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M R Stallcup

Publications and source records attributed to M R Stallcup.

At least 37 records · Page 2Linked to original sources
PubMed1999

Modulation of transcriptional activation and coactivator interaction by a splicing variation in the F domain of nuclear receptor hepatocyte nuclear factor 4alpha1.

Transcription factors, such as nuclear receptors, often exist in various forms that are generated by highly conserved splicing events. Whereas the functional significance of these splicing variants is often not known, it is known that nuclear receptors activate transcription through interaction with coactivators. The parameters, other than ligands, that might modulate those interactions, however, are not well characterized, nor is the role of splicing variants. In this study, transient transfection, yeast two-hybrid, and GST pulldown assays are used to show not only that nuclear receptor hepatocyte nuclear factor 4 alpha1 (HNF4alpha1, NR2A1) interacts with GRIP1, and other coactivators, in the absence of ligand but also that the uncommonly large F domain in the C terminus of the receptor inhibits that interaction. In vitro, the F domain was found to obscure an AF-2-independent binding site for GRIP1 that did not map to nuclear receptor boxes II or III. The results also show that a natural splicing variant containing a 10-amino-acid insert in the middle of the F domain (HNF4alpha2) abrogates that inhibition in vivo and in vitro. A series of protease digestion assays indicates that there may be structural differences between HNF4alpha1 and HNF4alpha2 in the F domain as well as in the ligand binding domain (LBD). The data also suggest that there is a direct physical contact between the F domain and the LBD of HNF4alpha1 and -alpha2 and that that contact is different in the HNF4alpha1 and HNF4alpha2 isoforms. Finally, we propose a model in which the F domain of HNF4alpha1 acts as a negative regulatory region for transactivation and in which the alpha2 insert ameliorates the negative effect of the F domain. A conserved repressor sequence in the F domains of HNF4alpha1 and -alpha2 suggests that this model may be relevant to other nuclear receptors as well.

Alternative Splicing↗
PubMed1999

Multiple signal input and output domains of the 160-kilodalton nuclear receptor coactivator proteins.

Members of the 160-kDa nuclear receptor coactivator family (p160 coactivators) bind to the conserved AF-2 activation function found in the hormone binding domains of nuclear receptors (NR) and are potent transcriptional coactivators for NRs. Here we report that the C-terminal region of p160 coactivators glucocorticoid receptor interacting protein 1 (GRIP1), steroid receptor coactivator 1 (SRC-1a), and SRC-1e binds the N-terminal AF-1 activation function of the androgen receptor (AR), and p160 coactivators can thereby enhance transcriptional activation by AR. While they all interact efficiently with AR AF-1, these same coactivators have vastly different binding strengths with and coactivator effects on AR AF-2. p160 activation domain AD1, which binds secondary coactivators CREB binding protein (CBP) and p300, was previously implicated as the principal domain for transmitting the activating signal to the transcription machinery. We identified a new highly conserved motif in the AD1 region which is important for CBP/p300 binding. Deletion of AD1 only partially reduced p160 coactivator function, due to signaling through AD2, another activation domain located at the C-terminal end of p160 coactivators. C-terminal coactivator fragments lacking AD1 but containing AD2 and the AR AF-1 binding site served as efficient coactivators for full-length AR and AR AF-1. The two signal input domains (one that binds NR AF-2 domains and one that binds AF-1 domains of some but not all NRs) and the two signal output domains (AD1 and AD2) of p160 coactivators played different relative roles for two different NRs: AR and thyroid hormone receptor.

Animals↗
PubMed1999

Constitutive activation of transcription and binding of coactivator by estrogen-related receptors 1 and 2.

In this report, we demonstrate that, in contrast to most previously characterized nuclear receptors, hERR1 and hERR2 (human estrogen receptor-related protein 1 and -2) are constitutive activators of the classic estrogen response element (ERE) as well as the palindromic thyroid hormone response element (TRE(pal)) but not the glucocorticoid response element (GRE). This intrinsically activated state of hERR1 and hERR2 resides in the ligand-binding domains of the two genes and is transferable to a heterologous receptor. In addition, we show that members of the p160 family of nuclear receptor coactivators, ACTR (activator of thyroid and retinoic acid receptors), GRIP1 (glucocorticoid receptor interacting protein 1), and SRC-1 (steroid receptor coactivator 1), potentiate the transcriptional activity by hERR1 and hERR2 in mammalian cells, and that both orphan receptors bind the coactivators in a ligand-independent manner. Together, these results suggest that hERR1 and hERR2 activate gene transcription through a mechanism different from most of the previously characterized steroid hormone receptors.

Acetyltransferases↗
PubMed1998

Structure and specificity of nuclear receptor-coactivator interactions.

Combinatorial regulation of transcription implies flexible yet precise assembly of multiprotein regulatory complexes in response to signals. Biochemical and crystallographic analyses revealed that hormone binding leads to the formation of a hydrophobic groove within the ligand binding domain (LBD) of the thyroid hormone receptor that interacts with an LxxLL motif-containing alpha-helix from GRIP1, a coactivator. Residues immediately adjacent to the motif modulate the affinity of the interaction; the motif and the adjacent sequences are employed to different extents in binding to different receptors. Such interactions of amphipathic alpha-helices with hydrophobic grooves define protein interfaces in other regulatory complexes as well. We suggest that these common structural elements impart flexibility to combinatorial regulation, whereas side chains at the interface impart specificity.

Amino Acid Sequence↗
PubMed1998

Enhancement of estrogen receptor transcriptional activity by the coactivator GRIP-1 highlights the role of activation function 2 in determining estrogen receptor pharmacology.

The human estrogen receptor (ER) contains two major activation functions (AFs) responsible for its transcriptional activity. One of these, activation function 2 (AF-2), located within the hormone-binding domain (HBD), has been shown to mediate the ligand-dependent transcriptional activity of ER as well as other members of the nuclear receptor superfamily. Recently, proteins interacting with the HBD of several nuclear receptors have been cloned. One of these proteins, glucocorticoid receptor interacting protein (GRIP-1), has been shown to interact with ER and was originally hypothesized to mediate its transcriptional activity through AF-2. However, we find in this study that the transcriptional activity of ER, containing mutations in the AF-2 core sequence, can be enhanced by coexpression of the coactivator GRIP-1, suggesting that this protein may not rely solely on the AF-2 domain for interaction. We propose, therefore, that the HBD of ER either contains multiple binding sites that are necessary for association with GRIP-1 or, alternatively, that this coactivator contacts the receptor in an undetermined region within the HBD. Importantly, these studies demonstrate also that mutations or deletion of AF-2 alter the ligand pharmacology of the receptor such that ER loses the ability to discriminate between agonists and antagonists. Interestingly, on these mutant receptors GRIP-1 still functions as a coactivator independent of the nature of the bound ligand. It is likely, therefore, that the C-terminal AF-2 domain may function as a molecular switch allowing the wild-type receptor to discriminate between agonists and antagonists as well as providing a surface with which associated proteins can interact.

Amino Acid Sequence↗
PubMed1998

Estrogen receptor activation function 1 works by binding p160 coactivator proteins.

Estrogen receptor-alpha contains two transactivation functions, a weak constitutive activation function (AF-1) and a hormone-dependent activation function (AF-2). AF-2 works by recruiting a large coactivator complex, composed of one or more p160s, CREB-binding protein (CBP)/p300, and P/CAF (p300 and CBP-associated factor), via direct contacts with the p160s. We report here that independent AF-1 activity also requires p160 contacts. Unlike AF-2, which binds signature NR boxes in the center of the p160 molecule, AF-1 binds to sequences near the p160 C terminus. We propose that the ability of AF-1 and AF-2 to interact with separate surfaces of the same coactivator is important for the ability of these transactivation functions to synergize.

Acetyltransferases↗
PubMed1998

Nuclear receptor-binding sites of coactivators glucocorticoid receptor interacting protein 1 (GRIP1) and steroid receptor coactivator 1 (SRC-1): multiple motifs with different binding specificities.

The activity of the AF-2 transcriptional activation function of nuclear receptors (NR) is mediated by the partially homologous transcriptional coactivators, glucocorticoid receptor interacting protein 1 (GRIP1)/transcriptional intermediary factor 2 (TIF2) and steroid receptor coactivator 1 (SRC-1). GRIP1 and SRC-1 bound nine different NRs and exhibited similar, but not identical, NR binding preferences. The most striking difference was seen with the androgen receptor, which bound well to GRIP1 but poorly to SRC-1. GRIP1 and SRC-1 contain three copies of the NR binding motif LXXLL (called an NR Box) in their central regions. Mutation of both NR Box II and NR Box III in GRIP1 almost completely eliminated functional and binding interactions with NRs, indicating that these two sites are crucial for most of GRIP1's NR binding activity. Interactions of GRIP1 with the estrogen receptor were more strongly affected by mutations in NR Box II, whereas interactions with the androgen receptor and glucocorticoid receptor were more strongly affected by NR Box III mutations. One isoform of SRC-1 has an additional NR Box (NR Box IV) at its extreme C terminus with an NR-binding preference somewhat different from that of the central NR-binding domain of SRC-1. GRIP1 has no NR Box in its C-terminal region and therefore no C-terminal NR-binding function. In summary, GRIP1 and SRC-1 have overlapping NR-binding preferences, but specific NRs display both coactivator and NR Box preferences that may contribute to the specificity of hormonal responses.

Amino Acid Sequence↗
PubMed1998

The DNA-binding and tau2 transactivation domains of the rat glucocorticoid receptor constitute a nuclear matrix-targeting signal.

Using an ATP-depletion paradigm to augment glucocorticoid receptor (GR) binding to the nuclear matrix, we have identified a minimal segment of the receptor that constitutes a nuclear matrix targeting signal (NMTS). While previous studies implicated a role for the receptor's DNA-binding domain in nuclear matrix targeting, we show here that this domain of rat GR is necessary, but not sufficient, for matrix targeting. A minimal NMTS can be generated by linking the rat GR DNA-binding domain to either its tau2 transactivation domain in its natural context, or a heterologous transactivation domain derived from the Herpes simplex virus VP16 protein. The transactivation and nuclear matrix-targeting activities of tau2 are separable, as transactivation mutants were identified that either inhibited or had no apparent effect on matrix targeting of tau2. A functional interaction between the NMTS of rat GR and the RNA-binding nuclear matrix protein hnRNP U was revealed in cotransfection experiments in which hnRNP U overexpression was found to interfere with the transactivation activity of GR derivatives that possess nuclear matrix-binding capacity. We have therefore ascribed a novel function to a steroid hormone transactivation domain that could be an important component of the mechanism used by steroid hormone receptors to regulate genes in their native configuration within the nucleus.

Adenosine Triphosphate↗
PubMed1997

Yeast hormone response element assays detect and characterize GRIP1 coactivator-dependent activation of transcription by thyroid and retinoid nuclear receptors.

The mouse glucocorticoid receptor-interacting protein (GRIP1) is a member of the ERAP160 family of nuclear receptor (NR) coactivators (including SRC-1 and TIF2) which function as bridging proteins between ligand-activated NRs bound to cognate hormone-response elements (HREs) and the transcription initiation apparatus (TIA). Although these coactivators bind to several NRs, studies overexpressing these coactivators with these NRs in mammalian cells have not uniformly observed a corresponding enhancement of ligand-dependent transactivation. Here, we show that GRIP1 interacts in vitro in a ligand-dependent manner with thyroid receptor, retinoic acid receptor, and retinoid X receptor. Additionally, in yeast (Saccharomyces cerevisiae) GRIP1 coactivator protein markedly increased the ability of these full-length class II NRs to transactivate beta-galactosidase reporter genes containing cognate HREs. The magnitude of GRIP1 enhancement of liganded NR homodimer was dependent upon NR subtype and HRE configuration. For most HRE configurations, thyroid receptor and retinoic acid receptor homodimers were essentially unresponsive or very weakly active in the absence of GRIP1, but GRIP1 dramatically restored the ligand-dependent function of these NRs. Although GRIP1 exerted no significant effect on NR homodimers in the absence of their cognate ligands, it increased the transactivation of unliganded NR heterodimers. Whether GRIP1 increased ligand-dependent transactivation of a heterodimer to levels greater than that of the cognate homodimer was determined by HRE configuration and copy number. Compared with the limitations of yeast two-hybrid and mammalian coexpression systems, the yeast HRE-assay systems described in this report facilitated both the detection of putative mammalian NR coactivator function and the elucidation of their mechanisms of transactivational enhancement.

Animals↗
PubMed1997

GRIP1, a transcriptional coactivator for the AF-2 transactivation domain of steroid, thyroid, retinoid, and vitamin D receptors.

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 alpha, vitamin D receptor, retinoic acid receptor alpha, and retinoid X receptor alpha. 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.

Amino Acid Sequence↗
PubMed1997

Identification of amino acids in the tau 2-region of the mouse glucocorticoid receptor that contribute to hormone binding and transcriptional activation.

The tau 2-region of steroid hormone receptors is a highly conserved region located at the extreme N-terminal end of the hormone-binding domain. A protein fragment encoding tau 2 has been shown to function as an independent transcriptional activation domain; however, because this region is essential for hormone binding, it has been difficult to determine whether the tau 2-region also contributes to the transactivation function of intact steroid receptors. In this study a series of amino acid substitutions were engineered at conserved positions in the tau 2-region of the mouse glucocorticoid receptor (mGR, amino acids 533-562) to map specific amino acid residues that contribute to the hormone-binding function, transcriptional activation, or both. Substitution of alanine or glycine for some amino acids (mutations E546G, P547A, and D555A) reduced or eliminated hormone binding, but the transactivation function of the intact GR and/or the minimum tau 2-fragment was unaffected for each of these mutants. Substitution of alanine for amino acid S561 reduced transactivation activity in the intact GR and the minimum tau 2-fragment but had no effect on hormone binding. The single mutation L550A and the double amino acid substitution L541G+L542G affected both hormone binding and transactivation. The fact that the S561A and L550A substitutions each caused a loss of transactivation activity in the minimum tau 2-fragment and the full-length GR indicated that the tau 2-region does contribute to the overall transactivation function of the full-length GR. Overall, the N-terminal portion of the tau 2-region (mGR 541-547) was primarily involved in hormone binding, whereas the C-terminal portion of the tau 2-region (mGR 548-561) was primarily involved in transactivation.

Amino Acid Sequence↗
PubMed1996

GRIP1, a novel mouse protein that serves as a transcriptional coactivator in yeast for the hormone binding domains of steroid receptors.

The yeast two-hybrid system was used to isolate a clone from a 17-day-old mouse embryo cDNA library that codes for a novel 812-aa long protein fragment, glucocorticoid receptor-interacting protein 1 (GRIP1), that can interact with the hormone binding domain (HBD) of the glucocorticoid receptor. In the yeast two-hybrid system and in vitro, GRIP1 interacted with the HBDs of the glucocorticoid, estrogen, and androgen receptors in a hormone-regulated manner. When fused to the DNA binding domain of a heterologous protein, the GRIP1 fragment activated a reporter gene containing a suitable enhancer site in yeast cells and in mammalian cells, indicating that GRIP1 contains a transcriptional activation domain. Overexpression of the GRIP1 fragment in mammalian cells interfered with hormone-regulated expression of mouse mammary tumor virus-chloramphenicol acetyltransferase gene and constitutive expression of cytomegalovirus-beta-galactosidase reporter gene, but not constitutive expression from a tRNA gene promoter. This selective squelching activity suggests that GRIM can interact with an essential component of the RNA polymerase II transcription machinery. Finally, while a steroid receptor HBD fused with a GAL4 DNA binding domain did not, by itself, activate transcription of a reporter gene in yeast, coexpression of this fusion protein with GRIP1 strongly activated the reporter gene. Thus, in yeast, GRIP1 can serve as a coactivator, potentiating the transactivation functions in steroid receptor HBDs, possibly by acting as a bridge between HBDs of the receptors and the basal transcription machinery.

Amino Acid Sequence↗
PubMed1995

Growth inhibition of androgen-insensitive human prostate carcinoma cells by a 19-norsteroid derivative agent, mifepristone.

Mifepristone, also known as RU 486, is a 19-norsteroid derivative. Currently, mifepristone is being tested in clinical trials on meningioma and breast cancer. In this study we analyzed whether mifepristone could inhibit the growth of human prostate cancer cells including androgen-insensitive (PC-3 and DU145) and androgen-sensitive (LNCaP) cell lines. At 1-nM concentration, mifepristone exhibited a marginal stimulatory action on LN-CaP and PC-3 cells. Nevertheless, a dose-dependent growth inhibition on those same cell lines was observed at concentrations of 1 microM and 10 microM. Twenty-day exposure to the clinically achievable concentration of 1 microM mifepristone resulted in consistent inhibition of all three cell lines studied. Furthermore, this in vitro growth inhibition was reflected in an in vivo nude mouse system. Mifepristone at the dosage of 4 mg/100 g body weight completely suppressed the growth of PC-3 tumors for 21 days, although this was followed by a growth rate similar to that of the control tumor. To understand the possible mechanism of mifepristone inhibition, PC-3 cells were exposed to mifepristone in comparison with dexamethasone (Dex), progesterone, and 5 alpha-dihydrotestosterone (DHT), each at 1-microM concentration. The results demonstrated that while both DHT and Dex alone had essentially no effect on cell growth, progesterone alone resulted in a 20% growth inhibition, while mifepristone had more than 60% inhibition with a 16-day exposure. At an equal concentration, the degree of growth inhibition of PC-3 cells by mifepristone or progesterone was partially diminished by simultaneous exposure to Dex. In conclusion, our results demonstrated that the growth of androgen-insensitive prostate cancer cells can be directly inhibited by mifepristone in cultures. This in vitro inhibition is reflected in xenografted tumors.

Animals↗
PubMed1995

A somatic cell genetic method for identification of untargeted mutations in the glucocorticoid receptor that cause hormone binding deficiencies.

Mouse T lymphoma cell line W7MG1, which is killed by physiological concentrations of glucocorticoid agonists, was used as a convenient genetic system for isolating sublines containing mutant glucocorticoid receptors (GR) with hormone-binding deficiencies. Partially hormone-resistant cell clones were derived from chemically mutagenized cell populations by selecting for growth in moderate concentrations of dexamethasone (Dex) and then screening for failure to grow in high Dex concentrations. Such clones are likely to have mutant GR. In GR cDNA clones from the partially resistant cell sublines, three different functionally significant mutations in the hormone-binding domain were identified: Leu-569 changed to Phe (L569F), Leu-670 to Phe (L670F), and Met-672 to Ile (M672I). Dose-response analyses with Dex and affinity labeling studies with dexamethasone 21-mesylate in transiently transfected cells indicated that all three mutant GR species had hormone-binding deficiencies. However, at saturating Dex concentrations the mutant and wild type GR activated a hormone-inducible reporter gene to the same extent; thus, these three mutations did not affect the ability of GR to activate transcription of the reporter gene after hormone was bound. In dose-response curves conducted with several glucocorticoid agonists, mutations L670F and M672I caused no change in ligand-binding specificities, while mutation L569F caused a modest change in specificity. Quantitative hormone-binding studies conducted with mutant GR synthesized in cell-free reactions showed that mutant GR species L569F and M672I had reduced Dex-binding affinities both at 0 C and at 22 C in the presence of molybdate. In contrast, for the L670F mutant, which exhibited the severest deficiency in vivo, the hormone-binding deficiency in the cell-free system was evident only at 26-37 C and primarily in the absence of molybdate. We propose that the L670F GR is an activation-labile type of mutant, which binds hormone normally in the presence of heat shock protein 90 but loses hormone rapidly after dissociation from heat shock protein 90. These three mutations define two new subregions of the GR polypeptide that are important for hormone binding.

Amino Acid Sequence↗
PubMed1994

The hormone-binding role of 2 cysteines near the C terminus of the mouse glucocorticoid receptor.

Previous biochemical analyses with covalent affinity labels and thiol-blocking reagents suggested possible roles for one methionine residue and multiple cysteine residues in binding of steroid to the 250-amino acid hormone-binding domain at the C-terminal end of mammalian glucocorticoid receptors. To test the functional roles of these residues in the receptor's ability to bind hormone and active transcription of target genes, the mouse glucocorticoid receptor cDNA was specifically mutated to cause single amino acid substitutions for methionine 610 and for each of the 5 cysteines (at positions 628, 644, 649, 671, and 742) in the hormone-binding domain. Among these 6 residues, only mutations in cysteine 671 and cysteine 742 caused substantial reductions in function. In transient transfection assays, the concentration of dexamethasone required for half-maximal activation of a glucocorticoid-responsive reporter gene was increased by 10-40-fold by changing cysteine 742 to serine or cysteine 671 to serine or alanine. At saturating concentrations of dexamethasone, the mutant receptors activated the reporter gene to the same extent as the wild type receptor, indicating that the mutations affected only the hormone-binding function of the receptor and not its ability to bind DNA or activate transcription once the hormone was bound.

Amino Acid Sequence↗
PubMed1994

Abnormal processing of a recombinant feline leukemia virus envelope polyprotein and its interference with subgroup C virus infection.

Processing of the env polyprotein of a noninfectious feline leukemia virus (FeLV) recombinant, named r6gp, was examined in human-transfected cells. The r6gp provirus was previously generated in the frame of FeLV, subgroup B, GA clone with substitution of all but 40 C-terminal amino acid sequences of the surface glycoprotein (SU) from an endogenous FeLV provirus element (CFE-6). Although r6gp produced a normal size (85 kDa) env glycoprotein precursor, the product, unlike the precursor of the parental virus, was neither additionally glycosylated nor further processed into mature env proteins. Biochemical observations were consistent with the idea that the chimeric env polyprotein was trapped in the endoplasmic reticulum (ER) and were directly supported by immunofluorescence microscopy analyses. Interestingly, the residence of the chimeric protein in the ER specifically interfered with FeLV, subgroup C (Sarma) virus infection but not the parental FeLV-B virus infection. Since FeLV-C provirus sequences could be readily detected in the infected cells, it appeared that r6gp env expression did not block entry of the challenge virus. While FeLV-B and CFE-6 env genes share an extensive overall sequence homology, a variable region (region VI) of CFE-6 near the C-terminus of SU, which was retained in the r6gp construct, exhibits a considerably higher degree of homology to FeLV-C than FeLV-B. Thus, we propose that region VI is involved in conferring specificity for the env polyprotein oligomerization in the ER, and that co-oligomerization of the trapped r6gp env with FeLV-C is the reason for specific interference with FeLV-C infection. The results also demonstrate for the first time a functional abnormality of a recombinant FeLV env gene which is structurally similar to those commonly detected in FeLV-induced feline lymphosarcomas.

Amino Acid Sequence↗
PubMed1994

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

Four site-directed missense mutations were constructed at the N-terminal end of the mouse glucocorticoid receptor (GR) hormone binding domain. This small subdomain is highly conserved among the steroid hormone receptors and is within a larger subregion believed to be important for hormone binding, transcriptional activation, and hsp90 binding. The ability of mutant and wild type GR to activate a reporter gene in response to various concentrations of dexamethasone was examined in transiently transfected COS-7 cells. Mutant GR species V544G (valine-544 changes to glycine) and V549G activated the reporter gene to approximately the same extent as wild type GR, but required approx. 7 and 23 times greater hormone concentrations, respectively. In contrast, double mutant LL541/2GG (leucines changed to glycines) could not activate transcription even at 10 microM dexamethasone or deacylcortivazol, while E543A (glutamic acid to alanine) was functionally indistinguishable from wild type GR. GR mutants LL541/2GG and V549G had reduced abilities to bind covalently to affinity label dexamethasone 21-mesylate. The partially and fully functional mutant GR species had no deficiency in transcriptional transactivation activity in the presence of saturating concentrations of agonist.

Affinity Labels↗