Hoechst Marion Roussel, Inc. (C.R.-J., D.G.,
B.B.,
A.V., C.O., D.P.) F-93235 Romainville Cedex, France
Laboratoire de Biologie Structurale (J.-M.W., B.G., D.M.)
IGBMC, CNRS/INSERM/ULP/Collège de France F-67404 Illkirch
cedex, France
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In this work, we have extended our previous strategy (12 ) aimed at defining residues in the LBD of steroid receptors that are responsible for selective ligand recognition and AF2-induced activity. This strategy is based on the assumption that all NRs share a common fold, as has been confirmed upon crystallization of NR LBDs (13 14 ). Swapping homologous sequences between NR LBDs would therefore not alter the overall structure, but reveal residues involved in ligand binding specificity. Indeed, the present study suggests that a very limited number of residues, some of which are outside the ligand-binding pocket, determine glucocorticoid and progestin selectivity. These data are discussed in the view of the human progesterone receptor (hPR) crystal structure and the derived homology models of the GR and mutated LBDs.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
|
The hGR and hPR LBD sequences display 57% identity, the helical
part being more conserved, with the exception of helix H7 (Fig. 1). The
residues lining the ligand binding pocket of hPR, most of which are
hydrophobic, are conserved in hGR, with the exception of a small number
of residues (see Fig. 1
). Most of the variations observed between hGR
and hPR are accompanied by compensatory changes, e.g. Ile756
(helix H12) is facing Val571 (helix H3) and Trp600 (helix
H5) in hGR whereas the equivalent residue in hPR, Val912
(helix H12) is facing Leu726 (helix H3) and Trp755 (helix H5) (Fig. 1
).
The most variable regions are located in helix H5, loop 67, helix H7,
and in the region between helices H11 and H12. The segment encompassing
helices H6 and H7 corresponds to cassette 3, which is analyzed in more
detail below.
Distinct Sequences of GR and PR Determine Agonistic Ligand
Selectivity
The GR agonist DEX was able to bind to the GP3 chimera in the
presence of block g3 (641-DQ) alone or in combination with g4 and g5
(GP3g34 or GP3g345). In the presence of block g2, g5, or g6, no DEX
binding was observed. Both residues of block g3 were involved in DEX
binding, but only the GP3-L642Q mutant was able to transactivate in
response to DEX and therefore fully capable of reproducing the behavior
of GP3g3 (see Partial Dissociation of Ligand Binding and
Transactivation below) (Tables 2 and 3
).
RU 27987 was able to bind all the GP3 mutants except GP3g2
(635-TLPCM). However, this binding only induced transactivation in
GP3g3 and GP3g5 chimeras (see Partial Dissociation of Ligand
Binding and Transactivation below). The analysis of block g2
showed that two residues (Ser637 and Phe639) were implicated in the
lack of RU 27987 binding to GP3g2. Indeed,
GP3-S637P/F639M, like GP3g2, did not bind RU 27987 (Tables 2 and 3
).
Antagonists Are Marginally Affected by the GR-PR
Mutants
In contrast to agonists, the antagonist ligands (RU 38486 and RU
43044) displayed no major differences in binding upon block
substitution(s). In particular, RU 38486 was capable of binding to all
the chimeric receptors. The glucocorticoid antagonist RU 43044 bound to
hGR, but not to hPR or GP3. Introducing any of the hGR blocks except
block g2 restored binding. Residues in block g3 (641-DQ), g4 (644-KH),
g5 (647-LYVSS), and g6 (653-LHR) were shown to be involved in RU43044
binding. The presence of an aspartate and glutamine residue at
positions 641 and 642, respectively, were important for RU43044 binding
since GP3-S641D and GP3-L642Q were able to bind RU43044 (compare GP3g3,
GP3-S641D, GP3-L642Q, GP3g34, GP3g345; Table 2). However, the GP3-L642Q
mutant displayed a greater affinity for RU 43044 than did GP3-S641D.
Moreover, a leucine residue at position 647 (block g5) was also
necessary for RU 43044 binding (GP3g5, GP3g56, and GP3-W647L; Table 2
),
whereas mutants with a tryptophan at position 647 were unable to bind
RU 43044 (GP3g5-L647W and GP3g56-L647W, Table 2
). In addition, the
presence of a phenylanine residue at position 653 (block 6) was also
required for RU 43044 binding. Indeed, GP3g6 was able to bind RU 43044,
in opposition to GP3 and GP3-F653L (Table 2
).
Partial Dissociation of Ligand Binding and Transactivation
Our study revealed more clearly the residues involved in
transactivation. For example, the presence of a glutamine residue at
position 642 in block g3 was sufficient to induce transactivation in
response to DEX (GP3-L642Q, Table 3). The introduction of block g5
(647-LYVSS) enhanced DEX-induced transactivation (compare GP3g34 and
GP3g345, Table 3
). The single leucine residue at position 647 was
mostly responsible for the enhanced transcription in the context of
block g5 upon DEX binding (compare GP3-W647L, GP3g34, GP3g345, and
GP3g345-L647W; Table 3
). Note that despite the weak affinity of DEX for
GP3-W647L, this receptor exhibited a strong transactivation
capacity.
Residues in block g3 could also contribute to RU
27987-dependent transcriptional activity. Actually, Gal-hPR and
GP3g3 display the same scale of transcriptional activity in response to
RU 27987 binding. Nevertheless, the role of this block
needs further clarification since no RU
27987-induced transcriptional activity could be observed in
GP3g34 (Table 3). In addition, the presence of both Ser641 and Leu642
was necessary to induce transactivation (Table 3
). Beside block g3,
block g5 was suggested to be a determinant for RU
27987-dependent transactivation. Indeed, the mutants exhibiting
transactivation in response to RU 27987 binding contained
block g5 (GP3g5, GP3g56, and GP3g345; Table 3
). GP3g345 even displayed
an enhanced activation upon binding of RU 27987 when
compared with GAL-hPR, probably due to complex interactions of blocks
g3 and g5 with the ligand. Again, a leucine at position 647 was
sufficient to gain wild-type behavior for binding and transactivation
(compare GP3 that binds RU 27987 but does not
transactivate and the single mutant GP3-W647L; Table 3
). Most of the
L647W mutants were not activated by RU 27987 (GP3g5-L647W,
GP3g56-L647W; Table 3
). Only the GP3g345-L647W mutant exhibited a
RU 27987-induced transcriptional activity, probably as a
consequence of the presence of block g3. Indeed, Gal-hPR, GP3g3, and
GP3g345-L647W showed the same level of transcriptional activity in
response to RU 27987 binding (Table 3
).
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Two Residues (Ser637 and Phe639) Are Involved in RU
27987 Binding
The GP3 chimera, unlike GR, displayed RU 27987
binding as a consequence of the substitution of block g2 (635-TLPCM) by
block p2 (KESSF) (Table 2). More precisely, the presence of Ser637 and
Phe639 was directly responsible for this ligand binding. The double
mutation GP3-S637P/F639M was sufficient to inhibit binding of RU
27987, whereas each single mutation did not affect the affinity
for RU 27987. A similar moderate effect on progesterone
binding has been observed for the hPR-S792A mutant (position 637 of
hGR) (15 ).
These data are consistent with the proposed three-dimensional model of
GP3 (Fig. 3) in which block 2 is close to the ligand-binding pocket,
suggesting that any mutation in this segment could potentially affect
the receptors capacity to bind the ligand. Note that residue 639
points toward the ligand cavity, unlike residue 637, which is located
at the surface of the protein. The presence of a proline at this later
position introduces a greater rigidity to this region, resulting most
likely in a decrease of RU 27987 binding. However, the
affinity of the point mutants for RU 27987 was decreased
when compared with GP3, suggesting that additional residues outside p2
contribute to the proper positioning of the ligand in the binding
pocket.
Ligand Discrimination and Transactivation Modulation by Leu647
Introducing block g5 (647-LYVSS) in GP3 led to mutants able to
discriminate between the two glucocorticoid ligands, RU 43044 and DEX
(GP3g5, GP3g56), whereas they conserved the capacity to bind the
progestin ligand RU 27987 (Table 2). The leucine residue
at position 647 was shown to be mostly responsible for the selectivity
provided by block g5 toward RU 43044. In contrast, in the context of
block g5, mutants with a tryptophan residue at this position did not
bind RU 43044 (compare GP3, GP3g5, GP3-W647L, and GP3g5-L647W).
Interestingly, no difference in ligand specificity is observed between
GP3g34, GP3g345, and GP3g345-L647W. The ability of these receptors to
bind RU 43044 is most likely due to the presence of block g3, which
seems to play a dominant role; its presence always leads to
glucocorticoid (DEX and RU 43044) binding. However, residues in block
g4 (644-KH) and g6 (653-LHR) were also involved in discriminating
between the two glucocorticoid ligands.
Note that all the chimeric receptors studied were able to bind RU
38486, but did not always bind RU 43044 (Table 3), suggesting that the
region accommodating the 11ß moiety (RU 38486) was conserved
throughout the chimeric receptors studied, unlike the one responsible
for 19-aryl moiety (RU 43044) binding (16 ). This is in agreement with
results indicating that Gly722 of hPR is the only major requirement for
RU 38486 binding to steroid receptor. This residue is conserved in hGR
(567) and hAR (708) but not in hMR or hER (17 ).
The presence of Leu647 (block g5) instead of a tryptophan was also
necessary for RU 27987-induced transactivation of GP3g5
and GP3g56 (Table 3). The L647W mutation in GP3g345 decreased, but did
not abolish transactivation in response to both RU 27987
and DEX, probably due to the presence of g3 residues (Table 3
; see
below). Note that a good transactivation level in GP3-W647L is achieved
despite a weak affinity observed for DEX. This could result of high DEX
doses (1 µM) used in transactivation assay, leading to
saturation of the receptor. Altogether, these results show that the
hydrophobic amino acid at position 647 is critical for determining
ligand selectivity and transactivation capacity. Since Met639 separates
this residue from the ligand binding pocket in hGR, it can only act
indirectly, affecting other residues in its vicinity (i.e.
Leu621, Met634, Met639, and Met646) through steric mediated
interactions (Fig. 3
).
The Leu642Gln Mutant Acquires Glucocorticoid Binding and
Concomitantly Restores Transactivation Capacity
In the GP3 model, Ser641 points toward the surface of the protein
whereas Leu642 is partially accessible to the solvent and is in Van der
Waals contact with the ligand as observed in the hPR complex (7 ). A
glutamine residue at this position could either be involved in hydrogen
bonds with the ligand or point toward the surface providing some extra
space so that bulkier ligands could be accommodated as agonists.
Glucocorticoid binding and transactivation could be restored by the
mutation L642Q (Tables 2 and 3
). Both residues of block g3 (641-DQ)
were similarly involved in DEX binding, but only GP3-L642Q restored
transactivation upon glucocorticoid binding (Tables 2
and 3
). However,
the affinity of the mutants for DEX and RU 43044 was decreased when
compared with GAL-hGR, suggesting that residues outside this block also
contribute to the proper positioning of the ligand in the
ligand-binding pocket. Actually, residues at position 647 (block 5) and
653 (block 6) were implicated in RU 43044 binding. These results are
consistent with data showing a wild-type transactivation efficiency
upon DEX binding for hGR-D641V and a 3-fold decreased affinity for 1
µM DEX (18 ), suggesting a minor role for this residue in
determining the DEX affinity for hGR as well as the DEX-induced
transactivation. However, as shown in Table 3
, the transactivation
efficiency of GP3g3 was 2-fold higher than that observed with
hGR-L642Q, highlighting the role of residue 641 in maximal
transactivation capacity.
All the mutants displaying an affinity for DEX were also able to bind
RU 27987. The mixed progestin/glucocorticoid specificity
observed with GP3g3, GP3g34, and GP3g345 (Table 2) could be explained
by the simultaneous presence of residues Ser637/Phe639 (block p2) and
residues from block g3 (Figs. 1
and 3
).
Despite the fact that most of the single mutations behaved similarly to their cognate blocks, the results obtained from the combination of different blocks did not always match the predictions of binding and transactivation properties based on the additive effect of single mutants, suggesting a more complex network of interactions. Some simple conclusions can nevertheless be made: 1) residues at positions 642 (block 3) and 647 (block 5) are crucial when activation is considered, 2) residues at positions 637 and 639 (block 2) as well as positions 642 (block 3) and 647 (block 5) are important for ligand binding specificity.
Implication in Steroid Receptor Ligand Binding Specificity and
Their Transactivation Capacity
Binding of the cognate ligand in the AR, GR, MR, and PR receptors
involves the recognition of the A-ring C3-ketone group present in all
the natural ligands. This group is anchored by two highly conserved
glutamine and arginine residues among these receptors (Gln570 and
Arg611 in hGR) as revealed in the progesterone complex (7 ). The
residues contacting the D-ring moieties in helix H3 and the loop1112
region are rather conserved between PR, GR, and MR, unlike the AR (Fig. 1; Asn564, Cys736, Thr739 of hGR) (19 ).
In contrast, residues encompassing helices 6 and 7, differ notably
among the steroid NRs and most likely determine the size/shape of the
ligand-binding pocket responsible for the ligand selectivity.
Especially residue 647, as in hGR, is of great importance in delimiting
the size/length of the binding cavity. This residue is located on the
opposite face of the receptor relative to H12. A smaller or bulkier
residue at position 647 could influence the orientation of the
activation helix, which may be displaced from its active position. This
may be achieved either by disrupting contacts between this helix and
the ligand, as has been shown for progestins in the MR context (19 ), or
by pushing H12 away, as in the ER/raloxifene antagonist complex
(5 ).
Altogether, these data suggest that the presence of a leucine or
histidine residue in hGR and hMR, respectively (Fig. 1), instead of a
tryptophan in hPR, alters the compactness of the binding cavity. This
specific amino acid difference in the LBD might be responsible for the
antagonistic activity of progestins on the GR and MR receptors and for
their agonist action on PR.
In conclusion, the GR-PR chimera LBD swap experiments described in this
work revealed that a region encompassing helices H6 and H7 is the
principal determinant of progestin and glucocorticoid binding
specificity. Among the 34 residues belonging to the identified cassette
(cassette 3; Fig. 1), five residues are shown to be involved either in
ligand binding specificity (residues at positions 637, 639, and 641 in
hGR) and/or in the transcriptional capacity of the chimeric receptors
(residues at positions 642 and 647 in hGR). Thus, residue at position
642 is involved in the binding and signal transduction of
glucocorticoids. Furthermore, the present data suggest also that the
residue at position 647 of hGR may play a pivotal role in conferring
the capacity to respond to ligands as agonists. The recently solved
crystal structures of the ER
/estradiol and PR/progesterone complexes
revealed the anchoring of steroid ligands to their cognate receptor (5 7 ). The present study is complementary to those results and provides an
additional insight on how NR LBDs, as exemplified by the steroid
family, embody ligand specificity and modulate transcriptional capacity
in response to ligand binding.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Three-Dimensional Model Construction
The hGR model has been generated with the academic version of
the MODELLER package (version 4.0) (21 ). The homology model
construction is based on the sequence alignment shown in Fig. 1 and was
initially based on the hRAR
crystal structure and then recalculated
taking the recently solved PR structure as a template. All the loops
were kept as generated by MODELLER. Most of the residues (98%) had
their backbone dihedral angles located in the favored or most favored
region as calculated by PROCHECK (22 ). In addition, the side
chains were all in a favorable conformation suggesting that,
altogether, the three-dimensional GR model is satisfactory for the
mutant analysis described in this work. The purpose of this study was
to analyze the spatial position of the mutants in the light of the
three-dimensional model to understand the importance of the different
blocks (see Results) and infer which residue(s) in each
block (p1 to p6 or g1 to g6 for hPR and hGR, respectively; Fig. 1
) are
most likely involved in binding. No further optimization was done. The
graphics packages used are O (23 ) and Quanta (Quanta 96 4.1/Charmm
23.1, MSI Inc., San Diego, CA).
The GP3 model has been obtained by replacing cassette 3 of hGR with the equivalent residues of hPR. GP3 follows the hGR numbering.
Construction of GAL-hGR and GAL-hPR
DNA coding for the hinge region and LBD of hGR (amino acids
682777) (24 ) and hPR (amino acids 632933 of hPRB isoform) (25 ) was
PCR amplified using Deep Vent DNA polymerase (New England Biolabs, Inc.Beverly, MA) from hG0 and hPR1 (kind gift of
Dr. H. Gronemeyer). The following oligonucleotides were used as primers
(XhoI and BamHI sites are
underlined):
hGR primer: 5'-ATTCCTCGAGCTA1456TGAACCTGGAAGCTCGA-3'
hGR reverse primer: 5'-CCATGGGGATCCT2317CACTTTTGATGAAACAG-3'
hPR primer: 5'-ATTCCTCGAGCTA1893TGGTCCTTGGAGGTCGA-3'
hPR reverse primer: 5'-CCATGGGGATCCT2785CACTTTTTATGAAAGAG-3'
DNA fragments amplified with these primers were inserted into the pG4Mpoly expression vector (Ref. 26 , kind gift of Dr. H. Gronemeyer), which contains the coding sequence for the first 147 amino acids of the yeast transcription factor GAL4, corresponding to its DBD. In the resulting GAL-hGR and GAL-hPR recombinant vectors, DNA coding for the hinge region and LBD were in frame cloned with DNA coding for GAL4 DBD.
Construction of GAL-hGR5c, GAL-hPR5c, and Chimeric
Receptors
Four restriction sites (BclI, BglII,
PstI, and BstXI) were introduced at homologous
positions after deletion of one PstI site on GAL-hGR and
deletion of one BclI site and one BstXI on
GAL-hPR. The insertion of these restriction sites left unchanged the
coding sequence of hGR and hPR. These mutations were introduced using
the double PCR technique. The sense oligonucleotides used for the
double PCR technique are indicated below (restriction sites are
underlined):
hGR, BclI+, PstI: 5'-C1765TGGATGATCAAATGACCCTATTGCAGTACTCC-3'
hGR, BglII+: 5'-C1861TGTGTTTTGCTCCAGATCTGATTATTAAT-3'
hGR, PstI+: 5'-G1954AGTTACACAGGCTGCAGGTATCTTATGAA-3'
hGR, BstXI+: 5'-T2143TTTATCAACTGACCAAACTCTTGGATTCT-3'
hPR, BclI: 5'-A2050TTCCACCACTGATTAACCTGTTAATGAGC-3'
hPR, BclI+: 5'-T2224TACATATTGATGATCAGATAACTCTCATT-3'
hPR, BglII+: 5'-C2326TGTATTTTGCACCAGATCTAATACTAAAT-3'
hPR, PstI+: 5'-G2419AGTTTGTCAAGCTGCAGGTTAGCCAAGAA-3'
hPR, BstXI+: 5'-T2611ATCAACTTACCAAACTTCTGGATAACTTGCAT-3'
Chimeric receptors were obtained by swapping cassettes. Introduction of the third cassette of GAL-hGR in GAL-hPR leads to GP3 chimera.
Mutation of at least three consecutive amino acid residues in GP3 has been done by replacing the PstI-BglII cassette with a synthetic cassette containing the appropriate mutations (bold). The following oligonucleotides, and their complementary strands, were used for substitution of the P3 cassette of GP3 after annealing and digesting with PstI and BglII (underlined):
g2: 5'-AGATCTAATACTAAATGAACAGCGGATGACTCTACCCTGCATGTATTCATTATGCCTTACCATGTGGCAGATCCCACAGGAGTTTGTCAAGCTGCAG-3'
g5: 5'-AGATCTAATACTAAATGAACAGCGGATGAAAGAATCATCATTCTATTCATTATGCCTTACCATGCTGTATGTTTCCTCTGAGTTTGTCAAGCTGCAG-3'
g34: 5'-AGATCTAATACTAAATGAACAGCGGATGAAAGAATCATCATTCTATGACCAATGTAAACACATGTGGCAGATCCCACAGGAGTTTGTCAAGCTGCAG-3'
g345: 5'-AGATCTAATACTAAATGAACAGCGGATGAAAGAATCATCATTCTATGACCAATGTAAACACATGCTGTATGTTTCCTCTGAGTTTGTCAAGCTGCAG-3'
g6: 5'-AGATCTAATACTAAATGAACAGCGGATGAAAGAATCATCATTCTATTCATTATGCCTTACCATGTGGCAGATCCCACAGGAGTTACACAGGCTGCAG-3'
g56: 5'-AGATCTAATACTAAATGAACAGCGGATGAAAGAATCATCATTCTATTCATTATGCCTTACCATGCTGTATGTTTCCTCTGAGTTACACAGGCTGCAG-3'
GP3 mutants containing less than three point mutations were obtained by site-directed mutagenesis using the Transformer Site-Directed Mutagenesis Kit (CLONTECH Laboratories, Inc., Palo Alto, CA) according to the instructions of the manufacturer. For all the mutations, HindIII primer (5'-GGCGAATTCAAGCTTGAAGCAAGC-3', HindIII site underlined) was used as selection primer. The following oligonucleotides were used as mutation primers (mutated codons underlined):
g3: 5'-CATCATTCTATGACCAATGCCTTACCATG-3'
g4: 5'-CTATTCATTATGCAAACACATGTGGCAGATCC-3'
S637P: 5'-GATGAAAGAACCCTCATTCTATTC-3'
F639M: 5'-GAAAGAATCATCAATGTATTCATTATGC-3'
S641D: 5'-CATCATTCTATGACTTATGCCTTACCATG-3'
L642Q: 5'-CATCATTCTATTCACAATGCCTTACCATG-3'
W647L: 5'-GCCTTACCATGCTGCAGATCCCAC-3'
F653L: 5'-CCCACAGGAGTTAGTCAAGCTGC-3'
S637P/F639M: 5'-GATGAAAGAACCCTCAATGTATTCATTATGC-3'
g345-L647W: 5'-GTAAACACATGTGGTATGTTTCC-3'
g5-L647W: 5'-GCCTTACCATGTGGTATGTTTCC-3'
The resulting constructs, GAL-GR and GAL-PR, as well as GP3 mutants were controlled by sequencing.
Cell Culture
HeLa and COS-1 cells were maintained in DMEM (Life Technologies, Inc., Gaithersburg, MD) containing 4.5 mg/ml
glucose supplemented with 400 µM L-glutamine
(Sigma, St. Louis, MO), 500 µM pyruvate
(Life Technologies, Inc.), 20 U/ml penicillin
(Sigma), 20 µg/ml streptomycin (Sigma), and
10% FCS (Multiser, Cytosystems, Castle Hill, Australia). For
transactivation and binding assays, cells were grown in the same medium
with the exception of FCS replaced by dextran-coated charcoal-treated
serum. Culture were maintained at 37 C in a humidified atmosphere of
5% CO2.
Transfection
COS-1 and HeLa cells were transfected by the calcium phosphate
coprecipitation procedure as described previously (27 ) except for the
2 x HEPES-buffered saline solution (280 mM NaCl, 50
mM HEPES, 1.5 mM
Na2HPO4, pH 7.12).
Transactivation Assay
For transfections, 2 x 105 HeLa
cells were seeded in six-well plates. After 24 h, cells were
transfected with receptor coding vector (17 mer)5-glob-luc (Ref. 28 ,
kind gift of Dr. P. Balaguer) and CMV-ßGal (kind gift of Dr. H.
Gronemeyer), 0.1 µg each. The total amount of DNA was adjusted to 3
µg with pBluescript II KS+
(Stratagene, La Jolla, CA). After 18 h, cells were
treated with vehicle or hormones (1 µM) from a 1
mM stock in ethanol. After a further 24 h, cells were
lysed in 25 mM Tris-phosphate, pH 7.8, 1 mM
EDTA, 10 mM MgCl2, 15% glycerol, 1%
Triton X-100, 2 mM dithiothreitol, 0.2 mM
phenylmethylsulfonylfluoride.
Determination of luciferase activity was performed using the Luciferase Assay System according to the instructions of the manufacturer (Promega Corp., Madison, WI). ß-Galactosidase activities were determined with HeLa extracts using the Galacto-Light reporter assay according to the instructions of the manufacturer (Perkin-Elmer Corp., Norwalk, CT). Luciferase and ß-galactosidase activities were measured on a Top Count Scintillation Counter (Packard Instruments, Meriden, CT). Results were expressed as arbitrary luciferase units relative to arbitrary ß-galactosidase units, to correct possible variations in transfection efficiencies.
Ligand Binding Assay
COS-1 cells transfected in 162-cm2 flasks
with 40 µg of the chimera encoding DNA were harvested 72 h later
using PBS/EDTA 5 mM. After homogenization in 10
mM Tris, pH 7.4, 250 mM sucrose, 0.1
mM phenylmethylsulfonylfluoride, 20 mM
dithiothreitol, cells were centrifuged at 105,000 x g
for 30 min. The supernatant obtained was used as follows: 125-µl
aliquots of the cytosol were incubated for 24 h at 4 C with
increasing concentrations (0.5 to 20 nM) of the
relevant tritiated ligand. Nonspecific binding was evaluated in
parallel incubations with the 3H-ligand in the
presence of a 100-fold excess of the corresponding nonlabeled compound.
Separation of bound and unbound ligand was achieved by the
dextran-charcoal method. Briefly, a 100-µl aliquot of incubated
cytosol was stirred for 10 min with an equal volume of a
dextran-charcoal (0.625%1.25%) suspension and centrifuged for 10
min at 800 x g. The bound radioactivity of a 100-µl
supernatant sample was counted. Scatchard plot analysis was used to
determine the association constant (Ka,
109
M-1) and the concentration
of binding sites (N, femtomoles/mg protein). For each experiment, an
assay was performed on mock-transfected cells to deduce specific
binding (Ne) due to endogenous PR and GR (100 fmol/mg protein). The
number of transiently expressed specific binding sites (N-Ne) is
obtained by subtracting the number of endogenous specific binding sites
(Ne) from the total number of specific binding sites (N). An
association constant (Ka,
109
M-1) value was calculated
when N-Ne > 100 fmol/mg protein. We assumed that a weak binding
(w) was present when 50 < N-Ne < 100 fmol/mg protein;
however, the observed binding could not clearly be attributed to
chimera as a consequence of the presence of endogenous receptors. No
binding (-) was observed when N-Ne < 50 fmol/mg protein. In our
experiments, RU 38486 can be considered as a proof of correct
expression level. Indeed, a Ka could always be
calculated for this ligand with all the mutants studied. This excludes
the hypothesis of low expression level for the mutants displaying weak
binding.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
FOOTNOTES |
---|
This work was supported by Grant BIO2-CT930473 from the EC BIOTECH.
1 Present address: Laboratoire de cristallographie et
modélisation des matériaux minéraux et biologiques,
boulevard des Aiguillettes, BP239, F-54506 Vandoeuvre les Nancy cedex,
France.
Received for publication June 28, 1999. Revision received March 15, 2000. Accepted for publication March 21, 2000.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|