-58 replaced by Arg, a four-amino acid deletion (Leu-68/Arg-69/Thr-70/Pro-71), Arg-135 replaced by Leu, and Arg-135 replaced by Trp. Eight additional mutants were prepared to provide complementary structure-function information. The four-amino acid deletion mutant failed to bind 11-cis-retinal. However, each of the Thr-58 and Arg-135 mutants bound 11-cis-retinal to form a pigment with a visible absorbance maximum (lambda max) of 500 nm. Upon illumination, each pigment was converted to a metarhodopsin II-like spectral form (lambda max = 380 nm). However, each of these spectrally normal ADRP mutants was defective in activating guanine nucleotide exchange by transducin. These results identify a defect in the signal transduction pathway in spectrally normal mutant rhodopsins that cause ADRP.
Assuntos
Genes Dominantes , Retinose Pigmentar/genética , Rodopsina/genética , Rodopsina/metabolismo , Transducina/metabolismo , Sequência de Aminoácidos , Animais , Bovinos , Linhagem Celular , Genes Sintéticos , Humanos , Cinética , Dados de Sequência Molecular , Mutagênese Insercional , Mutagênese Sítio-Dirigida , Estrutura Secundária de Proteína , Rodopsina/química , Segmento Externo da Célula Bastonete/metabolismo , Homologia de Sequência de Aminoácidos , Transducina/isolamento & purificação , TransfecçãoRESUMO
An assay for the antileukaemic agent cytosine-beta-D-arabinoside (ara-C) has been developed using capillary zone electrophoresis. Solid-phase extraction and on-capillary peak concentration are used to improve the detection limit. The electrophoretic separation time is less than 5 min. The limit of detection for ara-C in plasma is 0.5 microM (signal-to-noise ratio = 3). The assay has been validated for the determination of ara-C in human plasma over the concentration range 1-10 microM. The calibration curve was linear with a correlation coefficient r2 = 0.996. At an ara-C concentration of 8 microM the intra-day coefficient of variation was 9.1% and the inter-day coefficient of variation was 12.3%. At an ara-C concentration of 2 microM the coefficients of variation were 15.2 and 12.0%, respectively.
Assuntos
Citarabina/sangue , Eletroforese/métodos , HumanosRESUMO
Variation in skin-to-kidney center distance has been shown to have a significant influence on quantification of renal function with the gamma camera. Several techniques to compensate for this variability have been proposed in adults, yet it has been suggested that depth correction is not necessary for quantitative renography in children. Skin-to-kidney center distances were measured from computed tomograms in 53 supine pediatric patients. Nearly 40% of the kidneys examined varied more than 1 cm from the average renal depth, and 8% deviated more than 2 cm. Right kidney depth differed from left kidney depth by more than 1 cm in less than 10% of the patients. Measurements were in agreement with regression equations based on lateral scintigraphy in children, but were consistently underestimated by nomograms developed for skin-to-kidney center distance in adults. Failure to recognize interindividual variability in skin-to-kidney center distance can introduce significant errors in quantitative pediatric renography.