RESUMEN
HIV persistence during combination antiretroviral therapy (cART) is the principal obstacle to cure. Mechanisms responsible for persistence remain uncertain; infections may be maintained by persistence and clonal expansion of infected cells or by ongoing replication in anatomic locations with poor antiretroviral penetration. These mechanisms require different strategies for eradication, and determining their contributions to HIV persistence is essential. We used phylogenetic approaches to investigate, at the DNA level, HIV populations in blood, lymphoid, and other infected tissues obtained at colonoscopy or autopsy in individuals who were on cART for 8 to 16 years. We found no evidence of ongoing replication or compartmentalization of HIV; we did detect clonal expansion of infected cells that were present before cART. Long-term persistence, and not ongoing replication, is primarily responsible for maintaining HIV. HIV-infected cells present when cART is initiated represent the only identifiable source of persistence and is the appropriate focus for eradication.
Asunto(s)
Infecciones por VIH/virología , VIH/fisiología , Replicación Viral , Adolescente , Adulto , Fármacos Anti-VIH/farmacología , Fármacos Anti-VIH/uso terapéutico , Terapia Antirretroviral Altamente Activa , Niño , Femenino , VIH/clasificación , VIH/efectos de los fármacos , VIH/genética , Infecciones por VIH/tratamiento farmacológico , Humanos , Masculino , Especificidad de Órganos , Filogenia , ARN Viral , Análisis de Secuencia de ADN , Replicación Viral/efectos de los fármacos , Adulto JovenRESUMEN
BACKGROUND: Gastro-oesophageal reflux disease (GERD) and gastric acid hypersecretion respond well to suppression of gastric acid secretion. However, clinical management and research in diseases of acid secretion have been hindered by the lack of a non-invasive, accurate and reproducible tool to measure gastric acid output (GAO). Thus, symptoms or, in refractory cases, invasive testing may guide acid suppression therapy. AIM: To present and validate a novel, non-invasive method of GAO analysis in healthy subjects using a wireless pH sensor, SmartPill (SP) (SmartPill Corporation, Buffalo, NY, USA). METHODS: Twenty healthy subjects underwent conventional GAO studies with a nasogastric tube. Variables impacting liquid meal-stimulated GAO analysis were assessed by modelling and in vitro verification. Buffering capacity of Ensure Plus was empirically determined. SP GAO was calculated using the rate of acidification of the Ensure Plus meal. Gastric emptying scintigraphy and GAO studies with radiolabelled Ensure Plus and SP assessed emptying time, acidification rate and mixing. Twelve subjects had a second SP GAO study to assess reproducibility. RESULTS: Meal-stimulated SP GAO analysis was dependent on acid secretion rate and meal-buffering capacity, but not on gastric emptying time. On repeated studies, SP GAO strongly correlated with conventional basal acid output (BAO) (r = 0.51, P = 0.02), maximal acid output (MAO) (r = 0.72, P = 0.0004) and peak acid output (PAO) (r = 0.60, P = 0.006). The SP sampled the stomach well during meal acidification. CONCLUSIONS: SP GAO analysis is a non-invasive, accurate and reproducible method for the quantitative measurement of GAO in healthy subjects. SP GAO analysis could facilitate research and clinical management of GERD and other disorders of gastric acid secretion.
Asunto(s)
Endoscopía Capsular/métodos , Ácido Gástrico/metabolismo , Modelos Teóricos , Adulto , Femenino , Ácido Gástrico/fisiología , Determinación de la Acidez Gástrica , Humanos , Concentración de Iones de Hidrógeno , Masculino , Persona de Mediana Edad , Reproducibilidad de los Resultados , Adulto JovenRESUMEN
D(1)-like (D(1), D(5)) and D(2)-like (D(2), D(3), D(4)) dopamine receptors interact in the kidney to produce a natriuresis and a diuresis. Disruption of D(1) or D(3) receptors in mice results in hypertension that is caused, in part, by a decreased ability to excrete an acute saline load. We studied D(1)-like and D(2)-like receptor interaction in anesthetized spontaneously hypertensive rats (SHR) by the intrarenal infusion of Z-1046 (a novel dopamine receptor agonist with rank order potency of D(3)> or =D(4)>D(2)>D(5)>D(1)). Z-1046 increased glomerular filtration rate (GFR), urine flow, and sodium excretion in normotensive Wistar-Kyoto rats but not in SHRs. The lack of responsiveness to Z-1046 in SHRs was not an epiphenomenon, because intrarenal cholecystokinin infusion increased GFR, urine flow, and sodium excretion to a similar extent in the two rat strains. We conclude that renal D(1)-like and D(2)-like receptor interaction is impaired in SHRs. The impaired D(1)-like and D(2)-like receptor interaction in SHRs is not caused by alterations in the coding sequence of the D(3) receptor, the D(2)-like receptor expressed in rat renal tubules that has been shown to be involved in sodium transport. Because the diuretic and natriuretic effects of D(1)-like receptors are, in part, caused by an interaction with D(2)-like receptors, it is possible that the decreased Z-1046 action in SHRs is secondary to the renal D(1)-like receptor dysfunction in this rat strain.
Asunto(s)
Hipertensión/metabolismo , Riñón/metabolismo , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D2/metabolismo , Animales , Transporte Biológico/fisiología , Colecistoquinina/administración & dosificación , Modelos Animales de Enfermedad , Diuresis/efectos de los fármacos , Agonistas de Dopamina/administración & dosificación , Tasa de Filtración Glomerular/efectos de los fármacos , Tasa de Filtración Glomerular/fisiología , Infusiones Intraarteriales , Riñón/efectos de los fármacos , Pruebas de Función Renal , Masculino , Naftoles/administración & dosificación , Natriuresis/efectos de los fármacos , Natriuresis/fisiología , Ratas , Ratas Endogámicas SHR , Ratas Endogámicas WKY , Receptores de Dopamina D1/agonistas , Receptores de Dopamina D2/agonistas , Receptores de Dopamina D2/biosíntesis , Receptores de Dopamina D2/genética , Receptores de Dopamina D3 , Análisis de Secuencia de ADN , Sodio/metabolismoAsunto(s)
Receptores de Colecistoquinina/química , Receptores de Colecistoquinina/fisiología , Secuencia de Aminoácidos , Animales , Humanos , Datos de Secuencia Molecular , Receptores de Colecistoquinina/clasificación , Receptores de Colecistoquinina/metabolismo , Relación Estructura-Actividad , Terminología como AsuntoAsunto(s)
Ácido Gástrico/metabolismo , Mucosa Gástrica/fisiología , Reflujo Gastroesofágico/fisiopatología , Neuropéptidos/fisiología , Neurotransmisores/fisiología , Úlcera Péptica/fisiopatología , Estómago/fisiología , Adulto , Animales , Mucosa Gástrica/inervación , Mucosa Gástrica/fisiopatología , Humanos , Modelos Biológicos , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa , Estómago/inervación , Estómago/fisiopatologíaRESUMEN
Recently, we reported that the mutation of His(207) to Phe located in the second extracellular loop of the cholecystokinin B receptor strongly affected cholecystokinin (CCK) binding (Silvente-Poirot, S., Escrieut, C., and Wank, S. A. (1998) Mol. Pharmacol. 54, 364-371). To characterize the functional group in CCK that interacts with His(207), we first substituted His(207) to Ala. This mutation decreased the affinity and the potency of CCK to produce total inositol phosphates 302-fold and 456-fold without affecting the expression of the mutant receptor. The screening of L-alanine-modified CCK peptides to bind and activate the wild type and mutant receptors allowed the identification of the interaction of the C-terminal Asp(8) of CCK with His(207). The H207A-CCKBR mutant, unlike the wild type receptor, was insensitive to substitution of Asp(8) of CCK to other amino acid residues. This interaction was further confirmed by mutating His(207) to Asp. The affinity of CCK for the H207D-CCKBR mutant was 100-fold lower than for the H207A-CCKBR mutant, consistent with an electrostatic repulsion between the negative charges of the two interacting aspartic acids. Peptides with neutral amino acids in position eight of CCK reversed this effect and displayed a gain of affinity for the H207D mutant compared with CCK. To date, this is the first report concerning the identification of a direct contact point between the CCKB receptor and CCK.
Asunto(s)
Ácido Aspártico/metabolismo , Colecistoquinina/metabolismo , Histidina/metabolismo , Receptores de Colecistoquinina/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Células COS , Colecistoquinina/química , Mutagénesis Sitio-Dirigida , Receptor de Colecistoquinina B , Receptores de Colecistoquinina/química , Receptores de Colecistoquinina/genética , Relación Estructura-ActividadRESUMEN
Semliki Forest Virus (SFV) vectors allow the subcloning of a gene of interest directly in the expression vector, thus avoiding the need to select and purify viral recombinants, making this viral expression system attractive over many others for mammalian protein expression. We now describe a novel and generally applicable method for infection of non-permissive mammalian cells with SFV, that greatly enhances the utility of this expression system. We demonstrate that the hygroscopic polymer poly (ethylene glycol), PEG, promotes the infectivity of cells by SFV under conditions that did not promote cell-cell fusion. We also found that the PEG-induced infection and expression of an exogenous protein (green fluorescent protein, GFP) did not elevate the basal tyrosine kinase activity, induce a stress-activated responses, or result in aberrant cell responses. Expression of GFP tagged-Vav, an activator of stress-activated protein kinase (SAPK/JNK), resulted in the expected induction of JNK activity and in the normal redistribution of Vav in response to engagement of the high affinity receptor for IgE (FcepsilonRI). Thus, our findings that PEG allows the infection of non-permissive cells by SFV makes this system extremely attractive for expression of proteins in mammalian cells, and studies on signal transduction and cellular localization in immune and non-immune cells.
Asunto(s)
Excipientes Farmacéuticos/farmacología , Polietilenglicoles/farmacología , Virus de los Bosques Semliki , Transducción de Señal/fisiología , Células 3T3/metabolismo , Células 3T3/virología , Animales , Células COS/metabolismo , Células COS/virología , Cricetinae , Vectores Genéticos , Proteínas Fluorescentes Verdes , Riñón/metabolismo , Riñón/virología , Leucemia Basofílica Aguda/metabolismo , Leucemia Basofílica Aguda/virología , Proteínas Luminiscentes/biosíntesis , Proteínas Luminiscentes/genética , Ratones , Proteínas Oncogénicas/biosíntesis , Proteínas Oncogénicas/genética , Señales de Clasificación de Proteína/biosíntesis , Señales de Clasificación de Proteína/genética , Señales de Clasificación de Proteína/fisiología , Proteínas Proto-Oncogénicas c-vav , Ratas , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Virus de los Bosques Semliki/genéticaRESUMEN
The cholecystokinin (CCK) receptor types A and B (CCKAR and CCKBR) are G protein-coupled receptors with approximately 50% amino acid identity; both have high affinity for the sulfated CCK octapeptide (CCK-8), whereas only the CCKBR has high affinity for gastrin. Previously, we identified five amino acids in the second extracellular loop (ECL) of the CCKBR that were essential for gastrin selectivity. Subsequent mutagenesis of one of these five amino acids (H207F) resulted in the loss of radiolabeled CCK-8 binding. CCK-8 stimulated total inositol phosphate accumulation in COS-1 cells transiently expressing the CCKBR-H207F with full efficacy and a 3044-fold reduced potency, which suggests that the loss of radioligand binding was caused by a loss in affinity. Alanine scanning mutagenesis was performed on the amino terminus near the top of transmembrane domain I (TMI) and on ECL1, two extracellular domains implicated in ligand binding by previous mutagenesis studies. 125I-Bolton-Hunter-CCK-8 binding to mutant receptors transiently expressed in COS-1 identified one nonconserved amino acid, R57A, at the top of TMI that caused a 21-fold reduction in CCK-8 affinity and four conserved amino acids, N115A, L116A, F120A and F122A, in the ECL1 that caused a 15.6-, 6-, 440-, and 8-fold reduction in affinity or efficacy. Alanine substitution of the equivalent amino acids in the CCKAR corresponding to each of the five amino acids in ECL1 and ECL2 affecting CCK-8 affinity for the CCKBR revealed only two mutations, L103A and F107A, that decreased CCK-8 affinity (68- and 2885-fold, respectively). These data suggest that CCK-8 interacts at multiple contact points in the extracellular domains of CCK receptors and that the CCKAR and CCKBR have distinct binding sites despite their shared high affinity for CCK-8.
Asunto(s)
Receptores de Colecistoquinina/agonistas , Sincalida/farmacología , Secuencia de Aminoácidos , Animales , Sitios de Unión , Células COS , Datos de Secuencia Molecular , Conformación Proteica , Receptores de Colecistoquinina/metabolismo , Sincalida/metabolismoRESUMEN
The CCK and gastrin families of peptides act as hormones and neuropeptides on central and peripheral receptors to mediate secretion and motility in the gastrointestinal tract in the physiological response to a normal meal. Thus far, two CCK receptors have been molecularly identified to mediate the actions of CCK and gastrin, CCK-A and CCK-B receptors (CCK-AR and CCK-BR, respectively). The regulation of CCK-AR and CCK-BR affinity by guanine nucleotides and the receptor activation of G protein-dependent stimulation of phospholipase C and adenylyl cyclase suggested that they were guanine nucleotide-binding protein-coupled receptors [G protein-coupled receptors (GPCRs)]; however, the eventual cloning of their cDNAs revealed their heptahelical structure and confirmed their membership in the GPCR superfamily. The gastrointestinal system is a rich source of neuroendocrine hormones that interact with a large number of GPCRs to regulate the complex tasks of digestion, absorption, and excretion of a meal. This article focuses on the CCK family of GPCRs, and its activities in the gastrointestinal system.
Asunto(s)
Fenómenos Fisiológicos del Sistema Digestivo , Proteínas de Unión al GTP/metabolismo , Receptores de Superficie Celular/metabolismo , Receptores de Colecistoquinina/metabolismo , Secuencia de Aminoácidos , Animales , Colecistoquinina/genética , Colecistoquinina/metabolismo , Gastrinas/genética , Gastrinas/metabolismo , Humanos , Datos de Secuencia Molecular , Mutación , Fragmentos de Péptidos/genética , Receptores de Colecistoquinina/genéticaRESUMEN
Helodermin and exendin-4, two peptides isolated from the salivary gland of the Gila monster, Heloderma suspectum, are approximately 50% homologous to vasoactive intestinal peptide (VIP) and glucagon-like peptide-1 (GLP-1), respectively, and interact with the mammalian receptors for VIP and GLP-1 with equal or higher affinity and efficacy. Immunohistochemical studies suggested the presence of helodermin-like peptides in mammals. To determine whether helodermin and exendin-4 are present in mammals and their evolutionary relationship to VIP and GLP-1, their cDNAs were first cloned from Gila monster salivary gland. Northern blots and reverse transcription-polymerase chain reaction of multiple Gila monster tissues identified approximately 500-base pair transcripts only from salivary gland. Both helodermin and exendin-4 full-length cDNAs were approximately 500 base pairs long, and they encoded precursor proteins containing the entire amino acid sequence of helodermin and exendin-4, as well as a 44- or 45-amino acid N-terminal extension peptide, respectively, having approximately 60% homology. The size and structural organization of these cDNAs indicated that they were closely related to one another but markedly different from known cDNAs for the VIP/GLP-1 peptide family previously identified in both lower and higher evolved species. Cloning of the Gila monster VIP/peptide histidine isoleucine, pituitary adenylate cyclase activating polypeptide, and glucagon/GLP-1 cDNAs and Southern blotting of Gila monster DNA demonstrate the coexistence of separate genes for these peptides and suggests, along with the restricted salivary gland expression, that helodermin and exendin-4 coevolved to serve a separate specialized function. Probing of a variety of rat and human tissues on Northern blots, human and rat Southern blots, and genomic and cDNA libraries with either helodermin- or exendin-4-specific cDNAs failed to identify evidence for mammalian homologues. These data indicate that helodermin and exendin-4 are not the precursors to VIP and GLP-1 and that they belong to a separate peptide family encoded by separate genes. Furthermore, the existence of as yet undiscovered mammalian homologues to helodermin and exendin-4 seems unlikely.
Asunto(s)
Neuropéptidos/química , Péptidos/química , Péptidos/genética , Péptido Intestinal Vasoactivo/química , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Pollos , Clonación Molecular , Cartilla de ADN , ADN Complementario , Exenatida , Glucagón , Péptido 1 Similar al Glucagón , Péptidos Similares al Glucagón , Humanos , Péptidos y Proteínas de Señalización Intercelular , Lagartos , Mamíferos , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Péptidos/metabolismo , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa , Reacción en Cadena de la Polimerasa , Precursores de Proteínas/química , Ratas , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Trucha , PonzoñasRESUMEN
Internalization of a variety of different heptahelical G protein-coupled receptors has been shown to be influenced by a number of different structural determinants of the receptors, including the carboxyl terminus. To investigate the role of the carboxyl terminus of cholecystokinin (CCK) receptors in receptor internalization, the rat wild type (WT) CCK-A receptor (WT CCKAR) and the rat WT CCK-B receptor (WT CCKBR) were truncated after amino acid residue 399 (CCKAR Tr399) and 408 (CCKBR Tr408), thereby deleting the carboxyl-terminal 45 and 44 residues, respectively. All WT and mutant CCK receptors were stably expressed in NIH/3T3 cells. Internalization of the CCKAR Tr399 was not significantly different from the WT CCKAR. In contrast, internalization of the CCKBR Tr408 was decreased to 26% compared with the WT CCKBR internalization of 92%. The mutation of all 10 serine and threonine residues (as potential phosphorylation sites) in the carboxyl terminus of the CCKBR to alanines (mutant CCKBR DeltaS/T) could account for the majority of this effect (39% internalization). All mutant receptors displayed similar ligand binding characteristics, G protein coupling, and signal transduction as their respective WT receptors, indicating that the carboxyl termini are not necessary for these processes. Thus, internalization of the CCKBR, unlike that of the CCKAR, depends on the carboxyl terminus of the receptor. These results suggest that, despite the high degree of homology between CCKAR and CCKBR, the structural determinants that mediate the interaction with the endocytic pathway reside in different regions of the receptors.
Asunto(s)
Receptores de Colecistoquinina/química , Receptores de Colecistoquinina/metabolismo , Sincalida/farmacología , Células 3T3 , Secuencia de Aminoácidos , Animales , Calcio/metabolismo , Colorantes Fluorescentes , Radioisótopos de Yodo , Cinética , Ligandos , Ratones , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Estructura Secundaria de Proteína , Ensayo de Unión Radioligante , Ratas , Receptor de Colecistoquinina A , Receptor de Colecistoquinina B , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Rodaminas , Eliminación de Secuencia , Sincalida/metabolismo , Especificidad por Sustrato , TransfecciónRESUMEN
A chimeric protein consisting of the cholecystokinin receptor type A (CCKAR) and the green fluorescent protein (GFP) was used for studying receptor localization, internalization, and recycling in live cells in real time in four different cell lines. Fusion of the C terminus of the CCKAR to the N terminus of the GFP did not alter receptor ligand binding affinity, signal transduction, or the pattern of receptor surface expression and receptor-mediated cholecystokinin (CCK) internalization. The use of a new GFP mutant with increased fluorescence allowed the continuous observation of CCKAR-GFP in stably expressing cell lines. Newly obtained biologically active fluorescent derivatives of CCK were used for simultaneous observation of receptor and ligand trafficking in CHO, NIH/3T3, and HeLa cells stably expressing the fluorescent CCKAR and in transiently transfected COS-1 cells. Receptor internalization was predominantly ligand dependent in HeLa, COS-1, and CHO cells, but was mostly constitutive in NIH/3T3 cells, suggesting the existence of cell-specific regulation of receptor internalization. The CCKAR antagonists, L-364,718 and CCK 27-32 amide potently inhibited spontaneous internalization of the receptor. The average sorting time of CCK and the receptor in the endosomes was about 25 min. The receptor recycled back to the cell membrane with an average time of 60 min. While the ligands sorted to lysosomes, no receptor molecules could be detected there, and no receptor degradation was observed during recycling. These results demonstrate the usefulness of GFP tagging for real time imaging of G protein-coupled receptor trafficking in living cells and suggest that this technique may be successfully applied to the study of the regulation and trafficking mechanisms of other receptors.
Asunto(s)
Endocitosis , Fosfatos de Inositol/metabolismo , Receptores de Colecistoquinina/fisiología , Sincalida/farmacología , Células 3T3 , Animales , Células CHO , Células COS , Cricetinae , Genes Reporteros , Proteínas Fluorescentes Verdes , Células HeLa , Humanos , Cinética , Proteínas Luminiscentes/biosíntesis , Ratones , Ratas , Receptor de Colecistoquinina A , Receptores de Colecistoquinina/biosíntesis , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/metabolismo , Sincalida/metabolismo , TransfecciónRESUMEN
Endocytosis of gastrin was studied in a number of gastrin-receptor-expressing cell lines by confocal laser scanning microscopy (CLSM) with the aid of a biologically active fluorescent derivative, rhodamine green heptagastrin. Rapid clustering (within 4-7 min) and internalization of fluorescent ligand upon binding at room temperature and 37 degrees C were observed in the rat pancreatic acinar carcinoma cell line AR42J, human gastric carcinomas AGS-P and SIIA, human colon carcinomas HCT116 and HT29, and in NIH/3T3 cells transfected with human and rat gastrin/cholecystokinin-B receptor cDNA. Internalization was inhibited by hypertonic medium. Fluorescent heptagastrin and transferrin colocalized in the same endocytic vesicles at different stages of internalization suggesting that endocytosis occurred predominantly through a clathrin-dependent mechanism. At 37 degrees C partial colocalization with the lysosomal marker neutral red was detected by CLSM, implying that internalized gastrin accumulated in the lysosomes. Immunoelectron microscopy studies with antibodies against gastrin revealed the presence of the internalized hormone in multivesicular vesicles and endosomes. Almost no hormone was detected in lysosomes with the antibodies to gastrin, suggesting that the degradation of the peptide is rapid in those vesicles. Continuous accumulation of fluorescent label was observed by CLSM in the presence of the protein synthesis inhibitor cycloheximide, suggesting that the gastrin receptor is recycled back to the cell membrane after hormone delivery to intracellular compartments. An estimated average recycling time for the receptor molecules was 1 h in NIH/3T3 cells.
Asunto(s)
Carcinoma de Células Acinares/metabolismo , Carcinoma/metabolismo , Endocitosis , Gastrinas/metabolismo , Neoplasias Gastrointestinales/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias Pancreáticas/metabolismo , Receptores de Colecistoquinina/metabolismo , Células 3T3 , Animales , Invaginaciones Cubiertas de la Membrana Celular/metabolismo , Cicloheximida/farmacología , Endocitosis/efectos de los fármacos , Humanos , Lisosomas/metabolismo , Ratones , Microscopía Confocal , Microscopía Inmunoelectrónica , Inhibidores de la Síntesis de la Proteína/farmacología , Ratas , Proteínas Recombinantes/metabolismo , Transfección , Transferrina/metabolismo , Células Tumorales CultivadasRESUMEN
Pituitary adenylate cyclase-activating polypeptide (PACAP), a neuropeptide belonging to the VIP/secretin/glucagon family, is present in the hypothalamus, anterior pituitary, and adrenal gland where it regulates hormone release, in the GI tract where it modulates motility, and in human tumoral cell lines where it shows a growth-promoting effect. It is now appreciated that alternative splicing of two exons of the rat PACAP-R gene generate four major rPACAP-R splice variants that are differentially expressed in tissues and variably coupled to intracellular second messengers. Because of the potential implications of these findings in human physiology, we cloned the hPACAP-R gene. Similar to the rat, two exons (SV-1 and SV-2) are alternatively spliced to account for four major hPACAP-R receptor splice variants. These splice variants (hPACAP-R-null, hPACAP-R-SV1, hPACAP-R-SV2, hPACAP-R-SV-3) were cloned from a human frontal cortex cDNA library, stably transfected in NIH/ 3T3 cells and each characterized for ligand affinity, stimulation of adenylate cyclase (AC) and phospholipase C (PLC), and ligand-induced expression of the proto-oncogenes, c-fos, and c-myc. Stably transfected NIH/3T3 cells expressing similar numbers of receptors of the four splice variants showed nearly identical responses for ligand affinity and potency for P-38- and P-27-stimulated increases in cAMP and total inositol phosphates. However, each receptor splice variant differed in their ligand-stimulated efficacy for total inositol phosphate stimulation. The hPACAP-R-SV2 showed the greatest efficacy for stimulating phospholipase C that was approximately seven-fold greater than the hPACAP-R-SV1, twofold greater than the hPACAP-R-Null, and 1.5-fold greater than the hPACAP-R-SV-3 splice variants. To determine whether the splice variants also differ in their ability to stimulate immediate early gene expression, c-fos and c-myc transcripts were assayed by Northern blot and quantified by densitometry. PACAP-38 increased c-fos and c-myc expression for all four of the receptor splice variants that paralleled the efficacy for PLC stimulation, with the the SV-2 splice variant showing the greatest stimulation. These results show that the hPACAP-R-SV2 exhibits enhanced efficacy for coupling to both PLC and activation of the protooncogenes, c-fos and c-myc suggesting a novel and potentially important mechanism for differentially activating signal transduction pathways that influence cellular growth and differentiation.
Asunto(s)
Empalme Alternativo , Regulación de la Expresión Génica , Genes Inmediatos-Precoces , Receptores de la Hormona Hipofisaria/fisiología , Transducción de Señal , Células 3T3 , Glándulas Suprarrenales/fisiología , Secuencia de Aminoácidos , Animales , Clonación Molecular , AMP Cíclico/metabolismo , Exones , Variación Genética , Humanos , Hipotálamo/fisiología , Fosfatos de Inositol/metabolismo , Ratones , Datos de Secuencia Molecular , Hipófisis/fisiología , Estructura Secundaria de Proteína , Ratas , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria , Receptores de la Hormona Hipofisaria/biosíntesis , Receptores de la Hormona Hipofisaria/química , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Activación Transcripcional , TransfecciónRESUMEN
Alternative splicing of two exons of the rat pituitary adenylate cyclase activating polypeptide (PACAP) receptor gene generates four major splice variants that are differentially expressed in specific tissues and variably coupled to intracellular second messengers. To evaluate the potential implications of these findings in human physiology, the human PACAP receptor gene was cloned. Alternative splicing about two exons of the gene allowed for four major splice variants that were subsequently identified on cDNA cloning. Each of the four splice variant cDNAs (null, SV-1, SV-2, and SV-3) was stably expressed in NIH/3T3 cells at similar receptor densities. For each splice variant, PACAP (both PACAP-38 and PACAP-27) had similar affinity and potency for stimulating either adenylate cyclase or phospholipase C. However, each receptor splice variant differed in their ligand-stimulated maximal response (efficacy) for total inositol phosphate accumulation with the SV-2 showing the greatest efficacy, followed by the null, SV-1, and SV-3 splice variants. Therefore, unlike the rat, PACAP binds and stimulates signal transduction with nearly equal affinity and potency for each of the receptor splice variants although with varying efficacy for the stimulation of phospholipase C. These results suggest a novel and potentially important mechanism for a single hormone to not only couple to dual signal transduction cascades but also elicit tissue-specific differential activation of phospholipase C in humans.
Asunto(s)
Adenilil Ciclasas/metabolismo , Empalme Alternativo , Variación Genética , Neuropéptidos/fisiología , Hipófisis/metabolismo , Transducción de Señal , Fosfolipasas de Tipo C/metabolismo , Células 3T3 , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Cartilla de ADN , Exones , Humanos , Fosfatos de Inositol/metabolismo , Cinética , Ratones , Datos de Secuencia Molecular , Neuropéptidos/química , Neuropéptidos/genética , Neurotransmisores/fisiología , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa , Reacción en Cadena de la Polimerasa , Estructura Secundaria de Proteína , Ratas , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Sistemas de Mensajero Secundario , TransfecciónRESUMEN
The two known receptors mediating the actions of cholecystokinin (CCK) and gastrin, CCK type A (CCKAR) and CCK type B (CCKBR) receptors, are G protein-coupled receptors having approximately 50% amino acid homology. Both the CCKAR and CCKBR have high affinity for sulfated CCK peptides, while only the CCKBR has high affinity for gastrin peptides. To determine the structural basis for the selectivity of the CCKBR for gastrin, we first constructed a series of CCKB/AR chimeras in which restriction endonuclease-defined segments of the CCKBR were replaced with the corresponding segments of the CCKAR. Chimeras transiently expressed in COS-1 cells were screened for the selective loss of gastrin affinity according to the displacement of 125I-labeled Bolton-Hunter-CCK-8 binding by gastrin-17-I and CCK-8. The sequence spanning from transmembrane domain III (TM III) to TM V was the only segment that resulted in the selective loss of gastrin affinity. This segment could account for 100 of the expected 300-fold lower affinity of gastrin-17-I observed for the control CCKAR compared to the control CCKBR. Using site-directed mutagenesis in this segment of the CCKBR, we identified a sequence of 5 amino acids in the second extracellular loop responsible for this 100-fold selective loss in gastrin affinity. 125I-labeled Bolton-Hunter-CCK-8 binding displacement by L365,260 (a CCKBR selective antagonist) was unaffected by the changes in these 5 amino acids. These results present for the first time the identification of the amino acid sequence of the CCKBR conferring the majority of the selectivity for gastrin.
Asunto(s)
Gastrinas/metabolismo , Compuestos de Fenilurea , Estructura Secundaria de Proteína , Receptores de Colecistoquinina/química , Receptores de Colecistoquinina/metabolismo , Secuencia de Aminoácidos , Animales , Benzodiazepinonas/farmacología , Sitios de Unión , Unión Competitiva , Línea Celular , Membrana Celular/metabolismo , Chlorocebus aethiops , Gastrinas/farmacología , Humanos , Indicadores y Reactivos , Cinética , Modelos Estructurales , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación Puntual , Receptor de Colecistoquinina B , Receptores de Colecistoquinina/agonistas , Proteínas Recombinantes de Fusión/agonistas , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Homología de Secuencia de Aminoácido , Sincalida/análogos & derivados , Sincalida/metabolismo , Succinimidas/metabolismo , TransfecciónRESUMEN
The cholecystokinin (CCK) and gastrin families of peptides act as hormones and neuropeptides on central and peripheral CCK receptors to mediate secretion and motility in the gastrointestinal (GI) tract in the physiological response to a normal meal. CCK and its receptors are also widely distributed in the central nervous system (CNS) and contribute to the regulation of satiety, anxiety, analgesia, and dopamine-mediated behavior. Although the wide distribution, myriad number of functions, and reported pharmacological heterogeneity of CCK receptors would suggest a large number of receptor subtypes, the application of modern molecular biological techniques has identified two CCK receptors, CCK-A receptor (CCK-AR) and CCK-B receptor (CCK-BR), that mediate the actions of CCK and gastrin; gastrin receptors have been found to be identical to CCK-BR. CCK-AR, found predominantly in the GI system and select areas of the CNS, have high affinity for CCK and the nonpeptide antagonist L-364,718, whereas CCK-BR, found predominantly in the CNS and select areas of the GI system, have high affinity for CCK and gastrin and the nonpeptide antagonist L-365,260. Both CCK-AR and CCK-BR are highly conserved between species, although there is some tissue-specific variation in expression. Recombinant receptor expression faithfully reproduces the native receptor pharmacology and signal transduction pathways, allowing direct comparisons of receptor function between species as well as serving as a convenient source of receptor. Our present knowledge of the chromosomal localization, receptor gene structure, and primary sequence will allow further studies in disease association, receptor regulation, and structure-function analysis.
Asunto(s)
Receptores de Colecistoquinina/genética , Receptores de Colecistoquinina/fisiología , Secuencia de Aminoácidos , Animales , Humanos , Datos de Secuencia Molecular , Receptores de Colecistoquinina/química , Distribución TisularRESUMEN
We have identified pituitary adenylate cyclase activating peptide (PACAP) receptors on small cell lung cancer cell line NCI-N417 in a previous study. In this study, the role of PACAP in the growth and signal transduction of non-small cell lung cancer cells was investigated. Northern blot analysis with a full-length human PACAP receptor cDNA probe revealed a major 7.5-kb hybridizing transcript when total RNA extracted from NCI-H838 cells was used. PACAP bound with high affinity (Kd = 1 nM) to a single class of sites (Bmax = 14,000/cell) when NCI-H838 cells were used. Specific 125I-labeled PACAP binding was inhibited with high affinity by PACAP-27 and PACAP-38, with moderate affinity by PACAP(6-38), and with low affinity by vasoactive intestinal polypeptide, PACAP(28-38), and PACAP(16-38). PACAP-27 elevated cAMP in a dose-dependent manner, and the increase in cAMP caused by PACAP was reversed by PACAP(6-38). PACAP-27, but not vasoactive intestinal polypeptide, elevated cytosolic Ca2+ in individual NCI-H838 cells. PACAP-27 stimulated arachidonic acid release, and the increase caused by PACAP was reversed by PACAP(6-38). PACAP-27 stimulated colony formation in NCI-H838 cells, whereas the PACAP antagonist PACAP(6-38) reduced colony formation in the absence or presence of exogenous PACAP-27. In nude mice bearing NCI-H838 xenografts, PACAP(6-38) slowed tumor growth significantly. These data suggest that biologically active type 1 PACAP receptors are present on human non-small cell lung cancer cells, which exhibit dual signal transduction pathways and regulate cell proliferation.
Asunto(s)
Carcinoma de Pulmón de Células no Pequeñas/patología , Neoplasias Pulmonares/patología , Receptores de la Hormona Hipofisaria/fisiología , Animales , Antineoplásicos/farmacología , Ácido Araquidónico/metabolismo , Sitios de Unión , Northern Blotting , Calcio/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , División Celular/fisiología , AMP Cíclico/metabolismo , Femenino , Humanos , Líquido Intracelular/metabolismo , Radioisótopos de Yodo , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Trasplante de Neoplasias , Neuropéptidos/metabolismo , Neuropéptidos/farmacología , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria , Receptores del Polipéptido Activador de la Adenilato-Ciclasa Hipofisaria , Receptores de la Hormona Hipofisaria/metabolismo , Sensibilidad y Especificidad , Trasplante Heterólogo , Células Tumorales CultivadasRESUMEN
Receptors for cholcystokinin (CCK) can be pharmacologically classified into at least two distinct subtypes, CCKAR and CCKBR. In an effort to determine whether the CCKA and CCKB receptors may be associated with certain CNS or gastrointestinal diseases, we have localized and compared the human and mouse chromosomal loci encoded by the CCKAR and CCKBR genes. The gene encoding the CCKA receptor maps to a syntenic region of human chromosome 4 and mouse chromosome 5. The CCKB receptor gene, on the other hand, resides on a syntenic region of human chromosome 11 and distal mouse chromosome 7. Localization of the CCK receptors with two dopamine receptors, DRD5 (4p15.1-p15.3) and DRD4 (11p15), provides the interesting possibility of coinvolvement in neuropsychiatric or CNS illnesses.