RESUMEN
The agouti gene codes for agouti signaling protein (ASP), which is temporally expressed in wild-type mouse follicular melanocytes where it induces pheomelanin synthesis. Studies using purified full-length agouti signaling protein has shown that it competes with (&agr;)-melanocyte stimulating hormone for binding to the melanocortin 1 receptor. We have investigated whether ASP binds exclusively to the melanocortin 1 receptor expressed on mouse melanocytes in primary culture, or additionally activates a receptor that has not been identified yet. We have compared the responses of congenic mouse melanocytes derived from C57 BL/6J-E(+)/E(+), e/e, or E(so)/E(so) mice to (alpha)-MSH and/or ASP. E(+)/E(+) melanocytes express the wild-type melanocortin 1 receptor, e/e melanocytes express a loss-of-function mutation in the melanocortin 1 receptor that results in a yellow coat color, and E(so)/E(so) is a mutation that causes constitutive activation of the melanocortin 1 receptor and renders melanocytes unresponsive to (alpha)-melanocyte stimulating hormone. Mouse E(+)/E(+) melanocytes, but not e/e or E(so)/E(so) melanocytes, respond to agouti signaling protein with decreased basal tyrosinase activity, and reduction in levels of tyrosinase and tyrosinase-related proteins 1 and 2. Only in E(+)/E(+) melanocytes does agouti signaling protein abrogate the stimulatory effects of (alpha)-melanocyte stimulating hormone on cAMP formation and tyrosinase activity. These results indicate that a functional melanocortin 1 receptor is obligatory for the response of mammalian melanocytes to agouti signaling protein.
Asunto(s)
Péptidos y Proteínas de Señalización Intercelular , Melanocitos/metabolismo , Proteínas/metabolismo , Receptores de Corticotropina/fisiología , Transducción de Señal/fisiología , Proteína de Señalización Agouti , Animales , Células Cultivadas , AMP Cíclico/biosíntesis , AMP Cíclico/metabolismo , Isoenzimas/metabolismo , Melanocitos/efectos de los fármacos , Melanocitos/enzimología , Ratones , Monofenol Monooxigenasa/metabolismo , Receptores de Melanocortina , alfa-MSH/farmacologíaRESUMEN
The importance of p53 in carcinogenesis stems from its central role in inducing cell cycle arrest or apoptosis in response to cellular stresses. We have identified a Drosophila homolog of p53 ("Dmp53"). Like mammalian p53, Dmp53 binds specifically to human p53 binding sites, and overexpression of Dmp53 induces apoptosis. Importantly, inhibition of Dmp53 function renders cells resistant to X ray-induced apoptosis, suggesting that Dmp53 is required for the apoptotic response to DNA damage. Unlike mammalian p53, Dmp53 appears unable to induce a G1 cell cycle block when overexpressed, and inhibition of Dmp53 activity does not affect X ray-induced cell cycle arrest. These data reveal an ancestral proapoptotic function for p53 and identify Drosophila as an ideal model system for elucidating the p53 apoptotic pathway(s) induced by DNA damage.
Asunto(s)
Genes Supresores de Tumor , Proteínas de Insectos/fisiología , Proteína p53 Supresora de Tumor/fisiología , Secuencia de Aminoácidos , Animales , Apoptosis/efectos de la radiación , Sitios de Unión , División Celular , Inhibidor p21 de las Quinasas Dependientes de la Ciclina , Ciclinas/metabolismo , Drosophila melanogaster , Fase G1 , Humanos , Proteínas de Insectos/química , Proteínas de Insectos/genética , Péptidos y Proteínas de Señalización Intracelular , Datos de Secuencia Molecular , Proteínas/metabolismo , Homología de Secuencia de Aminoácido , Proteína p53 Supresora de Tumor/química , Proteína p53 Supresora de Tumor/genética , Proteinas GADD45RESUMEN
Agouti-related protein (AGRP) is a recently discovered orexigenic neuropeptide that inhibits the binding and action of alpha-melanocyte-stimulating hormone derived from proopiomelanocortin (POMC) at the melanocortin 3 receptor (MC3R) and melanocortin 4 receptor (MC4R) and has been proposed to function primarily as an endogenous melanocortin antagonist. To better understand the interplay between the AGRP and melanocortin signaling systems, we compared their nerve fiber distributions with each other by immunohistochemistry and their perikarya distribution with MC3R and MC4R by double in situ hybridization. Although deriving from distinct cell groups, AGRP and melanocortin terminals project to identical brain areas. Both AGRP and melanocortin neurons selectively express the MC3R, which provides a neuroanatomical basis for a dual-input circuit with biological amplification and feedback inhibition. These studies highlight a broader complexity in POMC-mediated behavior in the brain.
Asunto(s)
Encéfalo/metabolismo , Proopiomelanocortina/metabolismo , Proteínas/metabolismo , Proteína Relacionada con Agouti , Animales , Autorradiografía , Encéfalo/ultraestructura , Inmunohistoquímica , Hibridación in Situ , Péptidos y Proteínas de Señalización Intercelular , Masculino , Fibras Nerviosas/metabolismo , Ratas , Ratas Sprague-Dawley , Receptor de Melanocortina Tipo 3 , Receptor de Melanocortina Tipo 4 , Receptores de Corticotropina/antagonistas & inhibidores , Receptores de Corticotropina/metabolismoRESUMEN
Defects in signaling by leptin, a hormone produced primarily by adipose tissue that informs the brain of the body's energy reserves, result in obesity in mice and humans. However, the majority of obese humans do not have abnormalities in leptin or its receptor but instead exhibit leptin resistance that could result from defects in downstream mediators of leptin action. Recently, two potential downstream mediators, agouti-related protein (Agrp) and its receptor, the melanocortin-4 receptor (Mc4r), have been identified. Agrp and Mc4r are excellent candidates for human disorders of body weight regulation and represent promising targets for pharmacological intervention in the treatment of these disorders.
Asunto(s)
Peso Corporal/fisiología , Proteínas/metabolismo , Proteína Relacionada con Agouti , Animales , Humanos , Hipotálamo/metabolismo , Péptidos y Proteínas de Señalización Intercelular , Leptina , Ratones , Ratones Transgénicos , Obesidad/fisiopatología , Proteínas/genética , Receptor de Melanocortina Tipo 4 , Receptores de Péptidos/genética , Receptores de Péptidos/metabolismoRESUMEN
Agouti protein and Agouti-related protein (Agrp) regulate pigmentation and body weight, respectively, by antagonizing melanocortin receptor signaling. A carboxyl-terminal fragment of Agouti protein, Ser73-Cys131, is sufficient for melanocortin receptor antagonism, but Western blot analysis of skin extracts reveals that the electrophoretic mobility of native Agouti protein corresponds to the mature full-length form, His23-Cys131. To investigate the potential role of the amino-terminal residues, we compared the function of full-length and carboxyl-terminal fragments of Agrp and Agouti protein in a sensitive bioassay based on pigment dispersion in Xenopus melanophores. We find that carboxyl-terminal Agouti protein, and all forms of Agrp tested, act solely by competitive antagonism of melanocortin action. However, full-length Agouti protein acts by an additional mechanism that is time- and temperature-dependent, depresses maximal levels of pigment dispersion, and is therefore likely to be mediated by receptor down-regulation. Apparent down-regulation is not observed for a mixture of amino-terminal and carboxyl-terminal fragments. We propose that the phenotypic effects of Agouti in vivo represent a bipartite mechanism: competitive antagonism of agonist binding by the carboxyl-terminal portion of Agouti protein and down-regulation of melanocortin receptor signaling by an unknown mechanism that requires residues in the amino terminus of the Agouti protein.
Asunto(s)
Péptidos y Proteínas de Señalización Intercelular , Melanóforos/metabolismo , Proteínas/metabolismo , Receptores de Corticotropina/metabolismo , Xenopus/metabolismo , Proteína de Señalización Agouti , Proteína Relacionada con Agouti , Secuencia de Aminoácidos , Animales , Unión Competitiva , Células Cultivadas , Regulación hacia Abajo/efectos de los fármacos , Cinética , Ratones , Datos de Secuencia Molecular , Fragmentos de Péptidos/farmacología , Pigmentación/efectos de los fármacos , Receptores de Corticotropina/antagonistas & inhibidores , Receptores de Melanocortina , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Transducción de Señal , Piel/metabolismo , alfa-MSH/antagonistas & inhibidoresRESUMEN
Agouti-related protein (AGRP) is an orexigenic neuropeptide that acts via central melanocortin receptors, and whose messenger RNA (mRNA) levels are elevated in leptin-deficient mice. Fasting associated with a decline in circulating leptin normally causes a 15-fold elevation of hypothalamic Agrp mRNA levels but has no effect in leptin-deficient mice. Chronic hyperleptinemia associated with the tubby and Cpe(fat) mutations has no effect on Agrp mRNA levels, but short term leptin administration causes a 17% reduction of Agrp mRNA levels in nonmutant mice and a 700% reduction in leptin-deficient mice. In young nonobese animals, melanocortin receptor blockade associated with the Ay mutation causes complete resistance to leptin-induced weight loss. Dual in situ hybridization reveals that Agrp-expressing neurons in the medial portion of the arcuate nucleus constitute a subpopulation different from Pomc-expressing neurons, and that a significant proportion of Agrp-expressing neurons (10-25%) coexpresses the leptin receptor, Lepr-b. Immunocytochemistry confirms distinct locations of AGRP- and POMC-expressing cell bodies, but reveals an overlapping distribution of their terminal fields in the arcuate nucleus, the paraventricular hypothalamus, and the dorsomedial hypothalamus. These results suggest that in the fed state, AGRP is normally suppressed by leptin, and that release of this suppression during fasting leads to increased ingestive behavior.
Asunto(s)
Péptidos y Proteínas de Señalización Intercelular , Proteínas/metabolismo , Receptores de Superficie Celular , Transducción de Señal , Proteína de Señalización Agouti , Proteína Relacionada con Agouti , Animales , Núcleo Arqueado del Hipotálamo/citología , Proteínas Portadoras/genética , Ayuno , Hipotálamo/metabolismo , Leptina , Ratones , Ratones Endogámicos C57BL , Mutación , Neuronas/metabolismo , Obesidad/genética , Proopiomelanocortina/genética , Proteínas/genética , Proteínas/farmacología , ARN Mensajero/metabolismo , Receptores de Corticotropina/antagonistas & inhibidores , Receptores de Corticotropina/fisiología , Receptores de Leptina , Receptores de Melanocortina , Pérdida de PesoRESUMEN
Agouti protein and Agouti-related protein (Agrp) are paracrine signaling molecules that act by antagonizing the effects of melanocortins, and several alternatives have been proposed to explain their mechanisms of action. Genetic crosses in a sensitized background uncover a phenotypic difference between overexpression of Agouti and loss of Mc1r function, demonstrate that a functional Mc1r is required for the pigmentary effects of Agouti, and suggest that Agouti protein can act as an agonist of the Mc1r in a way that differs from alpha-MSH stimulation. In vitro, Agouti protein inhibits melanocortin action by two mechanisms: competitive antagonism that depends on the carboxyterminus of the protein, and downregulation of melanocortin receptor signaling that depends on the aminoterminus. Our findings provide evidence of a novel signaling mechanism whereby alpha-MSH and Agouti protein function as independent ligands that inhibit each other's binding and transduce opposite signals through a single receptor.
Asunto(s)
Péptidos y Proteínas de Señalización Intercelular , Proteínas/fisiología , alfa-MSH/fisiología , Proteína de Señalización Agouti , Proteína Relacionada con Agouti , Animales , Color del Cabello/genética , Ratones , Proteínas/genética , Proteínas/farmacología , Receptores de Corticotropina/fisiología , Receptores de Melanocortina , Transducción de Señal , alfa-MSH/antagonistas & inhibidoresRESUMEN
Recent studies have identified several neuropeptide systems in the hypothalamus that are critical in the regulation of body weight. The lateral hypothalamic area (LHA) has long been considered essential in regulating food intake and body weight. Two neuropeptides, melanin-concentrating hormone (MCH) and the orexins (ORX), are localized in the LHA and provide diffuse innervation of the neuraxis, including monosynaptic projections to the cerebral cortex and autonomic preganglionic neurons. Therefore, MCH and ORX neurons may regulate both cognitive and autonomic aspects of food intake and body weight regulation. The arcuate nucleus also is critical in the regulation of body weight, because it contains neurons that express leptin receptors, neuropeptide Y (NPY), alpha-melanin-stimulating hormone (alpha-MSH), and agouti-related peptide (AgRP). In this study, we examined the relationships of these peptidergic systems by using dual-label immunohistochemistry or in situ hybridization in rat, mouse, and human brains. In the normal rat, mouse, and human brain, ORX and MCH neurons make up segregated populations. In addition, we found that AgRP- and NPY-immunoreactive neurons are present in the medial division of the human arcuate nucleus, whereas alpha-MSH-immunoreactive neurons are found in the lateral arcuate nucleus. In humans, AgRP projections were widespread in the hypothalamus, but they were especially dense in the paraventricular nucleus and the perifornical area. Moreover, in both rat and human, MCH and ORX neurons receive innervation from NPY-, AgRP-, and alpha-MSH-immunoreactive fibers. Projections from populations of leptin-responsive neurons in the mediobasal hypothalamus to MCH and ORX cells in the LHA may link peripheral metabolic cues with the cortical mantle and may play a critical role in the regulation of feeding behavior and body weight.
Asunto(s)
Núcleo Arqueado del Hipotálamo/química , Área Hipotalámica Lateral/química , Hormonas Hipotalámicas/fisiología , Melaninas/fisiología , Neuropéptidos/fisiología , Hormonas Hipofisarias/fisiología , Proteína Relacionada con Agouti , Animales , Núcleo Arqueado del Hipotálamo/citología , Conducta Alimentaria , Humanos , Área Hipotalámica Lateral/citología , Inmunohistoquímica , Péptidos y Proteínas de Señalización Intercelular , Masculino , Ratones , Neuropéptido Y/fisiología , Proteínas/fisiología , Ratas , Ratas Sprague-Dawley , Organismos Libres de Patógenos EspecíficosRESUMEN
Agouti protein and Agouti-related protein (Agrp) are paracrine-signaling molecules that normally regulate pigmentation and body weight, respectively. These proteins antagonize the effects of alpha-melanocyte-stimulating hormone (alpha-MSH) and other melanocortins, and several alternatives have been proposed to explain their biochemical mechanisms of action. We have used a sensitive bioassay based on Xenopus melanophores to characterize pharmacologic properties of recombinant Agouti protein, and have directly measured its cell-surface binding to mammalian cells by use of an epitope-tagged form (HA-Agouti) that retains biologic activity. In melanophores, Agouti protein has no effect in the absence of alpha-MSH, but its action cannot be explained solely by inhibition of alpha-MSH binding. In 293T cells, expression of the Mc1r confers a specific, high-affinity binding site for HA-Agouti. Binding is inhibited by alpha-MSH, or by Agrp, which indicates that alpha-MSH and Agouti protein bind in a mutually exclusive way to the Mc1r, and that the similarity between Agouti protein and Agrp includes their binding sites. The effects of Agouti and the Mc1r in vivo have been examined in a sensitized background provided by the chinchilla (Tyrc-ch) mutation, which uncovers a phenotypic difference between overexpression of Agouti in lethal yellow (Ay/a) mice and loss of Mc1r function in recessive yellow (Mc1re/Mc1re) mice. Double and triple mutant studies indicate that a functional Mc1r is required for the pigmentary effects of Agouti, and suggest that Agouti protein can act as an agonist of the Mc1r in a way that differs from alpha-MSH stimulation. These results resolve questions regarding the biochemical mechanism of Agouti protein action, and provide evidence of a novel signaling mechanism whereby alpha-MSH and Agouti protein or Agrp function as independent ligands that inhibit each other's binding and transduce opposite signals through a single receptor.
Asunto(s)
Péptidos y Proteínas de Señalización Intercelular , Proteínas/metabolismo , Receptores de Corticotropina/metabolismo , Proteína de Señalización Agouti , Proteína Relacionada con Agouti , Animales , Epítopos/metabolismo , Melanóforos/citología , Melanóforos/efectos de los fármacos , Melanóforos/metabolismo , Ratones , Ratones Endogámicos C57BL , Pigmentación/efectos de los fármacos , Pigmentación/fisiología , Unión Proteica , Proteínas/genética , Proteínas/farmacología , Receptores de Corticotropina/genética , Receptores de Corticotropina/fisiología , Receptores de Melanocortina , Proteínas Recombinantes/genética , Xenopus , alfa-MSH/metabolismo , alfa-MSH/farmacologíaRESUMEN
Expression of Agouti protein is normally limited to the skin where it affects pigmentation, but ubiquitous expression causes obesity. An expressed sequence tag was identified that encodes Agouti-related protein, whose RNA is normally expressed in the hypothalamus and whose levels were increased eightfold in ob/ob mice. Recombinant Agouti-related protein was a potent, selective antagonist of Mc3r and Mc4r, melanocortin receptor subtypes implicated in weight regulation. Ubiquitous expression of human AGRP complementary DNA in transgenic mice caused obesity without altering pigmentation. Thus, Agouti-related protein is a neuropeptide implicated in the normal control of body weight downstream of leptin signaling.
Asunto(s)
Proteínas/fisiología , Receptores de Corticotropina/antagonistas & inhibidores , Receptores de Péptidos/antagonistas & inhibidores , Glándulas Suprarrenales/metabolismo , Secuencia de Aminoácidos , Animales , Femenino , Humanos , Hipotálamo/metabolismo , Masculino , Hormonas Estimuladoras de los Melanocitos/antagonistas & inhibidores , Hormonas Estimuladoras de los Melanocitos/farmacología , Melanóforos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Ratones Obesos , Ratones Transgénicos , Datos de Secuencia Molecular , Obesidad/etiología , Compuestos Organofosforados/farmacología , Proteínas/química , Proteínas/genética , Proteínas/farmacología , ARN/genética , ARN/metabolismo , Receptor de Melanocortina Tipo 3 , Receptor de Melanocortina Tipo 4 , Receptores de Corticotropina/metabolismo , Receptores de Péptidos/metabolismo , Proteínas Recombinantes/metabolismo , Transducción de Señal , XenopusRESUMEN
Molecular and biochemical mechanisms that switch melanocytes between the production of eumelanin or pheomelanin involve the opposing action of two intercellular signaling molecules, alpha-melanocyte-stimulating hormone (MSH) and agouti signal protein (ASP). In this study, we have characterized the physiological effects of ASP on eumelanogenic melanocytes in culture. Following exposure of black melan-a murine melanocytes to purified recombinant ASP in vitro, pigmentation was markedly inhibited and the production of eumelanosomes was decreased significantly. Melanosomes that were produced became pheomelanosome-like in structure, and chemical analysis showed that eumelanin production was significantly decreased. Melanocytes treated with ASP also exhibited time- and dose-dependent decreases in melanogenic gene expression, including those encoding tyrosinase and tyrosinase-related proteins 1 and 2. Conversely, melanocytes exposed to MSH exhibited an increase in tyrosinase gene expression and function. Simultaneous addition of ASP and MSH at approximately equimolar concentrations produced responses similar to those elicited by the hormone alone. These results demonstrate that eumelanogenic melanocytes can be induced in culture by ASP to exhibit features characteristic of pheomelanogenesis in vivo. Our data are consistent with the hypothesis that the effects of ASP on melanocytes are not mediated solely by inhibition of MSH binding to its receptor, and provide a cell culture model to identify novel factors whose presence is required for pheomelanogenesis.
Asunto(s)
Péptidos y Proteínas de Señalización Intercelular , Oxidorreductasas Intramoleculares , Melanocitos/fisiología , Glicoproteínas de Membrana , Oxidorreductasas , Proteínas/fisiología , Proteína de Señalización Agouti , Animales , Células Cultivadas , AMP Cíclico/metabolismo , Regulación de la Expresión Génica , Humanos , Isomerasas/genética , Isomerasas/metabolismo , Melaninas/biosíntesis , Melaninas/genética , Hormonas Estimuladoras de los Melanocitos/farmacología , Melanocitos/citología , Ratones , Ratones Endogámicos C57BL , Monofenol Monooxigenasa/genética , Monofenol Monooxigenasa/metabolismo , Proteínas/genética , Proteínas/metabolismo , ARN Mensajero/metabolismo , Receptores de la Hormona Hipofisaria/genética , Proteínas Recombinantes de Fusión/farmacologíaRESUMEN
In mouse follicular melanocytes, the switch between eumelanin and pheomelanin synthesis is regulated by the extension locus, which encodes the melanocortin-1 receptor (MC1R) and the agouti locus, which encodes a novel paracrine-signaling molecule that inhibits binding of melanocortins to the MC1R. Human melanocytes express the MC1R and respond to melanotropins with increased proliferation and eumelanogenesis, but a potential role for the human homolog of agouti-signaling protein, ASIP, in human pigmentation has not been investigated. Here we report that ASIP blocked the binding of alpha-melanocyte-stimulating hormone (alpha-MSH) to the MC1R and inhibited the effects of alpha-MSH on human melanocytes. Treatment of human melanocytes with 1 nM-10 nM recombinant mouse or human ASIP blocked the stimulatory effects of alpha-MSH on cAMP accumulation, tyrosinase activity, and cell proliferation. In the absence of exogenous alpha-MSH, ASIP inhibited basal levels of tyrosinase activity and cell proliferation and reduced the level of immunoreactive tyrosinase-related protein-1 (TRP-1) without significantly altering the level of immunoreactive tyrosinase. In addition, ASIP blocked the stimulatory effects of forskolin or dibutyryl cAMP, agents that act downstream from the MC1R, on tyrosinase activity and cell proliferation. These results demonstrate that the functional relationship between the agouti and MC1R gene products is similar in mice and humans and suggest a potential physiologic role for ASIP in regulation of human pigmentation.
Asunto(s)
Péptidos y Proteínas de Señalización Intercelular , Melaninas/metabolismo , Melanocitos/citología , Melanocitos/efectos de los fármacos , Glicoproteínas de Membrana , Oxidorreductasas , Proteínas/farmacología , alfa-MSH/antagonistas & inhibidores , Proteína de Señalización Agouti , Animales , Western Blotting , Bucladesina/farmacología , División Celular/efectos de los fármacos , División Celular/fisiología , Células Cultivadas , Colforsina/farmacología , AMP Cíclico/metabolismo , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Regulación Enzimológica de la Expresión Génica , Humanos , Radioisótopos de Yodo , Melanocitos/metabolismo , Ratones , Mitógenos/farmacología , Monofenol Monooxigenasa/análisis , Monofenol Monooxigenasa/genética , Monofenol Monooxigenasa/metabolismo , Pigmentación/fisiología , Proteínas/análisis , Proteínas/genética , Proteínas/metabolismo , Proteínas/fisiología , Receptores de la Hormona Hipofisaria/metabolismo , Proteínas Recombinantes/farmacología , alfa-MSH/metabolismo , alfa-MSH/farmacologíaRESUMEN
Mouse agouti protein is a paracrine signaling molecule that has previously been demonstrated to be an antagonist of melanocortin action at several cloned rodent and human melanocortin receptors. In this study we report the effects of agouti-signaling protein (ASIP), the human homolog of mouse agouti, on the action of alpha-MSH or ACTH at the five known human melanocortin receptor subtypes (hMCR 1-5). When stably expressed in L cells (hMC1R, hMC3R, hMC4R, hMC5R) or in the adrenocortical cell line OS3 (hMC1R, hMC2R, hMC4R), purified recombinant ASIP inhibits the generation of cAMP stimulated by alpha-MSH (hMC1R, hMC3R, hMC4R, hMC5R) or by ACTH (hMC2R). However, dose-response and Schild analysis indicated that the degree of ASIP inhibition varied significantly among the receptor subtypes; ASIP is a potent inhibitor of the hMC1R, hMC2R, and hMC4R, but has relatively weak effects at the hMC3R and hMC5R. These analyses also indicated that the apparent mechanism of ASIP antagonism varied among receptor subtypes, with characteristics consistent with competitive antagonism observed only at the hMC1R, and more complex behavior observed at the other receptors. ASIP inhibition at these latter receptors, nonetheless, can be classified as surmountable (hMC3R, hMC4R and hMC5R) or nonsurmountable (hMC2R). Recombinant ASIP also inhibited binding of radiolabeled melanocortins, [125I-Nle4, D-Phe7] alpha-MSH and [125I-Phe2, Nle4]ACTH 1-24, to the hMCR 1-5 receptors, with a relative efficacy that paralleled the ability of ASIP to inhibit cAMP accumulation at the hMC1R, hMC2R, hMC3R, and hMC4R. These results provide new insight into the biochemical mechanism of ASIP action and suggest that ASIP may play an important role in modulating melanocortin signaling in humans.
Asunto(s)
AMP Cíclico/metabolismo , Péptidos y Proteínas de Señalización Intercelular , Proteínas/farmacología , Receptores de Corticotropina/efectos de los fármacos , Proteína de Señalización Agouti , Línea Celular , Relación Dosis-Respuesta a Droga , Humanos , Receptores de Corticotropina/antagonistas & inhibidores , Receptores de Corticotropina/genética , Receptores de Melanocortina , Proteínas Recombinantes/farmacologíaRESUMEN
The pleiotropic effects of the viable yellow mutation (Avy), an allele of the mouse agouti coat-color locus, include increased susceptibility to spontaneous and chemically induced tumors that affect a wide variety of tissues. As a first step toward understanding the molecular basis of this phenomenon, we established permanent fibroblast-like cell lines from newborn Avy/a and control congenic a/a mice and compared their growth characteristics in vitro. From the VY/WffC3Hf/Nctr and YS/WffCH3f/Nctr-Avy inbre strains, each of which carries the Avy allele on a congenic background, 38 clonal Avy/a and 16 clonal a/a lines were established. Regardless of inbred strain, all Avy/a cell lines exhibited a significant degree of spontaneous transformation, as assessed by focus formation in monolayer culture, whereas none of the a/a cell lines formed foci in prolonged cultures. To test whether changes in dosage of the Avy- or a-bearing chromosomes were related to these events, we analyzed each cell line with a closely linked molecular probe from the Emv-15 locus, which in the VY strain detects a restriction fragment length variant (RFLV) informative for the Avy- and a-bearing chromosomes. Most of the transformed foci maintained heterozygosity for RFLVs detected by the probe, but two of the transformants lost the a-associated RFLV, and at least one of the transformants exhibited amplification of the Avy-associated RFLV. When the transformants were analyzed with 5' sequences derived from the recently cloned agouti gene, three of eight transformants lost the a-associated RFLV, and two of the transformants showed amplification of the Avy-associated RFLV. Reverse transcriptase-polymerase chain reaction assays indicated that agouti RNA was detected in Avy/a, not a/a cell lines. Surprisingly, some of the Avy/a transformants lacked agouti RNA. These results suggest that deregulated expression of the Avy allele is required for the initiation but not for the maintenance of transformation of the Avy/a cell cultures. These cell lines may provide an in vitro culture system for studying the effect of the agouti gene on tumorigenicity as well as to potentially study other pleiotropic phenotypes.
Asunto(s)
Transformación Celular Neoplásica , Péptidos y Proteínas de Señalización Intercelular , Proteínas/genética , Proteína de Señalización Agouti , Alelos , Animales , Secuencia de Bases , Línea Celular , Cartilla de ADN/química , ADN de Neoplasias/genética , Fibroblastos , Dosificación de Gen , Regulación Neoplásica de la Expresión Génica , Ratones , Ratones Endogámicos , Datos de Secuencia Molecular , ARN Mensajero/genética , ARN Neoplásico/genéticaRESUMEN
The mouse agouti coat color gene encodes a novel paracrine signaling molecule whose pulsatile expression produces a characteristic pattern of banded pigment in individual hairs. Several spontaneous agouti alleles produce adult-onset obesity and diabetes, and have provided important single-gene animal models for alterations in energy metabolism. Utilizing linkage groups conserved between mice and humans, we have cloned the human homolog of the mouse agouti gene from a human chromosome 20 yeast artificial chromosome known to contain S-adenosyl homocysteine hydrolase (AHCY). The human agouti gene, named Agouti Signaling Protein (ASP), encodes a 132 amino acid protein, the mRNA for which is expressed in testis, ovary, and heart, and at lower levels in liver, kidney, and foreskin. As predicted by the interactions of mouse agouti with the extension gene (which encodes the melanocyte receptor for alpha-melanocyte stimulating hormone [alpha-MSH]), expression of ASP in transgenic mice produces a yellow coat, and expression of ASP in cell culture blocks the alpha-MSH-stimulated accumulation of cAMP in mouse melanoma cells. The localization of ASP relative to other loci on chromosome 20 excludes it as a candidate for the MODY1 locus, a gene responsible for one form of early-onset non-insulin-dependent diabetes mellitus or maturity-onset diabetes of the young. The expression of ASP in human tissues suggests a function for agouti homologs in species that do not exhibit the characteristic phenotype of banded hairs.
Asunto(s)
Péptidos y Proteínas de Señalización Intercelular , Proteínas/química , Proteínas/genética , Homología de Secuencia de Ácido Nucleico , Proteína de Señalización Agouti , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cromosomas Artificiales de Levadura/química , Cromosomas Humanos Par 20 , ADN Complementario/aislamiento & purificación , Diabetes Mellitus Tipo 2/genética , Femenino , Color del Cabello/genética , Humanos , Masculino , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Miocardio/química , Ovario/química , Pigmentación/genética , Pigmentación/fisiología , Proteínas/farmacología , ARN Mensajero/análisis , Testículo/química , alfa-MSH/antagonistas & inhibidoresRESUMEN
In a previous survey of endogenous proviruses among inbred mouse strains, the Xmv-10 provirus was found only in strains that carried the non-agouti (a) mutation (Frankel et al. J. Virol. 63: 1763-1774, 1989). To determine whether insertion of Xmv-10 caused the a mutation, we cloned a portion of Xmv-10 and its insertion site. Using a fragment of flanking cellular DNA as a Southern hybridization probe, we found that the Xmv-10 provirus was still present in revertant alleles of a to a(t) or AW. A restriction fragment length variant (RFLV) in cellular DNA at the Xmv-10 insertion site was found to be correlated with the presence or absence of the provirus among inbred strains of laboratory mice regardless of their agouti allele. This correlation did not extend to wild mice, however, in which none of the samples contained Xmv-10, yet one, Mus domesticus poschiavinus, contained the insertion site RFLV correlated with Xmv-10 in laboratory mice. Analysis of an intersubspecific backcross with RFLVs at the insertion sites of Xmv-10 and Emv-15 (an endogenous provirus associated with Ay) revealed the following genetic map information: cen-A-0.31 +/- 0.31 cM-Emv-15-0.62 +/- 0.27 cM-Xmv-10-tel. Haplotype analysis of inbred strains in which a was not associated with Xmv-10 and in which Ay was not associated with Emv-15 demonstrated that these "exceptions" were explained most simply by a single recombination that disturbed the linkage relationships evident in most inbred strains.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Elementos Transponibles de ADN/genética , ADN Viral/genética , Ligamiento Genético/genética , Ratones/genética , Mutagénesis Insercional/genética , Mutación/genética , Provirus/genética , Integración Viral/genética , Alelos , Animales , Secuencia de Bases , Ratones Endogámicos , Datos de Secuencia MolecularRESUMEN
The mouse agouti gene controls the deposition of yellow and black pigment in developing hairs. Several dominant alleles, including lethal yellow (Ay), result in the exclusive production of yellow pigment and have pleiotropic effects that include obesity and increased tumor susceptibility. In an interspecific backcross, we established genetic limits for the agouti gene and found that the Ay and the lethal non-agouti (ax) allele were not separated from a previously identified probe at the breakpoint of the Is1GsO chromosomal rearrangement. Using the Is1GsO probe, we isolated the agouti gene, and find that it has the potential to code for a secreted protein expressed in hair follicles and the epidermis, and that the level of expression correlates with the synthesis of yellow pigment. In the Ay mutation, there is a chromosomal rearrangement that results in the production of a chimeric RNA expressed in nearly every tissue of the body. The 5' portion of this chimeric RNA contains highly expressed novel 5' sequences, but the 3' portion retains the protein-coding potential of the nonmutant allele. We speculate that dominant pleiotropic effects of Ay are caused by ectopic activation of a signaling pathway similar to that used during normal hair growth.
Asunto(s)
Genes Letales , Color del Cabello/genética , Péptidos y Proteínas de Señalización Intercelular , Mutación , Proteínas/genética , Proteína de Señalización Agouti , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Cósmidos , Cruzamientos Genéticos , ADN/genética , Embrión de Mamíferos , Femenino , Hibridación in Situ , Masculino , Ratones , Ratones Mutantes , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Recombinación Genética , Mapeo Restrictivo , Homología de Secuencia de Ácido NucleicoRESUMEN
The region surrounding the agouti coat color locus on mouse Chromosome 2 contains several genes required for peri-implantation development, limb morphogenesis, and segmentation of the nervous system. We have applied radiation hybrid mapping, a somatic cell genetic technique for constructing long-range maps of mammalian chromosomes, to eight molecular markers in this region. Using a mathematical model to estimate the frequency of radiation-induced breakage, we have constructed a map that spans approximately 20 recombination units and 475 centirays8000. The predicted order of markers, Prn-p-Pygb-Emv-13-Psp-Xmv-10-Emv-15-Src-Ada, is consistent with a previously derived multipoint meiotic map for six of the eight markers and suggests that Xmv-10 may lie relatively close to one or more of the agouti recessive lethal mutations. The resolution of our map is approximately 40-fold higher than the meiotic map, but the median retention frequency of mouse DNA in hybrid cells, 0.12, is 4-fold lower than similar experiments with human chromosomes. From one of the radiation hybrid lines that contained a minimum amount of mouse DNA, 25 independent cosmids were isolated with a mouse-specific hybridization probe. Single-copy fragments from two of these cosmids were shown to originate from mouse Chromosome 2, and the meiotic map position of one was found to be within 10 recombination units of the region of interest. Our results indicate more precise map positions for Pygb and Xmv-10, demonstrate that radiation hybrid mapping can provide high-resolution map information for the mouse genome, and establish a new method for isolating large fragments of DNA from a specific subchromosomal region.
Asunto(s)
Mapeo Cromosómico/métodos , Ratones/genética , Animales , Línea Celular , ADN/genética , Desarrollo Embrionario y Fetal/genética , Marcadores Genéticos , Genoma , Células Híbridas/efectos de la radiación , Pigmentación de la Piel/genéticaRESUMEN
The thermotropic properties of aqueous phosphatidylethanolamine dispersions vary with hydration. Measured by EPR-spectroscopy freshly hydrated dimyristoylphosphatidylethanolamine dispersions exhibit a gel to liquid-crystalline phase transition at Tml = 48 degrees C. Dehydration could be induced by prolonged incubation of a hydrated sample at 4 degrees C. The phase transition temperature of the dehydrated phase was determined to be Tmh = 54 degrees C. From the measured phase transition curves we followed the dehydration with time and found a cooperative nucleation process. A 50% dehydration was reached after 5 days. This dehydration process could be prevented by gangliosides: 1.5 mol% of GT1b, 4 mol% of GM1 or 7 mol% of GD1a or GM3 but also 7 mol% of phosphatidic acid were able to stabilize the hydrated phase completely. The effect of gangliosides GM1, GM3, GD1a, GT1b and of the negatively charged phosphatidic acid on the phase behaviour of dimyristoylphosphatidylethanolamine (DMPE) dispersions were investigated. The phase transition temperature of freshly hydrated DMPE samples was successively decreased from 48 to 43 degrees C with increasing amounts of GD1a up to 10 mol% whereby the phase transition was significantly broadened. Gangliosides GM1, GM3 and GT1b as well as phosphatidic acid had minor effects. Dispersions of pure DMPE prepared below the transition temperature Tml form the dehydrated phase again with a melting temperature of Tmh = 54 degrees C. In the presence of 10 mol% GD1a or GT1b this value is reduced to Tml, the phase transition temperature of the hydrated phase. The reduction induced by GM3 is less pronounced. With GM1 or phosphatidic acid the samples remain partially dehydrated and the phase transition curves become biphasic up to 7 mol% ganglioside or phosphatidic acid.
Asunto(s)
Gangliósidos , Lípidos de la Membrana , Fosfatidiletanolaminas , Fenómenos Químicos , Química Física , Membrana Dobles de Lípidos , Relación Estructura-Actividad , Temperatura , AguaRESUMEN
We have investigated the effect of bulk viscosity on lipid translational diffusion using the excimer formation technique. In contrast to a study by Vaz et al. (1987), performed with the fluorescence recovery after photobleaching technique, we observed only a minor decrease of less than a factor of two for pyrene labelled phosphatidylcholine in glycerinated phosphatidylcholine bilayer membranes compared to an aqueous dispersion. Even the diffusion of pyrene labelled gangliosides with an oligosaccharide head-group that protrudes from the membrane surface is not strongly restricted by the increased bulk viscosity. We conclude that the viscosity of the fluid bounding the lipid bilayers is of minor importance for the diffusion of membrane lipids.