RESUMEN
Receptors for PDGF play an important role in cell proliferation and migration and have been implicated in certain cancers. The 44-amino acid E5 protein of bovine papillomavirus binds to and activates the PDGFß receptor (PDGFßR), resulting in oncogenic transformation of cultured fibroblasts. Previously, we isolated an artificial 36-amino acid transmembrane protein, pTM36-4, which transforms cells because of its ability to activate the PDGFßR despite limited sequence similarity to E5. Here, we demonstrated complex formation between the PDGFßR and three pTM36-4 mutants: T21E, T21Q, and T21N. T21Q retained wild type transforming activity and activated the PDGFßR in a ligand-independent manner as a consequence of binding to the transmembrane domain of the PDGFßR, but T21E and T21N were severely defective. In fact, T21N substantially inhibited E5-induced PDGFßR activation and transformation in both mouse and human fibroblasts. T21N did not prevent E5 from binding to the receptor, and genetic evidence suggested that T21N and E5 bind to nonidentical sites in the transmembrane domain of the receptor. T21N also inhibited transformation and PDGFßR activation induced by v-Sis, a viral homologue of PDGF-BB, as well as PDGF-induced mitogenesis and signaling by preventing phosphorylation of the PDGFßR at particular tyrosine residues. These results demonstrated that T21N acts as a novel inhibitor of the PDGFßR and validated a new strategy for designing highly specific short transmembrane protein inhibitors of growth factor receptors and possibly other transmembrane proteins.
Asunto(s)
Activadores de Enzimas/metabolismo , Fibroblastos/metabolismo , Mutación Missense , Proteínas Oncogénicas v-sis/metabolismo , Inhibidores de Proteínas Quinasas/metabolismo , Receptor beta de Factor de Crecimiento Derivado de Plaquetas , Sustitución de Aminoácidos , Animales , Papillomavirus Bovino 1/genética , Papillomavirus Bovino 1/metabolismo , Bovinos , Línea Celular , Transformación Celular Viral/genética , Fibroblastos/patología , Humanos , Masculino , Ratones , Proteínas Oncogénicas v-sis/genética , Proteínas Oncogénicas Virales/genética , Proteínas Oncogénicas Virales/metabolismo , Fosforilación/genética , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/agonistas , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/antagonistas & inhibidores , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/genética , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Transducción de Señal/genéticaRESUMEN
IL-27 (EBI3p28) is a recently discovered heterodimeric cytokine, which is functionally related to IL-23p40p19 and IL-12p40p35. IL-27 acts in synergy with IL-12 early during Th1 development from naive T cells. IL-27 functions through the WSX-1 and the gpl30 receptor subunits, which shares homology with the IL-12Rbeta2 subunit. We have previously reported that IL-23 is up-regulated in CD11b+ microglia/macrophages in the CNS during the early phase of experimental autoimmune encephalomyelitis (EAE), and thus may contribute to the early induction of EAE. In the present study, we examined the expression of IL-27 and its receptor in the CNS, spleen, and lymph nodes at different stages of EAE actively induced with myelin oligodendrocyte glycoprotein peptide(35-55). Our findings show that IL-27 EBI3 and p28 mRNA were up-regulated to a maximum level at the peak of disease in APC from the CNS and lymph nodes, but not in the spleen. Moreover, IL-27 receptor (WSX-1) expression was greatly up-regulated during the early stage of EAE in both the CNS and lymph nodes. Taken together, our data show that subunits of IL-27 and its receptor (WSX-1) mRNAs are markedly up-regulated in inflammatory cells in the CNS at the peak of disease. Thus, IL-27 produced by infiltrating cells in the CNS may regulate in a paracrine manner the Th1 response in EAE.
Asunto(s)
Sistema Nervioso Central/metabolismo , Encefalomielitis Autoinmune Experimental/metabolismo , Inflamación/metabolismo , Interleucinas/biosíntesis , ARN Mensajero/biosíntesis , Receptores de Citocinas/biosíntesis , Animales , Antígeno CD11b/metabolismo , Linfocitos T CD4-Positivos/metabolismo , Cinética , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Microglía/metabolismo , Proteínas Oncogénicas v-sis/metabolismo , Receptores de Interleucina , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células TH1 , Regulación hacia ArribaRESUMEN
Cell surface retention sequence binding protein-1 (CRSBP-1) is a cell surface binding protein for the cell surface retention sequence (CRS) motif of the v-sis gene product (platelet-derived growth factor-BB). It has been shown to be responsible for cell surface retention of the v-sis gene product in v-sis-transformed cells (fibroblasts) and has been hypothesized to play a role in autocrine growth and transformation of these cells. Here we demonstrate that the CRSBP-1 cDNA cloned from bovine liver libraries encodes a 322-residue type I membrane protein containing a 23-residue signal peptide, a 215-residue cell surface domain, a 21-residue transmembrane domain, and a 63-residue cytoplasmic domain. CRSBP-1 expressed in transfected cells is an approximately 120-kDa disulfide-linked homodimeric glycoprotein and exhibits dual ligand (CRS-containing growth regulators (v-sis gene product and insulin-like growth factor binding protein-3, IGFBP-3) and hyaluronic acid) binding activity. CRSBP-1 overexpression (by stable transfection of cells with CRSBP-1 cDNA) enhances autocrine loop signaling, cell growth, and tumorigenicity (in mice) of v-sis-transformed cells. CRSBP-1 expression also enhances autocrine cell growth mediated by IGFBP-3 in human lung carcinoma cells (H1299 cells), which express very little, if any, endogenous CRSBP-1 and exhibits a mitogenic response to exogenous IGFBP-3, stably transfected with IGFBP-3 cDNA. However, CRSBP-1 overexpression does not affect growth of normal and transformed cells that do not produce these CRS-containing growth regulators. These results suggest that CRSBP-1 plays a role in autocrine regulation of cell growth mediated by growth regulators containing CRS.