RESUMEN

INTRODUCTION: The aim of this paper was to examine any injuries from posterior behind armour blunt trauma ballistic impacts directly over the spine onto typical hard body armours. Due to the spine being close to the surface of the skin and a lack of any previous specific research into this topic, this study was designed to gain preliminary insight into the mechanisms involved and injuries caused. Pigs were chosen as the closest representative of human spine, tissue and skin, although their spines are deeper under the surface than humans. Baseline spine and ribs shots were conducted to ensure that the study was effective. METHOD: This study used a 65 kg cadaveric pig eviscerated torso and 7.62 NATO ammunition (7.62×51; L2A2; mean velocity=838 m/s, SD=4 m/s) impacting hard body armour plates over the spine. Injuries were inspected, and sections were removed for X-ray and micro-CT assessment. RESULTS: There was no visible soft tissue damage under the impact point on the armour over the spine, and no bony injuries were reported. Baseline rib shots resulted in multiple rib fractures; some showed minimal displacement of the bone. Baseline spine shot resulted in damage across the spine involving spinal cord and bone. CONCLUSION: No injuries were noted from the spinal impacts, and the rib shots resulted in injuries consistent with those previously reported. The anatomical differences between pigs and humans does not preclude that bony injuries could occur in a human from these types of spinal ballistic impacts.

Asunto(s)

Ropa de Protección , Esqueleto/lesiones , Traumatismos Vertebrales/patología , Heridas por Arma de Fuego/patología , Animales , Balística Forense , Porcinos , Traumatismos Torácicos , Heridas no Penetrantes

RESUMEN

When a person is shot, they are generally wearing clothing which will be damaged by the perforation of the bullet. There are relatively few reports of such textile damage in the literature and the effect of blood on the textile damage observed is not reported. The appearance of textile damage caused by bullet impacts is further compounded by the diverse nature of (i) fabrics used in apparel and (ii) ammunition types. In this work, the effect of blood on textile damage due to ballistic impact was investigated by the development of a specimen that incorporated blood. The specimens were impacted with two types of pistol ammunition that are commonly available (i) 9mm Luger HP (8.03g; Federal Premium® Law Enforcement; jacketed hollow-point) and (ii) .357 Magnum (10.24g; Express® Pistol and Revolver; Remington, R357M3, flat-nose soft-point). The resulting textile damage was compared to that in specimens without a bleeding layer. The interaction of blood with textile damage caused by a bullet-impact affected the appearance of the textile damage and resulted in the dispersion of the bullet wipe. These results are important in the content of evidence examined by a textile damage assessor compared to what might be seen in a typical re-creation event in a laboratory. The use of a bleeding layer in textile damage investigations due to ballistic impact resulted in a more realistic scenario.

Asunto(s)

Vestuario , Balística Forense , Hemorragia , Heridas por Arma de Fuego , Animales , Armas de Fuego , Modelos Animales , Porcinos

RESUMEN

INTRODUCTION: Body armour typically comprises a fabric garment covering the torso combined with hard armour (ceramic/composite). Some users wear only soft armour which provides protection from sharp weapons and pistol ammunition. It is usually recommended that body armour is worn against the body with no air-gaps being present between the wearer and the armour. However, air-gaps can occur in certain situations such as females around the breasts, in badly fitting armour and where manufacturers have incorporated an air-gap claiming improvements in thermophysiological burden. The effect of an air-gap on the ballistic protection and the back face signature (BFS) as a result of a non-perforating ballistic impact was determined. METHODS: Armour panels representative of typical police armour (400x400 mm) were mounted on calibrated Roma Plastilina No 1 and impacted with 9 mm Luger FMJ (9×19 mm; full metal jacket; Dynamit Nobel DM11A1B2) ammunition at 365±10 m/s with a range of air-gaps (0-15 mm). Whether or not the ammunition perforated the armour was noted, the BFS was measured and the incidence of pencilling (a severe, deep and narrow BFS) was identified. RESULTS: For 0° impacts, a critical air-gap size of 10 mm is detrimental to armour performance for the armour/ammunition combination assessed in this work. Specifically, the incidences of pencilling were more common with a 10 mm air-gap and resulted in BFS depth:volume ratios ≥1.0. For impacts at 30° the armour was susceptible to perforation irrespective of air-gap. CONCLUSIONS: This work suggested that an air-gap behind police body armour might result in an increased likelihood of injury. It is recommended that body armour is worn with no air-gap underneath.

Asunto(s)

Aire , Equipos de Seguridad , Heridas por Arma de Fuego/prevención & control , Humanos , Policia

RESUMEN

INTRODUCTION: Some military specialists wear body armour that is more similar to police armour and provides protection from ammunition fired from pistols. During ballistic testing, these armours are mounted on a standardised type of modelling clay and the back face signature (BFS; depth of depression) formed as a result of the non-perforating impact event on to the armour is measured. This study investigated the effect of impact angle on the BFS and on the deformation of the bullet. METHODS: Two commonly worn types of armour (HG1/A+KR1 and HG1+KR1) were considered that provide protection from pistol ammunition and sharp weapons. Armours were tested against two types of pistol ammunition (9 mm full metal jacket and 9 mm hollow point) at eight different impact angles (0°, 15°, 30°, 45°, 60°, 70°, 75° and 80°). RESULTS: Increased impact angles resulted in smaller BFSs. Impact angle also affected whether bullets were retained in the armour; as the impact angle increased, the probability of a round exiting the side of the armour increased. Bullet deformation was affected by impact angle. CONCLUSIONS: Understanding the deformation of bullets may assist with recreating a shooting incident and interpreting forensic evidence.

Asunto(s)

Armas de Fuego , Ropa de Protección , Heridas por Arma de Fuego/prevención & control , Heridas no Penetrantes/prevención & control , Humanos , Personal Militar , Policia

RESUMEN

Tissue simulants are typically used in ballistic testing as substitutes for biological tissues. Many simulants have been used, with gelatine amongst the most common. While two concentrations of gelatine (10 and 20 %) have been used extensively, no agreed standard exists for the preparation of either. Comparison of ballistic damage produced in both concentrations is lacking. The damage produced in gelatine is also questioned, with regards to what it would mean for specific areas of living tissue. The aim of the work discussed in this paper was to consider how damage caused by selected pistol and rifle ammunition varied in different simulants. Damage to gelatine blocks 10 and 20 % in concentration were tested with 9 mm Luger (9 × 19 full metal jacket; FMJ) rounds, while damage produced by .223 Remington (5.56 × 45 Federal Premium® Tactical® Bonded®) rounds to porcine thorax sections (skin, underlying tissue, ribs, lungs, ribs, underlying tissue, skin; backed by a block of 10 % gelatine) were compared to 10 and 20 % gelatine blocks. Results from the .223 Remington rifle round, which is one that typically expands on impact, revealed depths of penetration in the thorax arrangement were significantly different to 20 % gelatine, but not 10 % gelatine. The level of damage produced in the simulated thoraxes was smaller in scale to that witnessed in both gelatine concentrations, though greater debris was produced in the thoraxes.

Asunto(s)

Balística Forense/métodos , Gelatina , Modelos Biológicos , Traumatismos Torácicos/patología , Heridas por Arma de Fuego/patología , Animales , Porcinos

RESUMEN

INTRODUCTION: Behind armour blunt trauma (BABT) has been defined as a non-penetrating injury caused by the rapid deformation of body armour. There has been an increasing awareness of BABT as an injury mechanism in both the military and civilian worlds; whether BABT results in serious injuries is debatable. METHOD: A systematic review of the openly accessible literature was conducted using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses method to investigate those injuries classified as BABT and their severity. RESULTS: 50 sources were identified that included pertinent information relevant to this systematic review on BABT injuries. Typical injuries reported included skin contusion, laceration and penetration, rib fracture and contusions to lungs, kidneys, spleen and (rarely) the heart. No evidence of fatal injuries due to BABT was identified. CONCLUSIONS: Whether BABT can lead to life-threatening injuries when small-arms ammunition impacts body armour components designed to stop that ammunition is debatable. It should be emphasised that other data may be available in government reports that are not openly available. Further research should be considered that investigates developments in body armour, including initiatives that involve reducing burden, and how they affect BABT.

Asunto(s)

Ropa de Protección , Heridas no Penetrantes , Femenino , Humanos , Masculino , Personal Militar , Policia

RESUMEN

The phosphoprotein scaffold Dishevelled is an essential component of both Wnt signalling and of the signalsome that constitutes the supermolecular 'punctae' of assembled proteins often observed in fluorescence microscopy. The C-terminal region beyond the DEP domain displays unique and interesting character, exploited herein by careful analysis of the primary structure. Human Dishevelled-1, -2, -3 and fly Dishevelled (Dsh) sequences were downloaded and interrogated in silico. The C-terminus of Dishevelled-3 is revealed by FoldIndex(®) to be rich in ordered structure. It displays primary sequence that is unique and divergent in important ways from vertebrate isoforms as well as from the fly Dsh. The region is amphipathic, high in prolyl content, and harbours polyprolines. Dishevelled-3 displays some regions, where the proline content is >40%. Polyprolyl sequences (2-4 residues) likely constitute important sites of interaction with other Dishevelled isoforms. Several histidine-single amino acid repeats are notable. The 637,638/647,648 repeats of Dvl3 are essential for Wnt non-canonical, but not canonical signalling. Mutagenesis reveals that the C-terminal sequence is essential for the formation of punctae, made visible by fluorescence microscopy. These Dvl3-based signalsomes are very large (25-35 MDa-MW), supermolecular complexes that display dynamic reorganization in response to Wnt stimulation. Dishevelled-3 C-terminus is rich in structure and unique motifs, worthy of detailed analysis with modern molecular tools.

Asunto(s)

Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/genética , Secuencia de Aminoácidos , Histidina/química , Fosfoproteínas/química , Fosfoproteínas/genética , Prolina/química , Isoformas de Proteínas/química , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Proteínas Dishevelled , Histidina/metabolismo , Humanos , Datos de Secuencia Molecular , Fosfoproteínas/metabolismo , Prolina/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estructura Secundaria de Proteína , Alineación de Secuencia

RESUMEN

Wnt signalling in development operates via members of the Frizzleds, G-protein-coupled receptors that bind specific Wnt ligands and mediate signalling via distinct pathways. The Wnt/Ca(2+)/cGMP pathway mediated by Frizzled-2 was discovered recently. Activation of this pathway leads to increased intracellular concentrations of Ca(2+) and decreased intracellular concentrations of cGMP. The nature of the phosphodiesterase responsible for this Frizzled-2-mediated effect on cGMP levels was identified based on three separate criteria: (i) sensitivity to selective enzyme inhibitors, (ii) behaviour on chromatographic separation, and (ii) isolation by two-dimensional gels in tandem with direct mapping by MS of tryptic digests of the activity. On the basis of results from these three analyses, the cGMP-specific phosphodiesterase, PDE6, is demonstrated to be an effector for the Wnt/Ca(2+)/cGMP signalling pathway of development, which is mediated by Frizzled-2.

Asunto(s)

Calcio/metabolismo , GMP Cíclico/metabolismo , Regulación del Desarrollo de la Expresión Génica , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Hidrolasas Diéster Fosfóricas/fisiología , Receptores de Neurotransmisores/fisiología , Animales , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6 , Electroforesis en Gel Bidimensional , Receptores Frizzled , Immunoblotting , Mapeo Peptídico , Hidrolasas Diéster Fosfóricas/metabolismo , Ratas , Receptores Acoplados a Proteínas G , Receptores de Neurotransmisores/metabolismo , Transducción de Señal , Tripsina/farmacología , Visión Ocular , Proteínas Wnt

RESUMEN

AKAPs (A-kinase anchoring proteins) are members of a diverse family of scaffold proteins that minimally possess a characteristic binding domain for the RI/RII regulatory subunit of protein kinase A and play critical roles in establishing spatial constraints for multivalent signalling assemblies. Especially for G-protein-coupled receptors, the AKAPs provide an organizing centre about which various protein kinases and phosphatases can be assembled to create solid-state signalling devices that can signal, be modulated and trafficked within the cell. The structure of AKAP250 (also known as gravin or AKAP12), based on analyses of milligram quantities of recombinant protein expressed in Escherichia coli, suggests that the AKAP is probably an unordered scaffold, acting as a necklace on which 'jewels' of structure-function (e.g. the RII-binding domain) that provide docking sites on which signalling components can be assembled. Recent results suggest that AKAP250 provides not only a 'tool box' for assembling signalling elements, but may indeed provide a basis for spatial constraint observed for many signalling paradigms. The spatial dimension of the integration of cell signalling will probably reflect many functions performed by members of the AKAP family.

Asunto(s)

Proteínas Portadoras/química , Proteínas de Unión al GTP/química , Proteínas de Anclaje a la Quinasa A , Proteínas de Ciclo Celular , Línea Celular Tumoral , Escherichia coli/metabolismo , Humanos , Membrana Dobles de Lípidos/química , Microscopía Fluorescente , Modelos Biológicos , Proteína Quinasa C/metabolismo , Estructura Terciaria de Proteína , Proteínas/química , Proteínas/fisiología , Proteínas Recombinantes/química , Transducción de Señal , Relación Estructura-Actividad

RESUMEN

Wnt proteins signal via cell surface receptors termed Frizzleds. Frizzleds display many properties characteristic of members of the superfamily of G-protein-coupled receptors, including heptihelical hydropathy plots; an exofacial N-terminal region that is glycosylated; a cytoplasmic C-terminal region that includes canonical motifs for phosphorylation by protein kinase A, protein kinase C and casein kinase II; cytoplasmic domains that couple to heterotrimeric G proteins, as evidenced by a GTP-shift in receptor affinity; receptor-mediated responses sensitive to depletion of specific G protein subunits and receptor-mediated responses sensitive to bacterial toxins that target G proteins. Evidence from a variety of developmental systems demonstrates Wnt-Frizzled (Fz) signaling via pathways other than the Wnt/beta-catenin pathway linked to transcription controlled by Lef/Tcf. Prominent among these additional pathways is a Wnt-Fz pathway regulating intracellular [Ca(++)] and cyclic GMP levels. The essential role of heterotrimeric G proteins in Wnt-Fz signaling is highlighted.

Asunto(s)

Proteínas Proto-Oncogénicas/fisiología , Receptores de Superficie Celular/fisiología , Transducción de Señal/fisiología , Proteínas de Pez Cebra , Animales , Mamíferos , Modelos Biológicos , Proteína Quinasa C/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Receptores de Neurotransmisores/fisiología , Vertebrados , Proteínas Wnt

RESUMEN

Lithium is a monovalent cation used therapeutically to treat a range of affective disorders (1), although the cellular mechanisms of lithium regulation that might contribute to its therapeutic effects at the level of neurotransmitter receptors are not known. Herein we report the ability of lithium to stimulate the internalization of beta2-adrenergic receptors. Lithium treatment of A431 human epidermoid carcinoma cells resulted in a rapid, prominent desensitization and internalization of beta2-adrenergic receptors. The ability of these receptors to generate a cyclic AMP response was strongly inhibited by lithium, at concentrations therapeutic in humans. Receptors for the serotonin (5HT1c) and for opiates (mu-opioid), in sharp contrast, resisted the effects of lithium on internalization. These data provide the first receptor-based mechanism to be described for lithium that could explain, in part, the therapeutic effects of lithium on affective disorders.

Asunto(s)

Antagonistas de Receptores Adrenérgicos beta 2 , Antimaníacos/farmacología , Litio/farmacología , Receptores Adrenérgicos beta 2/metabolismo , Transducción de Señal/efectos de los fármacos , AMP Cíclico/biosíntesis , Endocitosis , Humanos , Cinética , Microscopía Fluorescente , Receptores Opioides mu/metabolismo , Receptores de Serotonina/metabolismo , Células Tumorales Cultivadas

RESUMEN

Wnts are secreted ligands with diverse roles in animal development. Wnts bind to cell surface membrane proteins termed Frizzleds. Molecular cloning of members of the Frizzled family revealed hydropathy plots with seven putative, transmembrane-spanning regions, conserved in Frizzleds characterized in mice, humans, flies, and worms. Understanding how Frizzled translates binding of their cognate Wnts into intracellular signals controlling aspects of development has been an elusive goal. Earlier observations gathered from a variety of model systems provided compelling, but indirect, support that the Frizzled receptors may be members of the superfamily of G-protein-coupled receptors that possess seven transmembrane-spanning domains. Search for a linkage between Frizzled and possible downstream heterotrimeric G-proteins has been advanced by the use of bacterial toxins, antisense DNA, and novel chimeric receptor constructs. New data establish that Frizzleds are indeed bona fide G-protein-coupled receptors. Frizzled-1 couples via G-proteins Go and Gq to the canonical beta-catenin-Lef-Tcf pathway. Frizzled-2 couples via Gq and Gt to downstream effectors including calcium mobilization. Frizzleds and G-proteins might once have been considered strange bedfellows, not likely partners in signaling. The new data, consistent with the properties known for virtually all members of the G-protein-coupled receptors, reveal a more classic romance of signaling elements controlling aspects of early development.

Asunto(s)

Proteínas de Unión al GTP/metabolismo , Proteínas de Unión al GTP/fisiología , Proteínas Proto-Oncogénicas/metabolismo , Transducción de Señal , Transactivadores , Proteínas de Pez Cebra , Animales , Técnicas de Cocultivo , Proteínas del Citoesqueleto/metabolismo , Dimerización , Ligandos , Modelos Biológicos , Unión Proteica , Proteínas Proto-Oncogénicas/fisiología , Receptores de Neurotransmisores/metabolismo , Proteínas Wnt , beta Catenina

RESUMEN

Suppression of the expression of the heterotrimeric G-protein Galpha(i2) in vivo has been shown to provoke insulin resistance, whereas enhanced insulin signaling is observed when Galpha(i2) is overexpressed in vivo. The basis for Galpha(i2) regulation of insulin signaling was explored in transgenic mice with targeted expression of the GTPase-deficient, constitutively active Q205L Galpha(i2) in fat and skeletal muscle. Phosphorylation of insulin receptor and IRS-1 in response to insulin challenge in vivo was markedly amplified in fat and skeletal muscle expressing Q205L Galpha(i2). The expression and activity of the protein-tyrosine phosphatase 1B (PTP1B), but not protein-tyrosine phosphatases SHP-1, SHP-2, and LAR, were constitutively decreased in tissues expressing the Q205L Galpha(i2), providing a direct linkage between insulin signaling and Galpha(i2). The loss of PTP1B expression may explain, in part, the loss of PTP1B activity in the iQ205L transgenic mice. Activation of Galpha(i2) in mouse adipocytes with lysophosphatidic acid was shown to decrease PTP1B activity, whereas pertussis toxin inactivates Galpha(i2), blocks lysophosphatidic acid-stimulated inhibition of PTP1B activity, and blocks tonic suppression of PTP1B activity by Galpha(i2). Elevation of intracellular cAMP in fat cells is shown to increase PTP1B activity, whereas either depression of cAMP levels or direct activation of Galpha(i2) suppresses PTP1B. These data provide the first molecular basis for the interplay between Galpha(i2) and insulin signaling, i.e. activation of Galpha(i2) can suppress both the expression and activity of PTP1B in insulin-sensitive tissues.

Asunto(s)

Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Insulina/farmacología , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Receptores de Superficie Celular , Tejido Adiposo/metabolismo , Animales , AMP Cíclico/metabolismo , Activación Enzimática , Subunidad alfa de la Proteína de Unión al GTP Gi2 , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/genética , Regulación Enzimológica de la Expresión Génica , Lisofosfolípidos/farmacología , Masculino , Ratones , Ratones Transgénicos , Músculo Esquelético/metabolismo , Fosfoserina , Proteína Tirosina Fosfatasa no Receptora Tipo 1 , Proteínas Proto-Oncogénicas/genética , Ratas , Proteínas Tirosina Fosfatasas Clase 4 Similares a Receptores , Receptores Citoplasmáticos y Nucleares/metabolismo , Transducción de Señal , Proteínas Virales/metabolismo

RESUMEN

Heterotrimeric G-proteins, including Galpha(i2), have been implicated in modulating glucose disposal and insulin signaling. This cross-talk between G-protein-coupled and tyrosine kinase-coupled signaling pathways is a focal point for the study of integration of cell signaling. Herein we study the role of Galpha(i2) in modulating glucose transport, focusing upon linkages to insulin signaling. Utilizing mice harboring a transgene that directs the expression of a constitutively activated, GTPase-deficient mutant of Galpha(i2) (Q205L) in adipose tissue, skeletal muscle, and liver, we demonstrate that Galpha(i2) regulates the translocation of the insulin-sensitive GLUT4 glucose transporter in skeletal muscle and adipose tissue. The expression of Q205L Galpha(i2) increased glucose transport and translocation of GLUT4 to the plasma membrane in vivo in the absence of insulin stimulation. Adipocytes from the Q205L Galpha(i2) mice displayed enhanced insulin-stimulated glucose transport and GLUT4 translocation to the plasma membrane to levels nearly twice that of those from littermate controls. Phosphatidylinositol 3-kinase and Akt activities were constitutively activated in tissues expressing the Q205L Galpha(i2). Studies of adipocytes from wild-type mice displayed short term activation of phosphatidylinositol 3-kinase, Akt, and GLUT4 translocation in response to activation of Galpha(i2) by lysophosphatidic acid, a response sensitive to pertussis toxin. These data provide an explanation for the marked glucose tolerance of the Q205L Galpha(i2) mice and demonstrate a linkage between Galpha(i2) and GLUT4 translocation.

Asunto(s)

Subunidades alfa de la Proteína de Unión al GTP Gi-Go/fisiología , Insulina/farmacología , Proteínas de Transporte de Monosacáridos/metabolismo , Proteínas Musculares , Proteínas Serina-Treonina Quinasas , Proteínas Proto-Oncogénicas/fisiología , Tejido Adiposo/metabolismo , Animales , Transporte Biológico/efectos de los fármacos , Subunidad alfa de la Proteína de Unión al GTP Gi2 , Glucosa/metabolismo , Transportador de Glucosa de Tipo 4 , Lisofosfolípidos/farmacología , Masculino , Ratones , Ratones Transgénicos , Músculo Esquelético/metabolismo , Fosfatidilinositol 3-Quinasas/fisiología , Proteínas Proto-Oncogénicas c-akt

RESUMEN

The frizzled receptors, which mediate development and display seven hydrophobic, membrane-spanning segments, are cell membrane-localized. We constructed a chimeric receptor with the ligand-binding and transmembrane segments from the beta2-adrenergic receptor (beta2AR) and the cytoplasmic domains from rat Frizzled-1 (Rfz1). Stimulation of mouse F9 clones expressing the chimera (beta2AR-Rfz1) with the beta-adrenergic agonist isoproterenol stimulated stabilization of beta-catenin, activation of a beta-catenin-sensitive promoter, and formation of primitive endoderm. The response was blocked by inactivation of pertussis toxin-sensitive, heterotrimeric guanine nucleotide-binding proteins (G proteins) and by depletion of Galphaq and Galphao. Thus, G proteins are elements of Wnt/Frizzled-1 signaling to the beta-catenin-lymphoid-enhancer factor (LEF)-T cell factor (Tcf) pathway.

Asunto(s)

Proteínas del Citoesqueleto/metabolismo , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Receptores de Neurotransmisores/metabolismo , Transducción de Señal , Transactivadores , Factores de Transcripción/metabolismo , Proteínas de Xenopus , Proteínas de Pez Cebra , Secuencia de Aminoácidos , Animales , Clonación Molecular , Embrión no Mamífero/metabolismo , Endodermo/fisiología , Receptores Frizzled , Regulación de la Expresión Génica/efectos de los fármacos , Genes Reporteros , Guanosina Trifosfato/metabolismo , Isoproterenol/metabolismo , Isoproterenol/farmacología , Ratones , Datos de Secuencia Molecular , Toxina del Pertussis , Propranolol/metabolismo , Propranolol/farmacología , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Receptores Adrenérgicos beta 2/química , Receptores Adrenérgicos beta 2/genética , Receptores Adrenérgicos beta 2/metabolismo , Receptores Acoplados a Proteínas G , Receptores de Neurotransmisores/química , Receptores de Neurotransmisores/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Transfección , Células Tumorales Cultivadas , Factores de Virulencia de Bordetella/farmacología , Proteínas Wnt , Xenopus , beta Catenina

RESUMEN

Protein-tyrosine phosphatase (PTP) 1B has been implicated in negative regulation of insulin action, although little is known of the ability of insulin to regulate PTP1B itself. The ability of insulin to regulate phosphorylation and activation of PTP1B was probed in vivo. Challenge with insulin in vivo provoked a transient, sharp increase in the phosphotyrosine content of PTP1B in fat and skeletal muscle that peaked within 15 min. Insulin stimulated a decline of 60--70% in PTP1B activity. In mouse adipocytes, the inhibition of PTP1B activity and increased tyrosine phosphorylation of the enzyme were blocked by the insulin receptor tyrosine kinase inhibitor AG1024. Phosphoserine content of PTP1B declined in response to insulin stimulation. Elevation of intracellular cyclic AMP provokes a sharp increase in PTP1B activity and leads to increased phosphorylation of serine residues and decreased tyrosine phosphorylation. Suppression of cyclic AMP levels or inhibition of protein kinase A leads to a sharp decline in PTP1B activity, a decrease in phosphoserine content, and an increase in PTP1B phosphotyrosine content. PTP1B appears to be a critical point for insulin and catecholamine counter-regulation.

Asunto(s)

Adipocitos/enzimología , Tejido Adiposo/enzimología , Insulina/farmacología , Músculo Esquelético/enzimología , Fosfotirosina/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Animales , Células Cultivadas , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Epidídimo , Cinética , Masculino , Ratones , Ratones Endogámicos , Modelos Biológicos , Fosforilación , Proteínas Tirosina Fosfatasas/antagonistas & inhibidores , Receptor de Insulina/antagonistas & inhibidores

RESUMEN

The cyclic AMP-dependent kinase-anchoring proteins (AKAPs) function as scaffolds for a wide-range of protein-protein interactions. The 250-kDa AKAP known as gravin plays a central role in organizing G-protein-coupled receptors to the protein kinases and phosphatases that regulate receptor function in desensitization, resensitization, and sequestration. Although gravin is critical for G-protein-linked receptor biology, the molecular features of the receptor necessary for interaction with this scaffold are not known. Herein, we map the regions of the beta(2)-adrenergic receptor that are required for binding to gravin. Intracellular loops 1, 2, and 3 appear not to participate in the binding of the receptor to the scaffold. In contrast, the C-terminal cytoplasmic region of the receptor (Arg-329 to Leu-413) competes readily for the binding of the beta(2)-adrenergic receptor by gravin, both using in vitro and in vivo assays. C-terminally truncated peptides with sequences ranging from Arg-329 to Leu-342 (13 aminoacyl residues), to Asn-352 (23 residues), to Tyr-366 (37 residues), to Asp-380 (51 residues), or to His-390 (61 residues), as well as N-terminally truncated peptides from Gln-391 to Leu-413 (23 residues) or Leu-381 to Leu-413 (33 residues) displayed no ability to block binding of receptor to gravin. The combination of Arg-329 to His-390 peptide and Gln-391 to Leu-413 peptide, however, reconstitutes a fragmented but full-length C-terminal region and also potently blocks the ability of gravin to bind the beta(2)-adrenergic receptor. The gravin-receptor interaction was examined in response to agonist by confocal microscopy. Remarkably, the association of the receptor with gravin was not disrupted during agonist-induced sequestration. The receptor-scaffold complex was maintained during agonist-induced sequestration. These data, in agreement with the biochemical data, reveal that gravin binds the receptor through the beta(2)-adrenergic receptor C-terminal cytoplasmic domain and that this interaction is maintained as the receptor is internalized. This is the first report of an AKAP scaffold protein translocating with its receptor, in this case a G-protein-coupled receptor.

Asunto(s)

Proteínas/metabolismo , Receptores Adrenérgicos beta 2/química , Receptores Adrenérgicos beta 2/metabolismo , Proteínas de Anclaje a la Quinasa A , Arginina/química , Arrestinas/metabolismo , Sitios de Unión , Proteínas de Ciclo Celular , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Regulación hacia Abajo , Quinasa 2 del Receptor Acoplado a Proteína-G , Humanos , Leucina/química , Sustancias Macromoleculares , Microscopía Fluorescente , Modelos Biológicos , Fragmentos de Péptidos/farmacología , Unión Proteica , Estructura Terciaria de Proteína , Receptores Adrenérgicos beta 2/genética , Proteínas Recombinantes de Fusión/metabolismo , Células Tumorales Cultivadas , Quinasas de Receptores Adrenérgicos beta , beta-Arrestinas

RESUMEN

The nonreceptor tyrosine kinase Src has been implicated in the switching of signaling of beta2-adrenergic receptors from adenylylcyclase coupling to the mitogen-activated protein kinase pathway. In the current work, we demonstrate that Src plays an active role in the agonist-induced desensitization of beta2-adrenergic receptors. Both the expression of dominant-negative Src and treatment with the 4-amine-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) inhibitor of Src kinase activity blocks agonist-induced desensitization. Agonist triggers tyrosine phosphorylation of the beta2-adrenergic receptor and recruitment and activation of Src. Because phosphorylation of the Tyr-350 residue of the beta2-adrenergic receptor creates a conditional, canonical SH2-binding site on the receptor, we examined the effect of the Y350F mutation on Src phosphorylation, Src recruitment, and desensitization. Mutant beta2-adrenergic receptors with a Tyr-to-Phe substitution at Tyr-350 do not display agonist-induced desensitization, Src recruitment, or Src activation. Downstream of binding to the receptor, Src phosphorylates and activates G-protein-linked receptor kinase 2 (GRK2), a response obligate for agonist-induced desensitization. Constitutively active Src increases GRK phosphorylation, whereas either expression of dominant-negative Src or treatment with the PP2 inhibitor abolishes tyrosine phosphorylation of GRK and desensitization. Thus, in addition to its role in signal switching to the mitogen-activated protein kinase pathway, Src recruitment to the beta2-adrenergic receptor and activation are obligate for normal agonist-induced desensitization.

Asunto(s)

Agonistas Adrenérgicos beta/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Receptores Adrenérgicos beta 2/fisiología , Sustitución de Aminoácidos , Animales , Sitios de Unión , Células CHO , Proteína Tirosina Quinasa CSK , Carcinoma de Células Escamosas , Cricetinae , Genes Reporteros , Proteínas Fluorescentes Verdes , Humanos , Yodocianopindolol/farmacología , Isoproterenol/farmacología , Proteínas Luminiscentes/análisis , Proteínas Luminiscentes/biosíntesis , Mutagénesis Sitio-Dirigida , Oligodesoxirribonucleótidos Antisentido/farmacología , Fosforilación , Fosfotirosina , Proteínas Tirosina Quinasas/genética , Receptores Adrenérgicos beta 2/química , Receptores Adrenérgicos beta 2/efectos de los fármacos , Proteínas Recombinantes/química , Proteínas Recombinantes/efectos de los fármacos , Proteínas Recombinantes/metabolismo , Transfección , Células Tumorales Cultivadas , Quinasas de Receptores Adrenérgicos beta , Dominios Homologos src , Familia-src Quinasas

RESUMEN

The ability of the cytoplasmic, full-length C-terminus of the beta 2-adrenergic receptor (BAC1) expressed in Escherichia coli to act as a functional domain and substrate for protein phosphorylation was tested. BAC1 was expressed at high-levels, purified, and examined in solution as a substrate for protein phosphorylation. The mobility of BAC1 on SDS-PAGE mimics that of the native receptor itself, displaying decreased mobility upon chemical reduction of disulfide bonds. Importantly, the C-terminal, cytoplasmic domain of the receptor expressed in E. coli was determined to be a substrate for phosphorylation by several candidate protein kinases known to regulate G-protein-linked receptors. Mapping was performed by proteolytic degradation and matrix-assisted laser desorption ionization, time-of-flight mass spectrometry. Purified BAC1 is phosphorylated readily by protein kinase A, the phosphorylation occurring within the predicted motif RRSSSK. The kinetic properties of the phosphorylation by protein kinase A displayed cooperative character. The activated insulin receptor tyrosine kinase, which phosphorylates the beta-adrenergic receptor in vivo, phosphorylates BAC1. The Y364 residue of BAC1 was predominantly phosphorylated by the insulin receptor kinase. GRK2 catalyzed modest phosphorylation of BAC1. Phosphorylation of the human analog of BAC1 in which Cys341 and Cys378 were mutated to minimize disulfide bonding constraints, displayed robust phosphorylation following thermal activation, suggesting under standard conditions that the population of BAC1 molecules capable of assuming the "activated" conformer required by GRKs is low. BAC1 was not a substrate for protein kinase C, suggesting that the canonical site in the second cytoplasmic loop of the intact receptor is preferred. The functional nature of BAC1 was tested additionally by expression of BAC1 protein in human epidermoid carcinoma A431 cells. BAC1 was found to act as a dominant-negative, blocking agonist-induced desensitization of the beta-adrenergic receptor when expressed in mammalian cells. Thus, the C-terminal, cytoplasmic tail of this G-protein-linked receptor expressed in E. coli acts as a functional domain, displaying fidelity with regard to protein kinase action in vivo and acting as a dominant-negative with respect to agonist-induced desensitization.

Asunto(s)

Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Proteína Quinasa C/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Receptores Adrenérgicos beta 2/genética , Secuencia de Aminoácidos , Animales , Clonación Molecular , Cricetinae , Escherichia coli/genética , Humanos , Datos de Secuencia Molecular , Fosforilación , Receptores Adrenérgicos beta 2/química , Receptores Adrenérgicos beta 2/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , Quinasas de Receptores Adrenérgicos beta

RESUMEN

Insulin activates a complex set of intracellular responses, including the activation of mitogen-activated protein kinases Erk1,2. The counterregulatory actions of insulin on catecholamine action are well known and include phosphorylation of the beta(2)-adrenergic receptor on Tyr(350), Tyr(354), and Tyr(364) in the C-terminal cytoplasmic domain, as well as enhanced sequestration of the beta(2)-adrenergic receptor. Both beta-adrenergic agonists and insulin provoke sequestration of beta(2)-adrenergic receptors in a synergistic manner. In the current work, cross-talk between insulin action and beta(2)-adrenergic receptors revealed that insulin activation of Erk1,2 was amplified via beta(2)-adrenergic receptors. In Chinese hamster ovary cells, expression of beta(2)-adrenergic receptors enhanced 5-10-fold the activation of Erk1,2 by insulin and prolonged the activation, the greatest enhancement occurring at 5 min post-insulin. The potentiation of insulin signaling on Erk1,2 was proportional to the level of expression of beta(2)-adrenergic receptor. The potentiation of insulin signaling requires the integrity of Tyr(350) of the beta(2)-adrenergic receptor, a residue phosphorylated in response to insulin. beta(2)-adrenergic receptors with a Y350F mutation failed to potentiate insulin activation of Erk1,2. Expression of the C-terminal domain of the beta(2)-adrenergic receptor (Pro(323)-Leu(418)) in cells expressing the intact beta(2)-adrenergic receptor acts as a dominant negative, blocking the potentiation of insulin activation of Erk1,2 via the beta(2)-adrenergic receptor. Blockade of beta(2)-adrenergic receptor sequestration does not alter the ability of the beta(2)-adrenergic receptor to potentiate insulin action on Erk1,2. We propose a new paradigm in which a G-protein-linked receptor, such as the beta(2)-adrenergic receptor, itself acts as a receptor-based scaffold via its binding site for Src homology 2 domains, facilitating signaling of the mitogen-activated protein kinase pathway by insulin.

Asunto(s)

Insulina/farmacología , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Receptores Adrenérgicos beta/metabolismo , Tirosina/metabolismo , Agonistas Adrenérgicos beta/farmacología , Animales , Células CHO , Cromonas/farmacología , Cricetinae , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Técnica del Anticuerpo Fluorescente , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Isoproterenol/farmacología , Microscopía Fluorescente , Morfolinas/farmacología , Mutación , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación/efectos de los fármacos , Estructura Terciaria de Proteína , Receptores Adrenérgicos beta/química , Receptores Adrenérgicos beta/genética , Proteínas Recombinantes de Fusión , Transducción de Señal/efectos de los fármacos , Transfección , Células Tumorales Cultivadas , Tirosina/genética