Genetic analysis of the N-terminal end of the glucocorticoid receptor hormone binding domain

Milhon, Jon; Kohli, Kulwant K.; Stallcup, Michael R.

doi:10.1016/0960-0760(94)90110-4

Cited by 17 publications

(11 citation statements)

References 46 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Such sequences have recently been shown to be important for protein-protein interactions between steroid receptors and coactivators (28 -30). Furthermore, a double mutation of this motif (L553G/ L554G) has been reported to eliminate the cell-free affinity labeling of GR by Dex-Mes and the biological activity of Dex in whole cells (37). Thus, we suspected that this same LXXLL sequence might be a critical component for hsp90 binding to GR.…”

Section: Gr Mutations and Truncations-examinationmentioning

confidence: 95%

“…Mouse GR with the Leu to Gly mutations at positions equivalent to 553 and 554 of rGR was transcriptionally inactive (37). We predict that the steroid binding capacity of this L553G/L554G double mutant will be relatively unchanged at 0°C but that prebound steroid will rapidly dissociate at elevated temperatures following activation and that trypsin digestion will not afford the 16-kDa tryptic fragment, due to inadequate intramolecular contacts of the hinged cover of the LBD.…”

mentioning

confidence: 99%

See 1 more Smart Citation

The Seven Amino Acids (547–553) of Rat Glucocorticoid Receptor Required for Steroid and Hsp90 Binding Contain a Functionally Independent LXXLL Motif That Is Critical for Steroid Binding

Giannoukos¹,

Silverstein²,

Pratt³

et al. 1999

Journal of Biological Chemistry

View full text Add to dashboard Cite

Hsp90 association with glucocorticoid receptors (GRs) is required for steroid binding. We recently reported that seven amino acids (547-553) overlapping the aminoterminal end of the rat GR ligand-binding domain are necessary for hsp90 binding, and consequently steroid binding. The role of a LXXLL motif at the COOH terminus of this sequence has now been analyzed by determining the properties of Leu to Ser mutations in fulllength GR and glutathione S-transferase chimeras. Surprisingly, these mutations decreased steroid binding capacity without altering receptor levels, steroid binding affinity, or hsp90 binding. Single mutations in the context of the full-length receptor did not affect the transcriptional activity but the double mutant (L550S/ L553S) was virtually inactive. This biological inactivity was found to be due to an increased rate of steroid dissociation from the activated mutant complex. These results, coupled with those from trypsin digestion studies, suggest a model in which the GR ligand-binding domain is viewed as having a "hinged pocket," with the hinge being in the region of the trypsin digestion site at Arg 651 . The pocket would normally be kept shut via the intramolecular interactions of the LXXLL motif at amino acids 550 -554 acting as a hydrophobic clasp.

show abstract

Section: Gr Mutations and Truncations-examinationmentioning

confidence: 95%

mentioning

confidence: 99%

The Seven Amino Acids (547–553) of Rat Glucocorticoid Receptor Required for Steroid and Hsp90 Binding Contain a Functionally Independent LXXLL Motif That Is Critical for Steroid Binding

Giannoukos¹,

Silverstein²,

Pratt³

et al. 1999

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…To probe their roles and extend previous studies done by Simons et al. [10] and Stallcup and coworkers [11,12], who evaluated the function of the LXXLL in helix 1, we developed a series of LXXLL mutants. LXXLL mutations in helix 1 were shown to be functionally defective under all conditions studied, whereas LXXLL mutations in helix 10 were fully functional in transactivation and transrepression only requiring higher concentrations of hormone.…”

Section: Introductionmentioning

confidence: 98%

Functional analysis of the LXXLL motifs of the human glucocorticoid receptor: Association with altered ligand affinity

Dong

Jewell

Bienstock

et al. 2006

The Journal of Steroid Biochemistry and Molecular Biology

View full text Add to dashboard Cite

“…Yeast protein extracts were prepared by a urea-sodium dodecyl sulfate (SDS) method (37). SDS-polyacrylamide gel electrophoresis (PAGE) and immunoblotting were performed with anti-GR antibody BuGR2 as described previously (33).…”

Section: Construction Of Plasmidsmentioning

confidence: 99%

GRIP1, a Transcriptional Coactivator for the AF-2 Transactivation Domain of Steroid, Thyroid, Retinoid, and Vitamin D Receptors

Hong

Kohli

Garabedian

et al. 1997

Molecular and Cellular Biology

526

351

View full text Add to dashboard Cite

After binding to enhancer elements, transcription factors require transcriptional coactivator proteins to mediate their stimulation of transcription initiation. A search for possible coactivators for steroid hormone receptors resulted in identification of glucocorticoid receptor interacting protein 1 (GRIP1). The complete coding sequence for GRIP1, isolated from a mouse brain cDNA library, contains an open reading frame of 1,462 codons. GRIP1 is the probable ortholog of the subsequently identified human protein transcription intermediary factor 2 (TIF2) and is also partially homologous to steroid receptor coactivator 1 (SRC-1). The full-length GRIP1 interacted with the hormone binding domains (HBDs) of all five steroid receptors in a hormone-dependent manner and also with HBDs of class II nuclear receptors, including thyroid receptor ␣, vitamin D receptor, retinoic acid receptor ␣, and retinoid X receptor ␣. In contrast to agonists, glucocorticoid antagonists did not promote interaction between the glucocorticoid receptor and GRIP1. In yeast cells, GRIP1 dramatically enhanced the transcriptional activation function of proteins containing the HBDs of any of the above-named receptors fused to the GAL4 DNA binding domain and thus served as a transcriptional coactivator for them. This finding contrasts with previous reports of TIF2 and SRC-1, which in mammalian cells enhanced the transactivation activities of only a subset of the steroid and nuclear receptors that they physically interacted with. GRIP1 also enhanced the hormone-dependent transactivation activity of intact glucocorticoid receptor, estrogen receptor, and mineralocorticoid receptor. Experiments with glucocorticoid receptor truncation and point mutants indicated that GRIP1 interacted with and enhanced the activity of the C-terminal AF-2 but not the N-terminal AF-1 transactivation domain of the glucocorticoid receptor. These results demonstrate directly that AF-1 and AF-2 domains accomplish their transactivation activities through different mechanisms: AF-2 requires GRIP1 as a coactivator, but AF-1 does not.Nuclear hormone receptors (NRs) are conditional transcription factors that play important roles in various aspects of cell growth, development, and homeostasis by controlling expression of specific genes (4,15,29,43). Members of the NR superfamily, which includes the five steroid receptors (SRs) as well as the receptors for thyroid hormone (TR), retinoic acid (RAR), and vitamin D (VDR), are structurally characterized by three distinct domains: an N-terminal transcriptional activation domain (AD), a central DNA binding domain (DBD), and a C-terminal hormone binding domain (HBD). Before the binding of hormone, SRs, which are sometimes called class I of the NR family, remain inactive in a complex with hsp90 and other stress family proteins. The binding of hormone induces critical conformational changes in SRs that cause them to dissociate from the inhibitory complex, bind as homodimers to specific DNA enhancer elements associated with target genes, and m...

show abstract

Genetic analysis of the N-terminal end of the glucocorticoid receptor hormone binding domain

Cited by 17 publications

References 46 publications

The Seven Amino Acids (547–553) of Rat Glucocorticoid Receptor Required for Steroid and Hsp90 Binding Contain a Functionally Independent LXXLL Motif That Is Critical for Steroid Binding

The Seven Amino Acids (547–553) of Rat Glucocorticoid Receptor Required for Steroid and Hsp90 Binding Contain a Functionally Independent LXXLL Motif That Is Critical for Steroid Binding

Functional analysis of the LXXLL motifs of the human glucocorticoid receptor: Association with altered ligand affinity

GRIP1, a Transcriptional Coactivator for the AF-2 Transactivation Domain of Steroid, Thyroid, Retinoid, and Vitamin D Receptors

Contact Info

Product

Resources

About