Defective Calmodulin-Mediated Nuclear Transport of the Sex-Determining Region of the Y Chromosome (SRY) in XY Sex Reversal

Helena Sim, Kieran Rimmer, Sabine Kelly, Louisa M. Ludbrook, Andrew H. A. Clayton and Vincent R. Harley

Human Molecular Genetics Laboratory (H.S., K.R., S.K., L.M.L., V.R.H.), Prince Henry’s Institute of Medical Research, Monash Medical Centre, Clayton, Melbourne, Victoria 3168, Australia; Department of Biochemistry and Molecular Biology (K.R.), University of Melbourne, Victoria 3010, Australia; and Ludwig Institute for Cancer Research (A.H.A.C.), Parkville, Victoria 3050, Australia

Address all correspondence and requests for reprints to: Vincent R. Harley, Prince Henry’s Institute of Medical Research, Level 4 Block E, Monash Medical Centre, 246 Clayton Road, Clayton, Victoria 3168, Australia. E-mail: Vincent.Harley{at}phimr.monash.edu.au.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The sex-determining region of the Y chromosome (SRY) plays a key role in human sex determination, as mutations in SRY can cause XY sex reversal. Although some SRY missense mutations affect DNA binding and bending activities, it is unclear how others contribute to disease. The high mobility group domain of SRY has two nuclear localization signals (NLS). Sex-reversing mutations in the NLSs affect nuclear import in some patients, associated with defective importin-ß binding to the C-terminal NLS (c-NLS), whereas in others, importin-ß recognition is normal, suggesting the existence of an importin-ß-independent nuclear import pathway. The SRY N-terminal NLS (n-NLS) binds calmodulin (CaM) in vitro, and here we show that this protein interaction is reduced in vivo by calmidazolium, a CaM antagonist. In calmidazolium-treated cells, the dramatic reduction in nuclear entry of SRY and an SRY-c-NLS mutant was not observed for two SRY-n-NLS mutants. Fluorescence spectroscopy studies reveal an unusual conformation of SRY.CaM complexes formed by the two n-NLS mutants. Thus, CaM may be involved directly in SRY nuclear import during gonadal development, and disruption of SRY.CaM recognition could underlie XY sex reversal. Given that the CaM-binding region of SRY is well-conserved among high mobility group box proteins, CaM-dependent nuclear import may underlie additional disease states.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
IN MAMMALS, THE SEX-determining region of the Y chromosome (SRY) is a dominant gene necessary and sufficient to initiate gonadal development leading to testis formation (1). In the absence of SRY, an ovary forms and a female develops. In chromosomally XY humans, mutations in the SRY gene gives rise to male-to-female sex reversal (2). Although only 15% of XY female patients carry mutations in SRY, almost all XY female patients with SRY mutations show complete gonadal dysgenesis (Swyer syndrome). Swyer syndrome XY patients develop as females with both female internal and external genitalia but lack ovarian function (3).

Human SRY contains a 80-amino-acid high mobility group (HMG) box, which is well-conserved across mammalian species. Because this DNA-binding motif also induces a bend in DNA (4, 5), SRY is often described as an architectural transcription factor. The two domains on either side of the HMG box exhibit little interspecies conservation and do not resemble any obvious conserved structure. Studies on the transcriptional function of SRY have been hampered by the lack of identified in vivo targets, and conflicting reports have been published. Using model transactivation assays in culture cells, SRY can act as a weak transcriptional activator or repressor (6).

The importance of the HMG box to SRY function is highlighted by the fact that most Swyer syndrome mutations of SRY are clustered within this motif (see Ref. 7 for a recent review). In some cases, mutations in the HMG box reduce DNA-binding and/or DNA-bending activities of the protein. However several SRY mutants show wild-type DNA binding and bending, suggesting that they may be affecting yet unidentified activities of SRY (8, 9, 10, 11).

The DNA binding HMG domain of SRY contains two independent nuclear localization signals (NLSs), one at each end (Fig. 1Go) (12, 13). The overlap of nuclear import and DNA binding regions is conserved in 90% of DNA binding proteins, possibly as a result of the evolution of the nucleus as the DNA-containing compartment (14). Importin-ß, which traffics cargo through the nuclear pore complex (15), binds the C-terminal NLS (c-NLS; amino acids 131–134) of SRY (16), which consists of a short stretch of basic residues, resembling the NLS of the simian virus 40 (SV40) large T antigen (Fig. 1Go) (17). The N-terminal NLS (n-NLS; amino acids 59–77) consists of two highly basic regions separated by 11 residues, resembling the bipartite NLS identified in nucleoplasmin (18). Recently, we showed that an import-defective SRY mutant encoded by an XY female with the amino acid substitution R133W had defective importin-ß binding, presumably as a direct consequence of the mutation in the c-NLS (Fig. 1Go and Table 1Go) (11). This represented the first documented case of a human disease explained at the molecular level by the impaired ability of a protein to accumulate in the nucleus. In contrast, two other import-defective and XY sex-reversing SRY proteins with missense mutations in their n-NLS, R75N and R76P, bind normally to importin-ß, suggesting a defect elsewhere in an import pathway (Fig. 1Go and Table 1Go) (11). Table 1Go summarizes the clinical and biochemical data of SRY from the three Swyer patients with known import defects carrying the R75N, R76P, and R133W mutations (19, 20, 21).



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Fig. 1. NLSs of SRY

The 80-amino-acid HMG domain has two NLSs. The n-NLS in SRY binds CaM in vitro (22 ). The c-NLS is recognized by importin-ß (Impß). Ovals (O) indicate the positions of sex-reversing amino acid substitution mutations of three XY females with XY gonadal dysgenesis in the NLS regions of SRY. Sequence alignment showing conservation of the CaM-binding domain and of the Impß-binding domain in the HMG box of human SOX proteins. The bipartite n-NLS and the simian virus 40 (SV40)-like c-NLS are boldly underlined.

 

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Table 1. Missense Swyer Syndrome Mutations Analyzed in this Study

 
The n-NLS region of SRY can interact with calmodulin (CaM) in a calcium-dependent manner in vitro (22), and, although some evidence exists for a role for CaM in nuclear import, a direct role is controversial (23, 24). Given that a CaM-dependent nuclear import pathway for transcription factors has been reported (24, 25), we hypothesize that the SRY-CaM interaction is required for SRY nuclear import and for subsequent testis determination to occur.


    RESULTS AND DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
SRY Colocalizes and Interacts with CaM in COS7 Cells
We previously showed by native gel electrophoresis that the SRY HMG box interacts with CaM in a calcium-dependent manner through a 25-amino-acid sequence located in helix 1 of the SRY HMG box, which encompasses the n-NLS (Fig. 1Go) (22). The sequence of this domain is highly conserved in other members of the SOX (SRY-related HMG box) family and has been shown to bind CaM also in SOX9 (Fig. 1Go) (26).

To further study the interaction of SRY and CaM, we first investigated the localization of SRY and CaM by indirect immunofluorescence in COS7 cells transiently transfected with SRY. Both SRY and CaM were located predominantly in the nuclei of SRY-expressing transfected cells (Fig. 2AGo). Western blotting was carried out on COS7 cells transiently transfected with the SRY expression vector or the empty control vector, which showed that the SRY antibody and the CaM antibody were specific for SRY and CaM, respectively (Fig. 2BGo). Immunoprecipitation pull-down experiments were subsequently carried out on the above-described cell lysates to examine whether SRY and CaM interact. SRY was detected by Western blotting in cell extracts that had been immunoprecipitated with a CaM antibody in the presence of calcium [Fig. 2CGo(i)]. The interaction was not observed when the immunoprecipitation was carried out in the presence of EGTA or when an unrelated monoclonal antibody was used. The reverse experiment was also performed using the SRY antibody to coimmunoprecipitate CaM, which was detected using a CaM monoclonal antibody [Fig. 2CGo(ii)]. These data suggest a physical interaction between SRY and CaM in cells.



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Fig. 2. SRY Interacts with CaM in Vivo

A, Immunostaining of SRY and CaM. Colocalization of SRY and CaM in the nuclei of SRY-transfected COS7 cells. Scale bar, 10 µm. B, Specificity of the anti-SRY antibody. Total cell lysates prepared from COS7 cells transfected with SRY (S) or empty vector control plasmids (C) (2 x 105 cells/well) were loaded into each well. The SRY antibody detected a protein band of 28 kDa only in SRY-expressing cells. The CaM antibody detected CaM (17 kDa) in both lanes. C, Coimmunoprecipitation of SRY and CaM. (i) COS7 cells transfected with SRY (S) or empty vector control plasmids (C) were lysed and immunoprecipitated with a CaM monoclonal antibody or a nonrelated antibody (nr), in the presence of Ca2+ or EGTA, and coimmunoprecipitated SRY was detected by Western blotting with a SRY antibody. Lane 6 represents 1/15 of the input of SRY-transfected COS7 cell lysate used for coimmunoprecipitation. (ii) This is the reverse experiment of (i), in which cell lysates were immunoprecipitated with a sheep polyclonal SRY antibody or a sheep preimmune IgG antibody, in the presence of Ca2+ or EGTA, and the coimmunoprecipitated CaM was detected by Western blotting with a CaM monoclonal antibody. Lane 6 represents 1/15 of the input of SRY-transfected COS7 cell lysate used for coimmunoprecipitation.

 
Sex-Reversing Mutations in the SRY HMG Domain Change the Structure of HMG/ CaM complexes, But Do Not Affect CaM Binding Kinetics
Wild-type and mutant SRY HMG proteins were expressed in Escherichia coli bacteria and purified by ion-exchange chromatography to approximately 90% purity by SDS-PAGE analysis as described previously (27). To compare CaM-binding activities of SRY HMG box proteins, we determined by fluorescence spectroscopy the binding affinity in solution of purified SRY HMG domain for Alexa488 dye-coupled CaM, by titrating in the HMG domain (Fig. 3AGo). Wild-type SRY showed saturable 1:1 binding to CaM with moderate affinity [dissociation constant (kd), 75 ± 10 nM] as did the c-NLS mutant, R133W (kd, 75 ± 10 nM). Affinities for CaM were surprisingly similar for both n-NLS mutants, R75N (kd, 100 ± 15 nM) and R76P (kd, 160 ± 20 nM). The affinities of all isoforms fell inside the range of known in vivo targets of CaM (28), implying that a direct interaction between the SRY HMG box and CaM is plausible. The similarity between wild-type SRY and mutant affinities suggests that these mutations did not affect binding kinetics between the HMG domain and CaM.



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Fig. 3. Fluorescence Measurements of the Interaction of SRY and Mutant HMG Box Proteins with CaM

A, The binding affinities (Kd) of SRY and mutant HMG box proteins for CaM. Fluorescence anisotropy was used to determine the binding affinities of SRY and its mutants for Alexa488 dye-coupled CaM in solution, by titrating in the HMG domains. Each Kd represents an average of three calculations taken from three fit curves derived from three separate sets of data points (see Materials and Methods). B, (i) Tryptophan accessibility to aqueous-phase acrylamide quenching in free and CaM-bound SRY and mutant HMG protein (in presence of 50 mM CaCI2). Conformation changes due to quenching by 0.5 M acrylamide were assessed by the shift in average emission wavelength. The results from three independent experiments were analyzed using an unpaired t test (***, P < 0.001). (ii) Tryptophan fluorescence spectra of the SRY.CaM complexes in the absence (solid lines) and presence (dotted lines, scaled up 5-fold) of 0.5 M acrylamide (50 mM CaCI2 was included in all reactions). Typical error variation, ± 1 nm. wt, Wild type.

 
Next, we investigated whether SRY mutations altered the structure of the HMG-CaM complex. The overall conformation of the wild-type and mutant SRY in the free and CaM-bound states was measured by tryptophan fluorescence spectroscopy. Tryptophan fluorescence emitted from proteins activated by UV excitation is sensitive to the microenvironment around the hydrophobic indole chromophore of tryptophan residues. Changes in tryptophan fluorescence can provide information concerning changes in protein structure and dynamics. The SRY HMG domain contains three {alpha}-helices, each contributing a tryptophan to the hydrophobic core, which stabilizes the domain (29). We have shown previously that the fluorescence emission of the SRY HMG box undergoes characteristic shifts in wavelength maximum upon CaM binding when compared with free SRY HMG box (22). In these studies, we measured the fluorescence properties of SRY HMG protein and mutants R75N, R76P, and R133W. Tryptophan residues within the proteins were selectively excited with UV radiation of 295-nm wavelength, and their emission was measured from 300–500 nm. Red shifts in wavelength maxima of the free protein form suggest that the n-NLS mutants R75N (350 nm) and R76P (349 nm) have a different conformation to the wild-type protein (346 nm) and to the c-NLS mutant R133W (345 nm). The addition of CaM in the presence of Ca2+ caused an increase in the HMG box fluorescence and a blue shift in the emission wavelength maxima of all four proteins, although a smaller blue shift was observed in the n-NLS mutants (data not shown). The addition of EGTA reversed these changes, suggesting that the Ca2+-dependent changes are reversible in both the wild-type SRY and all three mutant HMG proteins (data not shown). Hence, despite small changes in the protein conformation of the free form of the mutants, this did not affect their ability to bind CaM reversibly in the presence of Ca2+.

To investigate further the accessibility of tryptophan residues of SRY and mutant HMG proteins, the addition of acrylamide quenching agent to SRY and the mutant HMG proteins alone showed similar changes in fluorescence intensity (5-fold reduction) and wavelength maximum (red shift of 6 nm) (Fig. 3BGo). In contrast, in HMG.CaM complexes, a minimal blue shift in emission wavelength maximum (<2 nm; typical variation ± 1 nm) was observed for both wild-type SRY HMG.CaM and R133W HMG.CaM, whereas dramatic changes were observed for R75N HMG.CaM (10 nm) and for R76P.CaM (6 nm) (Fig. 3BGo). This suggests that the wild-type and R133W HMG boxes have similar tryptophan shielding properties, whereas in the R75N and R76P mutants, where larger shifts in average emission wavelengths was observed, differential shielding was evident. Hence, the conformations sensed by the tryptophan residues in SRY HMG.CaM complexes are different between n-NLS mutants, R75N and R76P, and wild-type or importin-ß-binding defective mutant R133W. This indicates that sex-reversing SRY mutations in the HMG domain change the structure of the HMG.CaM complex, which may in turn alter recognition by the nuclear import machinery.

Calmidazolium Chloride (CDZ) Inhibits Ca2+-Dependent Interaction between SRY and CaM in Vitro and in Vivo
A CaM antagonist, CDZ, is an imidazole compound that binds strongly to the hydrophobic surface of CaM, preventing the binding of Ca2+ and thereby preventing correct folding of active CaM (30, 31). To test whether CDZ antagonizes SRY.CaM interaction, SRY HMG and CaM were incubated in the presence EGTA or in the presence of calcium and increasing CDZ, and complexes were resolved by native gel electrophoresis. As previously reported (22), stained protein bands were not observed in lanes containing SRY HMG alone because SRY HMG migrates toward the cathode. The migration of Ca2+-bound CaM was retarded compared with CaM in the presence of EGTA, and the Ca2+-CaM.SRY HMG protein interaction band was only observed when the reactions were carried out in the presence of calcium (Fig. 4AGo). Increasing concentrations of CDZ inhibited the formation of the SRY HMG.CaM complex in vitro in a dose-dependent manner, exhibiting the greatest effect at 30 µM (Fig. 4AGo). In addition, increasing CDZ resulted in a corresponding increase in the formation of a faster migrating CaM-CDZ protein band (solid circle, Fig. 4AGo). To test whether CDZ affects SRY and CaM binding in vivo, SRY-expressing COS7 cell lysates were immunoprecipitated with either the SRY or the CaM antibody in the presence or absence of CDZ. CDZ reduced the interaction between SRY and CaM, resulting in less coimmunoprecipitated SRY or CaM detected (Fig. 4BGo).



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Fig. 4. A CaM Antagonist, CDZ, Inhibits Ca2+-Dependent Complex Formation of SRY HMG and CaM in Vitro

A, Reaction mixtures were made to a final volume of 20 µl, typically containing 35 µM CaM and/or 40 µM purified SRY HMG box proteins. Increasing amounts of CDZ were added and all reactions were carried out in the presence of 5 mM CaCl2 or 1 mM EGTA. Reactions were incubated on ice for 1 h before native polyacrylamide gel electrophoresis. Proteins were visualized by Neuhoff staining. Arrows indicate the positions of apo CaM, Ca2+-CaM, and Ca2+-CaM.SRY HMG protein complexes. The solid circle marks the position of the CaM-CDZ protein bands. B, SRY-expressing COS7 cell lysates were immunoprecipitated with either the SRY or the CaM antibody (Ab) in the presence or absence of CDZ. CDZ greatly reduced the binding of CaM with SRY and vice versa.

 
CDZ Inhibits SRY Nuclear Import
To test whether the interaction between SRY and CaM has a physiological role in cells, we measured SRY nuclear import by indirect immunofluorescence in SRY-transfected cells treated with CDZ (Fig. 5AGo, left panel). SRY is normally localized exclusively in the nucleus of untreated, transfected COS7 cells. Upon addition of CDZ, the subcellular location of SRY protein immunofluorescence changed from predominantly nuclear (Fn/c 13.5) to both nuclear and cytoplasmic (Fn/c 2.4), indicating that a CaM-dependent nuclear import pathway was inhibited (Fig. 5AGo, left panel). Treatment of cells with EGTA also inhibited SRY interaction with CaM (data not shown), as for SOX9 (26), demonstrating that Ca2+-bound CaM is required for SRY nuclear import.



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Fig. 5. Qualitative and Quantitative Measurement of SRY and Mutant Nuclear Localization when Treated with the CaM Antagonist CDZ

A, pcDNA3-SRY wild-type (wt, 1.5 µg) or pcDNA3-SRY mutant (R75N, R76P, or R133W) were transiently transfected into COS7 cells using Fugene 6. Twenty-four hours after transfection, the cells were treated with 5 µM CDZ for an additional 24 h before immunostaining. Exogenous SRY expression was detected with a green fluorescent Alexa-488 dye-coupled secondary antibody, whereas 4',6-diamidino-2-phenylindole-stained DNA determined the location of the nuclei. SRY fluorescence was visualized by confocal microscopy and quantitated as previously described (26 ). Measurements represent the average of two separate transfections (n = 50). Nuclear accumulation of SRY is expressed as fluorescence in the nucleus over that in the cytoplasm (Fn/c) where background fluorescence has been subtracted. The average Fn/c values are indicated in the top right-hand corner of each panel. Scale bar, 10 µM. Results relative to untreated SRY wild-type-transfected cells (Fn/c given value of 100%) are shown for control and CDZ-treated SRY- and mutant-transfected cells. B, pEGFPC1-IGFBP-3 NLS (1.5 µg) and pEGFP-PTHrP were transiently transfected into COS7 cells and processed, visualized, and quantitated as described above. Fn/c values were used to graph the results in the lower panel. Scale bar, 10 µM. GFP, Green fluorescent protein.

 
Defective CaM-Mediated SRY Nuclear Import in Two Cases of XY Sex Reversal
SRY mutants from three XY females tested displayed impaired nuclear import (11). R133W showed defective importin-ß binding, whereas R75N and R76P had normal importin-ß binding activity (Table 1Go, Ref. 11). In contrast to wild-type SRY, we observed no effect of CDZ upon nuclear accumulation in the R75N mutant in transiently transfected cells (Fig. 5AGo), suggesting that the CaM-mediated nuclear import pathway is not active in this mutant. In the absence of CDZ, the R76P mutant shows approximately 50% (7.3/13.5) of wild-type SRY nuclear accumulation (Table 1Go and Ref. 11). CDZ treatment further reduced the nuclear accumulation of the R76P mutant (Fn/c 2.7) to a level similar to that of CDZ-treated wild type SRY (Fn/c 2.4) (Fig. 5AGo). This implies that, in untreated cells, the CaM-mediated pathway is only partially active for R76P. In contrast, CDZ treatment of the R133W mutant decreased nuclear accumulation to levels below those of wild-type SRY, consistent with importin-ß binding also being defective in this mutant. Hence, at least two critical factors are needed for nuclear import of SRY, importin-ß and CaM.

To rule out that the above-described data were nonspecific effects of CDZ, two other proteins with reported importin-ß-mediated nuclear import, IGF-binding protein-3 (IGFBP-3) (32) and PTHrP (33), were tested. Both were unaffected by CDZ treatment (Fig. 5BGo).

A Model for Nuclear Import of SRY in Normal Males and XY Females
The model in Fig. 6Go summarizes our current thinking of SRY nuclear import mechanisms. Sex is determined at wk 7 of gestation in the XY embryo when SRY is first expressed in the somatic cells of the genital ridge (Fig. 6AGo). SRY is normally imported into the cell nucleus by two mechanisms, one facilitated by SRY binding to CaM and the other involving SRY binding to importin-ß (Fig. 6BGo). In certain XY females, SRY mutations (shown as asterisks) affect SRY nuclear import, preventing gene activation either by blocking SRY-importin-ß binding or by blocking SRY-CaM binding. The DNA-binding and -bending activities of the c-NLS SRY mutant R133W are normal and its n-NLS/CaM-dependent import pathway is functional, as demonstrated by a reduction in nuclear import upon CDZ treatment (Fig. 5AGo). Thus, for R133W, XY sex reversal is the direct consequence of reduced importin-ß binding and subsequent nuclear import mediated by the c-NLS/importin-ß pathway (Fig. 6CGo). In XY females carrying the R75N and R76P mutations, the c-NLS/importin-ß pathway is probably functional, as both mutants bind importin-ß normally (11). Therefore, reduced nuclear import of R75N and R76P is most likely due to an impaired n-NLS/CaM-dependent import pathway. In the case of R76P, the HMG structure is similar to that of wild-type SRY, as indicated by fluorescence, DNA-binding, DNA-bending, and importin-ß-mediated import activities. This suggests that defective CaM recognition could be the sole molecular defect in this mutant, leading to inefficient CaM-mediated import (Fig. 6DGo). The fact that mutant SRY proteins encoded by XY females show deficient CaM-dependent nuclear import imply that this activity of SRY is necessary for sex determination. If a threshold level of SRY protein in the nucleus of primordial Sertoli cells at the time of testis determination is not reached, XY sex reversal can occur.



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Fig. 6. Model of Nuclear Import of SRY in Normal Males and XY Females

A, A wk-7 XY embryo with the genital ridge indicated by an arrow. B, SRY is normally imported into the cell nucleus by binding to CaM or to importin-ß. C, In certain XY females, SRY mutations (shown as asterisks) reduce SRY nuclear import either by blocking SRY-importin-ß binding or, D, by blocking SRY-CaM binding. The R133W clinical mutation disrupts binding between the c-NLS and importin-ß; however, nuclear import mediated by the n-NLS appears to be unaffected. In comparison, c-NLS/importin-ß-dependent nuclear import is active in the n-NLS mutants R75N and R76P; however, anomalous conformation of the mutant HMG.CaM complexes impair nuclear import mediated by the n-NLS.

 
Given that CaM binds distant members of the HMG domain superfamily (such as HMG1 and a SOX protein) (11, 26) and that the human genome contains over 100 genes encoding HMG domain proteins, it is very likely that other clinical syndromes could involve defects in analogous CaM recognition mechanisms.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cell Types and Culture
Monkey epithelial COS7 cells were cultured as a monolayer in DMEM (GIBCO, Rockville, MD), supplemented with 10% (vol/vol) fetal calf serum, 1% L-glutamine, and 1% (vol/vol) penicillin/streptomycin.

Transient Transfection
COS7 cells were transfected by FuGene 6 as recommended by the manufacturers (Roche Diagnostics, Indianapolis, IN). The CaM antagonist CDZ (Sigma Aldrich, St. Louis, MO), was added 24 h after transfection and incubated for an additional 24 h before further processing.

Immunohistochemistry
After transfection and drug treatment, immunohistochemistry was carried out as described previously (34). The primary antibodies used include the affinity-purified sheep antihuman SRY (1:400) and monoclonal anti-CaM (1:500) (Upstate Biotechnology, Lake Placid, NY) antibodies. The secondary antibodies used include Alexa 488-conjugated donkey antisheep IgG and donkey antimouse IgG and Alexa 647-conjugated donkey antimouse IgG antibodies (Molecular Probes, Eugene, OR). DNA was stained with 0.1 µg/ml of 4',6-diamidino-2-phenylindole or To-Pro 3 (Molecular Probes). Confocal laser scanning microscopy (FV500; Olympus, Melville, NY) was used for quantitation of nuclear accumulation of SRY protein, and image analysis was performed by using NIH ImageJ (public domain software). Briefly, measurements were taken of the density of fluorescence from the cytoplasm and the nucleus with the background fluorescence subtracted from the equation: Fn/c = (n – bkgdn)/(cp – bkgdcp), where n = nucleus and bkgdn = background in the nucleus, cp = cytoplasm and bkgdcp = background in the cytoplasm.

Preparation of Antibodies against Human SRY
Antibodies against human SRY were generated in sheep using a peptide (PP4), which corresponds to the C-terminal 15 amino acids of the human SRY protein. SRY antibodies were affinity-purified from sheep serum using the PP4 peptide covalently coupled to cyanogen bromide-activated Sepharose-4B (Amersham Pharmacia, Uppsala, Sweden).

Immunoprecipitation
Total cell extracts prepared from transfected cells in RIPA buffer (150 mM NaCl; 50 mM Tris-HCl, pH 7.5; 0.25% sodium deoxycholate; 0.1% Nonidet P-40; 1 mM NaF; 1 mM sodium orthovanadate; 1 mM phenylmethylsulfonyl fluoride; 10 µg/ml aprotinin; and 10 µg/ml leupeptin) were precleared by addition of protein G-Sepharose beads (25 µl/reaction) at 4 C for 30 min, before being subjected to immunoprecipitation with 1 µg of monoclonal CaM antibody (Upstate Biotechnology). Antibody/cell lysates were incubated at 4 C overnight. Next, 25 µl preequilibrated protein G beads were added and samples incubated for an additional 4 h. Immunoprecipitates were then washed five times with RIPA buffer before protein separation by SDS-PAGE and subjected to immunodetection with SRY antibody.

SRY and Mutant HMG Protein Production and Purification
Recombinant wild-type SRY and mutant HMG proteins (amino acids 59–136) were expressed and purified from E. coli as described previously (27).

CaM Production and purification
The pKK-CaM3 bacterial expression vector was a generous gift from Dr. E. E. Strehler (Mayo Clinic College of Medicine, Rochester, MN). Recombinant human CaM was expressed and purified from E. coli as described previously (35).

Native Polyacrylamide Gel Electrophoresis
Gel and buffer composition were essentially as for SDS-PAGE in standard Tris-glycine buffers but lacking sodium dodecyl sulfate and consisting of a 12.5% separating gel and a 5% stacking gel (22). Reaction mixtures were made to a final volume of 20 µl, typically containing 35 µM CaM and 40 µM purified SRY HMG box protein in 20 mM Tris (pH 6.8), 10% glycerol, 5 mM CaCl2, 1 mM dithiothreitol, and 0.1% bromophenol blue. Reactions were incubated for 1 h on ice before electrophoresis. After electrophoresis, gels were stained overnight with Neuhoff stain (36).

Tryptophan Fluorescence Analysis of SRY and Mutant HMG Box/CaM Interaction
Fluorescence spectra were recorded at 20 C on a SPEX Fluorolog-{tau}2 frequency domain spectrofluorometer (SPEX Instruments, Edison, NJ) using 0.5-ml quartz cells. The solutions contained 2 µM of purified SRY HMG protein, and 2 µM CaM.SRY HMG box protein was incubated with CaM on ice for 30 min in 25 mM Tris (pH 7.0), 0.1 M KCl, and 50 mM CaCI2 before fluorescence measurements were taken. Excitation of tryptophan residues was accomplished using an excitation wavelength of 295 nm from a 450-W Xenon lamp. Emission spectra were measured in the wavelength range of 300–500 nm and through a polarizer oriented at the magic angle (54.7°). The spectral band pass of excitation and emission was 5 nm. Spectra were fully corrected for the wavelength response of the detection system. The cell block was maintained at 20 C with a circulating water bath.

Acrylamide quenching was performed using an excitation wavelength of 295 nm to avoid tyrosine excitation and distortion by acrylamide. An aliquot of 4 M acrylamide was added to 400 µl of a 2 µM protein sample to a final concentration of 0.5 M, and the fluorescence intensity was recorded at 350 nm with a SPEX Fluorolog {tau}2 frequency domain spectrofluorometer. The fluorescence was corrected for dilution and inner-filtering by acrylamide.

Fluorescence Anisotrophy Determination of Binding Affinities between SRY and Mutant HMG Proteins with Ca2+-CaM
Fluorescence anisotropy measurements were performed at 20 C using a SPEX Fluorolog {tau}2 frequency domain spectrofluorometer. For the anisotrophy measurements of Alexa488 fluorophore-coupled CaM (Molecular Probes), the excitation wavelength was 495 nm, and the emission wavelength was 520 nm. Samples containing 20 nM CaM-Alexa488 and an increasing titration of the SRY HMG proteins were buffered in 25 mM Tri-HCl (pH 7.6), 1 mM CaCl2, 0.1 M KCl. The percentage of bound CaM was calculated from the anisotropy readings: %CaMBound = 100*(A-Afree)/A100%Bound – Afree). The concentration of SRY HMG box was increased until CaM binding was saturated. The data points generated were used to derive nonlinear fit curves using the Xmgrace computer program (Free Software Foundation, Cambridge, MA). With the hyperbolic equation: {theta} = Bmax[HMG box]/[HMG box] + Kd), where {theta} is the %CaMBound of the fit curve, Bmax is the maximum %CaMBound value ascribed to the fit curve, and Kd is the dissociation constant. Three separate sets of data points were used to derive three fit curves and therefore three Kd values. The three Kd values were then used to calculate the mean Kd within SD limits: SD = (n{Sigma}x2 ({Sigma}x)2/n2)1/2.


    ACKNOWLEDGMENTS
 
We thank Sue Panckridge for artwork and Dr. R. C. Baxter and Dr. M. T. Gillespie for the generous gifts of the pEGFP-IGFBP-3 NLS and pEGFP-PTHrP plasmid constructs.


    FOOTNOTES
 
This work was supported by National Health and Medical Research Council (Australia) Grant 198713.

First Published Online March 3, 2005

Abbreviations: CaM, Calmodulin; CDZ, calmidazolium chloride; c-NLS, C-terminal NLS; HMG, high mobility group; IGFBP-3, IGF-binding protein-3; NLS, nuclear localization signal; n-NLS, N-terminal NLS; SOX, SRY-related HMG box; SRY, sex-determining region of the Y chromosome.

Received for publication August 25, 2004. Accepted for publication February 22, 2005.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS AND DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Sinclair AH, Berta P, Palmer MS, Hawkins JR, Griffiths BL, Smith MJ, Foster JW, Frischauf AM, Lovell-Badge R, Goodfellow PN 1990 A gene from the human sex-determining region encodes a protein with homology to a conserved DNA-binding motif. Nature 346:240–244[CrossRef][Medline]
  2. Berta P, Hawkins JR, Sinclair AH, Taylor A, Griffiths BL, Goodfellow PN, Fellous M 1990 Genetic evidence equating SRY and the testis-determining factor. Nature 348:448–450[CrossRef][Medline]
  3. Hawkins JR 1994 Sex determination. Hum Mol Genet 3:1463–1467[Abstract]
  4. Harley VR, Clarkson MJ, Argentaro A 2003 The molecular action and regulation of the testis-determining factors, SRY (sex-determining region on the Y chromosome) and SOX9 [SRY-related high-mobility group (HMG) box 9]. Endocr Rev 24:466–487[Abstract/Free Full Text]
  5. Giese K, Pagel J, Grosschedl R 1994 Distinct DNA-binding properties of the high mobility group domain of murine and human SRY sex-determining factors. Proc Natl Acad Sci USA 91:3368–3372[Abstract/Free Full Text]
  6. Parker KL, Schedl A, Schimmer BP 1999 Gene interactions in gonadal development. Annu Rev Physiol 61:417–433[CrossRef][Medline]
  7. Knower KC, Kelly S, Harley VR 2003 Turning on the male—SRY, SOX9 and sex determination in mammals. Cytogenet Genome Res 101:185–198[CrossRef][Medline]
  8. Jager RJ, Harley VR, Pfeiffer RA, Goodfellow PN, Scherer G 1992 A familial mutation in the testis-determining gene SRY shared by both sexes. Hum Genet 90:350–355[Medline]
  9. Tho SP, Zhang YY, Hines RS, Hansen KA, Khan I, McDonough PG 1998 Analysis of DNA binding activity of recombinant SRY protein from XY sex reversed female mutant for SRY (n=4). J Soc Gynec Invest 5(Suppl 1): 138A
  10. Mitchell CL Harley VR 2002 Biochemical defects in eight SRY missense mutations causing XY gonadal dysgenesis. Mol Genet Metab 77:217–225[CrossRef][Medline]
  11. Harley VR, Layfield S, Mitchell CL, Forwood JK, John AP, Briggs LJ, McDowall SG, Jans DA 2003 Defective importin beta recognition and nuclear import of the sex-determining factor SRY are associated with XY sex-reversing mutations. Proc Natl Acad Sci USA 100:7045–7050[Abstract/Free Full Text]
  12. Poulat F, Girard F, Chevron MP, Goze C, Rebillard X, Calas B, Lamb N, Berta P 1995 Nuclear localization of the testis determining gene product SRY. J Cell Biol 128:737–748[Abstract]
  13. Sudbeck P, Scherer G 1997 Two independent nuclear localization signals are present in the DNA-binding high-mobility group domains of SRY and SOX9. J Biol Chem 272:27848–27852[Abstract/Free Full Text]
  14. Cokol M, Nair R, Rost B 2000 Finding nuclear localization signals. EMBO Rep 1:411–415[Abstract/Free Full Text]
  15. Stewart M 2003 Structural biology. Nuclear trafficking. Science 302:1513–1514[Abstract/Free Full Text]
  16. Forwood JK, Harley V, Jans DA 2001 The C-terminal nuclear localization signal of the sex-determining region Y (SRY) high mobility group domain mediates nuclear import through importin ß1. J Biol Chem 276:46575–46582[Abstract/Free Full Text]
  17. Makkerh JP, Dingwall C, Laskey RA 1996 Comparative mutagenesis of nuclear localization signals reveals the importance of neutral and acidic amino acids. Curr Biol 6:1025–1027[CrossRef][Medline]
  18. Beals CR, Sheridan CM, Turck CW, Gardner P, Crabtree GR 1997 Nuclear export of NF-ATc enhanced by glycogen synthase kinase-3. Science 275:1930–1934[Abstract/Free Full Text]
  19. Battiloro E, Angeletti B, Tozzi MC, Bruni L, Tondini S, Vignetti P, Verna R, D’Ambrosio E 1997 A novel double nucleotide substitution in the HMG box of the SRY gene associated with Swyer syndrome. Hum Genet 100:585–587[CrossRef][Medline]
  20. Zhi L, Xu Z, Xu G 1996 Molecular analysis of SRY gene of seven 46, XY females. Chin Med J Genet 12:258–261
  21. Affara NA, Chalmers IJ, Ferguson-Smith MA 1993 Analysis of the SRY gene in 22 sex-reversed XY females identifies four new point mutations in the conserved DNA binding domain. Hum Mol Genet 2:785–789[Abstract]
  22. Harley VR, Lovell-Badge R, Goodfellow PN, Hextall PJ 1996 The HMG box of SRY is a calmodulin binding domain. FEBS Lett 391:24–28[CrossRef][Medline]
  23. Corneliussen B, Holm M, Waltersson Y, Onions J, Hallberg B, Thornell A, Grundstrom T 1994 Calcium/calmodulin inhibition of basic-helix-loop-helix transcription factor domains. Nature 368:760–764[CrossRef][Medline]
  24. Taules M, Rodriguez-Vilarrupla A, Rius E, Estanyol JM, Casanovas O, Sacks DB, Perez-Paya E, Bachs O, Agell N 1999 Calmodulin binds to p21(Cip1) and is involved in the regulation of its nuclear localization. J Biol Chem 274:24445–24448[Abstract/Free Full Text]
  25. Sweitzer TD, Hanover JA 1996 Calmodulin activates nuclear protein import: a link between signal transduction and nuclear transport. Proc Natl Acad Sci USA 93:14574–14579[Abstract/Free Full Text]
  26. Argentaro A, Sim H, Kelly S, Preiss S, Clayton A, Jans DA, Harley VR 2003 A SOX9 defect of calmodulin-dependent nuclear import in campomelic dysplasia/autosomal sex reversal. J Biol Chem 278:33839–33847[Abstract/Free Full Text]
  27. Kelly S, Yotis J, Macris M, Harley V 2003 Recombinant expression, purification and characterisation of the HMG domain of human SRY. Protein Pept Lett 10:281–286[CrossRef][Medline]
  28. Persechini A, Cronk B 1999 The relationship between the free concentrations of Ca2+ and Ca2+-calmodulin in intact cells. J Biol Chem 274:6827–6830[Abstract/Free Full Text]
  29. Werner MH, Bianchi ME, Gronenborn AM, Clore GM 1995 NMR spectroscopic analysis of the DNA conformation induced by the human testis determining factor SRY. Biochemistry 34:11998–12004[CrossRef][Medline]
  30. el-Saleh SC, Solaro RJ 1987 Calmidazolium, a calmodulin antagonist, stimulates calcium-troponin C and calcium-calmodulin-dependent activation of striated muscle myofilaments. J Biol Chem 262:17240–17246[Abstract/Free Full Text]
  31. Smedley M, Stanisstreet M 1984 Effect of the calmodulin inhibitor R24571 (calmidazolium) on rat embryos cultured in vitro. Experientia 40:992–994[Medline]
  32. Schedlich LJ, Le Page SL, Firth SM, Briggs LJ, Jans DA, Baxter RC 2000 Nuclear import of insulin-like growth factor-binding protein-3 and -5 is mediated by the importin beta subunit. J Biol Chem 275:23462–23470[Abstract/Free Full Text]
  33. Cingolani G, Bednenko J, Gillespie MT, Gerace L 2002 Molecular basis for the recognition of a nonclassical nuclear localization signal by importin ß. Mol Cell 10:1345–1353[CrossRef][Medline]
  34. Preiss S, Argentaro A, Clayton A, John A, Jans DA, Ogata T, Nagai T, Barroso I, Schafer AJ, Harley VR 2001 Compound effects of point mutations causing campomelic dysplasia/autosomal sex reversal upon SOX9 structure, nuclear transport, DNA binding, and transcriptional activation. J Biol Chem 276:27864–27872[Abstract/Free Full Text]
  35. Rhyner JA, Koller M, Durussel-Gerber I, Cox JA, Strehler EE 1992 Characterization of the human calmodulin-like protein expressed in Escherichia coli. Biochemistry 31:12826–12832[CrossRef][Medline]
  36. Neuhoff V, Stamm R, Pardowitz I, Arold N, Ehrhardt W, Taube D 1990 Essential problems in quantification of proteins following colloidal staining with coomassie brilliant blue dyes in polyacrylamide gels, and their solution. Electrophoresis 11:101–117[CrossRef][Medline]




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