Cloning and Expression of the Glucocorticoid Receptor from the ...

0021-972X/97/$03.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1997 by The Endocrine Society

Vol. 82, No. 2 Printed in U.S.A.

Cloning and Expression of the Glucocorticoid Receptor from the Squirrel Monkey (Saimiri boliviensis boliviensis), a Glucocorticoid-Resistant Primate* PHILIP D. REYNOLDS, STEVEN J. PITTLER,

AND

JONATHAN G. SCAMMELL

Departments of Pharmacology (P.D.R., J.G.S.), Comparative Medicine (J.G.S.), and Biochemistry and Molecular Biology (S.J.P.), University of South Alabama College of Medicine, Mobile, Alabama 36688 ABSTRACT New World primates such as the squirrel monkey have elevated cortisol levels and glucocorticoid resistance. We have shown that the apparent binding affinity of the glucocorticoid receptor in squirrel monkey lymphocytes is 5-fold lower than that in human lymphocytes (apparent Kd, 20.9 ⫾ 1.8 and 4.3 ⫾ 0.2 nmol/L, respectively; n ⫽ 3), consistent with previous studies in mononuclear leukocytes isolated from the two species. As a first step in understanding the mechanism of decreased binding affinity in New World primates, we used reverse transcription-PCR to clone the glucocorticoid receptor from squirrel monkey liver and have compared the sequence to receptor sequences obtained from owl monkey liver, cotton-top tamarin B95-8 cells, and human lymphocytes. The squirrel monkey glucocorticoid receptor is approximately 97% identical in nucleotide and amino acid sequence to the human receptor. The ligand-binding domain (amino acids 528 – 777) of the squirrel monkey glucocorticoid receptor contains four amino acid differences (Ser551 to Thr, Ser616 to Ala, Ala618 to Ser, and Ile761 to Leu), all of which are present in owl monkey and cotton-top tamarin receptors. The DNA-binding domain (amino acids 421– 486) is completely conserved among human, squirrel monkey, owl monkey, and cotton-top tamarin receptors. Twenty-two differences from the

human sequence were found in the N-terminal region (amino acids 1– 421) of the squirrel monkey receptor. None of the substitutions in the ligand-binding domain matched mutations known to influence binding affinity in other species. To determine whether the substitutions per se were responsible for decreased affinity, squirrel monkey and human glucocorticoid receptors were expressed in the TNT Coupled Reticulocyte Lysate System. Expressions of human and squirrel monkey glucocorticoid receptors and a squirrel monkey receptor in which Phe774 was mutated to Leu (F774L) were similar. When expressed in the TNT System, squirrel monkey and human glucocorticoid receptors had similar, high affinity binding for dexamethasone (apparent Kd, 5.9 ⫾ 1.2 and 4.3 ⫾ 0.5 nmol/L, respectively; n ⫽ 3), whereas the squirrel monkey F774L receptor had lower affinity binding (apparent Kd, 20.4 ⫾ 2.0 nmol/L; n ⫽ 3). Thus, substitutions within the ligand-binding domain of the squirrel monkey glucocorticoid receptor cannot account for the decreased binding affinity of these receptors in squirrel monkey cells. Rather, the binding affinity is probably influenced by the expression of cytosolic factors that affect glucocorticoid receptor function. (J Clin Endocrinol Metab 82: 465– 472, 1997)

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and affinity for cortisol (6), and 3) a decreased rate of clearance of cortisol from the circulation (5, 7). Squirrel monkeys, however, do not show clinical signs of glucocorticoid excess; they have normal levels of plasma electrolytes (Na⫹, K⫹, and Cl⫺) (4). Thus, these markedly elevated cortisol levels are likely to have arisen to compensate for a generalized glucocorticoid hyposensitivity. Such elevated cortisol levels are shared with other New World primates, such as the common marmoset (Callithrix jacchus), the cotton-top tamarin (Saguinus oedipus), and, to a lesser extent, the owl monkey (Aotus sp.) (3). The mechanisms responsible for glucocorticoid hyposensitivity in New World primates are not fully understood and are under active investigation. Chrousos et al. (3) demonstrated a greater than 20-fold higher apparent Kd for the glucocorticoid receptors in squirrel monkey mononuclear leukocytes than in human cells, suggesting that a decreased binding affinity may contribute to glucocorticoid resistance. Additional information has been obtained from B95-8 cells, which are an Epstein-Barr virus (EBV)-transformed cell line derived from the cotton-top tamarin (8). Brandon et al. (9) used this cell line to clone and sequence the cotton-top tamarin glucocorticoid receptor and found a number of alterations compared with the human receptor, including an additional arginine within the DNA-binding domain and three amino

LUCOCORTICOIDS play an essential role in regulating a number of developmental and physiological processes. Disruption of glucocorticoid action is incompatible with life (1). Cellular sensitivity to glucocorticoids is influenced by a number of factors, including the level of glucocorticoid receptors, their affinity for ligand, their rate of translocation to the nucleus after activation, and their ability to transactivate a response (2). Disruption at each of these levels can lead to decreased sensitivity to glucocorticoids, most often leading to compensatory increases in plasma cortisol levels (2). Some animal species of the Americas have elevated plasma cortisol levels and have been proposed as models for the in vivo study of glucocorticoid sensitivity (2, 3). One of the most striking examples is the squirrel monkey (Saimiri sp.) (3), in which 100-fold higher free plasma cortisol levels are maintained by 1) elevated ACTH and cortisol synthesis (4, 5), 2) a corticosteroid-binding globulin with decreased capacity Received September 16, 1996. Revision received November 1, 1996. Accepted November 5, 1996. Address all correspondence and requests for reprints to: Jonathan G. Scammell, Ph.D., Department of Pharmacology, University of South Alabama, Mobile, Alabama 36688. * This work was supported in part by NIH-National Center for Research Resources Grant RR-01254.

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acid differences in the ligand-binding domain. More recently, this group demonstrated that the cytosol of B95-8 cells contains an inhibitor of glucocorticoid receptor binding that may also contribute to lower binding affinity (10). It has been reported that B95-8 cells overexpress the ␤ isoform of the glucocorticoid receptor (2), an isoform of the receptor that does not bind hormone but inhibits normal glucocorticoid induction of gene transcription (11, 12). However, it is possible that transformation and long term culture of B95-8 cells may have caused the altered expression of factors that influence glucocorticoid sensitivity. Thus, it is not yet known whether the hyposensitivity of New World primates to glucocorticoids is due to structural changes in the receptor protein or the expression of factors that affect binding affinity and/or transcriptional activation. In this study, our goal was to determine whether changes in the primary structure are responsible for the decreased affinity of the squirrel monkey glucocorticoid receptor. We developed a squirrel monkey B lymphoblast cell line that expresses a glucocorticoid receptor with low binding affinity, similar to that seen in freshly prepared mononuclear leukocytes from squirrel monkey. We cloned and sequenced the squirrel monkey glucocorticoid receptor and compared it to sequences of the receptor from other New World primates. Finally, we examined the binding affinity of the squirrel monkey receptor expressed in an in vitro transcription-translation system free of factors potentially operating to alter affinity in vivo. Materials and Methods Cell lines B95-8 cells were obtained from the American Type Culture Collection (Rockville, MD). A human EBV-transformed B lymphoblast cell line (HL) (13) was kindly provided by Dr. David D. Brandon (Oregon Health Sciences University, Portland, OR). A permanent squirrel monkey B lymphoblast cell line was established by transformation of mononuclear cells from a male squirrel monkey with EBV as previously described (14). These cells were CD20 positive and exhibited a karyotype consistent with the subspecies Saimiri boliviensis boliviensis. All cell lines were maintained in RPMI 1640 (Life Technologies, Grand Island, NY) supplemented with 10% FCS (Hyclone Laboratories, Logan, UT), 50 U/mL penicillin G, and 0.05 mg/mL streptomycin (Sigma Chemical Co., St. Louis, MO).

Intact cell binding studies Intact cell binding was performed essentially as previously described (13). Human and squirrel monkey EBV-transformed lymphocytes were adjusted to 1 ⫻ 106 cells/mL in fresh medium and incubated in a shaking incubator with 0 – 40 nmol/L [3H]dexamethasone (83 Ci/mmol; Amersham Corp., Arlington Heights, IL) in the presence and absence of 20 ␮mol/L radioinert dexamethasone (Sigma) at 25 C for 4 h. Cells were then washed three times with 5 mL ice-cold phosphate-buffered saline and resuspended in 0.5 mL phosphate-buffered saline. Five milliliters of scintillation cocktail were added to each tube, and the samples were counted. Data were analyzed by nonlinear regression analysis and visualized by the method of Scatchard (15) using GraphPad PRISM version 2.0 software (GraphPad Software, San Diego, CA). Differences between means were analyzed by Student’s t test.

with RNA STAT-60 (Tel-Test “B”, Friendswood, TX). Tissues were provided by the Tissue Resource of the University of South Alabama Primate Research Laboratory. For first strand cDNA synthesis, 2 ␮g total RNA were combined with 5 ␮g oligo(deoxythymidine)15, denatured at 75 C for 5 min, and annealed at room temperature for 10 min. Reverse transcription was carried out by a modification of the Reverse Transcription System (Promega Corp., Madison, WI) in a 20-␮L reaction containing 69 U AMV reverse transcriptase, 1 mmol/L of each deoxynucleotide, 20 U ribosomal RNAsin, 5 mmol/L MgCl2, 50 mmol/L KCl, and 0.1% Triton X-100 in 10 mmol/L Tris-HCl (pH 8.8) at 42 C for 1 h. Reactions were terminated by incubation at 75 C for 10 min, and the products were stored at ⫺20 C.

PCR amplification and thermal cycle dideoxy-DNA sequencing Primers for PCR and sequencing were selected based on homologous regions of the human (16), cotton-top tamarin (9), guinea pig (17), rat (18), and mouse (19) glucocorticoid receptor sequences (Table 1). PCR reactions were performed in 25 ␮L containing 50 ng of each primer, 10 –50 ng template cDNA, 2.5 U Taq polymerase (Promega), 2.5 U Taq Extender (Stratagene, La Jolla, CA), 0.2 mmol/L of each deoxy-NTP in 10 mmol/L KCl, 10 mmol/L (NH4)2SO4, 2 mmol/L MgSO4, 0.1% Triton X-100, 0.1 mg/mL nuclease-free BSA, and 20 mmol/L Tris-HCl (pH 8.8). Each reaction was overlayed with 35 ␮L mineral oil and denatured for 3 min at 95 C. PCR was performed in a Robocycler Gradient 40 (Stratagene) for 30 cycles, with each cycle consisting of 1 min at 95 C, 1 min at 55 C, and 2 min at 72 C. A final incubation of 8 min at 72 C completed the primer extension reaction. Full-length sequences of the glucocorticoid receptor cDNAs from squirrel monkey and owl monkey liver and human and B95-8 lymphoblasts were obtained with a Circumvent Thermal Cycle Dideoxy DNA Sequencing Kit (New England Biolabs, Beverly, MA). The reaction mixtures of 15 ␮L contained 150 ng 2.4 kilobases double stranded cDNA, 10 ng primer (Table 1), 2 U VentR (exo⫺) DNA polymerase, and 25 ␮Ci [␣-35S]deoxy-ATP (1500 Ci/mmol; New England Nuclear, Boston, MA) in 10 mmol/L KCl, 10 mmol/L (NH4)2SO4, 5 mmol/L MgSO4, 0.2% Triton X-100, and 20 mmol/L Tris-HCl (pH 8.8). After the addition of aliquots (3.2 ␮L) to tubes containing individual dideoxy-NTPs, the samples were overlaid with 20 ␮L mineral oil, heated to 95 C for 5 min, and then subjected to 95 C for 1 min, 55 C for 1 min, and 72 C for 1.5 min for 20 cycles. Stop/loading dye solution was added, and the reactions were heated to 80 C for 2 min followed by electrophoresis on 6% polyacrylamide-7 mol/L urea sequencing gels.

In vitro transcription and translation Primers for cloning were generated by the addition of BamHI and XbaI sites to GR-7 and GR-6 (Table 1), respectively. Full-length cDNAs were generated from total RNA from human lymphoblasts and squirrel monkey liver in 50 ␮L containing 50 ng template cDNA, 400 ng each of modified GR-7 and GR-6 primers, 1.25 U cloned Pfu DNA polymerase (Stratagene), and 0.2 mmol/L of each deoxy-NTP in 10 mmol/L KCl, 10 TABLE 1. Synthetic oligonucleotide primers used in PCR and sequencing reactions Sequence

Positiona

5⬘-GCCACTACAGGAGTCTCACAA-3⬘ 5⬘-GTGATGATTTCAGCTAACATCT-3⬘ 5⬘-GCAGGGTAGAGTCATTCTCTGCTC-3⬘ 5⬘-GAGCCAAGAGCTATTTGATG-3⬘ 5⬘-AGGCAACCATTCTTATTAAGGCAG-3⬘ 5⬘-AGTTGATATTCACTGATGGAC-3⬘ 5⬘-GGAGGACAGATGTACCACTATGACATG-3⬘ 5⬘-TTGTGAGACTCCTGTAGTGGC-3⬘ 5⬘-GTCATAGTGGTACATCTGTCCTCC-3⬘ 5⬘-TGGGAATCCCAGGTCATTTCC-3⬘ 5⬘-AGGAGAGCTTACATCTGGTCTCAT-3⬘ 5⬘-GGAAATGACCTGGGATTCCCACAG-3⬘

1639–1659 2405–2384 2046–2023 2175–2194 2490–2467 118–138 1114–1140 1659–1639 1137–1114 447– 427 1347–1324 427– 450

Ribonucleic acid (RNA) isolation and complementary DNA (cDNA) synthesis

GR-2 GR-3 GR-4 GR-5 GR-6 GR-7 GR-9 GR-10 GR-12 GR-13 GR-14 GR-15

Total RNA was isolated from human lymphocytes and squirrel monkey (S. boliviensis boliviensis) and owl monkey (Aotus trivirgatus) liver

a The numbers indicate the position of the sequences within the human cDNA (16).

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mmol/L (NH4)2SO4, 2 mmol/L MgSO4, 0.1% Triton X-100, 0.1 mg/mL BSA, 5% dimethylsulfoxide, and 20 mmol/L Tris-HCl (pH 8.8). Each reaction was overlaid with 60 ␮L mineral oil and denatured for 3 min at 95 C. Conditions for PCR were 30 cycles of 1 min at 95 C, 1 min at 55 C, and 6 min at 72 C. After digestion with BamHI and XbaI, each PCR product was subcloned into the BamHI-XbaI-cut pGEM-7Zf(⫹) expression vector (Promega) to yield human glucocorticoid receptor (hGR)pGEM7 and squirrel monkey glucocorticoid receptor (smGR)-pGEM7 plasmids. To determine the proper orientation of the vector and to confirm the nucleotide sequence, the resulting constructs were sequenced by the dideoxy chain termination method (20) using the Sequenase version 2.0 DNA sequencing kit (U.S. Biochemical Corp., Cleveland, OH). Human and squirrel monkey glucocorticoid receptors were expressed in the TNT Coupled Reticulocyte Lysate System (Promega) according to the manufacturer’s instructions. After translation, to each 50 ␮L reaction were added 5 ␮L of an ATP-regenerating system (21) (50 mmol/L ATP, 250 mmol/L creatine phosphate, 20 mmol/L MgCl2, and 100 U/mL creatine phosphokinase) followed by a 20-min incubation at 30 C. Reactions were diluted 1:1 in HEDM buffer (20 mmol/L HEPES, 1 mmol/L dithiothreitol, 3 mmol/L ethylenediamine tetraacetate, and 40 mmol/L sodium molybdate, pH 7.4) and assayed for dexamethasone binding. Twenty-microliter aliquots were incubated for 16 h at 4 C with [3H]dexamethasone (0 – 40 nmol/L) in the presence and absence of 1 ␮mol/L radioinert dexamethasone. Separation of bound from unbound steroid was achieved by incubation with an equal volume of a charcoaldextran solution (0.5% Norit-A charcoal and 0.5% Dextran T-70 in HEDM buffer) at 4 C for 20 min. The data were analyzed by nonlinear regression and the Tukey-Kramer multiple comparison test. In some reactions, methionine in the translation reaction was replaced by 40 ␮Ci [35S]methionine (1000 Ci/mmol; New England Nuclear), and the translation products were separated by SDS-PAGE (22) and visualized by fluorography.

Results Intact cell binding studies

The characteristics of glucocorticoid receptor binding were determined in the HL human B lymphoblast cell line (13) and a continuous squirrel monkey B lymphoblast cell line developed by transformation of mononuclear cells with EBV. Typical saturation curves and Scatchard plots are shown in Fig. 1. The overall binding data are summarized in Table 2. The results are consistent with single classes of dexamethasone-binding sites in each cell line. The glucocorticoid receptor content was significantly higher in HL cells than in the squirrel monkey line, consistent with previous studies showing that New World primate lymphoblasts are resistant to EBV induction of glucocorticoid receptors (13, 23). More importantly, we found that the apparent equilibrium dissociation constant (Kd) of the glucocorticoid receptors in squirrel monkey lymphoblasts was significantly higher than that in HL cells. Thus, binding analysis in EBV-transformed squirrel monkey lymphoblasts reproduced previous findings in mononuclear leukocytes and cultured skin fibroblasts, showing that the squirrel monkey expresses a receptor with low binding affinity (3). Isolation and sequencing of squirrel monkey glucocorticoid receptor cDNA

The binding affinity of the glucocorticoid receptor has been shown to be affected by a number of different mutations in the ligand-binding domain (24) as well as by defective binding of heat shock proteins (2). As a first step in understanding whether mutations in the ligand-binding domain contribute to the decreased affinity of the squirrel monkey

FIG. 1. A, Typical saturation curves of [3H]dexamethasone binding to human or squirrel monkey lymphocytes; B, Scatchard plots of the glucocorticoid receptor studies. EBV-transformed human and squirrel monkey lymphocytes were adjusted to 1 ⫻ 106 cells/mL and incubated for 2 h at 25 C with the indicated concentrations of [3H]dexamethasone. Specific bound values were determined by subtracting nonspecific values from total values. The data were visualized by the method of Scatchard, using GraphPad PRISM software. HL, Human lymphocytes; SML, squirrel monkey lymphocytes. TABLE 2. Glucocorticoid receptor characteristics in human and squirrel monkey lymphocytes

Human lymphocytes Squirrel monkey lymphocytes

n

Bmax

Kd (nmol/L)

3 3

63,900 ⫾ 3,200 17,600 ⫾ 800a

4.3 ⫾ 0.2 20.9 ⫾ 1.8a

Values are the mean ⫾ SEM. n, Number of determinations; Bmax, receptor sites per cell. a P ⬍ 0.001, significantly different from the apparent Kd of the glucocorticoid receptors in human lymphocytes.

glucocorticoid receptor, we amplified and sequenced the squirrel monkey receptor. This was performed in parallel with reverse transcription-PCR of glucocorticoid receptor cDNAs from human HL cells and from owl monkey liver and cotton-top tamarin B95-8 cells, two other New World pri-

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mates with decreased receptor binding affinity and elevated cortisol levels (3, 10). Full-length cDNAs were amplified by PCR and cycle-sequenced on both strands. The strategy for sequencing the coding region is shown in Fig. 2. The nucleotide and deduced amino acid sequences of the squirrel monkey glucocorticoid receptor are shown in Fig. 3. The nucleotide sequence is approximately 97% identical to the human receptor sequence. Thirty of the 80 nucleotide differences lead to 27 changes in the deduced amino acid sequence of the squirrel monkey receptor. In Fig. 4, we compared the deduced amino acid sequences of the squirrel monkey, owl monkey, and cotton-top tamarin glucocorticoid receptors to that of the human receptor, all obtained in this study, as well as to the published guinea pig, mouse, and rat amino acid sequences (17–19). The sequence of the glucocorticoid receptor from HL cells matched that originally reported for the human receptor by Hollenberg et al. in 1985 (16). Identical sequences were obtained from cDNA from owl monkey liver and an owl monkey B lymphoblast cell line (data not shown). The ligand-binding domain (amino acids 528 –777) of the squirrel monkey receptor contains four amino acid substitutions, all of which are shared in the owl monkey and cotton-top tamarin glucocorticoid receptor (Fig. 4). All of the changes in the squirrel monkey and owl monkey receptors are conservative (Ser551 to Thr, Ser616 to Ala, Ala618 to Ser, and Ile761 to Leu). Another substitution, again conservative (Lys770 to Arg), was observed only in the cotton-top tamarin glucocorticoid receptor sequence, confirming previous analysis of this receptor (9). We found that the DNA-binding domain (amino acids 421– 486) was completely conserved among the primate receptors, including that of the cotton-top tamarin. We were unable to confirm the insertion of a complete codon for arginine at position 452 previously reported in the cotton-top tamarin receptor (9). A number of substitutions were noted in the N-terminal regions of the receptors. Most of these changes were common to all three primate species.

FIG. 2. cDNA sequencing strategy for all of the glucocorticoid receptors sequenced. The composite primate glucocorticoid receptor cDNA is presented at the top, with the numbers indicated in the upper line representing the human glucocorticoid receptor amino acid sequence. The numbers below the composite represent positions within the human glucocorticoid receptor nucleotide sequence (vertical arrows). Horizontal arrows show the direction and extent of sequencing of each of the glucocorticoid receptor cDNAs, which were sequenced in parallel. The oligonucleotides (Table 1) used to sequence are indicated above the horizontal arrows. DBD, DNA-binding domain; LBD, ligand-binding domain.

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Expression of the squirrel monkey glucocorticoid receptor

None of the amino acid changes that we detected in the ligand-binding domain of the squirrel monkey or owl monkey glucocorticoid receptors coincide with mutations in the human receptor known to affect receptor function (24). All of the amino acid changes are conservative, but even conservative mutations, such as Ile729 to Val, have been shown to significantly affect the binding affinity of the glucocorticoid receptor (25). To determine whether the substitutions in the primary sequence of the ligand-binding domain of New World primate glucocorticoid receptors result in decreased binding affinity, we examined the binding of human and squirrel monkey receptors expressed in the TNT Coupled Reticulocyte Lysate System. This or similar systems have been shown to generate high affinity rat and trout glucocorticoid receptors (21, 26). Using this system, we generated from hGR-pGEM7 and smGR-pGEM7 plasmids [35S]methionine-labeled products with apparent molecular masses of 94 and 92 kDa (Fig. 5, lanes 1 and 2), consistent with the expression of the fulllength glucocorticoid receptor and a smaller form of the receptor resulting from minor proteolysis or from translation initiation at Met27 (27). The relative abundance of the 94- and 92-kDa forms was similar for expressed human and squirrel monkey glucocorticoid receptors. No labeled products were observed when plasmid DNA was omitted from the reaction (Fig. 5, lane 3), whereas the control luciferase plasmid generated an [35S]methionine-labeled product with an approximate apparent molecular mass of 62 kDa (Fig. 5, lane 4), consistent with the expression of full-length luciferase. Binding analysis was performed on receptors expressed using the TNT System but without [35S]methionine labeling. We compared the binding of expressed human and squirrel monkey glucocorticoid receptors with that of squirrel monkey glucocorticoid receptor in which Phe774 was mutated to Leu (F774L). This mutant was generated serendipitously by a single Taq polymerase error (TTT to CTT, nucleotides 2452– 2454) and was detected during sequencing of individual squirrel monkey glucocorticoid receptor clones. Disruption of the phenyl group of Phe780 in the mouse glucocorticoid receptor (Phe774 of the human and squirrel monkey receptors) has been shown to decrease binding affinity (28). Saturation curves and Scatchard plots from a single experiment are shown in Fig. 6. The overall binding data are summarized in Table 3. The level of expression of human and squirrel monkey glucocorticoid receptors was somewhat variable, although overall there was no statistical difference in the expression of the different receptors. Consistent with the binding activity of the mouse glucocorticoid receptor mutant (F780A) (28), the squirrel monkey F774L receptor exhibited a 5-fold lower affinity for dexamethasone than that observed with expressed human receptor (Table 3). In contrast with the low affinity obtained from whole cell binding studies, squirrel monkey glucocorticoid receptor expressed in the TNT System exhibited a high affinity, similar to that of the human receptor.


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FIG. 3. Nucleotide sequence of the squirrel monkey glucocorticoid receptor cDNA and predicted amino acid sequence of the receptor protein. Nucleotides are numbered in the right margin. The sequence ATG ⫽ Met (amino acid 1) starts with nucleotide 133 according to the numbering of the human glucocorticoid receptor nucleotide sequence (16). Amino acids are numbered throughout the sequence on the upper line.

Discussion

These studies tested whether the decreased affinity of the squirrel monkey glucocorticoid receptor observed previously in isolated mononuclear leukocytes and fibroblasts (3) and confirmed in B lymphoblasts was due to amino acid substitutions in the ligand-binding domain. We sequenced

the squirrel monkey glucocorticoid receptor and found four amino acid substitutions in this region compared to the human receptor. These substitutions are shared with the glucocorticoid receptor of the cotton-top tamarin, another New World primate with similarly elevated plasma cortisol levels (3). However, the same four substitutions are found in the

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FIG. 4. Amino acid sequence comparison of squirrel monkey, owl monkey, cotton-top tamarin, guinea pig, rat, and mouse glucocorticoid receptors with that of the human receptor. Dashed lines denote amino acids that are identical to the human sequence. Asterisks indicate gaps in the sequences to accommodate additional amino acids in glucocorticoid receptors of different species. Numbers corresponding to amino acid positions for each species are indicated in the right margin.

owl monkey receptor, which in mononuclear leukocytes from owl monkey exhibited a higher binding affinity for dexamethasone than the squirrel monkey receptor (3). Using a B lymphoblastic cell line derived from owl monkey, we confirmed this higher binding affinity for the owl monkey receptor (29). In keeping with these findings, the elevation of cortisol in the owl monkey is less pronounced (3). These results suggest that, unlike the cause of some forms of glucocorticoid resistance in humans (24), the reason for the

decreased binding affinity of the squirrel monkey glucocorticoid receptor does not lie in substitutions in the ligandbinding domain of the receptor. Our in vitro transcriptiontranslation study supports this idea. When expressed in the TNT Reticulocyte Lysate System, the squirrel monkey glucocorticoid receptor had the same high binding affinity as the human receptor. The expression of a high affinity squirrel monkey receptor was not an artifact of the expression system, as a low affinity receptor was faithfully expressed and analyzed.


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FIG. 5. In vitro transcription-translation of human and squirrel monkey glucocorticoid receptors. The transcription of hGR-pGEM7 and smGR-pGEM7 plasmids was performed using the TNT Coupled Reticulocyte Lysate System followed by translation in the presence of [35S]methionine. Total lysates (5 ␮L) were resolved by SDS-PAGE and visualized by fluorography. Lane 1, Human glucocorticoid receptor; lane 2, squirrel monkey glucocorticoid receptor; lane 3, no plasmid; lane 4, luciferase control.

The reason why squirrel monkey cells show apparently low affinity glucocorticoid receptor binding is unknown. It may result from a low expression of heat shock protein 90 (hsp90) or from the expression of a mutant hsp90 (30, 31). It is well established that hsp90, in a heterocomplex with other proteins including hsp70, maintains the receptor in a high affinity state (30 –32). Glucocorticoid resistance in some human leukemic cell lines has been attributed to either the expression of an abnormal hsp90 or the low expression of hsp70 (33). Alternatively, squirrel monkeys may express other factors that affect hormone binding. Inhibitors that influence glucocorticoid and vitamin D receptor binding have been identified in cotton-top tamarin B95-8 cells (10, 34, 35). It is not yet known whether similar factors are expressed in squirrel monkey cells. The mechanisms of glucocorticoid resistance in the squirrel monkey are different from those reported for another New World mammal, the guinea pig (Cavia porcellus). The glucocorticoid receptor of the guinea pig also has low binding affinity for dexamethasone with a compensating elevation of circulating cortisol (36, 37). The ligand-binding domain of the guinea pig receptor differs at 24 residues from the human glucocorticoid receptor (17). Similar substitutions in only 4 of these residues (Gly612, Thr545, Glu672, and Leu755) in the guinea pig receptor are shared with either squirrel monkey or rat and mouse receptors, leaving 20 substitutions that might contribute to changes in receptor binding. Preliminary studies suggest that the unique substitution at position 539 (Tyr to His) is at least partly responsible for the low affinity of the guinea pig glucocorticoid receptor (39). Several differences were noted between the sequence of the glucocorticoid receptor that we obtained from cotton-top tamarin B95-8 cells and that reported previously (9). The most striking difference was our failure to confirm the insertion of an arginine in the interfinger region of the DNA-binding

FIG. 6. A, Saturation curves of [3H]dexamethasone binding to human and squirrel monkey glucocorticoid receptor and a mutant squirrel monkey glucocorticoid receptor (F774L), expressed in a TNT Coupled Reticulocyte Lysate System; B, Scatchard plots of the glucocorticoid receptor binding. Aliquots (20 ␮L) of diluted reticulocyte lysates were incubated for 16 h at 4 C with the indicated concentrations of [3H]dexamethasone. Specific bound values were determined by subtracting nonspecific values from total values. The data were visualized by the method of Scatchard using GraphPad PRISM software. SM F774L, Squirrel monkey glucocorticoid receptor in which Phe774 was mutated to Leu. TABLE 3. Receptor binding characteristics of expressed human, squirrel monkey, and squirrel monkey (F774L) glucocorticoid receptors

Human Squirrel monkey Squirrel monkey (F774L)

n

Bmax

Kd (nmol/L)

3 3 3

30.6 ⫾ 1.0 33.3 ⫾ 7.9 39.1 ⫾ 8.3

4.3 ⫾ 0.5 5.9 ⫾ 1.2 20.4 ⫾ 2.0*

Values are the mean ⫾ SEM. n, Number of determinations; Bmax, femtomoles per 5 ␮L lysate. a P ⬍ 0.001, significantly different from the apparent Kd values of human and squirrel monkey glucocorticoid receptors.

domain, although we noted most of the other reported substitutions elsewhere in the molecule. The reported insertion of an arginine at position 452 of the cotton-top tamarin receptor is intriguing because it occurs at the boundary of exon 3 and intron C of both glucocorticoid receptor genes studied (40, 41). The sequence (exon 3, GAA Ggtagtg.. intron C ..atagGA CAG,

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exon 4; donor and acceptor splice sites are in boldface) codes for Glu-Gly-Gln. If instead the second 5⬘-donor site is used, the sequence GAA GGT Agtg.. intron C ..atagGA CAG is generated coding for Glu-Gly-Arg-Gln, the sequence found in the reported cotton-top tamarin glucocorticoid receptor. We do not know the genomic structure of the cotton-top tamarin receptor, but these results suggest that the previously reported cDNA arose from alternate splicing. However, the frequency of this alternate splicing in B95-8 cells is apparently not high, as the sequence that we obtained from thermal cycle sequencing was unambiguous. Thus, glucocorticoid resistance in New World primates may result from a variety of changes in signaling pathways. It does not appear to result from substitutions in the ligandbinding domain of the glucocorticoid receptors, although alternate splicing leading to a change in the DNA-binding domain may play a role in some primate families. Rather, a change in the expression or nature of cytosolic factors important for normal glucocorticoid receptor function may have greater influence. The effect of the many substitutions in the N-terminal region on the transcriptional activity of the activated receptor has yet to be investigated.

14. 15. 16. 17. 18. 19. 20. 21.

22. 23. 24.

Acknowledgments The authors are grateful to Dr. Susan Gibson (Department of Comparative Medicine) for her help with these studies. We thank Dr. Christian Abee (Department of Comparative Medicine) for his helpful comments on the manuscript.

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