RESUMO
We have produced high-titre HIV-1 green fluorescent protein-expressing lentiviral (LV) vectors pseudotyped with strain 3908 Venezuelan equine encephalitis virus glycoprotein (VEEV-G) and used them to study transduction of: (1) rat embryonic motor neuron (MN) and striatal neuron primary cultures, (2) differentiated MN cell line NSC-34 and (3) adult rat striatum. In primary neuronal cultures, transduction with VEEV-G-pseudotyped LV was more efficient and more neuronal than with vesicular stomatitis virus glycoprotein (VSV-G)-pseudotyped LV. In NSC-34 cells clear retrograde transport of VEEV-G vector particles was observed. In the striatum at the injection site, transduction with the VEEV-G vectors driven by cytomegalovirus or phosphoglycerate kinase promoters exhibited a distinct neuronal tropism with no microglial and only a minor astroglial component, superior to that obtained with VSV-G-pseudotyped LV, irrespective of the promoter used. Neuronal transduction efficiency increased over time. Distal to the injection site transduction of mitral cells in the olfactory bulb, thalamic neurons and dopaminergic neurons in the substantia nigra pars compacta was detected. This, together with observations of retrograde axonal trafficking in vitro indicates that these vectors also possess low level of retrograde neuronal transduction capability in vivo. In this study, we demonstrate both strong neurotropism as well as sustainability of expression and minimal host immune response in vivo, making the VEEV-G-pseudotyped LV vectors potentially useful for gene therapy of neurodegenerative diseases.
Assuntos
Vírus da Encefalite Equina Venezuelana/genética , Glicoproteínas/genética , HIV-1/genética , Neurônios Motores/citologia , Doenças Neurodegenerativas/terapia , Animais , Células Cultivadas , Corpo Estriado/citologia , Corpo Estriado/fisiologia , Vetores Genéticos , Proteínas de Fluorescência Verde , Humanos , Lentivirus/genética , Neurônios Motores/fisiologia , Doenças Neurodegenerativas/genética , Ratos , Transdução GenéticaRESUMO
GnRH receptors (GnRH-Rs) are found in human cancers, including those of the breast, and GnRH can inhibit the growth of cell lines derived from such cancers. Although pituitary and extrapituitary GnRH-R transcripts appear identical, their functional characteristics may differ. Most extrapituitary GnRH-Rs have low affinity for GnRH analogs and may not activate PLC or discriminate between agonists and antagonists in the same way as pituitary GnRH-Rs. Here we have assessed whether GnRH-Rs expressed exogenously in breast cancer cells differ from those in gonadotropes. We found no evidence for endogenous GnRH-Rs in MCF7 cells, but after infection with adenovirus expressing the GnRH-R (Ad GnRH-R) at a multiplicity of infection of 10 or greater, at least 80% expressed GnRH-Rs. These had high affinity (K(d) for [(125)I]buserelin, 1.4 nM) and specificity (rank order of potency, buserelin>GnRH>>chicken GnRH-II) and mediated stimulation of [(3)H]IP accumulation. Increasing viral titer [from multiplicity of infection, 3-300] increased receptor number (10,000-225,000 sites/cell) and [(3)H]IP responses. GnRH stimulated ERK2 phosphorylation in Ad GnRH-R-infected cells, and this effect, like stimulation of [(3)H]IP accumulation, was blocked by GnRH-R antagonists. GnRH also inhibited [(3)H]thymidine incorporation into Ad GnRH-R-infected cells (but not control cells). This effect was mimicked by agonist analogs and inhibited by two antagonists. Thus, when exogenous GnRH-Rs are expressed at density comparable to that in gonadotropes, they are functionally indistinguishable from the endogenous GnRH-Rs in gonadotropes, and increasing expression of high affinity GnRH-Rs can dramatically enhance the direct antiproliferative effect of GnRH agonists on breast cancer cells.
Assuntos
Neoplasias da Mama/fisiopatologia , Receptores LHRH/fisiologia , Transdução de Sinais/fisiologia , Adenoviridae , Ligação Competitiva , Neoplasias da Mama/patologia , Divisão Celular/fisiologia , Feminino , Técnicas de Transferência de Genes , Hormônio Liberador de Gonadotropina/farmacologia , Humanos , Fosfatos de Inositol/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Fosforilação/efeitos dos fármacos , Recombinação Genética , Timidina/antagonistas & inibidores , Células Tumorais CultivadasRESUMO
Nonmammalian vertebrates express at least two forms of GnRH and distinct forms of GnRH receptor (GnRH-R) have coevolved with their ligands. Mammalian and nonmammalian GnRH-R have key structural differences (notably the lack of C-terminal tails in mammalian GnRH-R) and comparative studies are beginning to reveal their functional relevance. However, cellular context and receptor density influence G protein-coupled receptor function and may be important variables in such work using heterologous expression systems. Here we report a comparative study using alphaT4 cells (gonadotrope progenitors that lack endogenous GnRH-R) transfected with a mammalian (human) or nonmammalian (Xenopus laevis type I) GnRH-R. Because conventional transfection strategies proved inefficient, recombinant adenovirus expressing these receptors were constructed, enabling controlled and efficient GnRH-R expression. When expressed in alphaT4 cells at physiological density, these GnRH-Rs retain the pharmacology of their endogenous counterparts (as judged by ligand specificity in radioligand binding and inositol phosphate accumulation assays) but do not activate adenylyl cyclase and are not constitutively active. Moreover, the Xenopus GnRH-R rapidly desensitizes and internalizes in these cells, whereas the human GnRH-R does not, and the internalization rates are not dependent upon receptor number. These data extend studies in COS, HEK, and GH3 cells showing that other GnRH-R with C-terminal tails desensitize and internalize rapidly, whereas tail-less mammalian GnRH-R do not. Retention of these distinctions at physiological receptor density in gonadotrope lineage cells, supports the argument that the evolution of nondesensitizing mammalian GnRH-Rs is functionally relevant and related to the development of mammalian reproductive strategies.
Assuntos
Adenoviridae/genética , Expressão Gênica , Hormônio Liberador de Gonadotropina/análogos & derivados , Hipófise/metabolismo , Receptores LHRH/genética , Receptores LHRH/metabolismo , Xenopus laevis/metabolismo , Adenilil Ciclases/metabolismo , Animais , Ligação Competitiva , Busserrelina/metabolismo , Linhagem Celular , Ativação Enzimática , Hormônio Liberador de Gonadotropina/metabolismo , Humanos , Inositol 1,4,5-Trifosfato/metabolismo , Fosfatos de Inositol/metabolismo , Radioisótopos do Iodo , Cinética , Proteínas Recombinantes/metabolismo , Células-Tronco/metabolismo , TransfecçãoRESUMO
Sustained stimulation of G-protein-coupled receptors (GPCRs) typically causes receptor desensitisation, which is mediated by phosphorylation, often within the C-terminal tail of the receptor. The consequent binding of beta-arrestin not only prevents the receptor from activating its G protein (causing desensitisation), but can also target it for internalisation via clathrin-coated vesicles and can mediate signalling to proteins regulating endocytosis and mitogen-activated protein kinase (MAPK) cascades. GnRH acts via phospholipase C (PLC)-coupled GPCRs on pituitary gonadotrophs to stimulate a Ca(2+)-mediated increase in gonadotrophin secretion. The type I GnRH receptors (GnRH-Rs), found only in mammals, are unique in that they lack C-terminal tails and apparently do not undergo agonist-induced phosphorylation or bind beta-arrestin; they are therefore resistant to receptor desensitisation and internalise slowly. In contrast, the type II GnRH-Rs, found in numerous vertebrates, possess such tails and show rapid desensitisation and internalisation, with concomitant receptor phosphorylation (within the C-terminal tails) or binding of beta-arrestin, or both. The association with beta-arrestin may also be important for regulation of dynamin, a GTPase that controls separation of endosomes from the plasma membrane. Using recombinant adenovirus to express GnRH-Rs in Hela cells conditionally expressing a dominant negative mutant of dynamin (K44A), we have found that blockade of dynamin-dependent endocytosis inhibits internalisation of type II (xenopus) GnRH-Rs but not type I (human) GnRH-Rs. In these cells, blockade of dynamin-dependent internalisation also inhibited GnRH-R-mediated MAPK activation, but this effect was not receptor specific and therefore not dependent upon dynamin-regulated GnRH-R internalisation. Although type I GnRH-Rs do not desensitise, sustained activation of GnRH-Rs causes desensitisation of gonadotrophin secretion, and we have found that GnRH can cause down-regulation of inositol (1,4,5) trisphosphate receptors and desensitisation of Ca(2+) mobilisation in pituitary cells. The atypical resistance of the GnRH-R to desensitisation may underlie its atypical efficiency at provoking this downstream adaptive response. GnRH-Rs are also expressed in several extrapituitary sites, and these may mediate direct inhibition of proliferation of hormone-dependent cancer cells. Infection with type I GnRH-R-expressing adenovirus facilitated expression of high-affinity, PLC-coupled GnRH-R in mammary and prostate cancer cells, and these mediated pronounced antiproliferative effects of receptor agonists. No such effect was seen in cells transfected with a type II GnRH-R, implying that it is mediated most efficiently by a non-desensitising receptor. Thus it appears that the mammalian GnRH-Rs have undergone a period of rapidly accelerated molecular evolution that is of functional relevance to GnRH-Rs in pituitary and extrapituitary sites.
Assuntos
Hormônio Liberador de Gonadotropina/metabolismo , Mamíferos/metabolismo , Hipófise/metabolismo , Receptores LHRH/metabolismo , Transdução de Sinais/fisiologia , Animais , Arrestinas/metabolismo , Endocitose/fisiologia , Evolução Molecular , Humanos , Neoplasias/metabolismo , Ligação Proteica , Receptores LHRH/genética , Sequências Repetidas Terminais , beta-ArrestinasRESUMO
Sustained stimulation of G-protein coupled receptors (GPCRs) typically causes receptor desensitisation that is mediated by phosphorylation, often within the C-terminal tail of the receptor. The consequent binding of beta-arrestin not only prevents the receptor from activating its G-protein (causing desensitisation) but can also target it for internalisation via clathrin-coated vesicles and can mediate signalling to proteins regulating endocytosis and mitogen-activated protein kinase (MAPK) cascades. GnRH acts via phospholipase C coupled GPCRs on pituitary gonadotrophs. The type I GnRH-receptors (GnRH-Rs) found only in mammals, are unique in that they lack C-terminal tails and apparently do not undergo agonist-induced phosphorylation or bind beta-arrestin. They are therefore resistant to receptor desensitisation and internalise slowly. In contrast, the type II GnRH-Rs, found in numerous vertebrates, possess such tails and show rapid desensitisation and internalisation with concomitant receptor phosphorylation (within the C-terminal tails) and/or binding of beta-arrestin. The binding to beta-arrestin may also be important for association with dynamin, a GTPase that controls cleavage of endosomes from the plasma membrane. Using recombinant adenovirus to express GnRH-R, we have found that blockade of dynamin-dependent endocytosis inhibits internalisation of type II (Xenopus) GnRH-Rs but not type I (human) GnRH-Rs, revealing the existence of functionally distinct routes through which these receptors are internalised. Although type I GnRH-R do not rapidly desensitise, sustained activation of GnRH receptors does cause desensitisation of gonadotrophin secretion, an effect which must therefore involve adaptive responses distal to the receptor. One such response is the GnRH-induced down regulation of inositol 1, 4, 5 trisphosphate receptors that apparently underlies desensitisation of Ca2+ mobilisation in a gonadotroph-derived cell line. Although activation of other GPCRs can down-regulate inositol 1, 4, 5 trisphosphate receptors, the effect of GnRH is atypically rapid and pronounced, presumably because of the receptor's atypical resistance to desensitisation. GnRH-Rs are also expressed in several extra-pituitary sites and these may mediate direct inhibition of proliferation of hormone-dependent cancer cells. Infection with type I GnRH-R expressing adenovirus facilitated expression of high affinity, PLC-coupled GnRH-R in mammary and prostate cancer cells and these mediated pronounced antiproliferative effects of receptor agonists. No such effect was seen in cells transfected with a type II GnRH-R, implying that it is mediated most efficiently by a non-desensitising receptor. Thus it appears that the GnRH-Rs have undergone a period of rapidly accelerated molecular evolution that is of functional relevance to GnRH-R signalling in pituitary and extra-pituitary sites.
Assuntos
Receptores LHRH/fisiologia , Sequência de Aminoácidos , Animais , Arrestinas/metabolismo , Regulação para Baixo , Hormônio Liberador de Gonadotropina/metabolismo , Humanos , Dados de Sequência Molecular , Neoplasias/metabolismo , Hipófise/metabolismo , Receptores LHRH/química , Ovinos , Transdução de Sinais/fisiologia , beta-ArrestinasRESUMO
Desensitization and internalization of G-protein-coupled receptors can reflect receptor phosphorylation-dependent binding of beta-arrestin, which prevents G-protein activation and targets receptors for internalization via clathrin-coated vesicles. These can be pinched off by a dynamin collar, and proteins controlling receptor internalization can also mediate mitogen-activated protein kinase signaling. Gonadotropin-releasing hormone (GnRH) stimulates internalization of its receptors via clathrin-coated vesicles. Mammalian GnRH receptors (GnRH-Rs) are unique in that they lack C-terminal tails and do not rapidly desensitize, whereas non-mammalian GnRH-R have C-terminal tails and, where investigated, do rapidly desensitize and internalize. Using recombinant adenovirus expressing human and Xenopus GnRH-Rs we have explored the relationship between receptor internalization and mitogen-activated protein kinase signaling in HeLa cells with regulated tetracycline-controlled expression of wild-type or a dominant negative mutant (K44A) of dynamin. These receptors were phospholipase C-coupled and had appropriate ligand affinity and specificity. K44A dynamin expression did not alter human GnRH-R internalization but dramatically reduced internalization of Xenopus GnRH-R (and epidermal growth factor (EGF) receptor). Blockade of clathrin-mediated internalization (sucrose) abolished internalization of all three receptors. Both GnRH-Rs also mediated phosphorylation of ERK 2 and for both receptors, this was inhibited by K44A dynamin. The same was true for EGF- and protein kinase C-mediated ERK 2 phosphorylation. ERK 2 phosphorylation was also inhibited by a protein kinase C inhibitor but not affected by an EGF receptor tyrosine kinase inhibitor. We conclude that a) desensitizing and non-desensitizing GnRH-Rs are targeted for clathrin-coated vesicle-mediated internalization by functionally distinct mechanisms, b) GnRH-R signaling to ERK 2 is dynamin-dependent and c) this does not reflect a dependence on dynamin-dependent GnRH-R internalization.