RESUMO
PURPOSE: Human remyelination promoting IgM mAbs target oligodendrocytes (OLs) and function in animal models of multiple sclerosis (MS). However, their mechanism of action is unknown. This study seeks to identify the cellular mechanism of action of a recombinant human IgM on OL survival. METHODS: Binding of rHIgM22 to the surface of rat OLs was studied by co-localization with various markers. RHIgM22-mediated effects on apoptotic signaling in OLs, differentiation markers, and signaling molecules were detected by Western blotting and immunoprecipitation. RESULTS: RHIgM22 co-localized with integrin ß3 but not other integrin ß-chains in OLs. Downstream of integrin ß3 we identified Src family kinase (SFK) Lyn as a key player of rHIgM22-mediated actions in OLs. Lyn immunoprecipitated in a complex together with integrin αvß3 and PDGFαR. Lyn expression was 9-fold up-regulated and Lyn activation was 3-fold higher inrHIgM22-treated OL cultures compared with controls. RHIgM22 inhibited apoptotic signaling by greater than 10-fold reduction of caspase-3 and capsase-9 cleavage and reduced by 4-fold expression of differentiation markers MBP and MOG in OLs. SFK inhibitors PP2 and SU6656 inhibited Lyn activity and restored caspase-cleavage in OLs. A human IgM that did not promote remyelination and medium wereused as controls. CONCLUSIONS: rHIgM22 prevented apoptotic signaling andinhibited OL differentiation by Lyn implying thatIgM-mediated remyelination is due toprotection of OPC and OLs rather than promotion of OPC differentiation.
Assuntos
Apoptose/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Imunoglobulina M/farmacologia , Oligodendroglia/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Quinases da Família src/metabolismo , Animais , Animais Recém-Nascidos , Encéfalo/citologia , Caspase 3/metabolismo , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Feminino , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Humanos , Imunoglobulina M/uso terapêutico , Imunoprecipitação/métodos , Indóis/farmacologia , Integrina beta3/metabolismo , Fosforilação/efeitos dos fármacos , Gravidez , Pirimidinas/farmacologia , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/farmacologia , Proteínas Recombinantes/uso terapêutico , Sulfonamidas/farmacologiaRESUMO
Molecular mechanisms for the regulation of islet amyloid polypeptide (IAPP) gene expression remain unclear. In the present study, we investigated the effects of glucose and forskolin on IAPP gene regulation in the INS-1 islet beta-cell line. Both glucose and forskolin increased the level of expression of this gene, as measured by Northern blot analysis, and increased IAPP gene transcription in a time- and concentration-dependent manner, as demonstrated in a reporter gene assay. Although inhibition of protein kinase A activity with H-89 eliminated the effect of forskolin on this gene, the glucose effect was unaffected. This supported the predominant use of a protein kinase A-independent signaling pathway for glucose regulation of the IAPP gene. Electrophoretic mobility shift assay further indicated that glucose and forskolin regulated expression of this gene by targeting different elements of the promoter. Mutation of the cAMP regulatory element flanking the IAPP coding region resulted in the loss of most of the forskolin-stimulated IAPP gene promoter activity, whereas glucose-enhanced IAPP gene transcription was unaffected. These results demonstrate parallel and distinct regulatory pathways involved in glucose- and forskolin-induced IAPP gene expression in this model beta-cell system.
Assuntos
Amiloide/genética , Colforsina/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Glucose/farmacologia , Transdução de Sinais , Sulfonamidas , Inibidores de Adenilil Ciclases , Northern Blotting , Carcinoma Ductal Pancreático , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Humanos , Polipeptídeo Amiloide das Ilhotas Pancreáticas , Isoquinolinas/farmacologia , Neoplasias Pancreáticas , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Acetato de Tetradecanoilforbol/farmacologia , Células Tumorais CultivadasRESUMO
Modifications of mucosal phospholipids have been detected in samples from patients with Helicobacter pylori-positive gastritis. These alterations appear secondary to increased phospholipase A2 activity (PLA2). The cytosolic form of this enzyme (cPLA2), normally involved in cellular signaling and growth, has been implicated in cancer pathogenesis. The aim of this study was to investigate cPLA2 expression and PLA2 activity in the gastric mucosae of patients with and without H. pylori infection. In gastric biopsies from 10 H. pylori-positive patients, cPLA2 levels, levels of mRNA as determined by reverse transcriptase PCR, levels of protein as determined by immunohistochemistry, and total PLA2 activity were higher than in 10 H. pylori-negative gastritis patients. To clarify whether H. pylori had a direct effect on the cellular expression of cPLA2, we studied cPLA2 expression in vitro with different human epithelial cell lines, one from a patient with larynx carcinoma (i.e., HEp-2 cells) and two from patients with gastric adenocarcinoma (i.e., AGS and MKN 28 cells), incubated with different H. pylori strains. The levels of cPLA2, mRNA, and protein expression were unchanged in Hep-2 cells independently of cellular adhesion or invasion of the bacteria. Moreover, no change in cPLA2 protein expression was observed in AGS or MKN 28 cells treated with wild-type H. pylori. In conclusion, our study shows increased cPLA2 expression and PLA2 activity in the gastric mucosae of patients with H. pylori infection and no change in epithelial cell lines exposed to H. pylori.
Assuntos
Mucosa Gástrica/enzimologia , Gastrite/microbiologia , Infecções por Helicobacter/enzimologia , Infecções por Helicobacter/microbiologia , Helicobacter pylori/fisiologia , Fosfolipases A/metabolismo , Adulto , Idoso , Citosol/enzimologia , Células Epiteliais/enzimologia , Gastrite/enzimologia , Humanos , Pessoa de Meia-Idade , Fosfolipases A2 , Células Tumorais CultivadasRESUMO
Differences in the molecular structure or organ distribution of receptors can limit the usefulness of a given species for drug studies. In this work, we have studied cholecystokinin (CCK) receptors in cynomolgus monkey, an animal model useful for preclinical testing. The type A CCK receptor cDNA was cloned and predicted to encode a 428 amino acid peptide that was 98% identical to the human receptor. Only 2 of the 10 residues that were distinct from the human receptor were not present in other cloned CCK receptor species. A Chinese hamster ovary cell line that stably expressed this receptor was developed. The cynomolgus receptor expressed in this environment was functionally indistinguishable from the human receptor, binding CCK with high affinity [inhibition constant (K(I)) = 1.8 +/- 0.5 nM] and exhibiting a potent intracellular calcium signaling response to this hormone (EC(50) = 6.6 +/- 2.1 pM). Like the human type A CCK receptor, this receptor was expressed prominently in monkey gallbladder and stomach and was expressed in low levels in brain and pancreas. The type B CCK receptor cDNA was cloned from stomach and brain (450 residue receptor that is 96% identical to the human receptor), where it was highly expressed yet was undetectable in gallbladder or pancreas. This work confirms the relevance of the cynomolgus species for preclinical testing of drugs acting on the type A CCK receptor.
Assuntos
Macaca fascicularis/genética , Receptores da Colecistocinina/biossíntese , Receptores da Colecistocinina/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Células CHO , Clonagem Molecular , Cricetinae , Humanos , Macaca fascicularis/metabolismo , Dados de Sequência Molecular , RNA Mensageiro/biossíntese , Receptor de Colecistocinina A , Receptor de Colecistocinina B , Receptores da Colecistocinina/fisiologia , Homologia de Sequência de Aminoácidos , Distribuição TecidualRESUMO
Affinity labeling is a powerful tool to establish spatial approximations between photolabile residues within a ligand and its receptor. Here, we have utilized a cholecystokinin (CCK) analogue with a photolabile benzoylphenylalanine (Bpa) sited in position 24, adjacent to the pharmacophoric domain of this hormone (positions 27-33). This probe was a fully efficacious agonist that bound to the CCK receptor saturably and with high affinity (K(i) = 8.9 +/- 1.1 nm). It covalently labeled the CCK receptor either within the amino terminus (between Asn(10) and Lys(37)) or within the third extracellular loop (Glu(345)), as demonstrated by proteolytic peptide mapping, deglycosylation, micropurification, and Edman degradation sequencing. Truncation of the receptor to eliminate residues 1-30 had no detrimental effect on CCK binding, stimulated signaling, or affinity labeling through a residue within the pharmacophore (Bpa(29)) but resulted in elimination of the covalent attachment of the Bpa(24) probe to the receptor. Thus, the distal amino terminus of the CCK receptor resides above the docked ligand, compressing the portion of the peptide extending beyond its pharmacophore toward the receptor core. Exposure of wild type and truncated receptor constructs to extracellular trypsin damaged the truncated construct but not the wild type receptor, suggesting that this domain also may play a protective role. Use of these additional insights into molecular approximations provided key constraints for molecular modeling of the peptide-receptor complex, supporting the counterclockwise organization of the transmembrane helical domains.
Assuntos
Receptores da Colecistocinina/metabolismo , Sequência de Aminoácidos , Animais , Células CHO , Cricetinae , Hidrólise , Ligantes , Modelos Moleculares , Dados de Sequência Molecular , Marcadores de Fotoafinidade , Fotoquímica , Conformação Proteica , Receptores da Colecistocinina/químicaRESUMO
Intermolecular interaction represents an important theme in regulation of intracellular trafficking of organelles that can be interrupted by competitive overexpression of a relevant molecular domain. We attempted to identify the functional importance of intracellular domains of the cholecystokinin (CCK) receptor by their over-expression in receptor-bearing Chinese hamster ovary (CHO-CCKR) cell lines. Although clathrin-dependent endocytosis and recycling of this receptor are well-established (J Cell Biol 128:1029-1042, 1995), any influence of distinct receptor domains is not understood. In this work, constructs representing each of the intracellular domains of the CCK receptor were coexpressed with wild-type receptor, and stable clonal cell lines were selected. Each was characterized for ligand binding and agonist-stimulated biological activity (inositol 1,4,5-trisphosphate generation), desensitization, resensitization, receptor internalization, and recycling. Each cell line expressed normal CCK radioligand binding, signaling, internalization, and desensitization. Three independent cell lines that coexpressed the 25-residue second intracellular loop domain exhibited deficient resensitization. In morphological assessment of receptor trafficking, this construct was also shown to interfere with receptor recycling to the plasma membrane. As a control, recycling of an unrelated G protein-coupled receptor was demonstrated to occur normally in this cell line. These observations suggest that rather than representing passive cargo within an endosome, a receptor can influence its own trafficking within the cell.
Assuntos
Receptores da Colecistocinina/metabolismo , Sequência de Aminoácidos , Animais , Ligação Competitiva , Células CHO , Cricetinae , Relação Dose-Resposta a Droga , Imunofluorescência , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Fragmentos de Peptídeos/biossíntese , Fragmentos de Peptídeos/fisiologia , Conformação Proteica , Receptores da Colecistocinina/química , Transdução de SinaisRESUMO
Agonist-stimulated phosphorylation of guanine nucleotide-binding protein (G protein)-coupled receptors has been recognized as an important mechanism for desensitization by interfering with coupling of the activated receptor with its G protein. We recently described a mutant of the CCK receptor that modified two of five key sites of phosphorylation (S260,264A) and eliminated agonist-stimulated receptor phosphorylation, despite normal ligand binding and signaling (20). As expected, this nonphosphorylated mutant had impaired rapid desensitization but was ultimately able to be desensitized by normal receptor internalization. Here we demonstrate that this mutant receptor is also defective in resensitization, with abnormal recycling to the cell surface. To explore this, another receptor mutant was prepared, replacing the same serines with aspartates to mimic the charge of serine-phosphate (S260,264D). This mutant was expressed in a Chinese hamster ovary cell line and shown to bind CCK normally. It had accelerated kinetics of signaling and desensitization and was phosphorylated in response to agonist occupation, with all other normal sites of phosphorylation modified. It was internalized like wild-type receptors and was resensitized and trafficked normally. This provides evidence for an additional important function for phosphorylation of G protein-coupled receptors. Phosphorylation may induce a conformational change in the receptor to expose other potential sites of phosphorylation and to expose domains involved in the targeting and trafficking of endosomes. The hierarchical phosphorylation of these sites may play a key role in receptor regulation.
Assuntos
Endocitose/fisiologia , Transporte Proteico/fisiologia , Receptores da Colecistocinina/química , Receptores da Colecistocinina/metabolismo , Animais , Células CHO , Colecistocinina/farmacologia , Cricetinae , Endocitose/efeitos dos fármacos , Proteínas de Ligação ao GTP/metabolismo , Mutagênese/fisiologia , Fosforilação , Estrutura Terciária de Proteína , Transporte Proteico/efeitos dos fármacos , Receptores da Colecistocinina/genéticaRESUMO
Photoaffinity labeling is a powerful tool for the characterization of the molecular basis of ligand binding. We recently used this technique to demonstrate the proximity between a residue within the carboxyl-terminal half of a secretin-like ligand and the amino-terminal domain of the secretin receptor (Dong, M., Wang, Y., Pinon, D. I., Hadac, E. M., and Miller, L. J. (1999) J. Biol. Chem. 274, 903-909). In this work, we have developed another novel radioiodinatable secretin analogue ([Bpa6,Tyr10]rat secretin-27) that incorporates a photolabile p-benzoyl-L-phenylalanine (Bpa) residue into position 6 of the amino-terminal half of the ligand and used this to identify a specific receptor residue proximate to it. This probe specifically bound to the secretin receptor with high affinity (IC50 = 13.2 +/- 2.5 nM) and was a potent stimulant of cAMP accumulation in secretin receptor-bearing Chinese hamster ovary-SecR cells (EC50 = 720 +/- 230 pM). It covalently labeled the secretin receptor in a saturable and specific manner. Cyanogen bromide cleavage of this molecule yielded a single labeled fragment that migrated on an SDS-polyacrylamide gel at Mr = 19,000 that shifted to 10 after deglycosylation, most consistent with either of two glycosylated fragments within the amino-terminal tail. By immunoprecipitation with antibody directed to epitope tags incorporated into each of the two candidate fragments, the most distal fragment at the amino terminus was identified as the domain of labeling. The labeled domain was further refined to the first 16 residues by endoproteinase Lys-C cleavage and by cyanogen bromide cleavage of another receptor construct in which Val16 was mutated to Met. Radiochemical sequencing of photoaffinity-labeled secretin receptor fragments established that Val4 was the specific site of covalent attachment. This provides the first residue-residue contact between a secretin ligand and its receptor and will contribute substantially to the molecular understanding of this interaction.
Assuntos
Receptores dos Hormônios Gastrointestinais/metabolismo , Secretina/análogos & derivados , Sequência de Aminoácidos , Animais , Sítios de Ligação , Células CHO , Cricetinae , Ligantes , Dados de Sequência Molecular , Fenilalanina/análogos & derivados , Fenilalanina/metabolismo , Marcadores de Fotoafinidade/metabolismo , Estrutura Secundária de Proteína , Ratos , Receptores Acoplados a Proteínas G , Secretina/metabolismo , Relação Estrutura-Atividade , Valina/metabolismoRESUMO
The carboxy-terminal region of many guanine nucleotide-binding protein (G protein)-coupled receptors contains important regulatory sequences such as an NP(x)2-3Y motif, a site of fatty acid acylation, and serine- and threonine-rich domains. The type A CCK receptor contains all of these, yet their significance has not been examined. We have, therefore, constructed a series of receptor site mutants and truncations that interfere with each of these motifs and expressed each in Chinese hamster ovary cells where they were studied for radioligand binding, cell signaling, receptor internalization, and intracellular trafficking. Each construct was synthesized and transported appropriately to the cell surface, where CCK bound with high affinity, elicited an inositol 1,4, 5-trisphosphate response, and resulted in internalization and normal trafficking. Thus modification or elimination of each of these established sequence motifs had no substantial effect on any of these parameters of receptor and cellular function. However, an additional construct that truncated the carboxy terminus, eliminating an additional 15-amino-acid segment devoid of any currently recognized sequence motifs, resulted in a marked change in receptor trafficking, with all other parameters of receptor function normal. This mutant receptor construct was delayed at the stage of early endosomes, delaying its progress to the lysosome-enriched perinuclear compartment from the rapid time course followed by wild-type receptor and all of the other constructs. It is proposed that this region of the CCK receptor tail contains a new motif important for intracellular receptor trafficking.
Assuntos
Conformação Proteica , Receptores da Colecistocinina/química , Receptores da Colecistocinina/fisiologia , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Células CHO , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Colecistocinina/farmacologia , Cricetinae , Proteínas de Ligação ao GTP/metabolismo , Inositol 1,4,5-Trifosfato/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação Puntual , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Deleção de Sequência , Transdução de Sinais , Sincalida/farmacologia , TransfecçãoRESUMO
We have developed a biologically active analogue of cholecystokinin (CCK) that incorporates a photolabile benzoylphenylalanine (Bpa) moiety in the middle of its pharmacophoric domain, which efficiently establishes a covalent bond with an interacting domain of the CCK receptor. This probe incorporated L-Bpa in the position of Gly29 of the well characterized, radioiodinatable CCK analogue, D-Tyr-Gly-[(Nle28,31)CCK-26-33]. It was a potent pancreatic secretagogue (EC50 = 28 +/- 6 nM) that was equally efficacious with natural CCK, and bound to the CCK receptor with moderate affinity (IC50 = 450 +/- 126 nM). This was adequate to allow specific covalent labeling of the receptor. The labeled domain was within the cyanogen bromide fragment of the receptor including the top of TM6 (the sixth transmembrane domain), the third extracellular loop, and TM7 (the seventh transmembrane domain), as proven by direct Edman degradation sequencing. When this fragment was modified by the replacement of Val342 with Met to generate an additional site of cyanogen bromide cleavage, the labeled fragment was reduced in apparent size consistent with its representing the carboxyl-terminal portion of this fragment. Radiochemical sequencing of that fragment demonstrated covalent attachment of the probe to His347 and Leu348 in this domain. This represents the second experimentally demonstrated contact between a CCK analogue and this receptor, complementing the labeling of the domain just above TM1 (the first transmembrane domain) by a photolabile residue at the carboxyl terminus of CCK (Ji, Z. S., Hadac, E. M., Henne, R. M., Patel, S. A., Lybrand, T. P., and Miller, L. J. (1997) J. Biol. Chem. 272, 24393-24401). Both contacts are consistent with the conformational model of CCK binding proposed on the basis of the initial contact.
Assuntos
Colecistocinina/metabolismo , Receptores da Colecistocinina/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Células CHO , Cricetinae , Masculino , Sondas Moleculares , Dados de Sequência Molecular , Mutagênese , Ratos , Ratos Sprague-Dawley , Receptores da Colecistocinina/química , Receptores da Colecistocinina/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
The mouse cholecystokinin (CCK) receptor is functionally distinct from the extensively studied rat receptor on the basis of differences in binding and biological activity of phenethyl ester analogs of CCK. These are partial agonists at the rat receptor and full agonists at the mouse pancreatic receptor. To explore this, we cloned the cDNA for the mouse type A CCK receptor, established a receptor-bearing Chinese hamster ovary (CHO) cell line and characterized its binding and biological characteristics. Despite 25 differences in amino acid sequence from the rat receptor, including a seven-amino acid insertion in the third intracellular loop, mouse and rat receptors were functionally indistinguishable when expressed in CHO cells. Of note, in the mouse pancreatic cell environment, a stable analog of guanosine triphosphate significantly inhibited binding of CCK-OPE, whereas it had no effect on binding to the same receptor on the CHO-CCKM cell line or to the rat receptor in either environment of the acinar cell. This likely reflects a difference in coupling of the mouse receptor to its G protein in the natural environment of the acinar cell. This may relate to differences extrinsic to the receptor, in the stoichiometry or character of G proteins or in the composition or organization of the lipid environment of the mouse acinar cell membrane. Although this may require complementation of the unique sequence of the mouse receptor, that structure alone is insufficient to explain this phenomenon. Receptor microenvironment makes an important, yet often ignored, contribution to receptor function.
Assuntos
Receptores da Colecistocinina/agonistas , Sequência de Aminoácidos , Animais , Sequência de Bases , Células CHO , Colecistocinina/farmacologia , Clonagem Molecular , Cricetinae , Camundongos , Dados de Sequência Molecular , Ratos , Receptor de Colecistocinina A , Receptores da Colecistocinina/química , Receptores da Colecistocinina/genética , Relação Estrutura-AtividadeRESUMO
Receptor regulation is a key component of the phenomenon of desensitization in response to agonist stimulation which protects cells from overstimulation. Receptor internalization is one part of this response, often quantified by the portion of saturable ligand binding which becomes resistant to acidic washes. It is now clear that this can include receptor in multiple distinct cellular compartments. We have developed a morphological technique involving dual fluorescent probes to delineate the plasmalemma and the ligand-occupied receptor using confocal microscopy, with analysis involving three-dimensional reconstruction and quantitation of receptor movement through each compartment. When a radioiodinated cholecystokinin (CCK) analogue occupied its receptor on the CHO-CCKR cell line, it became progressively more resistant to dissociation with acidic medium. Quantitation of receptor internalization in these cells over time using this dynamic morphological technique correlated with the acid-resistant receptor fraction, and provided the additional information of the cellular compartments traversed. This technique will have multiple applications to explore the cell-specific handling of this and other ligand-occupied receptors.
Assuntos
Receptores da Colecistocinina/metabolismo , Animais , Transporte Biológico Ativo , Células CHO , Cricetinae , Corantes Fluorescentes , Processamento de Imagem Assistida por Computador , Cinética , Microscopia Confocal , Ensaio Radioligante , Ratos , Receptores da Colecistocinina/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
In an attempt to establish the relationship between the protein encoded by the recently cloned type A cholecystokinin (CCK) receptor cDNA and the two distinct plasmalemmal proteins on the rat pancreatic acinar cell that were previously described as candidates to represent this receptor, we have established a Chinese hamster ovary (CHO) cell line stably expressing large amounts of this recombinant protein and have used biochemical methods to characterize it directly. Upon affinity labeling, this protein migrated faster on a sodium dodecyl sulfate-polyacrylamide gel than the M(r) 85,000-95,000 molecule previously felt to represent the best candidate. However, deglycosylation with endoglycosidase F demonstrated that it had the same size core protein as that candidate, and this identification was further supported by protease peptide mapping. We postulated that the structural differences between the recombinant and the native proteins related to differences in glycosylation. Consistent with this, lectin-binding experiments demonstrated that both represented complex glycoproteins but that only the native receptor-bound Ulex europeus agglutinin I. Since this lectin binds to fucose residues that are added late in glycoprotein biosynthesis, it is possible that the distinct processing observed affected only that step. In spite of this structural difference, the type A CCK receptor-bearing CHO cell CCK receptor was functionally indistinguishable from the native acinar cell receptor. This included its ability to initiate signaling cascades, its sensitivity to stable GTP analogues, and its binding affinities for agonists and antagonists. The fidelity of this receptor expression system, while representing a 25-fold increase in receptor density over the native pancreatic acinar cell, should provide an ideal substrate for the examination of structure-function relationships within this molecule.
Assuntos
Lectinas de Plantas , Receptores da Colecistocinina/química , Receptores da Colecistocinina/metabolismo , Marcadores de Afinidade , Animais , Ligação Competitiva , Células CHO , Colecistocinina/metabolismo , Cricetinae , Glicosilação , Guanilil Imidodifosfato/metabolismo , Lectinas/metabolismo , Pâncreas/química , Ratos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Relação Estrutura-AtividadeRESUMO
Phosphorylation of G protein-coupled receptors is an established mechanism for desensitization in response to agonist stimulation. We previously reported phosphorylation of the pancreatic acinar cell cholecystokinin (CCK) receptor and the establishment of two-dimensional phosphopeptide mapping of its sites of phosphorylation (Ozcelebi, F., and Miller, L. J. (1995) J. Biol. Chem. 270, 3435-3441). Here, we use similar techniques to map sites of phosphorylation of the same receptor expressed on a stable receptor-bearing Chinese hamster ovary (CHO)-CCKR cell line. Like the native cell, the CHO-CCKR cell receptor was phosphorylated in response to agonist stimulation in a concentration-dependent manner; however, the time course was quite different. CHO-CCKR cell receptor phosphorylation increased progressively to a plateau after 15 min, while in the acinar cell it peaks within 2 min and returns to baseline over this interval. There were distinct qualitative and quantitative differences in the sites of phosphorylation of the two receptor systems. One site previously attributed to action of a staurosporine-insensitive kinase in the acinar cell was absent in the CHO-CCKR cell. Site-directed mutagenesis was utilized to eliminate predicted sites of protein kinase C action, but only two of four such sites affected the phosphopeptide map of this receptor. Chemical and radiochemical sequencing were performed on these and other phosphopeptides which were present in both the CHO-CCKR cells and agonist-stimulated pancreatic acinar cells to provide direct evidence for the phosphorylation sites actually utilized. Thus, these data support the usefulness and limitations of a model cell system in studying receptor phosphorylation and desensitization.
Assuntos
Pâncreas/metabolismo , Estrutura Secundária de Proteína , Receptores da Colecistocinina/metabolismo , Sequência de Aminoácidos , Animais , Células CHO , Colecistocinina/farmacologia , Cromatografia Líquida de Alta Pressão , Cricetinae , Eletroforese em Gel de Poliacrilamida , Modelos Estruturais , Dados de Sequência Molecular , Pâncreas/citologia , Mapeamento de Peptídeos , Fosfopeptídeos/química , Fosfopeptídeos/isolamento & purificação , Fosforilação , Ratos , Receptores da Colecistocinina/biossíntese , Receptores da Colecistocinina/química , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Sincalida/farmacologia , Acetato de Tetradecanoilforbol/farmacologia , TransfecçãoRESUMO
BACKGROUND & AIMS: Cholesterol gallstone disease and obesity are often associated and share the potential, yet unreported, common etiology of cholecystokinin (CCK) dysfunction. While cloning the human CCK-A receptor complementary DNA (cDNA), we found predominance of a 262-base pair coding region deletion in a cDNA library prepared from a patient with this phenotype. The aim of this study was to determine the abundance, functional significance, and mechanism for generating this gene product. METHODS: Relative abundance of CCK receptor gene products was determined using polymerase chain reaction and hybridization analysis. Constructs were expressed in COS cells and studied for radioligand binding and intracellular calcium responses. A human genomic clone for this receptor was sequenced, and the critical regions were compared with those of the patient. RESULTS: Ninety-three percent of the patient's CCK receptor transcripts contained the 262-base pair deletion, whereas only 1.5% +/- 0.9% of control patients had the deletion. This encoded a receptor that did not bind or signal. The deletion corresponded with the third exon; however, this sequence and flanking introns were normal in the patient. CONCLUSIONS: Abnormality of processing an apparently normal CCK receptor gene yields the predominant product with an absent third exon and encoding a nonfunctional receptor, probably reflecting a defective trans-acting splicing factor. An atypical lariat region in the third intron may explain the presence of small amounts of this product in control patients.
Assuntos
Colelitíase/genética , Obesidade/genética , Receptores da Colecistocinina/genética , Adulto , Sequência de Bases , Colesterol/metabolismo , Sequência Consenso , DNA Complementar , Éxons , Feminino , Humanos , Dados de Sequência Molecular , Receptores da Colecistocinina/metabolismoRESUMO
Receptor molecules play a major role in the desensitization of agonist-stimulated cellular responses. For G protein-coupled receptors, rapid desensitization occurs via receptor phosphorylation, sequestration, and internalization, yet the cellular compartments in which these events occur and their interrelationships are unclear. In this work, we focus on the cholecystokinin (CCK) receptor, which has been well characterized with respect to phosphorylation. We have used novel fluorescent and electron-dense CCK receptor ligands and an antibody to probe receptor localization in a CCK receptor-bearing CHO cell line. In the unstimulated state, receptors were diffusely distributed over the plasmalemma. Agonist occupation stimulated endocytosis via both clathrin-dependent and independent pathways. The former was predominant, leading to endosomal and lysosomal compartments, as well as recycling to the plasmalemma. The clathrin-independent processes led to a smooth vesicular compartment adjacent to the plasmalemma resembling caveolae, which did not transport ligand deeper within the cell. Potassium depletion largely eliminated clathrin-dependent endocytosis, while not interfering with agonist-stimulated receptor movement into subplasmalemmal smooth vesicle compartments. These cellular endocytic events can be related to the established cycle of CCK receptor phosphorylation and dephosphorylation, which we have previously described (Klueppelberg, U. G., L. K. Gates, F. S. Gorelick, and L. J. Miller. 1991. J. Biol. Chem. 266:2403-2408; Lutz, M. P., D. I. Pinon, L. K. Gates, S. Shenolikar, and L. J. Miller. 1993. J. Biol. Chem. 268:12136-12142). The rapid onset and peak of receptor phosphorylation after agonist occupation correlates best with a plasmalemmal localization, while stimulated receptor phosphatase activity correlates best with receptor residence in intracellular compartments. We postulate that the smooth vesicular compartment adjacent to the plasmalemma functions for the rapid resensitization of the receptor, while the classical clathrin-mediated endocytotic pathway is key for receptor downregulation via lysosomal degradation, as well as less rapid resensitization.
Assuntos
Receptores da Colecistocinina/metabolismo , Transdução de Sinais , Animais , Anticorpos , Células CHO/ultraestrutura , Compartimento Celular , Clatrina/farmacologia , Cricetinae , Endocitose/efeitos dos fármacos , Microscopia EletrônicaRESUMO
BACKGROUND: Structural characterization of pancreatic secretin receptors has been limited by difficulties in generating suitable radioligands and obtaining sufficient substrate. The aims of this study were to design, synthesize, and characterize high affinity radiolabeled analogues of secretin suitable for "intrinsic" photoaffinity labeling and to clone, express, and characterize the recombinant rat pancreatic secretin receptor. METHODS: The ability of synthetic analogues to stimulate amylase secretion by pancreatic acini was studied. Receptor complementary DNA (cDNA) was cloned by screening a rat pancreatic library with a probe based on the sequence of a neural cell secretin-binding protein. Competition binding and affinity labeling were performed with membranes prepared from rat pancreas and transfected cells. RESULTS: Two probes were fully efficacious secretagogues, which bound in a specific, high-affinity, rapid, and temperature-dependent manner. Only ([125I]Tyr10, pNO2-Phe22) rat secretin 27 covalently labeled a 50,000-62,000-molecular weight pancreatic membrane protein, with labeling inhibited in a concentration-dependent manner by secretin but not vasoactive intestinal polypeptide. Hybridization screening yielded a full-length cDNA identical to the neural clone. Photoaffinity labeling of this recombinant receptor identified a 57,000-62,000-molecular weight protein with specificity similar to that of native pancreas. Both native and recombinant receptors migrated at a molecular weight of 42,000 after endoglycosidase F deglycosylation. CONCLUSIONS: This study provides evidence for the molecular identity of the pancreatic secretin receptor and presents a novel probe important in structural characterization of its agonist-binding domain.
Assuntos
Marcadores de Afinidade/metabolismo , Pâncreas/química , Receptores dos Hormônios Gastrointestinais/análise , Secretina/metabolismo , Sequência de Aminoácidos , Animais , Membrana Celular/química , DNA Complementar/análise , Dados de Sequência Molecular , Peso Molecular , Reação em Cadeia da Polimerase , Ratos , Ratos Sprague-Dawley , Receptores Acoplados a Proteínas G , Receptores dos Hormônios Gastrointestinais/química , Receptores dos Hormônios Gastrointestinais/genética , Proteínas Recombinantes/análise , Relação Estrutura-AtividadeRESUMO
Through binding to cholecystokinin (CCK) A receptors, CCK is an important physiologic regulator of both gallbladder contraction and pancreatic enzyme secretion. In this work, we have used a combination of hybridization screening of a cDNA library and polymerase chain reaction to clone a 2.1 kb cDNA which encodes the human gallbladder CCKA receptor. Nucleotide sequence analysis revealed an open reading frame encoding a 428 amino acid protein, with seven putative transmembrane domains and a high degree of homology with the rat CCKA receptor. COS cells transfected with this cDNA clone bound CCK-8 and L-364,718 with high affinities appropriate for the CCKA receptor, and exhibited a transient increase in intracellular calcium in response to CCK. This should provide an important resource for the analysis of the role of this receptor in human physiology and pathophysiology.
Assuntos
Vesícula Biliar/metabolismo , Receptores da Colecistocinina/genética , Sequência de Aminoácidos , Animais , Ligação Competitiva , Células Cultivadas , Clonagem Molecular , DNA , Humanos , Dados de Sequência Molecular , Receptores da Colecistocinina/metabolismo , Sistemas do Segundo Mensageiro , Homologia de Sequência de Aminoácidos , TransfecçãoRESUMO
The snRNP E protein is one of several proteins associated with the U family of small nuclear RNAs that are involved in RNA processing. Isolation and characterization of the snRNP E protein cDNA sequences from mouse and chicken revealed a 100% conservation of the predicted amino acid sequence when compared to that of the human homologue. Further characterization of a genomic clone for the mouse snRNP E protein gene revealed that the 5' untranslated region and the immediate 5' upstream region have also been highly conserved: 72 and 70%, respectively. Conserved 5' regions include multiple copies of the CTTCCG hexamer sequence which are involved in regulating transcription of the human snRNP E protein gene. Mobility shift assays using corresponding DNA fragments from both human and mouse reveal that both fragments can compete for binding of at least one common transcription factor. These studies demonstrate that along with the amino acid sequence conservation between human and mouse, the snRNP E protein gene has also maintained a high DNA sequence conservation within its basal promoter structure.
Assuntos
Ribonucleoproteínas Nucleares Pequenas/genética , Transcrição Gênica , Animais , Sequência de Bases , Southern Blotting , DNA , Humanos , Camundongos , Dados de Sequência Molecular , Alinhamento de SequênciaRESUMO
A complex locus on human chromosome 1 brings together sequences homologous to a G protein and two components of the RNA processing machinery of eukaryotic cells. Specifically, the seventh intron of the human Gi3 alpha gene contains a fusion of a partial snRNP E protein pseudogene to a variant U6 snRNA gene. The novel U6 sequence contains nine point mutations and a one nucleotide deletion relative to the major U6 genes from humans. Unlike all other vertebrate U6 genes characterized to date, the variant U6 gene is efficiently transcribed by RNA polymerase III even in the absence of all natural flanking sequences. The union of elements from the signal transduction pathway and the RNA processing machinery suggests the possibility of functional interplay.