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Discordant Measures of Androgen-Binding Kinetics in Two Mutant Androgen Receptors Causing Mild or Partial Androgen Insensitivity, Respectively¹

Lady Davis Institute for Medical Research (D.L.S, L.K.B., L.P., M.A.T.), Sir M. B. Davis-Jewish General Hospital, Montreal, Quebec, Canada H3T 1E2; the Departments of Medicine (L.P., M.T.), Biology (L.P.), Human Genetics (L.P.), and Pediatrics (G.P., L.A., L.P.), McGill University, Montreal, Quebec, Canada H3A 1B1; Montreal Children’s Hospital (G.P., L.A.), Montreal, Quebec; Canada H3H IP3; and the Division of Endocrinology and Metabolism, University of Alberta (J.G.), Edmonton, Alberta, Canada T6G 2S2

Address all correspondence and requests for reprints to: Dr. Mark Trifiro, Lady Davis Institute, Sir M. B. Davis-Jewish General Hospital, 3755 Cote Street, Catherine Road, Montreal, Quebec, Canada H3T 1E2.

	Abstract

Top Abstract Introduction Subjects and Methods Results Discussion References

 
We have characterized two different mutations of the human androgen receptor (hAR) found in two unrelated subjects with androgen insensitivity syndrome (AIS): in one, the external genitalia were ambiguous (partial, PAIS); in the other, they were male, but small (mild, MAIS). Single base substitutions have been found in both individuals: E772A in the PAIS subject, and R871G in the MAIS patient. In COS-1 cells transfected with the E772A and R871G hARs, the apparent equilibrium dissociation constants (K_d) for mibolerone (MB) and methyltrienolone are normal. Nonetheless, the mutant hAR from the PAIS subject (E772A) has elevated nonequilibrium dissociation rate constants (k_diss) for both androgens. In contrast, the MAIS subject’s hAR (R871G) has k_diss values that are apparently normal for MB and methyltrienolone; in addition, the R871G hAR’s ability to bind MB resists thermal stress better than the hAR from the PAIS subject. The E772A and R871G hARs, therefore, confer the same pattern of discordant androgen-binding parameters in transfected COS-1 cells as observed previously in the subjects’ genital skin fibroblasts. This proves their pathogenicity and correlates with the relative severity of the clinical phenotype. In COS-1 cells transfected with an androgen-responsive reporter gene, trans-activation was 50% of normal in cells containing either mutant hAR. However, mutant hAR-MB binding is unstable during prolonged incubation with MB, whereas normal hAR-MB binding increases. Thus, normal equilibrium dissociation constants alone, as determined by Scatchard analysis, may not be indicative of normal hAR function.

An increased k_diss despite a normal K_d for a given androgen suggests that it not only has increased egress from a mutant ligand-binding pocket, but also increased access to it. This hypothesis has certain implications in terms of the three-dimensional model of the ligand-binding domain of the nuclear receptor superfamily.

	Introduction

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CONSTITUTIONAL human androgen receptor (hAR) mutations in 46,XY persons cause inadequate responses to androgen in various target organs, resulting in an array of phenotypes that constitute the androgen insensitivity syndrome (AIS) (1, 2, 3). Our classification has three categories (4): complete, when the external genitalia are female; partial (PAIS), when they are clinically ambiguous; and mild (MAIS), when they are male. At puberty, subjects with PAIS usually have gynecomastia, a high pitched voice, reduced sex hair, and impaired spermatogenesis. Those with MAIS may have some of these characteristics.

Amino acid substitution mutations in all three functional domains of the hAR may produce PAIS or MAIS (5, 6, 7, 8, 9). Virtually all of those in the ligand-binding domain (LBD) either reduce otherwise normal androgen binding (2) or yield defective androgen binding. The latter is usually expressed by a combination of abnormal biochemical properties, including a decreased affinity constant (increased K_d), an increased rate of androgen dissociation (k_diss), reduced androgen binding under the challenge of thermal stress (thermolability), or failure to up-regulate binding during prolonged incubation with androgen.

In this report we characterize two missense hAR mutations (E772A; R871G) and by DNA transfection studies document the concomitance of normal K_d 1) with abnormal k_diss values for different androgens in the E772A hAR, 2) normal k_diss values in the R871G hAR, and 3) decreased trans-activation properties for both mutants. We also document dysfunction of mutant hARs by demonstrating greater instability of their androgen-binding activity during prolonged incubation with androgen at 37 or 42 C (E772A > R871G). The implications of these data are considered in the context of the three-dimensional model recently proposed for the LBD of the nuclear receptor superfamily.

	Subjects and Methods

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Subjects

Subjects 3287 and 4007 were previously described in detail by Kaufman et al. (10).

PCR mutagenesis

Each mutation was recreated in the PSVhAR.BHEX expression vector (11, 12) by the overlap extension method (13) as previously described (14) using appropriate primers. Details are available upon request. To verify that the appropriate mutations were incorporated and that extraneous mutations were not, the insert was sequenced beyond the ligation points.

Cell culture and transfections

COS-1 cells and genital skin fibroblasts (GSF) were routinely maintained in Opti-MEM (Life Technologies, Burlington, Canada) with 5–10% FCS. COS-1 cells were electroporated as previously described (15), pooled, and plated at densities of 3 x 10⁵ cells/well in 24-well plates or 6 x 10⁵ cells/35-mm plate. One or 2 µg normal, E772A, or R871G plasmid were used per transfection in binding experiments, whereas for trans-activation experiments, 2 µg pMMTV-GH (MMTV, mouse mammary tumor virus) were transfected with 1 µg of each of the normal or mutant or plasmids. In all experiments, 2 µg pCMV-ß-galactosidase (CMV, cytomegalovirus) was included to control for transfection efficiency. Transfectants were assayed for androgen-binding activity 48 h later or 76 h later in the case of the GH assay.

Androgen binding assays

Tritiated androgens used were: mibolerone (MB; 7,17-dimethyl-19-nortestosterone; 80.6 Ci/mmol) and methyltrienolone (MT; 17ß-hydroxy-17-methyl-estra-4,9,11-triene-3-one; 83 Ci/mmol; Amersham, Oakville, Canada). The radioinert androgens used were MB and MT (DuPont, Mississauga, Canada). Androgen-binding activity was determined as previously described (14, 16).

Apparent equilibrium dissociation rate constants (K_d)

Quadruplicate wells of transfected COS-1 cells were labeled in MEM supplemented with 100 µM cycloheximide (Sigma Chemical Co., St. Louis, MO) and 0, 0.19, 0.38, 0.75, 1.5, or 3 nM [³H]androgen for 2 h at 37 C. Duplicate wells were labeled with the corresponding amount of hormone plus a 200-fold excess of radioinert androgen. Results were plotted as bound/free androgen vs. bound androgen and K_d values calculated from the slope of the Scatchard plots.

Nonequilibrium dissociation constants (k_diss)

Forty-eight hours after transfection, quadruplicate wells of transfected COS-1 cells were labeled with 3 nM [³H]androgen, and duplicate wells were labeled with 3 nM [³H]androgen plus a 200-fold excess of radioinert androgen for 2 h at 37 C. The incubation media were then replaced by media containing only a 200-fold excess of radioinert androgen as a chase. Cells were harvested at 0, 30, 60, 90, and 120 min and assayed for androgen-binding activity. Androgen-binding activities were plotted semilog-arithmically as a percentage of the androgen-binding activity remaining at each time point. The k_diss values were determined directly from the slopes of the lines.

Thermolability of hAR-MB complexes

Quadruplicate wells of transfected COS-1 cells were labeled with 3 nM [³H]MB and duplicate wells with 3 nM [³H]MB plus a 200-fold excess of radioinert MB at 37 C for 2 h to allow for the formation of hAR-MB complexes. One set of plates was assayed after the initial incubation time for androgen-binding activity. For other time points, medium in each well was replaced by fresh medium differing only in that it contained 100 µM cycloheximide. Binding assays were performed after 2, 4, and 6 h of incubation at 37 or 42 C.

GH assay

COS-1 cells were cotransfected with 1 µg mutant or normal pSVhAR.BHEX plasmid, 2 µg pCMV-ß-galactosidase, and 10 µg of the reporter construct pMMTV-GH (17). Four hours later, MEM and 10% FCS with 0, 0.15, 0.35, 0.6, 1.2, 2.5, or 5 nM [³H]MB, or the corresponding concentration of [³H]MB plus a 200-fold excess of radioinert MB were added to duplicate wells of a 24-well plate. Seventy-two hours after androgen addition, secreted hGH (50 µL sample) was quantitated using an immunoassay kit (Nichols Institute Diagnostics, San Juan Capistrano, CA), and the androgen-binding activity of the cells was determined.

Western blot analysis

Forty-eight hours after transfection, transfectants in duplicate 60-mm dishes (Becton Dickson Canada, Saint-Laurent, Canada) were incubated in MEM containing [³H]MB and 10% FCS or in the same medium without hormone for 2, 4, 8, and 18 h at 37 C. Cells were harvested, and 100 µg protein were subjected to SDS-PAGE and Western blotting as previously described (16). A monoclonal antibody F39.4.1 (18), directed to hAR amino acids 305–324 [numbered according to the method of Lubahn (19)], was used to detect hAR, whereas an anti- tubulin monoclonal antibody was used as a control for protein loading (Amersham, Arlington Heights, IL).

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Identification of mutations

Direct PCR sequencing revealed a single point mutation in the hAR-coding sequence in the GSF from each subject. An A to C transversion at nucleotide 2677 in exon 5 of the LBD in subject 3287 corresponds to a substitution of an alanine for a conserved glutamic acid at codon 772 (E772A; Fig. 1A). This mutation was also present in the mother and maternal half-sister of subject 3287 (not shown). An A to G transition was found in exon 8 at nucleotide 2973 in subject 4007, causing a glycine residue to be substituted for an unconserved arginine residue (R871G; Fig. 1B). This mutation was also found in the subject’s heterozygous sister 4008.

View larger version (36K): [in this window] [in a new window]   Figure 1. The location and context of the E772A (A) and R871G (B) mutations are shown compared to the amino acid sequences of the human progesterone (hPR), glucocorticoid (hGR), and mineralocorticoid (hMR) receptors. Dashes indicate conserved amino acids. Boxes outline the ß-turn and helixes of the structure predicted for the ligand-binding domain of the nuclear receptor superfamily (26 ); the open circles indicate residues in the immediate vicinity of the ligand.

Each mutation was incorporated separately into the pSVhAR.BHEX expression vector by PCR mutagenesis (13). The presence of the desired mutations was confirmed by DNA sequencing before their use in functional studies. Androgen binding assays were performed with each mutant hAR in transfected COS-1 cells. Normal levels of androgen-binding activity were obtained with both E772A and R871G, in accord with the data on GSF from 3287 and 4007 (10).

Apparent equilibrium dissociation rate constants (K_d) and nonequilibrium dissociation rates (k_diss)

Upon transfection in COS cells the E772A and R871G mutant hARs were found to have K_d values of 0.3–1 nM with all ligands tested (Table 1). In normal hAR experiments, K_d values ranged from 0.2 mM to 0.5 nM. In almost half of the experiments the confidence intervals of both normal and mutants clearly overlap.

To obtain maximal MB-binding activity, transfectants were exposed to MB at 37 C for 4 h. When shifted to 42 C in the presence of MB and 100 µM cycloheximide for 6 h, the E772A and R871G mutant hARs lost 84% and 72% of their MB-binding activities, respectively; normal hARs lost only 33% of their MB-binding activity (Fig. 2, bottom). In the control experiment, where the cells were incubated at 37 C, neither mutant receptor lost MB-binding activity at a greater rate than the normal hAR (Fig. 2, top).

View larger version (32K): [in this window] [in a new window]   Figure 2. Thermolability of hAR-MB complexes formed with MB in normal, E772A, and R871G COS-1 transfectants. Normal, E772A, or R871G mutant hARs were allowed to form complexes with MB at 37 C, then were incubated with MB in the presence of cycloheximide at 37 C (top panel) or 42 C (bottom panel) for 2, 4, or 6 h. hAR-MB complexes formed in the E772A and R871G transfectants were more thermolabile than normal at 42 C. Bars are the mean of MB-binding activity for four experiments for normal, E772A, and R871G hARs; error bars represent the SEM. Experiments on the same line were performed simultaneously.

After 72 h in the presence of various concentrations of [³H]MB, the E772A and R871G mutant hARs trans-activated the GH reporter gene to a lesser extent than the normal hAR (63–73% and 45–61% of normal, respectively; Fig. 3, top). In one experiment, the E772A hAR performed better than the R871G hAR; in another experiment, they were equally able to trans-activate the reporter gene. In terms of GH secreted per fmol MB bound/mg protein (Fig. 3, bottom), however, both mutants appeared to possess normal trans-activation potential.

View larger version (20K): [in this window] [in a new window]   Figure 3. Trans-activation potential of hARs in normal, E772A, and R871G COS-1 transfectants. Trans-activation potential for normal, E772A, and R871G hARs was measured as the ability of the hAR-MB complexes to activate transcription of GH from a cotransfected mouse mammary tumor virus-GH reporter gene. A, The E772A and R871G mutant hARs were inferior at trans-activating the reporter gene compared to normal hAR at given MB concentrations. B, Trans-activation potential of the normal, E772A, and R871G hARs were comparable when expressed as GH produced per fmol MB bound/mg protein.

Normal, E772A, and R871G transfectants were labeled with [³H]MB over 18 h. At various times MB-binding activity was determined, and cell lysates were harvested for Western analysis. Comigration of E772A and R871G mutant hAR protein with the normal hAR protein revealed that these mutant hARs were of normal size (110 kDa; Fig. 4). After exposure to MB for 18 h at 37 C, E772A and R871G hARs had lost 40% and 22% of the initial MB-binding activity, respectively, whereas the normal cells had gained 30%. Despite the concurrent loss of MB-binding activity over 18 h, there was no detectable loss of mutant hAR proteins as determined by densitometric analysis of the Western blots. Under the same conditions, normal hAR gave MB-binding activity that increased over 18 h with no apparent gain of immunoreactive protein.

View larger version (78K): [in this window] [in a new window]   Figure 4. Immunoblotting and MB-binding activity for normal, E772A, and R871G hARs in COS-1 transfectants. Cellular lysates were harvested from transfectants at various times after incubation with MB. MB-binding activity was determined concurrently (normal, 460; E772A, 580; R871G, 587 fmol/mg protein). Blots were reacted with the anti-hAR antibody F39.4.1 and subsequently with the monoclonal anti- tubulin antibody (yielding a band of 55 kDa) to control for equal protein loading.

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

The normal K_d values, despite increased k_diss values of E772A and R871G, thus appear discordant in contrast to the usual concordant relationship between K_d and k_diss; this merits further consideration. Increased k_diss values are usually concordant with increased K_d values (12, 17, 21, 22, 23). For normal K_d values to accompany increased k_diss values, an unusual shift in the association/dissociation ratios should occur. To maintain a normal equilibrium affinity for androgens, the increased off-rate of androgen from the mutant receptor should be compensated by an equally increased on-rate of the androgen to its receptor (10). Exchange studies suggest that this may indeed be the case. The E772A hAR in transfected COS cells exchanged MB and MT twice as fast as normal hAR (data not shown). The R871G hAR, whose k_diss values for MB and MT are normal, exchanged ligands at a rate equal to a normal receptor. The V866M mutation (15), which also has elevated k_diss rates and an abnormal K_d, demonstrates a slightly elevated exchange rate (1.8 x normal), but far less than the increased k_diss. In this case, an inability to raise the on-rate sufficiently will result in an abnormal K_d. Thus, simple determination of ligand affinity, as performed by Scatchard analysis (24), may not be an accurate indicator of steroid receptor function; additional kinetic and functional assays must be performed to assess the degree of impairment in the case of a suspected pathogenic mutation.

In point of fact we have shown that 1) both mutant hARs when complexed to MB in transfected COS-1 cells exhibited increased thermolability, E772A more so than R871G. Previously unbound mutant hAR were not thermolabile (data not shown); 2) after incubation with MB for 18 h, the R871G and E772A mutant hARs lost significant MB-binding activity, whereas the normal hAR was able to augment its MB-binding activity. Western blotting experiments revealed one immunoreactive band for normal and mutant hARs, indicating that proteolysis was not a factor in the loss of MB-binding activity. Thus, it appears that the mutant hARs progressively adopt a nonbinding conformational state upon prolonged incubation with MB, which does not appear to occur with the normal AR. 3) Both E772A and R871G mutant hARs were less capable of trans-activating a reporter gene than the normal hAR in terms of GH produced, but were indistinguishable from normal in terms of GH produced per unit ligand bound. Any putative differences in trans-activation potential between E772A and R871G, however, did not manifest themselves under the experimental conditions used.

The recent x-ray analysis of the LBD crystal structures of retinoid X (RXR), retinoic acid, thyroid hormone-1, estrogen, and progesterone (PR) receptors have shed some light on the possible conformational changes induced by ligand binding. Based on the crystallographic data of RXR-LBD and retinoic acid receptor--LBD, Wurtz et al. (20) have constructed a structural alignment of LBD sequences for steroid receptors (Fig. 5). According to their model, the ligand-binding pockets of nuclear receptors exhibit a common architecture and undergo similar transformation upon ligand binding. More recently, Brzozowski et al. (25) and Williams and Sigler (26) have complete x-ray crystallographic studies of the LBD of the estrogen receptor- and the PR. Here again, the overall structures of both remarkably resemble those of previous members of the steroid receptor superfamily. The canonical nuclear receptor LBD is composed of 12 -helixes and 2 antiparallel ß-strands. The RXR- and PR lack helix 2. Upon ligand binding it is proposed that helixes 10 and 11 become continuous, releasing helix 12, which then undergoes a large conformational movement, covering or acting as a lid to the ligand-binding pocket. Thus, a normal reactive receptor "cages" its ligand. A defective receptor may bind a ligand, but not undergo postligand conformational changes, and thus may not cage the ligand, allowing for faster ligand egress. This may explain the abnormal kinetics of both our mutants. E772A lies C-terminal in LBD helix 6, near a ß-strand, in close proximity to the active binding site. During ligand binding, helix 6 is proposed to interact with a loop, allowing for helix 12 to be repositioned over the active binding site. A mutation in helix 6 could thus prevent this series of conformational steps from occurring. It also lies near an area responsible for interactions with the 3-keto group of steroid ligands. R871G is at the C-terminal end of helix 10, which may interfere with the ability of helix 12 to move appropriately, allowing for certain ligands to be released and others not. The phenotypic differences between the MAIS and PAIS subjects could reflect the degree of departure from normal conformational changes their respective mutant hARs undergo upon ligand binding. Crystallographic studies of the hAR LBD could well provide insight into the unusual combination of normal K_d with abnormal k_diss in these mutant receptors.

View larger version (32K): [in this window] [in a new window]   Figure 5. Schematic representation of a nuclear receptor ligand-binding domain showing the location of the hAR E772A and R871G mutations (adapted from Ref. 32). The amino- (N) and carboxyl- (C) terminals of the LBD are indicated. The -helixes (H1 and H3-H12) are represented by boxes, and the ß-strands (S1 and S2) forming the ß-turn are shown by shaded arrows.

	Acknowledgments

 
We thank Marie Vasiliou for the initial identification of the R871G mutation, Kay Berckmans and Rhona Rosenzweig for secretarial assistance, and Dr. Robert Kaufhold for gathering the 3287 pedigree.

	Footnotes

 
¹ This work was supported by funds from the Medical Research of Canada, Fonds de la Recherche en Santé du Québec, and the Fonds pour la Formation de Chercheurs et l’Aide à la Recherche du Québec.

Received March 4, 1998.

Revised October 14, 1998.

Accepted November 2, 1998.

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