Expression of a truncated retroviral envelope gene enhances expression of normal cellular phenotypes

The envelope gene of Moloney murine leukemia virus (Mo-MLV) and its various functional domains have been studied extensively but not as much in terms of their biological effects on cell growth. In this study, we report the biological characterization of a truncated Mo-MLV envelope gene, LN11, which is devoid of a signal peptide. Its expression in various cell types, as compared to the control, enabled the transduced cells to assume a more normal phenotype, which is defined by an increase in contact inhibition and factor dependence, as well as reduced tumorigenicity. LN11-transduced fibroblasts exhibited a higher degree of contact inhibition, assumed a more flattened morphology and were more adherent compared to the control. In v-abl transformed hematopoietic cells, expression of LN11 resulted in slower cell growth, which was due to an enhanced dependence on exogenous growth factors. Enforced expression of LN11 also resulted in a slower rate of tumor development and a reduced tumor load. Thus, modification of a retroviral genome could have a significant impact on cell growth and development. This is one example where we need to consider the safety issue carefully when constructing retrovirus vectors for gene therapy.     

The product of the retroviral transforming gene v-myb is a truncated version of the protein encoded by the cellular oncogene c-myb

Avian myeloblastosis virus (AMV) is an oncogenic retrovirus that rapidly causes myeloblastic leukemia in chickens and transforms myeloid cells in culture. AMV carries an oncogene, v-myb, that is derived from a cellular gene, c-myb, found in the genomes of vertebrate species. We constructed a plasmid vector that allows expression of a portion of the coding region for v-myb in a procaryotic host. We then used the myb-encoded protein produced in bacteria to immunize rabbits. The antisera obtained permitted identification of the proteins encoded by both v-myb and chicken c-myb. The molecular weights of the products of v-myb and c-myb (45,000 and 75,000 respectively) indicate that the v-myb protein is an appreciably truncated version of the c-myb protein.     

NGF induction of NGF receptor gene expression and cholinergic neuronal hypertrophy within the basal forebrain of the adult rat

Chronic infusion of nerve growth factor (NGF) into the forebrain of the adult rat produced increases in NGF receptor (NGF-R) mRNA hybridization, NGF-R immunoreactivity, choline acetyltransferase (ChAT) mRNA hybridization, and neuronal hypertrophy, when compared with vehicle infusion or noninfused rat brain. In situ hybridization showed NGF induction of NGF-R gene expression, documented by increases in the number of NGF-R mRNA-positive cells within the medial septum, diagonal band, and nucleus basalis magnocellularis. NGF also produced hypertrophy of ChAT mRNA-positive neurons. These results suggest that NGF produces cholinergic neuronal hypertrophy through induction of NGF-R gene expression within the basal forebrain.     

NGF/PI3K signaling-mediated epigenetic regulation of delta opioid receptor gene expression

The G protein-coupled delta opioid receptor gene (dor) has been associated with neuronal survival, differentiation, and neuroprotection. Our previous study identified PI3K/Akt/NF-κB signaling is a main downstream signaling pathway in nerve growth factor (NGF)-induced temporal expression of the dor gene in the PC12 cell model. It is still unknown how NGF/PI3K signaling regulates the expression of the dor gene in the nucleus. In the current study, we investigated how PI3K signaling affected epigenetic modifications of histone H3 Lys⁹ (H3K9) in the 5′-UTR region of the rat dor gene locus. NGF treatment resulted in the global reversal of H3K9 trimethylation in cells. Moreover, the locus-specific reversal of H3K9 trimethylation and acetylation of H3K9 were dependent upon NGF/PI3K signaling and temporally well correlated with NGF-induced gene expression. These results indicate the importance of epigenetic modifications of H3K9, particularly the reversal of trimethylated H3K9, in the regulation of NGF/PI3K-dependent genes during neuronal differentiation.     

Identification of NGF receptor in chromatin of melanoma cells using monoclonal antibody to cell surface NGF receptor

A 230 KDa species of Nerve Growth Factor (NGF) receptor was immunoprecipitated from EcoRI-digested chromatin of melanoma cells using a monoclonal antibody to the 75 KDa cell surface NGF receptor. The chromatin NGF receptor was shown to exist tightly bound to DNase II-sensitive sequences which, upon growth factor binding, became resistant to DNase II digestion.     

Development of a cysteine-deprived and C-terminally truncated GLP-1 receptor

The glucagon-like peptide-1 receptor (GLP-1R) belongs to family B of the G-protein coupled receptors (GPCRs), and has become a promising target for the treatment of type 2 diabetes. Here we describe the development and characterization of a fully functional cysteine-deprived and C-terminally truncated GLP-1R. Single cysteines were initially substituted with alanine, and functionally redundant cysteines were subsequently changed simultaneously. Our results indicate that Cys¹⁷⁴, Cys²²⁶, Cys²⁹⁶ and Cys⁴⁰³ are important for the GLP-1-mediated response, whereas Cys²³⁶, Cys³²⁹, Cys³⁴¹, Cys³⁴⁷, Cys⁴³⁸, Cys⁴⁵⁸ and Cys⁴⁶² are not. Extensive deletions were made in the C-terminal tail of GLP-1R in order to determine the limit for truncation. As for other family B GPCRs, we observed a direct correlation between the length of the C-terminal tail and specific binding of ¹²⁵I-GLP-1, indicating that the membrane proximal part of the C-terminal is involved in receptor expression at the cell surface. The results show that seven cysteines and more than half of the C-terminal tail can be removed from GLP-1R without compromising GLP-1 binding or function.     

Anchorage-independent growth of fibroblasts that express a truncated IGF-I receptor

The purpose of this investigation was to study signaling by an insulin-like growth factor I receptor (IGF-I R) that lacks the extracellular portion of the receptor. We transfected IGF-I R-negative mouse embryo fibroblasts with a truncated IGF-I R consisting of only the transmembrane and cytoplasmic part of the beta subunit. Proliferation as assessed by counting cells was the same for vector only transfectants and the truncated receptor transfectants in defined medium containing EGF and PDGF. In contrast, anchorage-independent growth as measured by colony formation in soft agar was markedly increased for the truncated IGF-I R transfectants compared to the vector transfectants. MAP-kinase activity in the truncated IGF-I R transfectants was not higher than in the vector transfectants; however, PI 3-kinase activity was significantly higher in the IGF-I R transfectants. These results provide evidence that an IGF-I receptor consisting of only the transmembrane and cytoplasmic domain of the beta subunit can signal pathways leading to anchorage-independent growth.     

Generation of pigmented stripes in albino mice by retroviral marking of neural crest melanoblasts.

The pigment cells of the skin are derived from melanoblasts which originate in the neural crest. The dorsoventral migration of melanoblasts has been visualized in pigment stripes seen in aggregation chimeras, and the width of these bands has suggested that the entire pigmentation of the coat is derived from a small number of founder cells. We have generated mosaic mice by marking single melanoblasts in utero to gain information on the clonal history of pigment-forming cells. A retroviral vector carrying the human tyrosinase gene was constructed and microinjected into neurulating albino mouse embryos. Albino mice are devoid of pigmentation due to deficiency of tyrosinase. Thus, transduction of the wild-type gene into the otherwise normal melanoblasts should rescue the mutant phenotype, giving rise to patches of pigmentation, which correspond to the area colonized by the mitotic progeny of a marked clone. Mosaic animals derived from the injected embryos indeed showed pigmented bands with a width strikingly similar to the 'standard' stripes seen in aggregation chimeras. These results are consistent with the notion that the unit width bands seen in aggregation chimeras represent the clonal progeny of a single melanoblast and verify Mintz's (1967) conclusion that a few founder melanoblasts give rise to coat pigmentation. The pigment cells of the eye are of dual origin: the melanocytes in choroid and outer layer of the iris are derived from the neural crest and those in the pigment layer of the retina from the neuroepithelium of the optic cup. Marked clones in both lineages were observed in the eyes of many mosaic animals.     

Conformational dynamics in a truncated epidermal growth factor receptor ectodomain

Structural studies have revealed two forms of the monomeric epidermal growth factor receptor (EGFR) ectodomain: a compact (tethered) form stabilized by interdomain interactions and an extended (untethered) form in the presence of ligand. An important question is whether the ligand induces a conformational transition from a tethered to untethered form or whether there is a preexisting conformational equilibrium between tethered and untethered states. To distinguish between these two possibilities, we investigated a truncated receptor, EGFR501 (spanning residues 1-501), that contains the minimal elements required for high-affinity ligand binding in solution. Conformational transitions and dynamics were inferred by means of fluorescence from five internal tryptophan residues that are located within or close to the ligand-binding domains of EGFR501. A preexisting conformational equilibrium between tethered and untethered states in EGFR501 was deduced from (1) the nonlinear Arrhenius temperature dependence of fluorescence and (2) fluorescence polarization showing independently mobile domains. In contrast, the ligand-EGFR501 complex revealed a linear Arrhenius temperature dependence of fluorescence and increased fluorescence polarization due to a lack of significant interdomain motions. The data suggest that the role of the ligand is to trap the EGFR501 in the untethered state that is transiently formed in solution through a preexisting conformational equilibrium.     

Cleavage of K-FGF produces a truncated molecule with increased biological activity and receptor binding affinity

The K-FGF/HST (FGF-4) growth factor is a member of the FGF family which is efficiently secreted and contains a single N-linked glycosylation signal. To study the role of glycosylation in the secretion of K-FGF, we mutated the human K-fgf cDNA to eliminate the glycosylation signal and the mutated cDNA was cloned into a mammalian expression vector. Studies of immunoprecipitation from the conditioned medium of cells expressing this plasmid revealed that the lack of glycosylation did not impair secretion, however the unglycosylated protein was immediately cleaved into two NH2-terminally truncated peptides of 13 and 15 kD, which appeared to be more biologically active than the wild-type protein. These two proteins also showed higher heparin binding affinity than that of wt K-FGF. We have expressed in bacteria the larger of these two proteins (K140), in which the NH2-terminal 36 amino acids present in the mature form of K-FGF have been deleted. Mitogenicity assays on several cell lines showed that purified recombinant K140 had approximately five times higher biological activity than wild-type recombinant K-FGF. Studies of receptor binding showed that K140 had higher affinity than wt K-FGF for two of the four members of FGF receptor's family, specifically for FGFR-1 (flg) and FGFR-2 (bek). K140 also had increased heparin binding ability, but this property does not appear to be responsible for the increased affinity for FGF receptors. Thus removal of the NH2-terminal 36 amino acids from the mature K-FGF produces growth factor molecules with an altered conformation, resulting in higher heparin affinity, and more efficient binding to FGF receptors. Although it is not clear whether cleavage of K-FGF to generate K140 occurs in vivo, this could represent a novel mechanism of modulation of growth factor activity.     

Effects of NGF and glucocorticoid on NGF receptor immunolabeling of cultured rhesus adrenal chromaffin cells

Nerve growth factor (NGF) promotes the outgrowth of neurites from cultured adrenal chromaffin cells from adult rhesus monkeys, but little is known about the distribution, at the cellular level, of the NGF receptors (NGFR) responsible for this response. We examined changes in immunostaining for NGFR in chromaffin cells cultured for 4 weeks in the presence or absence of NGF, with or without dexamethasone (DEX), which inhibits neuritic outgrowth from these cells. Purified cultures of adrenal chromaffin cells from adult rhesus monkeys were grown for up to 9 weeks in NGF, DEX, NGF plus DEX, or control medium. Cells were immunolabeled with three different monoclonal antibodies directed against different epitopes of the human NGFR. Although the distribution of immunolabeling was not uniform from cell to cell, the overall intensity of NGFR immunolabeling varied dramatically between different growth conditions. Of greatest interest, DEX-treated cells stained the most intensely at all time points, while the intensity of immunolabeling was much fainter in NGF-treated cells and decreased with time in culture. In contrast to the intensity of labeling, the proportion of chromaffin cells with immunoreactivity increased with time in all treatment groups. Thus, GCs do not appear to antagonize the effects of NGF merely by decreasing the total number of immunoreactive NGFR on the surface of these cells.