RESUMEN
In this study we demonstrate that the photoconvertible monomeric Kikume green-red (mKikGR) protein is suitable to study trafficking of G protein-coupled receptors. Taking mKikGR-tagged mutants of the vasopressin V(2) receptor (V(2)R) as models, we analyzed whether the V(2)R-specific pharmacological chaperone SR121463B influences receptor folding on a co- or post-translational level. Misfolded mKikGR-tagged V(2)Rs were completely photoconverted in the early secretory pathway yielding a red receptor population (already synthesized receptors) and an arising green receptor population (newly synthesized receptors). Trafficking of both receptor populations could be rescued by treatment with SR121463B demonstrating that the substance can act co- and post-translationally.
Asunto(s)
Proteínas Luminiscentes/metabolismo , Chaperonas Moleculares/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Secuencia de Aminoácidos , Antagonistas de los Receptores de Hormonas Antidiuréticas , Color , Células HEK293 , Humanos , Proteínas Luminiscentes/genética , Chaperonas Moleculares/química , Datos de Secuencia Molecular , Morfolinas/química , Morfolinas/metabolismo , Pliegue de Proteína , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/genética , Receptores de Vasopresinas/química , Receptores de Vasopresinas/genética , Receptores de Vasopresinas/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Compuestos de Espiro/química , Compuestos de Espiro/metabolismoRESUMEN
The specific inhibition of the biosynthesis of target proteins is a relatively novel strategy in pharmacology and is based mainly on antisense approaches (e.g. antisense oligonucleotides or RNA interference). Recently, a novel class of substances was described acting at a later step of protein biosynthesis. The cyclic heptadepsipeptides CAM741 and cotransin were shown to inhibit selectively the biosynthesis of a small subset of secretory proteins by preventing stable insertion of the nascent chains into the Sec61 translocon complex at the endoplasmic reticulum membrane (Besemer, J., Harant, H., Wang, S., Oberhauser, B., Marquardt, K., Foster, C. A., Schreiner, E. P., de Vries, J. E., Dascher-Nadel, C., and Lindley, I. J. (2005) Nature 436, 290-293; Garrison, J. L., Kunkel, E. J., Hegde, R. S., and Taunton, J. (2005) Nature 436, 285-289). These peptides act in a signal sequence-discriminatory manner, which explains their selectivity. Here, we have analyzed the cotransin sensitivity of various G protein-coupled receptors in transfected HEK 293 cells. We show that the biosynthesis of the human endothelin B receptor (ET(B)R) is highly sensitive to cotransin, in contrast to that of the other G protein-coupled receptors analyzed. Using a novel biosynthesis assay based on fusions with the photoconvertible Kaede protein, we show that the IC(50) value of cotransin action on ET(B)R biosynthesis is 5.4 µm and that ET(B)R signaling could be completely blocked by treating cells with 30 µm cotransin. Taken together, our data add an integral membrane protein, namely the ET(B)R, to the small group of cotransin-sensitive proteins.
Asunto(s)
Péptidos Cíclicos/farmacología , Biosíntesis de Proteínas/efectos de los fármacos , Receptor de Endotelina B/biosíntesis , Células HEK293 , Humanos , Proteínas Luminiscentes/biosíntesis , Proteínas Luminiscentes/genética , Biosíntesis de Proteínas/genética , Receptor de Endotelina B/genética , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genéticaRESUMEN
The corticotropin-releasing factor receptor type 2a (CRF(2(a))R) belongs to the family of G protein-coupled receptors. The receptor possesses an N-terminal pseudo signal peptide that is unable to mediate targeting of the nascent chain to the endoplasmic reticulum membrane during early receptor biogenesis. The pseudo signal peptide remains uncleaved and consequently forms an additional hydrophobic receptor domain with unknown function that is unique within the large G protein-coupled receptor protein family. Here, we have analyzed the functional significance of this domain in comparison with the conventional signal peptide of the homologous corticotropin-releasing factor receptor type 1 (CRF(1)R). We show that the presence of the pseudo signal peptide leads to a very low cell surface receptor expression of the CRF(2(a))R in comparison with the CRF(1)R. Moreover, whereas the presence of the pseudo signal peptide did not affect coupling to the G(s) protein, G(i)-mediated inhibition of adenylyl cyclase activity was abolished. The properties mediated by the pseudo signal peptide were entirely transferable to the CRF(1)R in signal peptide exchange experiments. Taken together, our results show that signal peptides do not only influence early protein biogenesis. In the case of the corticotropin-releasing factor receptor subtypes, the use of conventional and pseudo signal peptides have an unexpected influence on signal transduction.
Asunto(s)
Adenilil Ciclasas/metabolismo , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Regulación de la Expresión Génica/fisiología , Señales de Clasificación de Proteína , Receptores de Hormona Liberadora de Corticotropina/biosíntesis , Transducción de Señal/fisiología , Adenilil Ciclasas/genética , Animales , Línea Celular , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/genética , Humanos , Ratas , Receptores de Hormona Liberadora de Corticotropina/genéticaRESUMEN
In this study, we compare for the first time the nanomechanical properties of lipid bilayer islands on flat and porous surfaces. 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC) and 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) bilayers were deposited on flat (silicon and mica) and porous silicon (pSi) substrate surfaces and examined using atomic force spectroscopy and force volume imaging. Force spectroscopy measurements revealed the effects of the underlying substrate and of the lipid phase on the nanomechanical properties of bilayers islands. For mica and silicon, significant differences in breakthrough force between the center and the edges of bilayer islands were observed for both phospolipids. These differences were more pronounced for DMPC than for DPPC, presumably due to melting effects at the edges of DMPC bilayers. In contrast, bilayer islands deposited on pSi yielded similar breakthrough forces in the central region and along the perimeter of the islands, and those values in turn were similar to those measured along the perimeter of bilayer islands deposited on the flat substrates. The study also demonstrates that pSi is suitable solid support for the formation of pore-spanning phospholipid bilayers with potential applications in transmembrane protein studies, drug delivery, and biosensing.
Asunto(s)
Membrana Dobles de Lípidos/química , Nanotecnología , Fosfolípidos/química , Silicio/química , Silicatos de Aluminio/química , Silicatos de Aluminio/metabolismo , Fenómenos Biomecánicos , Biomimética , Membrana Celular/química , Membrana Celular/metabolismo , Membrana Dobles de Lípidos/metabolismo , Microscopía de Fuerza Atómica , Fosfolípidos/metabolismo , Porosidad , Silicio/metabolismoRESUMEN
Prostaglandin E2 (PGE2), is a major prostanoid produced by the activity of cyclooxygenases (COX) in response to various physiological and pathological stimuli. PGE2 exerts its effects by activating four specific E-type prostanoid receptors (EP1, EP2, EP3, and EP4). In the present study, we analyzed the expression of the PGE2 receptor EP1 (mRNA and protein) in different regions of the adult rat brain (hippocampus, hypothalamus, striatum, prefrontal cerebral cortex, parietal cortex, brain stem, and cerebellum) using reverse transcription- polymerase chain reaction, Western blotting, and immunohistochemical methods. On a regional basis, levels of EP1 mRNA were the highest in parietal cortex and cerebellum. At the protein level, we found very strong expression of EP1 in cerebellum, as revealed by Western blotting experiments. Furthermore, the present study provides for the first time evidence that the EP1 receptor is highly expressed in the cerebellum, where the Purkinje cells displayed very high immunolabeling of their perikaryon and dendrites, as observed in the immunohistochemical analysis. Results from the present study indicate that the EP1 prostanoid receptor is expressed in specific neuronal populations, which possibly determine the region-specific response to PGE2.