Disrupted Amino- and Carboxyl-Terminal Interactions of the Androgen Receptor Are Linked to Androgen Insensitivity

James Thompson, Fahri Saatcioglu, Olli A. Jänne and Jorma J. Palvimo

Biomedicum Helsinki Institute of Biomedicine/Physiology (J.T., O.A.J., J.J.P.) Institute of Biotechnology (J.J.P.) and Department of Clinical Chemistry (O.A.J.) University of Helsinki FIN-00014 Helsinki, Finland
Biotechnology Centre of Oslo (F.S.) University of Oslo N-0316 Oslo, Norway


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We have compared the functional consequences of seven single-point mutations in the ligand-binding domain (LBD) of the androgen receptor (AR). The mutations span helices 3 to 11 and are present in patients suffering from androgen insensitivity syndromes (AIS) and other male-specific disorders. The mutants, except M742V, bound to androgen response elements in vivo and in vitro and showed a testosterone-dependent conformational change. With regard to functional activity, the mutant M742V had severely blunted ability to transactivate or exhibit the androgen-dependent amino/carboxyl-terminal (N/C) interaction; mutants F725L, G743V, and F754L showed reduced transactivation potential and attenuated N/C interaction; and mutants V715M, R726L, and M886V had minor functional impairments. The mutants belonging to the first two groups also displayed reduced response to coexpressed GRIP1. In addition, mutations of amino acids M894 and A896 in the putative core activation domain 2 (AF2) in helix 12 confirmed that this helix is important for N/C interactions. Thus, amino acids located between helices 3 and 4 (F725 and R726), in helix 5 (M742, G743, and F754), and in helix 12 (M894 and A896) play critical roles in mediating the N/C interaction of AR. The data also show that disrupted N/C interaction is a potential molecular abnormality in AIS cases in which LBD mutations have not resulted in markedly impaired ability to bind androgen.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The development and maintenance of male and some female sexual characteristics is dependent on the normal function of the androgen receptor (AR). Upon binding the natural androgens, 5{alpha}-dihydrotestosterone (DHT) and testosterone (T), AR elicits through a complex sequence of interactions the reproductive and homeostatic functions of the male phenotype. Subtle disruptions of the molecular structure and mechanisms of AR regulation can result in clinical phenotypes that span from mild (MAIS) and partial (PAIS) to complete androgen insensitivity syndromes (CAIS), and are also involved in the development of prostate cancer (1). Most of these phenotypes and syndromes result from single nucleotide substitutions in the AR gene (2).

The steroid receptors belong to the nuclear receptor superfamily and have a well characterized and conserved modular structure (3, 4). The N-terminal domain (NTD) is the least conserved region among these receptors and can vary in size from being <6% of the total protein for the vitamin D receptor to >50% for AR. Within the AR NTD resides the hormone-independent transcription activation function 1 (AF1) (3, 4, 5). Also located within the AR NTD are conserved FXXLF and WXXLF motifs (6) that form, in part, the interface for the interaction of NTD with the hormone-dependent activation function 2 (AF2) located in the C-terminal ligand-binding domain (LBD) (7). The AR LBD comprises 12 helices, and a ligand-binding pocket is formed by the helices 3, 4, 5, 7, 11, and 12 together with the ß-sheet preceding helix 6 (8, 9). The LBDs of steroid receptors function not only to bind ligands but also to stabilize homodimerization and orchestrate interactions with coregulators (3, 4). Unlike many other steroid receptors, the AF2 of AR is transcriptionally weak (7, 10). However, the ligand-dependent interaction between the NTD and the LBD region is needed for optimal AR function (6, 10, 11). Whether this N/C interaction is direct or partially bridged via coregulators remains to be clarified. The closely related p160 steroid receptor coactivators that include SRC-1, GRIP1/TIF2, and AIB1/ACTR/TRAM-1 (reviewed in Refs. 12, 13, 14, 15) have distinct regions that interact with the NTD and the LBD, thereby potentially bridging the N/C interaction of AR (6, 10, 11). The three conserved LXXLL motifs of p160 coactivators form amphipathic {alpha}-helices that bind to a hydrophobic groove formed by helices 3, 4, 5, and 12 of the LBDs, whereas regions outside these motifs are involved in contacting the NTDs (16, 17, 18, 19).

In this report, we have elucidated the molecular consequences of seven AR LBD mutations found in human patients (20). These amino acid substitutions are localized in or around helices 3, 4, 5, and 11. Our data indicate that many of these mutant ARs are defective in their N/C interactions, even though the mutations have no severe effects on ligand binding. In addition, by mutating AR amino acids M894 and A896, we show that the integrity of helix 12 (21) is important for AR N/C interaction.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Influence of the LBD Mutations on Transactivation by AR
Seven AR LBD point mutations that span helices 3–11 were selected (20) and recreated into mammalian expression vectors to compare the molecular mechanisms underlying some androgen-insensitive phenotypes and other male-specific disorders (Table 1Go). The AR mutants V715M, F725L, R726L, G743V, and M886V are previously shown to possess normal equilibrium dissociation constants for androgens, whereas M742V and F754L have 5- and 2-fold reduced androgen-binding affinity, respectively (24, 27). Whole-cell binding assays in COS-1 cells using [3H]mibolerone as a ligand showed that the dissociation constant (KD) values of different AR forms were indeed very similar, with the exception that the mutant M742V exhibited a KD twice as high as that of wild-type AR (data not shown). As a control, we used a helix 12-truncated version of AR that terminates at amino acid 889 (TRUNC) and is unable to bind steroid (data not shown).


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Table 1. Summary of the AR Mutations Examined

 
The wild-type and mutant receptors were cotransfected into COS-1 cells along with a probasin promoter (-285/+32)-driven luciferase reporter to investigate the effects of the substitutions on AR-dependent transcription in the presence of natural androgens, T or DHT. With T, transcriptional activities of R726L and M886V were slightly reduced, whereas mutants F725L, G743V, and F754L retained only 20–30% of wild-type AR activity (Fig. 1AGo). Interestingly, the mutant V715M was approximately twice as active as wild-type receptor at 1 nM T, and its activity was saturated already at 10 nM T. In contrast, mutant M742V was practically inactive at all T concentrations tested. Comparable results were obtained with a reporter driven by two androgen response elements (AREs) in front of a minimal TATA sequence (pARE2TATA-LUC) (data not shown). When DHT was used as the androgen, transcriptional activities of mutant receptors in relation to wild-type AR were generally 20–30% higher than in the presence of T (Fig. 1BGo). It is of particular note that the activity of M742V reached approximately 30% of that of wild-type receptor at 100 nM DHT. The effects of synthetic androgens, mibolerone and methyltrienolone, were also tested on the mutants showing severely compromised transcriptional activity with T. In the presence of the latter two steroids, the activities of F725L, M742V, G743V, and F754L in relation to wild-type AR were higher than those obtained in the presence of DHT (Fig. 1CGo). In sum, androgens activated all mutant receptors but the resulting activities varied considerably with different androgens. M742V especially exemplifies this phenomenon; it is almost inactive with T, but mibolerone can rescue its transcriptional activity to >=50% of that of wild-type AR. Immunoblotting with an AR-specific antibody demonstrated that the wild-type and mutant receptors were expressed at similar levels in COS-1 cells (Fig. 1DGo).



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Figure 1. Transcriptional Activation by Wild-Type and Mutated ARs

COS-1 cells were transfected with the expression vectors encoding wild-type and mutated ARs (2 ng) along with pPB(-285/+32)-LUC (200 ng), and pCMVß (50 ng) (see Materials and Methods). A, Twenty-four hours after transfection, the cells received fresh medium containing indicated concentrations of T for the subsequent 24 h. B, The same experiment as in panel A, except for DHT replaced T. C, Transcriptional activities of wild-type AR and mutants F725L, M742V, G743V, and F754L in the presence of 0.1, 1, and 10 nM T, mibolerone (MB), or methyltrienolone (R1881). Luciferase activities of the cell extracts were adjusted to the transfection efficiency using ß-galactosidase activity. In panels A and B, the activity of wild-type AR in the presence of 100 nM T or 100 nM DHT, respectively, is set as 100. In panel C, the activity of wild-type AR in the presence of 10 nM T is set as 100. The mean ± SD values from at least three independent experiments are shown. D, An immunoblot analysis of mutant ARs expressed in COS-1 cells. The cells on 12-well plates were transfected with the expression vectors encoding wild-type and mutated ARs (2 ng/well). Twenty-four hours after transfection, the cells received fresh medium containing 100 nM T and were cultured for an additional 24 h. Whole cell extracts were prepared from cells pooled from three wells as described in Materials and Methods. Extracts (20 µg protein/lane) were subjected to electrophoresis under denaturing conditions followed by immunoblotting with an AR-specific K333 antibody (56 ). (TRUNC, AR that terminates at amino acid 889.)

 
The effects of ectopic GRIP1 (glucocorticoid receptor interacting protein 1) expression to the transcriptional activities of mutant receptors were also examined. All the mutants responded to coexpressed GRIP1 in the presence of 100 nM T; wild-type AR and mutants V715M, R726L, and M886V displayed a >=4-fold relative increase in the transcriptional activity, whereas mutants F725L, M742V, G743V, and F754L showed somewhat lower (~3-fold) coactivation by GRIP1 (Fig. 2Go). The helix 12-truncated AR that is totally transcriptionally inactive failed to respond to GRIP1 coexpression.



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Figure 2. Influence of GRIP1 on Transcriptional Activation by Wild-Type and Mutated ARs

COS-1 cells were transfected with the expression vectors for wild-type or mutated ARs (2 ng), pPB(-285/+32)-LUC (200 ng), pCMVß (50 ng) along with (+) or without (-) pSG5-GRIP1 (300 ng). The -GRIP1 cells received 150 ng empty pSG5 and 150 ng pBluescript SK (Stratagene). Twenty-four hours after transfection, the cells received fresh medium with 100 nM T for the subsequent 24 h. Luciferase activities in cell extracts were normalized using ß-galactosidase activity. The activity of AR in the presence of T without GRIP1 is set as 100, and the mean ± SD values from at least three independent experiments are shown. The fold increase in AR activity elicited by the presence of GRIP1 is depicted above the bars.

 
Effects of LBD Mutations on the Conformation of AR
We have previously shown that the agonist-induced conformation of AR protects the LBD from partial proteolysis, yielding 30-kDa trypsin- or chymotrypsin-resistant fragments (30). In contrast, receptor forms that are incapable of hormone binding are completely cleaved under the same conditions. To study the influence of the mutations on AR conformation, the mutants were translated in vitro in the presence of [35S]methionine, incubated with or without 100 nM T, and subsequently digested with trypsin. All the mutants, except M742V, were resistant to digestion in the presence of T and showed the same approximately 30 kDa-limit fragments as the wild-type AR (Fig. 3AGo), indicating that the binding of T stabilizes their conformation. The helix 12-truncated receptor that cannot bind androgens (data not shown, Ref. 31) showed no resistance to trypsin (Fig. 3AGo). The behavior of the M742V mutant in this assay is consistent with the transactivation data, suggesting that it binds T very weakly and/or its conformation in the presence of T is distinct and less stable than those of the other AR forms. Since M742V showed clearly detectable transcriptional activity in the presence of both DHT and mibolerone, the influence of these two steroids on the M742V conformation was studied. In agreement with the ability of these androgens to rescue its transactivation, both DHT and mibolerone were capable of conferring trypsin resistance on M742V (Fig. 3BGo). However, the latter two androgens protected the wild-type AR clearly more efficiently from trypsin than was the case with the M742V mutant.



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Figure 3. Protease Resistance of Native and Mutated ARs

A, In vitro translation of wild-type and mutated AR proteins was carried out in the presence of [35S]methionine, and 25-µl portions of the translation mixture were digested with trypsin (6 ng/µl) in the presence of 100 nM testosterone (+T) or vehicle (-T) for 30 min. Upper panels, Labeled AR proteins at time point 0 min. Lower panels, Resultant trypsin digestion pattern of each mutant. B, Comparison of the ability of T, DHT, or mibolerone (MB) (100 nM each) to confer trypsin resistance on the M742V mutant and wild-type AR (WT). Reaction products were analyzed on 10% SDS-PAGE gels and visualized by fluorography. Arrows depict the ~30-kDa protease-resistant fragments. Migration of molecular mass markers is shown on the left.

 
DNA Binding of AR Mutants
We next investigated whether the point mutations influence the DNA-binding ability of the receptor forms. Mutated ARs were expressed in COS-1 cells in the presence and absence of T, and whole cell extracts were analyzed by electrophoretic mobility shift assay (EMSA). The extracts were incubated with 32P-labeled intronic ARE of the C3(1) gene, and receptor-DNA complexes were resolved. In the absence of androgen in the culture medium, only minimal binding of wild-type AR to the ARE was detectable, whereas extracts derived from cells grown in the presence of T showed strong AR-ARE complexes (Fig. 4AGo). This was also the case with mutants V715M, F725L, R726L, and M886V. In contrast, DNA binding of M742V was barely stimulated by T. Inclusion of androgen had a lesser effect on the DNA complex formation by G743V and F754L than with the other mutants (Fig. 4AGo). Helix 12-truncated AR did not bind to ARE under these conditions. Immunoblotting of the cell extracts showed that the less avid DNA binding of M742V, G743V, or F754L was not due to a lower expression of these mutants in COS-1 cells (Fig. 4AGo).



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Figure 4. Binding of Native and Mutant ARs to ARE

A, Upper panels, Equal amounts of whole cell extracts (10 µg protein) from COS-1 cells expressing wild-type and mutated ARs were preincubated with 100 nM testosterone (+T) or vehicle (-T) at 22 C for 30 min before incubation (60 min) with 32P-labeled C3(1 )-ARE at 22 C. Protein-DNA complexes were separated on 4% nondenaturing polyacrylamide gels and detected by autoradiography. Lower panels, Immunoblot analysis of the cell extracts subjected to EMSA analysis; K333 (56 ) was used as the primary antibody. B, ARs (7 µl) synthesized by in vitro translation in reticulocyte lysates were preincubated with 100 nM testosterone (+T) or vehicle (-T) and subjected to EMSA analysis as described in panel A. Note that although somewhat variable amounts of incubation mixtures were loaded, as illustrated by the nonspecific bands (NS) migrating between the AR-ARE complex and free probe, it does not hamper interpretation of the data. An arrowhead depicts the positions of specific AR-ARE complexes and F refers to the free probe. The experiments were repeated twice with essentially identical results.

 
We have previously shown that in vitro-translated holo-AR-DNA complex migrates on EMSA gels faster than that of apo-AR, which is interpreted to reflect a more compact structure of the holo-AR (32). To complement the experiments performed with COS-1 cell extracts, receptors were produced in rabbit reticulocyte lysates and subjected to EMSA analyses. The in vitro-translated receptors were preincubated in the presence of T or vehicle before the addition of 32P-labeled ARE. In agreement with our previous results (30, 32), receptors produced in the reticulocyte lysate displayed a considerable DNA-binding activity even without androgen (Fig. 4BGo). Whether this is due to residual androgen(s) in the lysates or to some other factors is not clear at this time. In any event, differences between the mobility of holo- and apo-AR complexes were detectable for wild-type AR and mutants V715M, R726L, and M886V. T elicited a less pronounced or no effect on the migration of protein-DNA complexes formed by F725L, M742V, G743V, and F754L. The helix 12-truncated AR form was again severely compromised in its ability to interact with DNA in this assay. These data show collectively that mutants with a weak DNA-binding activity in COS-1 cells also have diminished conformational change when expressed in reticulocyte lysates and exposed to T, probably reflecting the same phenomenon—an improper folding of the LBD in response to androgen.

The Function of AR LBD in Isolation
The AR LBD contains an AF2 domain that functions in yeast but is barely active in mammalian cells (7, 10). To investigate the consequences of the single amino acid substitutions in more detail, we inserted each mutation into the AR LBD fused in-frame to Gal4 DNA-binding domain (Gal4). COS-1 cells transfected with the Gal4-LBD constructs and immunoblotting of the cell extracts with Gal4 antibody revealed that the encoded proteins were expressed to similar levels (data not shown). Transcriptional activities of the Gal4-LBD proteins were examined in COS-1 cells using a Gal4 response element-driven luciferase reporter (pG5-LUC). The effect of cotransfected GRIP1 was also investigated. In agreement with our previous results (7, 10), the AF2 of wild-type LBD remained very weak in the presence of T, and none of the mutant LBDs showed transcriptional activity in this assay (Fig. 5Go, and data not shown). Coexpression of GRIP1 in the absence of androgen did not influence the activities of the Gal4-LBDs (data not shown), whereas in the presence of hormone, GRIP1 activated wild-type AF2 as well as V715M, R726L, and M886V by >=28-fold. In contrast, mutants F725L, M742V, and G743V responded only marginally to coexpressed GRIP1 (5- to 7-fold activation), and F754L showed an intermediate level (~16-fold) of activation (Fig. 5Go).



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Figure 5. The Effect of GRIP1 on the Transcriptional Activation by Wild-Type and Mutated LBDs

COS-1 cells were transfected with the expression vectors encoding wild-type and mutated LBDs fused to Gal4 DBD (100 ng), pG5-LUC reporter (200 ng), pCMVß (50 ng) in the presence (+) or absence (-) of pSG5-GRIP1 (120 ng). For -GRIP1 cells, 60 ng of pSG5 plus 60 ng of pBluescript SK were added. Twenty-four hours after transfection, the cells received fresh medium with 100 nM T, and the culture was continued for additional 24 h. LUC activities, normalized by the ß-galactosidase activity, are expressed relative to that of wild-type LBD in the presence of androgen but without GRIP1 (WT LBD + testosterone = 1). The mean ± SD values from at least three independent experiments are shown. The fold increase in the AR LBD activity elicited by GRIP1 is depicted above the bars.

 
In addition to the patient mutations, M894 and A896 in helix 12 were mutated to investigate their role in the function of AR LBD. The nonpolar amino acid residue Met at codon 894 was changed either to an acidic (Asp) or to a small nonpolar residue (Ala), and Ala at codon 896 was substituted with bulkier residues, either Val or Leu. M894A and M894D mutants exhibited no response to coexpressed GRIP1, and both A896L and A896V were activated only by about 5-fold, in comparison to the approximately 30-fold activation of wild-type LBD (Fig. 5Go). Thus, the LBD mutants fall into three categories in terms of their responses to GRIP1: 1) very little or no activation (mutants F725L, M742V, G743V, M894A, M894D, A896L, and A896V); 2) partial activation (F754L); and 3) activation comparable to wild-type AR LBD (V715M, R726L, and M886V).

The Effects of the LBD Mutations on the N/C Interaction
The N- and C-terminal regions of AR interact in an androgen-dependent fashion to coordinate molecular events that are important for the function of the receptor (6, 10, 33). It is possible to reconstitute the AR N/C interaction by coexpressing a Gal4 DNA-binding domain (DBD) fused to the AR LBD and a fragment containing AR NTD fused to the VP16 activation domain (AD) (10). Using this two-hybrid system in COS-1 cells, we investigated how substitutions in the AR LBD influence the N/C interaction and what is the effect of coexpressed GRIP1.

In the absence of coexpressed GRIP1, mutants V715M, R726L, and M886V showed N/C interaction similar to that of wild-type AR (Fig. 6Go, open bars). In contrast, mutants G743V and F754L had 20–30% and mutants F725L and M742 had only <=10% of the activity of wild-type receptor LBD. Of the helix 12 mutants, M894A showed 50% of wild-type N/C interaction, whereas mutants A896L and A896V had only <=25% of the wild-type LBD activity. Interestingly, conversion of M894 to Asp totally abolished N/C interaction as assessed by the two-hybrid system (Fig. 6Go). Coexpression of GRIP1 enhanced the NTD interaction of all the receptor LBDs by approximately 3- to 8-fold, except for M894D that showed no rescue of N/C interaction. Differences in the GRIP1 interactions of the mutants as isolated LBDs and when coexpressed with the NTD are highlighted by the helix 12 mutant M894A; GRIP1 failed to activate its LBD in isolation, but M894A maintained approximately 50% of the wild-type activity to interact with the NTD, which was rescued to the level of the wild-type LBD by coexpressed GRIP1 (Fig. 6Go). Mutants F725L, M742V, G743V, and F754L showed severely reduced responses to GRIP1 as isolated LBDs; nevertheless, GRIP1 stimulated their N/C interaction by a factor that was similar to wild-type LBD, suggesting that the reduced transcriptional activity of the mutants is mainly due to compromised N/C interaction rather than impaired GRIP1 response. These results also indicate that amino acids in the loop region between helices 3 and 4, together with those in helices 5 and 12, play important roles in the N/C interaction.



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Figure 6. The Influence of Point Mutations in the AR LBD on the N/C Terminal Interaction

COS-1 cells were transfected with the expression vectors for Gal4 DBD fusions of wild-type and mutated LBDs (100 ng), pVP16-rAR-(5–538) (100 ng, an AR NTD expression vector), pG5-LUC reporter (200 ng), pCMVß (50 ng), and with (+) or without (-) pSG5-GRIP1 (120 ng) as depicted. The total amount of DNA transfected was balanced by adding empty expression vectors when appropriate. For -GRIP1 cells, 60 ng of pSG5 plus 60 ng of pBluescript SK were added. The indicated AR LBDs were present both in the presence and absence of coexpressed GRIP1. Twenty-four hours after transfection, the cells received fresh medium with 100 nM T, and the culture was continued for 24 h. Normalized LUC activities are expressed relative to that of wild-type LBD in the presence of androgen but without GRIP1 (WT LBD + NTD = 100). The mean ± SD values from at least three independent experiments are shown. The fold increase in the N/C interaction activity elicited by GRIP1 is depicted above the bars.

 
The Influence of LBD Mutations on AR-Mediated Repression of Nuclear Factor (NF)-{kappa}B
Activation of AR by ligand binding can also lead to transcriptional repression of AR target genes (34, 35, 36). Even though transrepression does not usually involve the interaction of AR with specific DNA elements (34, 35, 36), our previous data show that transactivation and transrepression are influenced in a dissimilar fashion by AIS mutations in the AR DNA-binding domain (36). One well characterized group of target genes that are repressed by AR are those under the positive control by members of the nuclear factor-{kappa}B (NF-{kappa}B) family. We have shown that AR attenuates transactivation by RelA in a dose- and androgen-dependent fashion (34, 36). To investigate whether the LBD mutants differ in their abilities to inhibit RelA-dependent transcription, COS-1 cells were cotransfected with the expression vectors for wild-type or mutated ARs along with RelA and a NF-{kappa}B-regulated reporter (p{kappa}B6tk-LUC). All the AR mutants, except the helix 12-truncated form and M742V, were similar to the wild-type receptor in their ability to repress RelA-activated transcription in a T-dependent fashion (Fig. 7Go). It is of note that even though T could not stimulate M742V in transactivation assays, it still partially activated the mutant’s transrepressive function (Fig. 7Go), which was approximately 50% of that of wild-type AR. These results agree with our previous data showing that transactivation and transrepression are separate functional entities and that AR LBD plays a minor role in the inhibition of RelA function (34, 36).



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Figure 7. The Ability of AR Mutants to Repress RelA-Induced Transactivation

COS-1 cells were transfected with the expression vectors encoding full-length wild-type and mutated ARs (20 ng) along with 30 ng RelA (pCMV-p65), p{kappa}B6tk-LUC reporter (150 ng), and pCMVß (50 ng). All transfections contained equal amounts of DNA. Twenty-four hours after transfection, the cells received fresh medium with (+) or without (-) 100 nM T for the subsequent 24 h. Normalized LUC activities are expressed relative to reporter gene activity elicited by RelA alone (RelA = 100). The mean ± SD values from at least three independent experiments are shown.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We have evaluated the molecular consequences of seven AR point mutations linked to patients with AIS or prostate cancer, which did not exhibit a markedly impaired androgen-binding activity. The mutants F725L and M742V were found in PAIS patients (1, 24). These residues are conserved among the steroid receptors, except that there is a Leu in the estrogen receptor sequence at the position corresponding to M742 (Fig. 8Go). Both mutants are severely impaired in their N/C interactions and ability to transactivate. The effects of the F725L substitution on AR activity and domain interaction are comparable to those observed in a recent study (11). F725 resides in a loop region between helices 3 and 4, and the corresponding residue F367 in ER{alpha} is reported to form part of the interface for the recognition by GRIP1 nuclear receptor box II peptide (37, 38). F725 might be involved in helix positioning, and changing Phe to Leu reduces structural rigidity, and the coordination of the helices 3, 4, and 5 is compromised. Interestingly, a mutation in a neighboring amino acid (N727K) was recently shown to lead to a similar disruption between receptor domains and GRIP1/TIF2 (39).



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Figure 8. Alignment of LBD Sequences that Correspond to Steroid Receptor Helices 3–5 and Helices 11–12

AR amino acid residues mutated in this investigation are indicated by the bold text above the sequence alignments. Mutations, except for those in helix 12, were selected from the AR mutations database (20 ). (ER, Estrogen receptor; GR, glucocorticoid receptor; PR, progesterone receptor; MR, mineralocorticoid receptor.)

 
M742 resides in helix 5 and, according to the AR LBD crystal structure, is directly involved in forming the ligand-binding pocket and contacting the ligand (9). Although Val at codon 742 fits into this pocket, the mutated residue enlarges the pocket and thereby affects ligand binding (9). Interestingly, our results show that the transcriptional activity of M742V differs significantly with different androgens; the mutant is not activated by T, whereas the bulkier androgens, DHT and mibolerone, can markedly rescue its transcriptional activity. Consistent with these data, DHT and mibolerone, but not T, are able to elicit a conformational change in the receptor. Thus, our functional data showing that the M742 residue is one of the amino acid residues critical in distinguishing between different androgens agree well with the AR LBD structural model (9). The M742 mutant also binds poorly to DNA. Since F725L binds efficiently to ARE under the same conditions, even though it shows very little N/C interaction, the lack of association between LBD and NTD does not alone explain the inability of M742V to bind DNA.

The mutants G743V and F754L reside in helix 5 (Fig. 8Go), and they have been reported to cause PAIS/CAIS and PAIS, respectively (25, 26, 27). The latter mutation has also been identified in prostate cancer (28). Neither G743 nor F754 residues are conserved among the nuclear receptors, suggesting that they possess an AR-specific role. Both mutants showed severely compromised N/C interactions and a reduced ability to transactivate from the probasin promoter. The latter result with G743V is in agreement with previous data obtained using the mouse mammary tumor virus promoter (25). G743 and F754 bound to AREs more efficiently than the M742V mutant, albeit their receptor-DNA complex formation was weaker than that of wild-type AR in the presence of T. The compromised N/C interaction of the helix 5 mutants is probably due to distortions in helix-helix associations within the LBD. The substitutions in helix 5 may perturb its conformation in such a way that they weaken the helix 3-helix 5 interaction, which appears to be important for the bending of helix 3 and receptor activation (40). Structural models predict that the bent helix 3 is involved together with helix 12 in the formation of a surface(s) for coactivator binding (4). Taken together, our data indicate that helix 5 of the AR LBD plays a central role in N/C contacts. Our results also suggest that the LBD surfaces participating in interactions with the AR NTD resemble those shown to be involved in LBD-coactivator interactions of other receptors.

GRIP1 is able to bind through its three nuclear receptor boxes (LXXLL motifs) to AR LBD (18) and, independently of these motifs, to the AF1 region of AR (17, 41). However, in comparison to other steroid receptors, AR shows clear preference for certain LXXLL motifs (42). Mutations of the nuclear receptor boxes eliminated completely the coactivator function of GRIP1 on many nuclear receptors, such as thyroid hormone receptor, but they reduced coactivation on AR only by about 40% (17). Although some mutations in the AR LBD, such as those in helix 5, reduced significantly the response of Gal4-LBDs to GRIP1, the coactivator was nevertheless able to enhance the N/C interaction of the mutant receptors as well as their transcriptional activity as full-length proteins. These data together with previous reports (11, 16, 17, 43) indicate that AR LBD plays a minor role in GRIP1 interactions.

V715M and R726L have been identified in prostate cancer (22, 23), and M886V is associated with oligospermic infertility (29), a condition regarded as mild androgen insensitivity. V715M resides in helix 3, R726L in the loop between helices 3 and 4, and M886V in helix 11 (Fig. 8Go). The mutations did not affect the stability of receptor-DNA complexes, all three showed N/C interactions similar to that of wild-type AR, and the AF2s of the three mutants were activated by GRIP1 as well as or better than that of wild-type receptor. The transcriptional activities of M886V and R726L as full-length receptors on the probasin promoter were, however, slightly reduced. Unlike in a previous report (29), we detected no reduction in the ability of M886V to interact with AREs, nor was there impairment in its N/C interactions. V715M is reported to be activated by adrenal androgens and progesterone more efficiently than the native receptor (22), which may have some bearing on the progression of prostate cancer. With regard to predisposition to prostate cancer, our data add an important feature to V715M function, in that this substitution renders the receptor more sensitive to low androgen concentrations. Even though the R726L mutant had in this work properties very similar to those of wild-type AR, others have shown it to be activated by estradiol to a greater extent than the wild-type receptor (23), which is perhaps the reason for the association of this mutation to a 6-fold increased risk of prostate cancer (44). Some other mutants found in prostate cancer, such as T877A and L701H (45, 46), also alter the ligand-binding specificity of AR, but no detailed structural and/or functional characterization of the mutants has been carried out.

In addition to V903M mutation in a person with PAIS, five other mutations (M895T, I898T, P904S, P904H, L907F) within helix 12 of AR have thus far been reported in patients with CAIS (2, 47, 48, 49, 50). Mutations at codon 894 demonstrated that Met is involved in the N/C interaction and also in the interaction with GRIP1; M894D showed no association with the NTD, and it was not activated by GRIP1 in either the N/C interaction assay or as Gal4-LBD. As a full-length receptor, the corresponding mutant is transcriptionally inactive (43). Even though the LBD containing the M894A substitution was totally unresponsive to GRIP1, it retained about one-half of the wild-type N/C interaction. This result is in line with the finding that the transcriptional activity of the corresponding full-length form is reduced only by 20% (43). Bevan et al. (51) have recently shown that mutation of both M894 and the neighboring M895 to Ala abolishes the activity of AR. However, mutagenesis of single amino acid residues was not performed in that study. Mutation of A896 to Leu or Val reduced the N/C terminal interactions to one third of wild-type level. As LBDs, both A896L and A896V harbored very little response to GRIP1, but similar to M894A, their interaction with NTD was rescued by coexpressed GRIP1. In agreement with our results, He et al. (11) have recently demonstrated that several AR LBD substitutions, such as I898T (helix 12) and K720A (helix 3), weaken the interaction of LBD with both GRIP1/TIF2 and NTD. I898T exhibits attenuated N/C interaction but interacts well with GRIP1/TIF2, whereas K720A has lost the ability to bind the p160 coactivator but retained the domain interaction activity (11). The behavior of the latter mutant is similar to that of M894A in this study. Thus, the surfaces of AR LBD for GRIP1/TIF2 and NTD interaction overlap but are not identical.

The data of this work, together with the results obtained by mutagenesis of other amino acids of AR LBD found in AIS (11, 52), support the notion that disrupted N/C interaction is a potential molecular defect in many AIS patients, in whom the LBD mutations have not resulted in severe impairment in androgen-binding properties. Many of these substitutions may also alter the ability of AR to interact with coactivators such as those of the p160 family. It should be pointed out, however, that defect(s) in coactivator protein(s) can also lead to CAIS, as illustrated in a recent study (53).


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Plasmid Constructions
For studies using the mammalian two-hybrid system, the pM-LBD that expresses Gal4 DBD fusion of wild-type human AR LBD (amino acids 624–919) has been described (7). The individual point mutations were incorporated into the LBD using the Quick Change Site-Directed Mutagenesis Kit (Stratagene, La Jolla, CA). The following upper and lower oligonucleotides (mutated codons underlined) were used:

PCR was performed for 12 cycles in accordance with the manufacturer’s instructions. Mutation incorporation was checked by sequencing both strands using the Pharmacia ALFexpress DNA sequencing system (Amersham Pharmacia Biotech, Buckinghamshire, UK). As a negative control we created an AF2 region deletion mutant termed pM-LBD-TRUNC (residues 624–889) that was created using PCR and the following primers: upper, 5'-CGTAGGATCCGGATG624ACTCTGGGAGCCCGGA-3'; lower, 5'-TGACGTCGAC-TCA889CACGCTCACCATGTG-3'. Upper primer creates a BamHI site at amino acid residue 624 (underlined) and lower primer creates a SalI site at amino acid residue 889 (underlined). The PCR fragments were inserted into BamHI/SalI-cleaved pM vector (CLONTECH Laboratories, Inc., Palo Alto, CA). The LBD helix 12 (core AF2) mutants fused to GAL4 DBD were created using single-stranded mutagenic primers corresponding to amino acid residues 890–902 of AR (43). The pVP16-rAR-(5–538), which expresses amino acid residues 5–538 of rAR, has been described previously (10). Full-length AR mutants were created by digesting the pM-LBD mutants with Tth111I and XbaI and inserted the fragment into the Tth111I/XbaI site of full-length human AR expression vector pCMV-hAR (54). For in vitro studies, full-length mutant sequences were transferred into pSG5 vector (Stratagene) by removing a Tth111I/BamHI sequence from a pSG5-hAR construct (32) and ligating the corresponding restriction fragment from the pCMV-hAR mutant forms. All sequences were confirmed by Pharmacia ALFexpress DNA sequencing system. pPB(-285/+32)-LUC containing nucleotides -285 to +32 of the rat probasin promoter driving firefly luciferase coding region has been described (34). pCMV-RelA and p{kappa}B6tk-LUC were kindly provided by Dr. Patrick Baeuerle (55). pG5-LUC was obtained from Promega Corp. (Madison, WI) and pSG5-GRIP1 was a gift from Dr. Michael Stallcup.

Cell Culture and Transfections
COS-1 cells (from American Type Culture Collection, Manassas, VA) were maintained in DMEM containing penicillin (25 U/ml), streptomycin (25 U/ml), and 10% FBS. Transfections were performed using FuGENE 6 reagent (Roche Molecular Biochemicals, Mannheim, Germany) according to the manufacturer’s instructions. In brief, 30 x 103 COS-1 cells were seeded on 12-well plates 24 h before transfection. Four hours before transfection cells received fresh medium containing 10% charcoal-stripped FBS. One hundred fifty nanograms of reporter plasmid, 50 ng of pCMVß (CLONTECH Laboratories, Inc.), and indicated amounts of expression constructs were transfected. Eighteen hours after transfection, the cells received fresh medium containing 2% charcoal-stripped FBS and 100 nM T or vehicle. After a 30-h culture, the cells were harvested, and then lysed in reporter lysis buffer (Promega Corp.), after which the luciferase (LUC) and ß-galactosidase activities were assayed as described previously (10).

Immunoblot Analysis of Extracts from Transfected Cells
Transfected COS-1 cells were washed twice with PBS and the pellets were resuspended in SDS-PAGE sample buffer, heated at 95 C for 5 min, and analyzed on 10% or 12% SDS-polyacrylamide gels. The proteins were transferred onto a nitrocellulose membrane (Amersham Pharmacia Biotech). Membranes were then immunoblotted with a mouse monoclonal Gal4 DBD antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or with an antiserum K333 raised against full-length rat AR (56) as needed. The membranes were subsequently incubated with either a horseradish phosphatase-conjugated goat antimouse or antirabbit IgG antibody (Zymed Laboratories, Inc., South San Francisco, CA), and immunocomplexes were detected using the ECL Western blot reagents (Amersham Pharmacia Biotech) according to the manufacturer’s instructions.

Partial Proteolytic Digestion of in Vitro-Translated Human AR Mutant Constructs
[35S]Methionine-labeled ARs were translated using 0.5 µg cDNA in pSG5 backbone and the TNT T7 Quick Coupled Transcription/Translation System (Promega Corp.) according to the manufacturer’s instructions in the presence of 25 µM ZnCl2 at 30 C for 90 min. Twenty-five microliters of the resulting proteins were incubated for 10 min in the presence of 100 nM T or vehicle at 30 C before being digested with trypsin (6 ng/µl). Samples were taken at time points 0 min and 25 min. The reactions were stopped by adding 1.5 µl of the digestion mixture to 23.5 µl of 2 x SDS-PAGE sample buffer, followed by a 5-min incubation at 95 C. Samples were analyzed on 10% SDS-PAGE gels. After the electrophoresis, the gels were fixed in methanol (45%, vol/vol)-acetic acid (10%, vol/vol), treated with Amplify (Amersham Pharmacia Biotech), dried, and visualized by fluorography.

EMSA
For preparation of whole cell extracts, 100 x 103 COS-1 cells on six-well plates were transfected with 1.5 µg of wild-type or mutated AR cDNAs (pCMV-hAR) using the FuGENE 6 reagent. Twenty-four hours after transfection, the cells received fresh medium with or without 100 nM T and were grown for additional 24 h. Subsequently, the cells were washed twice with 2 ml of ice-cold PBS and harvested, after which whole-cell extracts were prepared by using a buffer containing 20 mM Tris-HCl (pH 7.5), 400 mM KCl, 15% (vol/vol) glycerol, 1 mM EDTA, 2 mM dithiothreitol, and 1:200 protease inhibitor cocktail (P-8340, Sigma, St. Louis, MO ) with or without 10-7 M T. Aliquots of cell extracts (10 µg of protein) were taken for DNA-binding reactions performed at 22 C as described previously (57). Alternatively, the different AR forms were produced by translation in vitro as described above. After a 30-min incubation at 22 C in the absence or presence of 100 nM T, 32P-labeled C3(1)-ARE element (32) was added to reactions, and incubations were continued for 60 min. The mixtures were then loaded onto a 4% nondenaturing polyacrylamide gel (29:1), which had been prerun at 200V for 30 min. Electrophoresis was carried out in 0.25 x Tris-borate EDTA (TBE) at 22 C for 135 min. The gel was subsequently dried and subjected to autoradiography.

Whole-Cell Steroid Binding Assay
For whole-cell steroid-binding assays, 30 x 103 COS-1 were seeded and transfected with 0.4 µg of each pCMV-hAR mutant expression vector. Twenty-four hours after transfection, cells received fresh DMEM medium containing 10% charcoal-stripped FBS and were grown for an additional 24 h. Subsequently, the cells were washed once with PBS and the medium changed to DMEM without serum containing a range of [3H]mibolerone concentrations (0.1–60 nM). For measurements of nonspecific binding, duplicate wells contained [3H]mibolerone with a 200-fold molar excess of nonradioactive mibolerone. After 2 h of incubation at 37 C and 5% CO2, medium was collected for radioactivity measurements, and the cells were washed three times with ice-cold PBS (1 ml/well), harvested, and collected by centrifugation at 2,000 x g for 10 min at 4 C. The cell pellets were resuspended in 0.5 M NaOH and incubated at 56 C for 15 min. The radioactivity of the solubilized pellets and collected media was measured, and the method of Scatchard was employed to determine binding constants essentially as described by Isomaa et al. (58).


    ACKNOWLEDGMENTS
 
The excellent technical assistance of Leena Pietilä, Birgit Tiilikainen, Seija Mäki, Kati Saastamoinen, and Pirjo Kilpiö is gratefully acknowledged. We thank Thomas Slagsvold for AF2 constructs and Michael Stallcup for the GRIP1 expression plasmid.


    FOOTNOTES
 
Address requests for reprints to: Jorma J. Palvimo, Ph.D., Biomedicum Helsinki, Institute of Biomedicine/Physiology, P.O. Box 63 (Haartmaninkatu 8), University of Helsinki, FIN-00014 Helsinki, Finland. E-mail: jorma.palvimo{at}helsinki.fi

This work was supported by grants from the Medical Research Council (Academy of Finland), the Finnish Foundation for Cancer Research, Norwegian Cancer Society, the Sigrid Jusélius Foundation, Biocentrum Helsinki, the Helsinki University Central Hospital, and the Association for the Cure of Cancer of Prostate (CaP CURE).

Received for publication December 27, 2000. Accepted for publication February 27, 2001.


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