Analysis of the v-myb structural components important for transactivation of gene expression.

In order to define the domains of the v-myb protein that are important for transactivation of gene expression, we have studied transactivation by the v-myb gene and a set of v-myb deletion mutants using transient transfection assays in NIH 3T3 cells. Analysis of the set of v-myb deletion products demonstrated that a previously unidentified region in the carboxyl-terminal portion of the protein is required for transactivation. This region lies between amino acids 295-356 with respect to the 5' end of the v-myb gene. Switching the v-myb DNA binding domain with the DNA binding domain of the rat glucocorticoid receptor (rGR) switched the cis-element requirement for v-myb action: only reports containing glucocorticoid response elements were activated by myb-rGR fusion proteins. The carboxyl terminal region essential for transactivation by the intact v-myb gene was also necessary for transactivation by the rGR-fusion gene. Carboxyl-terminal deletion mutations that encompassed the novel transactivation region were able to block wild-type v-myb transactivation when tested in transient co-expression assays. In an unexpected sidelight to our studies, we could demonstrate that the lacZ gene present in the prokaryotic vector sequences contained a DNA element that fortuitously can act as a v-myb-dependent enhancer element, and that v-myb protein can bind to this element in vitro. The lacZ enhancer contains the myb consensus DNA binding site YAAC(G/T)G.     

Complex human glucocorticoid receptor dim mutations define glucocorticoid induced apoptotic resistance in bone cells

A mutation in the D-loop of the second zinc finger of the DNA-binding domain of the human glucocorticoid receptor (hGR), A458T (GR(dim)), has been suggested to be essential for dimerization and DNA binding of the GR, and genetically altered GR(dim) mice survive, whereas murine GR knockout mice die. Interestingly, thymocytes isolated from the GR(dim) mice were reported to be resistant to glucocorticoid-induced apoptosis. To further evaluate the dim mutations in glucocorticoid-induced apoptosis, we stably expressed either the hGR(dim) (A458T) or the hGR(dim4) (A458T, R460D, D462C, and N454D) mutant receptors in human osteosarcoma (U-2 OS) cells that are devoid of hGR and unresponsive to glucocorticoids. We analyzed these cell lines by comparison with a stable expression hGRα U-2 OS cell line, which undergoes apoptosis after glucocorticoid treatment. Transient reporter gene assays with glucocorticoid response element-driven vectors revealed that the hGR(dim) mutation had diminished steroid responsiveness and cells carrying the hGR(dim4) mutation were unresponsive to steroid, whereas glucocorticoid-induced nuclear factor κB repression was unaffected by either mutation. Interestingly, both the hGR(dim) and hGR(dim4) receptors readily formed dimers as measured by immunoprecipitation. Examination of GR-mediated apoptosis showed that hGR(dim) cells were only partially resistant to apoptosis, whereas hGR(dim4) cells were completely resistant to glucocorticoid-induced cell death despite remaining sensitive to other apoptotic stimuli. Global gene expression analysis revealed that hGR(dim4) cells widely regulated gene expression but differentially regulated apoptotic mRNA when compared with cells expressing wild-type hGRα. These studies challenge conclusions drawn from previous studies of GR dim mutants.     

Mutations in squirrel monkey glucocorticoid receptor impair nuclear translocation

To identify the determinants of impaired glucocorticoid receptor (GR) signaling in a model of glucocorticoid resistance, cloned GR from Guyanese squirrel monkeys (gsmGR) was tagged with enhanced green fluorescent protein, and nuclear translocation was examined in transfected COS1 cells. In keeping with evidence that gsmGR transactivational competence is impaired, we found that nuclear translocation is likewise diminished in gsmGR relative to human GR (hGR). Experiments with GR chimeras revealed that replacement of the gsmGR ligand binding domain (LBD) with that from hGR increased translocation. Truncated gsmGR constructs lacking the LDB after amino acid 552 also showed increased translocation even in the absence of cortisol. Three back-mutations of gsmGR to hGR (Thr551Ser, Ala616Ser, and Ser618Ala) in the LBD confirmed that these amino acids play a role in diminished translocation.     

AUUUA motifs in the 3'UTR of human glucocorticoid receptor alpha and beta mRNA destabilize mRNA and decrease receptor protein expression

An association between a gene polymorphism of the human glucocorticoid receptor (hGR) gene and rheumatoid arthritis has recently been suggested. This polymorphism contains an A to G mutation in the 3'UTR of exon 9beta, which encodes the 3'UTR of the mRNA of the hGRbeta isoform. The hGRbeta isoform can act as a dominant negative inhibitor of hGRalpha, and therefore may contribute to glucocorticoid resistance. The A to G mutation is located in an AUUUA motif, which is known to destabilize mRNA. In the present study, the importance of the mutation in this AUUUA motif was further characterized and mutations in other AUUUA motifs in the 3'UTR of hGRbeta and hGRalpha mRNA were studied. hGRbeta and hGRalpha expression vectors, carrying mutations in one AUUUA motif or all AUUUA motifs were transiently transfected into COS-1 cells. Each transfected vector was analyzed for the mRNA expression level, the mRNA turnover rate and the protein expression level. The naturally occurring mutation in the 3'UTR of hGRbeta mRNA increased mRNA stability and protein expression. Mutation of two other AUUUA motifs in the 3'UTR of hGRbeta, or mutation of all four AUUUA motifs resulted in a similar effect. Mutation of the most 5' AUUUA motif did not alter hGRbeta mRNA expression or mRNA stability. Mutation of all 10 AUUUA motifs in the 3'UTR of hGRalpha mRNA increased hGRalpha mRNA expression and mRNA stability as well as expression of the receptor protein level. Thus, the naturally occurring mutation in an AUUUA motif in the 3'UTR of hGRbeta mRNA results not only in increased mRNA stability, but also in increased receptor protein expression, which may contribute to glucocorticoid resistance. A similar role is suggested for two other AUUUA motifs in the 3'UTR of hGRbeta mRNA and for the 10 AUUUA motifs that are present in the 3'UTR of hGRalpha.     

Impaired transactivation of the glucocorticoid receptor cloned from the Guyanese squirrel monkey

Squirrel monkeys are among a diverse group of New World primates that demonstrate unusually high levels of circulating corticosteroids and glucocorticoid receptor (GR) insensitivity. Recent evidence suggests that overexpression of an immunophilin impairs dexamethasone binding to GR in the Bolivian squirrel monkey (Saimiri boliviensis). Here we describe the cloning, expression, and functional characterization of GR from the closely related Guyanese squirrel monkey (S. sciureus). The cloned Guyanese squirrel monkey GR (gsmGR) cDNA closely resembles human GR (hGR) cDNA, and yields a high affinity dexamethasone binding receptor when expressed in COS-1 cells. Transactivation analysis of hGR and gsmGR expressed in CV-1 cells and cultured squirrel monkey kidney (SMK) cells indicates that: (1) SMK cells elaborate a functional high activity GR from human GR cDNA; (2) gsmGR is an order of magnitude less efficient than hGR at transactivation in CV-1 and SMK cells; and (3) maximal transactivation by gsmGR is attenuated in both cell lines. Glucocorticoid resistance in S. sciureus is at least partly attributable to a naturally occurring mutation in the GR gene that results in impaired GR transactivation.     

Glucocorticoid receptor expression in receptorless mutants isolated from the human leukemic cell line CEM-C7

The molecular basis for the loss of steroid binding activity in receptorless (r-) glucocorticoid-resistant (dexr) mutants isolated from the glucocorticoid-sensitive (dexs) cell line CEM-C7 was investigated. Although there was little binding of the reversibly associating ligand [3H]dexamethasone in r- mutants, labeling with the covalent affinity ligand [3H] dexamethasone 21-mesylate revealed significant amounts of a 92 kilodalton human glucocorticoid receptor (hGR) protein. Immunoblots of hGR protein in r- and normal cells showed that r- mutants expressed approximately half the amount of immunoreactive hGR protein seen in dexs cells. Comparison of the genomic organization of the hGR genes in normal and mutant cells revealed no discernable differences in the structure, or dosage, indicating that the r- phenotype was not the result of gross deletion or rearrangement of the hGR genes. In addition, r- cells expressed the same 7 kilobase mRNA as normal cells. More importantly, the amount of hGR mRNA expressed in r- cells was never significantly less, and in some cases was greater than, that seen in normal cells, indicating that the decrease in immunoreactive hGR protein seen in r- cells is not the result of loss of hGR mRNA expression. Taken together with the known mutation rate of the hGR gene(s) in these cells, these results suggest that the hGR genes in dexs CEM-C7 cells are allelic and that dexs cells express both a normal hGR protein and one with an altered steroid binding site. Furthermore, they suggest that the r- phenotype is acquired as the result of mutation within the coding region of the originally functional allele, leading to loss of ligand binding and expression of immunoreactive product.     

Genetic variability of the coding region for the prion protein gene (PRNP) in gayal (Bos frontalis)

The gayal (Bos frontalis) is a rare semi-wild bovid species in which bovine spongiform encephalopathy (BSE) has not been reported. Polymorphisms of the prion protein gene (PRNP) have been correlated significantly with resistance to BSE. In this study, the coding region of PRNP was cloned and characterized in samples from 125 gayal. A total of ten single nucleotide polymorphisms (SNPs), including six silent mutations (C60T, G75A, A108T, G126A, C357T and C678T) and four mis-sense mutations (C8A, G145A, G461A and C756G), corresponding to amino acids T3K, G49S9, N154S and I252M were identified, revealing high genetic diversity. Three novel SNPs including C60T, G145A and C756G, which have not been reported previously in bovid species, were retrieved. There also was one insertion–deletion (187Del24) at the N-terminal octapeptide repeat region. Alignment of nucleotide and amino acid sequences showed a high degree of similarity with other bovid species. Using phylogenetic analyses it was revealed that gayal has a close genetic relationship with Zebu cattle. In short, preliminary information is provided about genotypes of the PRNP in gayal. This could assist with the study of the pathogenesis of transmissible spongiform encephalopathies and cross species transmission as well as a molecular breeding project for gayal in China.     

Stable overproduction of intact glucocorticoid receptors in mammalian cells using a selectable glucocorticoid responsive dihydrofolate reductase gene

We have generated several mammalian cell lines that stably express high levels of intact glucocorticoid receptor. These cells were created by cotransfecting a glucocorticoid-dependent dihydrofolate reductase (DHFR) gene into DHFR-deficient Chinese hamster ovary (CHO) cells together with a plasmid directing the expression of human glucocorticoid receptor. Using this approach, transfection frequencies indicate that the inclusion of glucocorticoid receptor cDNA increased the efficiency of DHFR transformation greater than 10-fold over nonreceptor control DNA. When a stably cotransfected line (designated MG/hGR) was subjected to short term growth in cytotoxic concentrations of the antifolate methotrexate, these cells strongly resisted growth inhibition when dexamethasone was present in the medium. This effect was steroid specific and was inhibited by the glucocorticoid antagonist RU38486. In an effort to exploit the methotrexate-induced coamplification properties of the DHFR gene as a means of creating cell lines having increased levels of glucocorticoid receptor, MG/hGR cells were chronically exposed to a relatively low concentration of methotrexate (50 nM). After this treatment a resistant line was isolated (MG/hGR/MTX50) that displayed complete dependence on exogenous glucocorticoid for growth. To investigate the molecular basis for the enhanced ability of MG/hGR/MTX50 cells to resist the cytotoxic effects of methotrexate in the presence of dexamethasone, glucocorticoid receptor protein in these cells was characterized and compared to parental CHO cells and methotrexate sensitive MG/hGR cells. Affinity labeling with [3H]dexamethasone mesylate and Western blot analysis with antiglucocorticoid receptor antiserum revealed that nontransfected CHO cells have virtually undetectable levels of glucocorticoid receptor protein whereas cotransfected MG/hGR cells contain at least 3 times more intact monomeric receptor protein of Mr 94,000. Correspondingly, analysis of receptor protein in MG/hGR/MTX50 cells indicated that these cells contain 8 to 10 times more glucocorticoid receptor than nontransfected CHO cells. Scatchard analysis of steroid binding curves revealed that these increases correspond to 6,600, 22,000 and 63,000 dexamethasone binding sites per cell for nontransfected CHO cells, cotransfected MG/hGR cells, and MG/hGR/MTX50 cells, respectively. Sedimentation profiles of native receptor in transfected and methotrexate-resistant cells further support the progressive increase in receptor content and demonstrate that glucocorticoid receptor exists in cotransfected cels as an oligomeric complex under hypotonic conditions (9S complex in the presence of 20 mM sodium molybdate, 7S in the absence of molybdate), which dissociates to a monomeric 4S species in the presence of 0.4 M KCl. These physicochemical properties are indistinguishable from those observed for the endogenous hamster glucocorticoid receptor and suggest that stably transfected human glucocort     

Site-directed mutagenesis of rabbit LAT1 at amino acids 219 and 234

The availability of amino acids in the brain is regulated by the blood-brain barrier (BBB) large neutral amino acid transporter type 1 (LAT1) isoform, which is characterized by a high affinity (low Km) for substrate large neutral amino acids. The hypothesis that brain amino acid transport activity can be altered with single nucleotide polymorphisms was tested in the present studies with site-directed mutagenesis of the BBB LAT1. The rabbit has a high Km LAT1 large neutral amino acid transporter, as compared to the low Km neutral amino acid transporter at the human or rat BBB. The rabbit LAT1 was cloned from a rabbit brain capillary cDNA library. Alignment of the amino acid sequences of rabbit, human, and rat LAT1 revealed two radical amino acid residues that differ in the rabbit relative to the rat or human LAT1. The G219D mutation had a modest effect on the Km and Vmax of tryptophan transport via cloned rabbit LAT1 in frog oocytes, but the W234L variant reduced the Km by 64% and the Vmax by 96%. Conversely, LAT1 transport of either tryptophan or phenylalanine was nearly normalized when the double mutation W234L/G219D variant was produced. These studies show that marked changes in the affinity and capacity of the LAT1 are caused by single nucleotide polymorphisms and that phenotype can be restored with a double mutation.