The mouse glucocorticoid receptor: mapping of functional domains by cloning, sequencing and expression of wild-type and mutant receptor proteins.

Context: Interindividual variations in glucocorticoid sensitivity have been associated with manifestations of cortisol excess or deficiency and may be partly explained by polymorphisms in the human glucocorticoid receptor (hGR) gene. We studied a 43-yr-old female, who presented with manifestations consistent with tissue-selective glucocorticoid hypersensitivity. We detected a novel, single, heterozygous nucleotide (G → C) substitution at position 1201 (exon 2) of the hGR gene, which resulted in aspartic acid to histidine substitution at amino acid position 401 in the amino-terminal domain of the hGRα. We investigated the molecular mechanisms of action of the natural mutant receptor hGRαD401H. Methods-Results: Compared with the wild-type hGRα, the mutant receptor hGRαD401H demonstrated a 2.4-fold increase in its ability to transactivate the glucocorticoid-inducible mouse mammary tumor virus promoter in response to dexamethasone but had similar affinity for the ligand (dissociation constant = 6.2 ± 0.6 vs. 6.1 ± 0.6 nm) and time to nuclear translocation (14.75 ± 0.25 vs. 14.25 ± 1.13 min). The mutant receptor hGRαD401H did not exert a dominant positive or negative effect upon the wild-type receptor, it preserved its ability to bind to glucocorticoid response elements, and displayed a normal interaction with the glucocorticoid receptor-interacting protein 1 coactivator. Conclusions: The mutant receptor hGRαD401H enhances the transcriptional activity of glucocorticoid-responsive genes. The presence of the D401H mutation may predispose subjects to obesity, hypertension, and other manifestations of the metabolic syndrome.

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Molecular and genetic characterization of natural variants of HIV-1 Nef gene from North India and its functional implication in down-regulation of MHC-I and CD-4

Current HIV Research ◽

10.2174/1570162x18666200925160755 ◽

2020 ◽

Vol 18 ◽

Author(s):

J. Singh ◽

L. Ronsard ◽

M. Pandey ◽

R. Kapoor ◽

V.G. Ramachandran ◽

...

Keyword(s):

Biological Activities ◽

Sequence Similarity ◽

Eukaryotic Expression ◽

North India ◽

Cell Surface Expression ◽

Functional Domains ◽

Wild Type ◽

Down Regulation ◽

Subtype B ◽

Hiv 1

Background: HIV-1 Nef is an important accessory protein with multiple effector functions. Genetic studies of HIV-1 Nef gene shows extensive genetic diversity and the functional studies have been carried out mostly with Nef derived from regions dominated by subtype B (North America & Europe). Objective: This study was carried out to characterize genetic variations of the Nef gene from HIV-1 infected individuals from North-India and to find out their functional implications. Methods: The unique representative variants were sub-cloned in eukaryotic expression vector and further characterized with respect to their ability to down regulate cell surface expression of CD4 and MHC-1molecules. Results: The phylogenetic analysis of Nef variants revealed sequence similarity with either consensus subtype B or B/C recombinants. Boot scan analysis of some of our variants showed homology to B/C recombinant and some to wild type Nef B. Extensive variations were observed in most of the variants. The dN/dS ratio revealed 80% purifying selection and 20% diversifying selection implying the importance of mutations in Nef variants. Intracellular stability of Nef variants differed greatly when compared with wild type Nef B and C. There were some variants that possessed mutations in the functional domains of Nef and responsible for its differential CD4 and MHC-1 down regulation activity. Conclusion: We observed enhanced biological activities in some of the variants, perhaps arising out of amino acid substitutions in their functional domains. The CD4 and MHC-1 down-regulation activity of Nef is likely to confer immense survival advantage allowing the most rare genotype in a population to become the most abundant after a single selection event.

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Glucocorticoid Receptor Phosphorylation Differentially Affects Target Gene Expression

Molecular Endocrinology ◽

10.1210/me.2007-0219 ◽

2008 ◽

Vol 22 (8) ◽

pp. 1754-1766 ◽

Cited By ~ 157

Author(s):

Weiwei Chen ◽

Thoa Dang ◽

Raymond D. Blind ◽

Zhen Wang ◽

Claudio N. Cavasotto ◽

...

Keyword(s):

Glucocorticoid Receptor ◽

Transcriptional Activation ◽

Leucine Zipper ◽

Target Genes ◽

Divalent Cations ◽

Phosphorylation Site ◽

Transcriptional Response ◽

Receptor Occupancy ◽

Wild Type ◽

Interacting Protein

Abstract The glucocorticoid receptor (GR) is phosphorylated at multiple sites within its N terminus (S203, S211, S226), yet the role of phosphorylation in receptor function is not understood. Using a range of agonists and GR phosphorylation site-specific antibodies, we demonstrated that GR transcriptional activation is greatest when the relative phosphorylation of S211 exceeds that of S226. Consistent with this finding, a replacement of S226 with an alanine enhances GR transcriptional response. Using a battery of compounds that perturb different signaling pathways, we found that BAPTA-AM, a chelator of intracellular divalent cations, and curcumin, a natural product with antiinflammatory properties, reduced hormone-dependent phosphorylation at S211. This change in GR phosphorylation was associated with its decreased nuclear retention and transcriptional activation. Molecular modeling suggests that GR S211 phosphorylation promotes a conformational change, which exposes a novel surface potentially facilitating cofactor interaction. Indeed, S211 phosphorylation enhances GR interaction with MED14 (vitamin D receptor interacting protein 150). Interestingly, in U2OS cells expressing a nonphosphorylated GR mutant S211A, the expression of IGF-binding protein 1 and interferon regulatory factor 8, both MED14-dependent GR target genes, was reduced relative to cells expressing wild-type receptor across a broad range of hormone concentrations. In contrast, the induction of glucocorticoid-induced leucine zipper, a MED14-independent GR target, was similar in S211A- and wild-type GR-expressing cells at high hormone levels, but was reduced in S211A cells at low hormone concentrations, suggesting a link between GR phosphorylation, MED14 involvement, and receptor occupancy. Phosphorylation also affected the magnitude of repression by GR in a gene-selective manner. Thus, GR phosphorylation at S211 and S226 determines GR transcriptional response by modifying cofactor interaction. Furthermore, the effect of GR S211 phosphorylation is gene specific and, in some cases, dependent upon the amount of activated receptor.

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Platelet-derived growth factor (PDGF)-dependent association of phospholipase C-gamma with the PDGF receptor signaling complex

Molecular and Cellular Biology ◽

10.1128/mcb.10.5.2359-2366.1990 ◽

1990 ◽

Vol 10 (5) ◽

pp. 2359-2366

Author(s):

D K Morrison ◽

D R Kaplan ◽

S G Rhee ◽

L T Williams

Keyword(s):

Growth Factor ◽

Tyrosine Phosphorylation ◽

Serine Phosphorylation ◽

Platelet Derived Growth Factor ◽

Pdgf Receptor ◽

Wild Type ◽

Receptor Proteins ◽

Signaling Complex

We investigated the interaction of phospholipase C-gamma (PLC-gamma) with wild-type and mutant forms of the platelet-derived growth factor (PDGF) beta-receptor both in vivo and in vitro. After PDGF treatment of CHO cell lines expressing wild-type or either of two mutant (delta Ki and Y825F) PDGF receptors, PLC-gamma became tyrosine phosphorylated and associated with the receptor proteins. The receptor association and tyrosine phosphorylation of PLC-gamma correlated with the ability of these receptors to mediate ligand-induced phosphatidylinositol turnover. However, both the delta Ki and Y825F mutant receptors were deficient in transmitting mitogenic signals, suggesting that the PDGF-induced tyrosine phosphorylation and receptor association of PLC-gamma are not sufficient to account for the growth-stimulatory activity of PDGF. Wild-type and delta Ki mutant PDGF receptor proteins expressed with recombinant baculovirus vectors also associated in vitro with mammalian PLC-gamma. However, baculovirus-expressed c-fms, v-fms, c-src, and Raf-1 proteins failed to associate with PLC-gamma under similar conditions. Phosphatase treatment of the baculovirus-expressed PDGF receptor greatly decreased its association with PLC-gamma. This requirement for receptor phosphorylation was also observed in vivo, where PLC-gamma could not associate with a mutant PDGF receptor (K602A) defective in autophosphorylation. PLC-gamma also coimmunoprecipitated with two other putative receptor substrates, the serine-threonine kinase Raf-1 and the 85-kilodalton phosphatidylinositol-3' kinase, presumably through its association with the ligand-activated receptor. Furthermore, baculovirus-expressed Raf-1 phosphorylated purified PLC-gamma in vitro at sites which showed increased serine phosphorylation in vivo in response to PDGF. These results suggest that PDGF directly influences PLC activity by inducing the association of PLC-gamma with a receptor signaling complex, resulting in increased tyrosine and serine phosphorylation of PLC-gamma.

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Structure, Functional Domains and Subcellular Distribution of Glucocorticoid Receptor

Steroid Receptors in Health and Disease ◽

10.1007/978-1-4684-5541-0_5 ◽

1988 ◽

pp. 71-83

Author(s):

Jan Carlstedt-Duke ◽

Ann-Charlotte Wikstrom ◽

Jan-Ake Gustafsson

Keyword(s):

Glucocorticoid Receptor ◽

Subcellular Distribution ◽

Functional Domains

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Steroid binding and stabilization of glucocorticoid-receptor proteins from rat thymus

Biochemical Journal ◽

10.1042/bj1260011pa ◽

1972 ◽

Vol 126 (3) ◽

pp. 11P-11P ◽

Cited By ~ 5

Author(s):

P A Bell ◽

A Munck

Keyword(s):

Glucocorticoid Receptor ◽

Receptor Proteins ◽

Steroid Binding

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Deletion of the kinase insert sequence of the platelet-derived growth factor beta-receptor affects receptor kinase activity and signal transduction

Molecular and Cellular Biology ◽

10.1128/mcb.10.2.801-809.1990 ◽

1990 ◽

Vol 10 (2) ◽

pp. 801-809

Author(s):

L Severinsson ◽

B Ek ◽

K Mellström ◽

L Claesson-Welsh ◽

C H Heldin

Keyword(s):

Growth Factor ◽

Protein Tyrosine Kinase ◽

Catalytic Efficiency ◽

Kinase Domain ◽

Platelet Derived Growth Factor ◽

Tyrosine Kinase Domain ◽

Wild Type ◽

Mutant Receptor ◽

Beta Receptor ◽

Type Receptor

A characteristic feature of the platelet-derived growth factor (PDGF) beta-receptor is the presence of an insert sequence in the protein tyrosine kinase domain. A receptor mutant which lacks the entire insert of 98 amino acids was expressed in CHO cells, and its functional characteristics were compared with those of the wild-type receptor. The mutant receptor bound PDGF-BB with high affinity and mediated internalization and degradation of the ligand with efficiency similar to that of the wild-type receptor but did not transduce a mitogenic signal. It was found to display a decreased autophosphorylation after ligand stimulation and had a decreased ability to phosphorylate exogenous substrates; phosphofructokinase was not phosphorylated at all, whereas a peptide substrate was phosphorylated, albeit at a lower rate compared with phosphorylation by the wild-type receptor. Furthermore, the mutant receptor did not mediate actin reorganization but mediated an increase in c-fos expression. The data indicate that the insert in the kinase domain of the PDGF beta-receptor is important for the substrate specificity or catalytic efficiency of the kinase; the deletion of the insert interferes with the transduction of some, but not all, of the signals that arise after activation of the receptor.

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A mutant androgen receptor from patients with Reifenstein syndrome: identification of the function of a conserved alanine residue in the D box of steroid receptors

Molecular and Cellular Biology ◽

10.1128/mcb.13.12.7850-7858.1993 ◽

1993 ◽

Vol 13 (12) ◽

pp. 7850-7858

Author(s):

F Kaspar ◽

H Klocker ◽

A Denninger ◽

A C Cato

Keyword(s):

Androgen Receptor ◽

Dna Binding ◽

Steroid Receptors ◽

Regulatory Elements ◽

Binding Domain ◽

Dna Binding Domain ◽

Wild Type ◽

Binding Studies ◽

Mutant Receptor ◽

Alanine Residue

Reifenstein syndrome is an eponymic term that describes partial androgen-insensitive disorders. Androgen receptor isolated from five patients with this syndrome contains a specific mutation in the DNA binding domain of the receptor. This mutation converts an alanine to a threonine at position 596 next to the zinc catenation site at the second finger. The threonine 596 mutant receptor mediated normal androgen response at promoters with closely positioned multiple regulatory elements for the androgen receptor and other transcription factors. Promoters with single isolated androgen response elements were not transactivated by the mutant receptor. In in vitro receptor-DNA binding studies, interaction with DNA by the mutant receptor was achieved only in the presence of an anti-androgen receptor antibody. Exchanging alanine 596 in the wild-type androgen receptor with serine or valine produced mutants with properties indistinguishable from those of the naturally occurring threonine 596 mutant receptor. These results indicate that an alanine residue at position 596 contributes important structural and functional activities to the androgen receptor. In the androgen receptor from the patients with Reifenstein syndrome, in which this alanine is converted to a threonine, wild-type receptor properties can be restored by exchanging an additional threonine at position 602 to an alanine. An alanine residue at position 596 or 602 in the DNA binding domain of the androgen receptor is therefore important for the full function of this receptor. In all steroid receptors that bind the core sequence AGAACANNNTGTTCT, an alanine residue is also present at a position equivalent to alanine 596 in the androgen receptor.

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Analysis of MinC Reveals Two Independent Domains Involved in Interaction with MinD and FtsZ

Journal of Bacteriology ◽

10.1128/jb.182.14.3965-3971.2000 ◽

2000 ◽

Vol 182 (14) ◽

pp. 3965-3971 ◽

Cited By ~ 98

Author(s):

Zonglin Hu ◽

Joe Lutkenhaus

Keyword(s):

Escherichia Coli ◽

Cell Division ◽

Functional Domains ◽

Wild Type ◽

Terminal Domain ◽

Ring Assembly ◽

Z Ring ◽

Ftsz Assembly

ABSTRACT In Escherichia coli FtsZ assembles into a Z ring at midcell while assembly at polar sites is prevented by themin system. MinC, a component of this system, is an inhibitor of FtsZ assembly that is positioned within the cell by interaction with MinDE. In this study we found that MinC consists of two functional domains connected by a short linker. When fused to MalE the N-terminal domain is able to inhibit cell division and prevent FtsZ assembly in vitro. The C-terminal domain interacts with MinD, and expression in wild-type cells as a MalE fusion disrupts minfunction, resulting in a minicell phenotype. We also find that MinC is an oligomer, probably a dimer. Although the C-terminal domain is clearly sufficient for oligomerization, the N-terminal domain also promotes oligomerization. These results demonstrate that MinC consists of two independently functioning domains: an N-terminal domain capable of inhibiting FtsZ assembly and a C-terminal domain responsible for localization of MinC through interaction with MinD. The fusion of these two independent domains is required to achieve topological regulation of Z ring assembly.

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