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PCR expression mutagenesis: a high-throughput mutation assay applied to the glucocorticoid receptor ligand-binding domain
BBRC Biochemical and Biophysical Research Communications 321 (2004) 893–899 www.elsevier.com/locate/ybbrc
PCR expression mutagenesis: a high-throughput mutation assay applied to the glucocorticoid receptor ligand-binding domain Jun Chen *, John A. Blackford Jr., S. Stoney Simons Jr. * Steroid Hormones Section, NIDDK/LMCB, National Institutes of Health, Bethesda, MD, USA Received 25 June 2004 Available online 28 July 2004
Abstract Glucocorticoid receptors (GRs) are extensively studied members of the steroid hormone receptor superfamily that regulate the transcription rates of numerous genes. Notwithstanding, the role of each GR amino acid in the various steps of transactivation is still unknown. A recent report shows that linear DNA has the same capacity as super-helical plasmid DNA for gene expression in transient transfection assays. Based on this observation, we describe a high-throughput assay to analyze a large set of alanine point mutations that are introduced by two rounds of PCR. The PCR products are then directly transfected into cells. This PCR expression mutagenesis (PEM) technique is used to identify several new residues of the GR ligand binding domain that influence ligand binding and/or transactivation. PEM thus provides a quick method for screening large quantities of mutant proteins. In combination with automation, PEM provides a more rapid and efficient tool for probing the role of each amino acid in the biological functions of a given protein. Published by Elsevier Inc. Keywords: High-throughput point mutation analysis; PCR mutagenesis; Glucocorticoid receptors; Ligand binding domain; Transactivation activity; Ligand binding activity
Glucocorticoid receptors (GRs) are members of the superfamily of steroid/nuclear receptors. These receptors are versatile transcription factors that regulate the expression of a large number of genes that are required for the proper development, differentiation, and homeostasis of vertebrates. GRs are typical of the steroid receptors, which contain a transactivation domain in both the N- and C-terminal regions of the protein (called AF1 and AF2, respectively), a DNA binding domain (DBD) in the middle, and a ligand binding domain (LDB) in the C-terminal half of the receptor that includes the AF2 domain. After binding to biologically ac* Corresponding author. J. Chen, Pharmacology Department, C2040, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814, USA. Fax: 1-301-402-3572 (S.S. Simons Jr.). E-mail addresses: [email protected] (J. Chen), [email protected]. gov (S.S. Simons).
0006-291X/$ - see front matter. Published by Elsevier Inc. doi:10.1016/j.bbrc.2004.07.047
tive DNA sequences called hormone response elements (HREs), homo- and/or hetero-dimeric forms of the receptors interact with a variety of factors, including coactivators, corepressors, and components of the transcriptional machinery to alter the rates of transcription of target genes and eventually the levels of the translated gene products. The domains required for the above numerous biological functions are distributed throughout each steroid receptor protein, which has an average size of about 500 amino acids. The LBD is usually about 250 amino acids long and is particularly rich in functionally active domains. For example, the GR LBD encodes sequences that are important for receptor dimerization [1,2] and the binding of other proteins, such as hsp90, coactivators [1], and corepressors, in addition to the binding of specific ligands and the expression of transactivation activity [3,4]. Not surprisingly, the sequences for several of
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J. Chen et al. / Biochemical and Biophysical Research Communications 321 (2004) 893–899
these domains overlap, thus making it difficult to dissect out the important features of each region with the available methodologies. Deletion mutations usually affect multiple activities and are of limited use. Genetic screens are very informative but do not allow one to specify which residue is to be mutated. Site-directed point mutations are the most informative. However, the large size of the LBDs, coupled with the variety of amino acids that can be present at each position and the length of time required to prepare the mutant protein plasmids, has severely limited the number of mutations that have been examined. These restrictions have precluded a comprehensive examination of the LBD of any steroid receptor, not to mention the entire protein. A recent report [5] indicated to us that one could screen a large collection of GR point mutations simply by analyzing the biological properties of linear cDNA plasmids that are prepared by PCR. This is because linear DNA was shown to have the same capacity as plasmid DNA in terms of gene expression in transfection assays. Thus, the relatively time-consuming steps of constructing, cloning, purifying, and sequencing plasmids containing the cDNA of each mutant protein could be eliminated. The present study documents the reduction to practice of this prediction, thus demonstrating the feasibility of determining the biological consequences of mutating each amino acid of the GR C-terminal domain by a predictable method, which we call PCR expression mutagenesis (PEM). Incorporating PEM in a high-throughput procedure, we identify several new amino acid residues of GR that appear to be involved in ligand binding and/or transactivation.
of Renilla TK reporter (Promega) as an internal control and other plasmids or PCR products as indicated, and adjusted to a total of 0.3 lg/plate with herring sperm DNA. After incubating the cells at 37 C for 5 h, the transfection mixture was replaced with normal medium. The cells were incubated at 37 C overnight before being induced with the appropriate steroid for 24 h. The cells were lysed and assayed for reporter gene activity using the luciferase assay reagent according to the manufacturerÕs instructions (Promega). Luciferase activity were measured in an EG&G Berthhold luminometer (Microlumat LB 96 P) and normalized to Renilla expression to correct for differences in transfection efficiency. Western blotting. SDS–PAGE gels, transfer of the proteins to nitrocellulose, Western blotting (dilutions = 1:10,000 for anti-VP16, 1:10,000 for anti-Gal), and detection of the bands by enhanced chemiluminescence was performed as reported [8].
Results Use of PCR expression mutagenesis in high-throughput assays The critical, time-saving aspects of PCR expression mutagenesis (PEM) for use in saturation mutagenesis assays are shown in Fig. 1. Primers with point mutations (arrows labeled primers 3 and 4, with ‘‘X’’ indicating the position of the point mutation) are paired with wild type primers (1 and 2) to generate oligos 1–4 and 3–2, respectively, by PCR. The products are gel purified, mixed, and used as templates for a second round of PCR with primers 1 and 2 to generate a larger fragment containing a promoter and the fusion protein with the desired mu-
Materials and methods Unless otherwise indicated, all operations were performed at 0 C. Chemicals and plasmids. The following chemicals were purchased from the indicated sources: prestained molecular weight markers, LipofectAMINE plus, herring sperm DNA, and oligonucleotides, Invitrogen (Carlsbad, CA); acrylamide and bisacrylamide, National Diagnostics (Atlanta, GA); restriction enzymes and DNA polymerase, New England Biolabs (Beverly, MA), Invitrogen, and Promega; and FR-LUC reporter, Santa Cruz Biotechnology, (Santa Cruz, CA). VP16/GR407C [6] and GAL/GR525C [7] are described elsewhere. PCR amplification. For the first round of PCR, 20–100 ng of template DNA with 45 ll PCR SuperMix (Invitrogen) and 70 pmol of each primer was brought up to a total volume of 50 ll with water. PCR was performed according to the manufacturerÕs recommendations with an annealing temperature of 51.7 C. The PCR products were gel purified using Qaigen Gel Extraction kit and used as the template for the second round of PCR under the same conditions (20–50 ng of each template, 70 pmol of each primer, 45 ll PCR SuperMix, and 35 cycles with annealing temperature at 51.7 C). Transient transfection assays. Cos-7 and CV-1 cells were grown in 24-well dishes in DulbeccoÕs modified EagleÕs medium (Invitrogen) supplemented with 10% fetal calf serum. Cells were seeded 1 day before transfection at a density of 2 · 104 for CV-1 cells and 2 · 105 for Cos-7 cells. Cells were transfected using 1 ll of LipofectAMINE Plus reagent and 2 ll of LipofectAMINE with 50 ng of reporter plasmid, plus 10 ng
Fig. 1. Schematic flow diagram of the preparation of mutant GRs by PEM. Various domains in a plasmid template [promoter, VP16 activation domain (VP16AD), GAL4 DNA binding domain (GAL4DBD), GR, and intron/poly(A) sequence] are indicated along with the position of selected primers. The ‘‘X’’ in the primer and GR sequence indicates a point mutation. See text for details.
J. Chen et al. / Biochemical and Biophysical Research Communications 321 (2004) 893–899
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Fig. 2. Effect of physical state of transfected reporter gene on the efficiency of transactivation. Different amounts of VP16/GR407 DNA in a circular plasmid or as a linear PCR product without or with a poly(A) tail (shaded box) and without or with prior purification (purÕd) were transiently transfected into CV-1 cells along with the GREtkLUC reporter. The medium was adjusted to 1% EtOH ±1 lM Dex and the amount of luciferase activity (normalized to Renilla expression to adjust for transfection efficiency) was determined as described in Materials and methods and plotted either as relative luciferase activity or fold induction by 1 lM Dex.
tation. The expression of functional protein from transiently transfected plasmid vs. PCR product was examined in the context of the known ability of the VP16/GR407C chimera to transactivate a GREtkLUC reporter after transient transfection in CV-1 cells and treatment with the agonist dexamethasone (Dex) [6]. The GR fragment of 407–795 (=407C) contains the DNA binding domain necessary to bind to the DNA sequence of the GRE while potent transactivation is conveyed by the presence of the VP16 activation domain. At the same time, we examined the effect of including the
poly(A) termination signal in the PCR product, of purifying the PCR product, and of varying the amount of PCR product that is transfected. As shown in Fig. 2, 100 ng of crude PCR product gives about the same fold induction as the purified PCR product and the plasmid DNA regardless of whether or not the poly(A) tail is present. The total activity is lower but, because the basal activity in the absence of steroid is also lower, the fold induction, and thus the sensitivity of the assay, is the same for 100 ng of either crude linear PCR product without poly(A) tail or super-helical plasmid DNA. There-
Fig. 3. Design of assays for properties of mutant GRs. (A) Cartoons of GR and TIF2 constructs used in assays. Location of helix 5 and helix 12 is shown within the GR LBD of the full-length GR, VP16/GR407C, and GAL/GR525C (number indicates the amino terminal residue of the GR fragment, which extends to the carboxyl terminus, or ‘‘C’’). Various other domains of GR, and the regions of VP16 (wavy lines), GAL (shaded), and coactivator TIF2 are shown, with the numbers above the cartoon indicating the amino acid position of each boundary. (B) Flow sheet for identifying the biological consequence of each GR point mutant. See text for details.
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fore, we used 100 ng of crude PCR product without poly(A) tail in all future experiments. Approach for saturation mutagenesis analysis of segments of GR LBD To test the utility of PEM for rapidly analyzing the role of a large number of amino acids in the biological activities of a protein, we decided to use PEM to examine all of the residues in helices 5 and 12 of the rat GR. These helices are of particular interest due to their contributions to the conserved hydrophobic core of the LBD, to the transactivation activity of the AF2 domain of GRs, to intramolecular contacts, and to the binding specificity of steroids, of coactivators like TIF2, and of corepressors (Fig. 3A) [1,2,6,9–13]. We employed the approach of alanine-scanning [14] to examine role of each amino acid in helices 5 and 12. Therefore, we used PEM to convert each amino acid to alanine except for residues 623, 625, and 772, which exist as alanine in the wild type rat GR. The two previously characterized constructs of VP16/GR407C [6] and GAL/GR525C [7,15,16] in Fig. 3A were selected to separate the potential effect of each mutation on the variety
Fig. 4. Transactivation activity of GAL/GR525C mutants with different concentrations of Dex. Triplicate samples of CV-1 cells are transiently transfected with the GAL-responsive FRLuc reporter and 0.1 lg of unpurified PCR product of GAL/GR525C with the indicated point mutations and then treated with 1 or 10 lM Dex. The fold induction was determined and plotted as in Fig. 2.
of LBD activities in the ordered series of experiments outlined in Fig. 3B in which possible reasons for inactivity in the first assay are sequentially eliminated. The ability of GAL/GR525C to display steroid-dependent induction of the FRLuc reporter allows us to assess the effects of point mutations on steroid binding affinity and transactivation activity independent of DNA binding. Unresponsive mutants in this assay are then systematically examined for defects in steroid binding affinity, intrinsic transactivation activity, coactivator-induced transactivation, and protein expression, each of which could afford a relatively inactive GR. Analysis of GR helix 5 and 12 mutations As set forth in Fig. 3B, the first assays of the mutant GRs are in the context of GAL/GR525C at two concentrations of Dex: 1 and 10 lM (Fig. 4). For the purposes of a rapid screening assay, we classify all mutations that show P50% of the activity of the wild type as having negligible effects. By this criterion, the ability of approximately half of the mutant receptors to display close to wild type induction capacity with normally saturating Dex concentrations of 1 lM argues that changing these amino acids to alanine does not seriously alter either ligand binding or intrinsic transactivation activity. The activity of F624A and L626A, relative to the wild type
Fig. 5. Interaction of VP16/GR407C mutants with GAL/TIF2.4 in a mammalian two-hybrid assay. Triplicate samples of CV-1 cells were transiently transfected with FRLuc and 0.1 lg of unpurified PCR product of VP16/GR407C with the indicated point mutations and then treated with 1 lM Dex. The fold induction was determined and plotted as in Fig. 2.
J. Chen et al. / Biochemical and Biophysical Research Communications 321 (2004) 893–899
GR525C, was somewhat higher in the presence of 10 lM Dex than 1 lM Dex, suggesting that these mutations reduce the affinity of GR for Dex. The 11 mutations that produce <50% of wild type GR induction activity with 10 lM Dex in Fig. 4 could reflect a loss of intrinsic transactivation activity, a reduced capacity to associate with coactivators such as TIF2, an inability to bind steroid, or the absence of protein expression. A mammalian two-hybrid assay with GAL/ TIF2.4 and VP16/GR407C is used to examine the association of GR with the coactivator TIF2 [17]. The P50% activity in Fig. 5 for Dex induction of the VP16/ GR407 constructs with GAL/TIF2.4 coupled with a very low activity in Fig. 4 for W618A, L621A, I779A, and possibly L626A and E773A, suggests that the ability of these mutant GRs to interact with coactivators is normal but their intrinsic transactivation activity is low. Conversely, the low fold induction by Dex in Fig. 5 for M619A, W628A, R629A, M770A, L771A, E773A, I774A, and I775A suggests that their negligible activation in Fig. 4 could be due to a reduced affinity of these mutants for coactivators. Interestingly, F624A appears to affect the affinity of both steroid (Fig. 4) and coactivator (Fig. 5). To obtain independent evidence for these conclusions regarding reduced coactivator affinity, we examined the activity of VP16/GR407C with a GREtkLUC reporter (Fig. 6). Good activity here but not in Fig. 5 indicates that the mutant receptor binds steroid and can cause steroid-dependent transactivation but only with the help of a fused heterologous transactiva-
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Fig. 7. Levels of protein expression of transiently transfected GAL/ GR525C mutants in Cos-7 cells.
Table 1 Summary of properties of mutant GRs examined by PEM Property
Mutation
Wild type GR
617, 620, 622, 627 769, 776, 777, 778, 780 624, 626
Mutant has lower affinity for agonist steroid Mutant has reduced intrinsic transactivation activity Mutant has reduced affinity for coactivators Mutant binds steroid poorly, if at all Mutant protein is not expressed or is not stable
618, 621, 626 773, 779 624, 628 770, 773, 774 619, 629 771, 775 None
tion domain. By this criterion, we conclude that W628A, M770A, I774A, and probably F624A and E773A have significantly lowered affinity for coactivators. The remaining mutants to be accounted for are M619A, R629A, L771A, and I775A. Western blots show that each of these mutants is expressed at near wild type levels (Fig. 7). Therefore, as diagramed in Fig. 3B, we conclude that the major consequence of the alanine mutation at these four positions is to greatly lower the affinity of Dex for the receptor. A summary of all of the consequences of the mutations examined is given in Table 1.
Discussion
Fig. 6. Transactivation activity of VP16/GR407C mutants upon binding to a GRE-regulated reporter. Triplicate samples of CV-1 cells were transiently transfected with GREtkLUC and 0.1 lg of unpurified PCR product of VP16/GR407C with the indicated point mutations and then treated with 1 lM Dex. The fold induction was determined and plotted as in Fig. 2.
We describe here a method for rapidly determining the functional consequences of many different single point mutations in a biologically active protein. The major time-saving element of this assay is that PCR products, instead of cloned plasmids, can be used in transiently transfected cells. For this reason, we call this procedure PCR expression mutagenesis or PEM. It should be appreciated that the presence of PCR-generated mutations can be safely ignored because their very low abundance in the bulk PCR product will not alter the properties of the much larger amount of desired point mutant. The specific biological assays of the PCR products containing individual point mutants will be dictated by the protein being examined. In the present case, our assays were selected to examine several
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of the known properties of the rat GR LBD. The validity of the PEM methodology is demonstrated by the fact that the mutation of several residues in helices 5 and 12 of GR affords phenotypes that are identical to those of previously characterized mutations in the same position [3,4]. Given the number of biological functions that are mediated by the GR LBD, it is not surprising that some mutations altered more than one response, even in the context of truncated GRs. Thus, the F624A mutation slightly reduces the affinity for Dex and decreases coactivator affinity. Similarly L626A affects both Dex affinity and the intrinsic transactivation activity of GR (Table 1). The recent publication of X-ray structures of ligandbound GR LBDs [1,2] allows a structure–function comparison of the various mutations that we have described. Using the coordinates of Bledsoe et al. [1], we find that all of the mutations that affect steroid binding are in vicinity of the bound steroid (data not shown). Mutations affecting transactivation are both near the steroid and on the surface of the receptor LBD (data not shown and Fig. 8). Interestingly, of the four residues that influence transactivation and are on the surface of the GR LBD, only three (M770, E773, and I774 of rat GR) interact with the TIF2 peptide [1]. The location of I779, which affects intrinsic transactivation, does not contact the TIF2 peptide and suggests that either other regions of TIF2 or a different molecule might interact here. The fact that I779 is identified in the present study as being involved in coactivator-independent intrinsic transactivation means that the association with a molecule other than a coactivator may be the dominant role of this residue. It should be noted that the other surface amino acid implicated in intrinsic transactivation (E773) is near I779 and could bind to the same putative non-coactivator molecule, which could then contact TIF2.
One caveat of the assays that we have used, but not of the PEM method itself, is that they involve truncated instead of full-length GRs. This is an intrinsic limitation of any assay that uses fragments of a protein to determine the effects of mutations on one particular property in the absence of the many other properties of the full-length protein. However, such results with truncated proteins are very useful in guiding the interpretation of assays involving the same mutation in the context of the full-length protein. Also, it should be noted that our conclusions from experiments with truncated receptors are the same as the published results from full-length GRs [3,4]. A second caveat is that the effects of each mutation have been classified on the basis of qualitative responses. If the mutant protein has P50% of the wild type GR response, then we have concluded that there is no significant effect of the mutation. While this classification is suitable during an initial screening, more detailed studies in the context of the full-length protein are clearly needed to determine whether such relatively minor changes might not have more significant consequences on whole cell biological activities. Despite these obvious limitations, it appears that the PEM method represents a useful new approach for reducing the time that is needed to analyze the consequences of saturation point mutagenesis on the biological properties of a given protein in the above and similar screening assays [18,19].
Acknowledgment We thank Greti Aguilera (NICHD, NIH) for critically reviewing the manuscript.
References
Fig. 8. Position of selected point mutations that affect GR transactivation activity in the published space-filling model of GR-LBD complex with TIF2 peptide [1]. Color code for residues: 770, purple; 773, cyan; 774, dark blue; 779, red; and TIF2, orange. See text for details.
[1] R.K. Bledsoe, V.G. Montana, T.B. Stanley, C.J. Delves, C.J. Apolito, D.D. McKee, T.G. Consler, D.J. Parks, E.L. Stewart, T.M. Willson, M.H. Lambert, J.T. Moore, K.H. Pearce, H.E. Xu, Cell 110 (2002) 93–105. [2] B. Kauppi, C. Jakob, M. Farnegardh, J. Yang, H. Ahola, M. Alarcon, K. Calles, O. Engstrom, J. Harlan, S. Muchmore, A.K. Ramqvist, S. Thorell, L. Ohman, J. Greer, J.A. Gustafsson, J. Carlstedt-Duke, M. Carlquist, J. Biol. Chem. 278 (2003) 22748– 22754. [3] S.S. Simons Jr., Vitamins and Hormones 48 (1994) 49–130. [4] S.S. Simons Jr., in: L.P. Freedman (Ed.), The Molecular Biology of Steroid and Nuclear Hormone Receptors, 1998, pp. 35–104. [5] K.F. Sykes, S.A. Johnston, Nat. Biotechnol. 17 (1999) 355–359. [6] Q. Wang, J.A. Blackford Jr., L.-N. Song, Y. Huang, S.S. Simons Jr., Mol. Endocrinol. 18 (2004) 1376–1395. [7] S. Kaul, J.A. Blackford Jr., S. Cho, S.S. Simons Jr., J. Biol. Chem. 277 (2002) 12541–12549. [8] J. Chen, Y. He, S.S. Simons Jr., Biochemistry 43 (2004) 245–255. [9] J.-M. Wurtz, W. Bourguet, J.-P. Renaud, V. Vivat, P. Chambon, D. Moras, H. Gronemeyer, Nat. Struct. Biol. 3 (1996) 87–94.
J. Chen et al. / Biochemical and Biophysical Research Communications 321 (2004) 893–899 [10] A. Marimuthu, W. Feng, T. Tagami, H. Nguyen, J.L. Jameson, R.J. Fletterick, J.D. Baxter, B.L. West, Mol. Endocrinol. 16 (2002) 271–286. [11] V. Perissi, L.M. Staszewski, E.M. McInerney, R. Kurokawa, A. Krones, D.W. Rose, M.H. Lambert, M.V. Milburn, C.K. Glass, M.G. Rosenfeld, Genes Dev. 13 (1999) 3198–3208. [12] X. Hu, M.A. Lazar, Nature 402 (1999) 93–96. [13] X. Hu, Y. Li, M.A. Lazar, Mol. Cell. Biol. 21 (2001) 1747–1758. [14] J.A. Wells, Methods Enzymol. 202 (1991) 390–411.
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[15] S. Cho, J.A. Blackford, Jr., B.L. Kagan, S.S. Simons, Jr. Mol. Endo. (2004) in press. [16] S. Cho, J.A. Blackford, Jr., S.S. Simons, Jr. (submitted). [17] Y. He, D. Szapary, S.S. Simons Jr., J. Biol. Chem. 277 (2002) 49256–49266. [18] P.J. Carter, G. Winter, A.J. Wilkinson, A.R. Fersht, Cell 38 (1984) 835–840. [19] A.I. Shulman, C. Larson, D.J. Mangelsdorf, R. Ranganathan, Cell 116 (2004) 417–429.
PCR expression mutagenesis: a high-throughput mutation assay applied to the glucocorticoid receptor ligand-binding domain Jun Chen *, John A. Blackford Jr., S. Stoney Simons Jr. * Steroid Hormones Section, NIDDK/LMCB, National Institutes of Health, Bethesda, MD, USA Received 25 June 2004 Available online 28 July 2004
Abstract Glucocorticoid receptors (GRs) are extensively studied members of the steroid hormone receptor superfamily that regulate the transcription rates of numerous genes. Notwithstanding, the role of each GR amino acid in the various steps of transactivation is still unknown. A recent report shows that linear DNA has the same capacity as super-helical plasmid DNA for gene expression in transient transfection assays. Based on this observation, we describe a high-throughput assay to analyze a large set of alanine point mutations that are introduced by two rounds of PCR. The PCR products are then directly transfected into cells. This PCR expression mutagenesis (PEM) technique is used to identify several new residues of the GR ligand binding domain that influence ligand binding and/or transactivation. PEM thus provides a quick method for screening large quantities of mutant proteins. In combination with automation, PEM provides a more rapid and efficient tool for probing the role of each amino acid in the biological functions of a given protein. Published by Elsevier Inc. Keywords: High-throughput point mutation analysis; PCR mutagenesis; Glucocorticoid receptors; Ligand binding domain; Transactivation activity; Ligand binding activity
Glucocorticoid receptors (GRs) are members of the superfamily of steroid/nuclear receptors. These receptors are versatile transcription factors that regulate the expression of a large number of genes that are required for the proper development, differentiation, and homeostasis of vertebrates. GRs are typical of the steroid receptors, which contain a transactivation domain in both the N- and C-terminal regions of the protein (called AF1 and AF2, respectively), a DNA binding domain (DBD) in the middle, and a ligand binding domain (LDB) in the C-terminal half of the receptor that includes the AF2 domain. After binding to biologically ac* Corresponding author. J. Chen, Pharmacology Department, C2040, Uniformed Services University of the Health Sciences, 4301 Jones Bridge Road, Bethesda, MD 20814, USA. Fax: 1-301-402-3572 (S.S. Simons Jr.). E-mail addresses: [email protected] (J. Chen), [email protected]. gov (S.S. Simons).
0006-291X/$ - see front matter. Published by Elsevier Inc. doi:10.1016/j.bbrc.2004.07.047
tive DNA sequences called hormone response elements (HREs), homo- and/or hetero-dimeric forms of the receptors interact with a variety of factors, including coactivators, corepressors, and components of the transcriptional machinery to alter the rates of transcription of target genes and eventually the levels of the translated gene products. The domains required for the above numerous biological functions are distributed throughout each steroid receptor protein, which has an average size of about 500 amino acids. The LBD is usually about 250 amino acids long and is particularly rich in functionally active domains. For example, the GR LBD encodes sequences that are important for receptor dimerization [1,2] and the binding of other proteins, such as hsp90, coactivators [1], and corepressors, in addition to the binding of specific ligands and the expression of transactivation activity [3,4]. Not surprisingly, the sequences for several of
894
J. Chen et al. / Biochemical and Biophysical Research Communications 321 (2004) 893–899
these domains overlap, thus making it difficult to dissect out the important features of each region with the available methodologies. Deletion mutations usually affect multiple activities and are of limited use. Genetic screens are very informative but do not allow one to specify which residue is to be mutated. Site-directed point mutations are the most informative. However, the large size of the LBDs, coupled with the variety of amino acids that can be present at each position and the length of time required to prepare the mutant protein plasmids, has severely limited the number of mutations that have been examined. These restrictions have precluded a comprehensive examination of the LBD of any steroid receptor, not to mention the entire protein. A recent report [5] indicated to us that one could screen a large collection of GR point mutations simply by analyzing the biological properties of linear cDNA plasmids that are prepared by PCR. This is because linear DNA was shown to have the same capacity as plasmid DNA in terms of gene expression in transfection assays. Thus, the relatively time-consuming steps of constructing, cloning, purifying, and sequencing plasmids containing the cDNA of each mutant protein could be eliminated. The present study documents the reduction to practice of this prediction, thus demonstrating the feasibility of determining the biological consequences of mutating each amino acid of the GR C-terminal domain by a predictable method, which we call PCR expression mutagenesis (PEM). Incorporating PEM in a high-throughput procedure, we identify several new amino acid residues of GR that appear to be involved in ligand binding and/or transactivation.
of Renilla TK reporter (Promega) as an internal control and other plasmids or PCR products as indicated, and adjusted to a total of 0.3 lg/plate with herring sperm DNA. After incubating the cells at 37 C for 5 h, the transfection mixture was replaced with normal medium. The cells were incubated at 37 C overnight before being induced with the appropriate steroid for 24 h. The cells were lysed and assayed for reporter gene activity using the luciferase assay reagent according to the manufacturerÕs instructions (Promega). Luciferase activity were measured in an EG&G Berthhold luminometer (Microlumat LB 96 P) and normalized to Renilla expression to correct for differences in transfection efficiency. Western blotting. SDS–PAGE gels, transfer of the proteins to nitrocellulose, Western blotting (dilutions = 1:10,000 for anti-VP16, 1:10,000 for anti-Gal), and detection of the bands by enhanced chemiluminescence was performed as reported [8].
Results Use of PCR expression mutagenesis in high-throughput assays The critical, time-saving aspects of PCR expression mutagenesis (PEM) for use in saturation mutagenesis assays are shown in Fig. 1. Primers with point mutations (arrows labeled primers 3 and 4, with ‘‘X’’ indicating the position of the point mutation) are paired with wild type primers (1 and 2) to generate oligos 1–4 and 3–2, respectively, by PCR. The products are gel purified, mixed, and used as templates for a second round of PCR with primers 1 and 2 to generate a larger fragment containing a promoter and the fusion protein with the desired mu-
Materials and methods Unless otherwise indicated, all operations were performed at 0 C. Chemicals and plasmids. The following chemicals were purchased from the indicated sources: prestained molecular weight markers, LipofectAMINE plus, herring sperm DNA, and oligonucleotides, Invitrogen (Carlsbad, CA); acrylamide and bisacrylamide, National Diagnostics (Atlanta, GA); restriction enzymes and DNA polymerase, New England Biolabs (Beverly, MA), Invitrogen, and Promega; and FR-LUC reporter, Santa Cruz Biotechnology, (Santa Cruz, CA). VP16/GR407C [6] and GAL/GR525C [7] are described elsewhere. PCR amplification. For the first round of PCR, 20–100 ng of template DNA with 45 ll PCR SuperMix (Invitrogen) and 70 pmol of each primer was brought up to a total volume of 50 ll with water. PCR was performed according to the manufacturerÕs recommendations with an annealing temperature of 51.7 C. The PCR products were gel purified using Qaigen Gel Extraction kit and used as the template for the second round of PCR under the same conditions (20–50 ng of each template, 70 pmol of each primer, 45 ll PCR SuperMix, and 35 cycles with annealing temperature at 51.7 C). Transient transfection assays. Cos-7 and CV-1 cells were grown in 24-well dishes in DulbeccoÕs modified EagleÕs medium (Invitrogen) supplemented with 10% fetal calf serum. Cells were seeded 1 day before transfection at a density of 2 · 104 for CV-1 cells and 2 · 105 for Cos-7 cells. Cells were transfected using 1 ll of LipofectAMINE Plus reagent and 2 ll of LipofectAMINE with 50 ng of reporter plasmid, plus 10 ng
Fig. 1. Schematic flow diagram of the preparation of mutant GRs by PEM. Various domains in a plasmid template [promoter, VP16 activation domain (VP16AD), GAL4 DNA binding domain (GAL4DBD), GR, and intron/poly(A) sequence] are indicated along with the position of selected primers. The ‘‘X’’ in the primer and GR sequence indicates a point mutation. See text for details.
J. Chen et al. / Biochemical and Biophysical Research Communications 321 (2004) 893–899
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Fig. 2. Effect of physical state of transfected reporter gene on the efficiency of transactivation. Different amounts of VP16/GR407 DNA in a circular plasmid or as a linear PCR product without or with a poly(A) tail (shaded box) and without or with prior purification (purÕd) were transiently transfected into CV-1 cells along with the GREtkLUC reporter. The medium was adjusted to 1% EtOH ±1 lM Dex and the amount of luciferase activity (normalized to Renilla expression to adjust for transfection efficiency) was determined as described in Materials and methods and plotted either as relative luciferase activity or fold induction by 1 lM Dex.
tation. The expression of functional protein from transiently transfected plasmid vs. PCR product was examined in the context of the known ability of the VP16/GR407C chimera to transactivate a GREtkLUC reporter after transient transfection in CV-1 cells and treatment with the agonist dexamethasone (Dex) [6]. The GR fragment of 407–795 (=407C) contains the DNA binding domain necessary to bind to the DNA sequence of the GRE while potent transactivation is conveyed by the presence of the VP16 activation domain. At the same time, we examined the effect of including the
poly(A) termination signal in the PCR product, of purifying the PCR product, and of varying the amount of PCR product that is transfected. As shown in Fig. 2, 100 ng of crude PCR product gives about the same fold induction as the purified PCR product and the plasmid DNA regardless of whether or not the poly(A) tail is present. The total activity is lower but, because the basal activity in the absence of steroid is also lower, the fold induction, and thus the sensitivity of the assay, is the same for 100 ng of either crude linear PCR product without poly(A) tail or super-helical plasmid DNA. There-
Fig. 3. Design of assays for properties of mutant GRs. (A) Cartoons of GR and TIF2 constructs used in assays. Location of helix 5 and helix 12 is shown within the GR LBD of the full-length GR, VP16/GR407C, and GAL/GR525C (number indicates the amino terminal residue of the GR fragment, which extends to the carboxyl terminus, or ‘‘C’’). Various other domains of GR, and the regions of VP16 (wavy lines), GAL (shaded), and coactivator TIF2 are shown, with the numbers above the cartoon indicating the amino acid position of each boundary. (B) Flow sheet for identifying the biological consequence of each GR point mutant. See text for details.
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fore, we used 100 ng of crude PCR product without poly(A) tail in all future experiments. Approach for saturation mutagenesis analysis of segments of GR LBD To test the utility of PEM for rapidly analyzing the role of a large number of amino acids in the biological activities of a protein, we decided to use PEM to examine all of the residues in helices 5 and 12 of the rat GR. These helices are of particular interest due to their contributions to the conserved hydrophobic core of the LBD, to the transactivation activity of the AF2 domain of GRs, to intramolecular contacts, and to the binding specificity of steroids, of coactivators like TIF2, and of corepressors (Fig. 3A) [1,2,6,9–13]. We employed the approach of alanine-scanning [14] to examine role of each amino acid in helices 5 and 12. Therefore, we used PEM to convert each amino acid to alanine except for residues 623, 625, and 772, which exist as alanine in the wild type rat GR. The two previously characterized constructs of VP16/GR407C [6] and GAL/GR525C [7,15,16] in Fig. 3A were selected to separate the potential effect of each mutation on the variety
Fig. 4. Transactivation activity of GAL/GR525C mutants with different concentrations of Dex. Triplicate samples of CV-1 cells are transiently transfected with the GAL-responsive FRLuc reporter and 0.1 lg of unpurified PCR product of GAL/GR525C with the indicated point mutations and then treated with 1 or 10 lM Dex. The fold induction was determined and plotted as in Fig. 2.
of LBD activities in the ordered series of experiments outlined in Fig. 3B in which possible reasons for inactivity in the first assay are sequentially eliminated. The ability of GAL/GR525C to display steroid-dependent induction of the FRLuc reporter allows us to assess the effects of point mutations on steroid binding affinity and transactivation activity independent of DNA binding. Unresponsive mutants in this assay are then systematically examined for defects in steroid binding affinity, intrinsic transactivation activity, coactivator-induced transactivation, and protein expression, each of which could afford a relatively inactive GR. Analysis of GR helix 5 and 12 mutations As set forth in Fig. 3B, the first assays of the mutant GRs are in the context of GAL/GR525C at two concentrations of Dex: 1 and 10 lM (Fig. 4). For the purposes of a rapid screening assay, we classify all mutations that show P50% of the activity of the wild type as having negligible effects. By this criterion, the ability of approximately half of the mutant receptors to display close to wild type induction capacity with normally saturating Dex concentrations of 1 lM argues that changing these amino acids to alanine does not seriously alter either ligand binding or intrinsic transactivation activity. The activity of F624A and L626A, relative to the wild type
Fig. 5. Interaction of VP16/GR407C mutants with GAL/TIF2.4 in a mammalian two-hybrid assay. Triplicate samples of CV-1 cells were transiently transfected with FRLuc and 0.1 lg of unpurified PCR product of VP16/GR407C with the indicated point mutations and then treated with 1 lM Dex. The fold induction was determined and plotted as in Fig. 2.
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GR525C, was somewhat higher in the presence of 10 lM Dex than 1 lM Dex, suggesting that these mutations reduce the affinity of GR for Dex. The 11 mutations that produce <50% of wild type GR induction activity with 10 lM Dex in Fig. 4 could reflect a loss of intrinsic transactivation activity, a reduced capacity to associate with coactivators such as TIF2, an inability to bind steroid, or the absence of protein expression. A mammalian two-hybrid assay with GAL/ TIF2.4 and VP16/GR407C is used to examine the association of GR with the coactivator TIF2 [17]. The P50% activity in Fig. 5 for Dex induction of the VP16/ GR407 constructs with GAL/TIF2.4 coupled with a very low activity in Fig. 4 for W618A, L621A, I779A, and possibly L626A and E773A, suggests that the ability of these mutant GRs to interact with coactivators is normal but their intrinsic transactivation activity is low. Conversely, the low fold induction by Dex in Fig. 5 for M619A, W628A, R629A, M770A, L771A, E773A, I774A, and I775A suggests that their negligible activation in Fig. 4 could be due to a reduced affinity of these mutants for coactivators. Interestingly, F624A appears to affect the affinity of both steroid (Fig. 4) and coactivator (Fig. 5). To obtain independent evidence for these conclusions regarding reduced coactivator affinity, we examined the activity of VP16/GR407C with a GREtkLUC reporter (Fig. 6). Good activity here but not in Fig. 5 indicates that the mutant receptor binds steroid and can cause steroid-dependent transactivation but only with the help of a fused heterologous transactiva-
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Fig. 7. Levels of protein expression of transiently transfected GAL/ GR525C mutants in Cos-7 cells.
Table 1 Summary of properties of mutant GRs examined by PEM Property
Mutation
Wild type GR
617, 620, 622, 627 769, 776, 777, 778, 780 624, 626
Mutant has lower affinity for agonist steroid Mutant has reduced intrinsic transactivation activity Mutant has reduced affinity for coactivators Mutant binds steroid poorly, if at all Mutant protein is not expressed or is not stable
618, 621, 626 773, 779 624, 628 770, 773, 774 619, 629 771, 775 None
tion domain. By this criterion, we conclude that W628A, M770A, I774A, and probably F624A and E773A have significantly lowered affinity for coactivators. The remaining mutants to be accounted for are M619A, R629A, L771A, and I775A. Western blots show that each of these mutants is expressed at near wild type levels (Fig. 7). Therefore, as diagramed in Fig. 3B, we conclude that the major consequence of the alanine mutation at these four positions is to greatly lower the affinity of Dex for the receptor. A summary of all of the consequences of the mutations examined is given in Table 1.
Discussion
Fig. 6. Transactivation activity of VP16/GR407C mutants upon binding to a GRE-regulated reporter. Triplicate samples of CV-1 cells were transiently transfected with GREtkLUC and 0.1 lg of unpurified PCR product of VP16/GR407C with the indicated point mutations and then treated with 1 lM Dex. The fold induction was determined and plotted as in Fig. 2.
We describe here a method for rapidly determining the functional consequences of many different single point mutations in a biologically active protein. The major time-saving element of this assay is that PCR products, instead of cloned plasmids, can be used in transiently transfected cells. For this reason, we call this procedure PCR expression mutagenesis or PEM. It should be appreciated that the presence of PCR-generated mutations can be safely ignored because their very low abundance in the bulk PCR product will not alter the properties of the much larger amount of desired point mutant. The specific biological assays of the PCR products containing individual point mutants will be dictated by the protein being examined. In the present case, our assays were selected to examine several
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of the known properties of the rat GR LBD. The validity of the PEM methodology is demonstrated by the fact that the mutation of several residues in helices 5 and 12 of GR affords phenotypes that are identical to those of previously characterized mutations in the same position [3,4]. Given the number of biological functions that are mediated by the GR LBD, it is not surprising that some mutations altered more than one response, even in the context of truncated GRs. Thus, the F624A mutation slightly reduces the affinity for Dex and decreases coactivator affinity. Similarly L626A affects both Dex affinity and the intrinsic transactivation activity of GR (Table 1). The recent publication of X-ray structures of ligandbound GR LBDs [1,2] allows a structure–function comparison of the various mutations that we have described. Using the coordinates of Bledsoe et al. [1], we find that all of the mutations that affect steroid binding are in vicinity of the bound steroid (data not shown). Mutations affecting transactivation are both near the steroid and on the surface of the receptor LBD (data not shown and Fig. 8). Interestingly, of the four residues that influence transactivation and are on the surface of the GR LBD, only three (M770, E773, and I774 of rat GR) interact with the TIF2 peptide [1]. The location of I779, which affects intrinsic transactivation, does not contact the TIF2 peptide and suggests that either other regions of TIF2 or a different molecule might interact here. The fact that I779 is identified in the present study as being involved in coactivator-independent intrinsic transactivation means that the association with a molecule other than a coactivator may be the dominant role of this residue. It should be noted that the other surface amino acid implicated in intrinsic transactivation (E773) is near I779 and could bind to the same putative non-coactivator molecule, which could then contact TIF2.
One caveat of the assays that we have used, but not of the PEM method itself, is that they involve truncated instead of full-length GRs. This is an intrinsic limitation of any assay that uses fragments of a protein to determine the effects of mutations on one particular property in the absence of the many other properties of the full-length protein. However, such results with truncated proteins are very useful in guiding the interpretation of assays involving the same mutation in the context of the full-length protein. Also, it should be noted that our conclusions from experiments with truncated receptors are the same as the published results from full-length GRs [3,4]. A second caveat is that the effects of each mutation have been classified on the basis of qualitative responses. If the mutant protein has P50% of the wild type GR response, then we have concluded that there is no significant effect of the mutation. While this classification is suitable during an initial screening, more detailed studies in the context of the full-length protein are clearly needed to determine whether such relatively minor changes might not have more significant consequences on whole cell biological activities. Despite these obvious limitations, it appears that the PEM method represents a useful new approach for reducing the time that is needed to analyze the consequences of saturation point mutagenesis on the biological properties of a given protein in the above and similar screening assays [18,19].
Acknowledgment We thank Greti Aguilera (NICHD, NIH) for critically reviewing the manuscript.
References
Fig. 8. Position of selected point mutations that affect GR transactivation activity in the published space-filling model of GR-LBD complex with TIF2 peptide [1]. Color code for residues: 770, purple; 773, cyan; 774, dark blue; 779, red; and TIF2, orange. See text for details.
[1] R.K. Bledsoe, V.G. Montana, T.B. Stanley, C.J. Delves, C.J. Apolito, D.D. McKee, T.G. Consler, D.J. Parks, E.L. Stewart, T.M. Willson, M.H. Lambert, J.T. Moore, K.H. Pearce, H.E. Xu, Cell 110 (2002) 93–105. [2] B. Kauppi, C. Jakob, M. Farnegardh, J. Yang, H. Ahola, M. Alarcon, K. Calles, O. Engstrom, J. Harlan, S. Muchmore, A.K. Ramqvist, S. Thorell, L. Ohman, J. Greer, J.A. Gustafsson, J. Carlstedt-Duke, M. Carlquist, J. Biol. Chem. 278 (2003) 22748– 22754. [3] S.S. Simons Jr., Vitamins and Hormones 48 (1994) 49–130. [4] S.S. Simons Jr., in: L.P. Freedman (Ed.), The Molecular Biology of Steroid and Nuclear Hormone Receptors, 1998, pp. 35–104. [5] K.F. Sykes, S.A. Johnston, Nat. Biotechnol. 17 (1999) 355–359. [6] Q. Wang, J.A. Blackford Jr., L.-N. Song, Y. Huang, S.S. Simons Jr., Mol. Endocrinol. 18 (2004) 1376–1395. [7] S. Kaul, J.A. Blackford Jr., S. Cho, S.S. Simons Jr., J. Biol. Chem. 277 (2002) 12541–12549. [8] J. Chen, Y. He, S.S. Simons Jr., Biochemistry 43 (2004) 245–255. [9] J.-M. Wurtz, W. Bourguet, J.-P. Renaud, V. Vivat, P. Chambon, D. Moras, H. Gronemeyer, Nat. Struct. Biol. 3 (1996) 87–94.
J. Chen et al. / Biochemical and Biophysical Research Communications 321 (2004) 893–899 [10] A. Marimuthu, W. Feng, T. Tagami, H. Nguyen, J.L. Jameson, R.J. Fletterick, J.D. Baxter, B.L. West, Mol. Endocrinol. 16 (2002) 271–286. [11] V. Perissi, L.M. Staszewski, E.M. McInerney, R. Kurokawa, A. Krones, D.W. Rose, M.H. Lambert, M.V. Milburn, C.K. Glass, M.G. Rosenfeld, Genes Dev. 13 (1999) 3198–3208. [12] X. Hu, M.A. Lazar, Nature 402 (1999) 93–96. [13] X. Hu, Y. Li, M.A. Lazar, Mol. Cell. Biol. 21 (2001) 1747–1758. [14] J.A. Wells, Methods Enzymol. 202 (1991) 390–411.
899
[15] S. Cho, J.A. Blackford, Jr., B.L. Kagan, S.S. Simons, Jr. Mol. Endo. (2004) in press. [16] S. Cho, J.A. Blackford, Jr., S.S. Simons, Jr. (submitted). [17] Y. He, D. Szapary, S.S. Simons Jr., J. Biol. Chem. 277 (2002) 49256–49266. [18] P.J. Carter, G. Winter, A.J. Wilkinson, A.R. Fersht, Cell 38 (1984) 835–840. [19] A.I. Shulman, C. Larson, D.J. Mangelsdorf, R. Ranganathan, Cell 116 (2004) 417–429.