Europcatr Jourtral of Phannncology Q 1492
-
Molecular
Pharmacology
Swriotr. 227 ( 1992) 353-356
Elsevier Science Publishers B.V. All rights reserved 0922-410~/92/$OS.O0
EJPMOL X0095
Short communication
kcleotide
sequence of estrogen receptor cDNA from Sprague-Dawley rat
Elena Spreafico, Ezio Bettini, Giuseppe Pollio and Adriana Milana
Mokcular
Pharmacology
Loboraroty,
Inslimfe of Phannarolo~ical
Sciences, litritwsity
Maggi of Milan, Milatr, Ita!,
Received 24 July 1992. accepted 1 September 1992
The cDNA of tbc Sprague-Dawlcy rat estrogen receptor was scqucnccd. With rcspcct to the published Wistar rat estrogen receptor sequence, 3 single amino acid difference (tryptophan instead of asparagine) was found in the hormone binding site. Since tryptophan was found at the same position in chicken, human and mouse cslrogcn receptors, it is proposed that the Wistar rat could represent an interesting natural mutant for estrogen rcccptor studies. Estrogen rcccptors; Polymerax chain reaction; (Sprague-Dawley rat)
I. Introduction In the past few years the comprehension of the mechanism of action of steroid receptors has been facilitated by the availability of recombinant DNA technologies. To date, most of the intracellular receptors have been sequenced and a series of studies were conducted in order to clarify structural-functional relationships. A schematic view of some of the functional domains identifiable in the members of the steroid receptors family indicates that the hormone binding site is generally located at the COOH terminal, whereas the DNA binding domain is in the central position and the NH,-terminal portion is important for a number of functions among which is to be cited the interaction with transcription factors (O’Malley, 1990). With regard to the estrogen receptor, the sequences of human (Green et al., 19863, chicken (Krust et al., 1986), mouse (White et al., 1987) and Wistar rat (Koike et al., 1987) cDNA were reported recently. In the present study the sequence of the SpragueDawley rat was determined. A difference between the Wistar and Sprague-Dawley estrogen receptor sequences was found. The interest of the present study resides in the fact that the discrepancy concerns one single amino acid present in the hormone binding site
Corrcspondcncc10: Adriana Muggi. Institute of Phnrmacoloyical Sciences.Via BulzareUi 9, 2(!!2Y Milan, Italy. Tel. 3Y-2-2il4HH375: Fi~x OY-2.2Y404961,
which is very well conserved among all the species analyzed to the present time.
2. Materials and methods
2.1. Animals Female Sprague-Dawley rats (Charles River, Calco, Italy) were kept in an environmentally controlled room (ligli on from 6:00 am. to 8:00 p.m., temperature 20°C) and provided with food and drinking water ad libitum. 2.2. Total RNA preparation
and
PCR
Total cytoplasmic RNA was purified from hypothalamus or uterus of female rats according to Chirgwin ct al. (1979). Single-strand cDNA was synthssizcd from 10 pg of total RNA using oligo-dT,,_,, as described by Gubler and Hoffman (1983). The cDNA amplification was performed in a volume of 100 ~1 in the prcscncc of: 1X buffer (SO mM KCI, 10 mM Tris-KCI, 1.5 mM MgCl,, 0.01% Triton X-100 pH 9.0 a: 25°C) (Promega. Madison, WI, USA), 2.5 units of Taq polymerase (Promega, Madison, WI, USA), 200 PM of each deoxynucleotide. The oligonucleotides were added at a final concentration of 50 pmo1/100 ~1. On the basis of the published sequence (Koikc ct al., 1987), two sets of oligonucleotides wcrc synthcsizcd: \i: in the first set, the 2lmcrs were complcmentaty to the coding strand: the first Zlmcr corresponded to the region (- 179)-
a
i
0
A
C
,A 2
[3H]-ESTl?ADIOL
4
6
(nM) P
000 BOUND (fmol/mgprot) Fig. 2. [‘H]Estradiol binding in cytosol of rat hypothalamus. A: Different concentrations of [“H]E, were used in the absence (total binding, q ) or presence (non-specific binding, A 1 of cold @-estradiol. Specific binding (e) was calculated as the difference between total and non-specific binding. B: Scatchard plot of specifically bound [“HI&. B,,,, = 8.4 fmol/mg protein; K, = 0.37 nM.
amino acid, however, did not affect the hydrophilicity profile of the entire receptor protein. Since the difference found occurs in the hormone binding site (Kumar et al., 19861, a series of saturation analyses were performed in order to establish whether this mutation had any functional consequence. The results obtained are shown in fig. 2; in the Sprague-Dawley rat the estrogen receptor bound [‘HIP-estradiol with a Kd = 0.37 nM, the same affinity described for human (Green et al., 1986) and chicken (Krust et al., 1986) receptors.
4. Discussion The present study demonstrates a difference of two nucleotides between the estrogen receptor sequence of the Sprague-Dawley rat and the sequence published for the Wistar rat. The first difference occurs in the 5’ flanking sequence and should not bear any functional consequence since: this region has never been associated with any regulatory function. The second, howcvcr, occurs in the hormone binding region and could bear functional consequences since tryptophan and asparaginc may diffcrcntially influence the activity of this segment of rcccptor. Interestingly, when an intcrspccics comparison was made, it was found that in chicken (Krust ct al., 1986), mouse (White c.t al., 19871
and man (Green et al., 19863 the amino acid 488 of estrogen receptor is very well conserved and is invariably tryptophan. On the basis of this finding, it is not surprising to observe that the affinity of the SpragucDawlcy estrogen receptor is identical to the affinity reported for this receptor in other species (Green et al., 1986). Therefore, the Wistar rat seems to be the on!y one showing asparaginc at this position. The Sprague-Dawley strain originates from a male of unknown origin and a female of Wistar descent, the Sprague-Dawley stocks in existence today are practically continued as outbred population (Baker et al., 1.979). Therefore, it is unlikely that the presence of tryptophan in position 488 is due to any retromutation; it simply must have been inherited from the male ancestor. In spite of the fact that the hydrophilicity pattern of the hormone binding site is the same in the presence of both amino acids, it would still be very interesting to study in more detail the effect of the mutation present in the Wistar rat. In fact, previous studies done in the Wistar rat have reported a lower affinity (1.5 nM) of estrogen receptor for 6.‘HIPestradiol (Gesell and Roth, 1981), suggesting that the mutation described could somehow influence the estrogen receptor hormone binding activity. On the basis of the results presented, the Wistar rat could represent an interesting natural mutant for studies of the relationships between estrogen receptor structure and function.
Acknowledgements We are grateful to Simona Frigerio for her technical support. This work was partially scpported by CNR Target Projects ‘Genetic Enpinecring’ and ‘Biotechnology and Bioinstrumenlalion’.
References Baker, H.J.. J.R. Lindsey and S.H. Wcisbruth. 1’170. The Lahl~raI1)ry Rat, Vol. I (Academic Press. New York) p. 2Y. Bettini E. and A. Maggi. 1901. A rapid method for the quanritaticm of estrogen receptors in small amounts of tissue. 9. Immunol. Methods 144, X7. Bradford, H.H., lY7h, A rapid and sensitive method for quantirication of microgram quantities of prolcin ulilizinp the principle :tf protein-dye binding, Anal. Biochem. 72, 24X. Chirgwig, J.M., A.E. Przyhyla. R.J. MacDlmald and W.J. RU(hx. IY7Y. Isolation of biologically active ribimuclcic acid flom sourcec enriched in rihonucleasc, Biochemistry 18. S204. &sell, S.M. and G.S. Roth, 19x1, Decrease in rat Ulerine cslroecn receptors during aging: physic and immunochcmical properties. Endocrinology l(9Y. 1502. Green, S.. P. Waher. V. Kumar. A. Krust. J.-M. Borne& P. Argos and I’. Cltamhon. I‘)#(>. Human ocs~ro~gen rcccptor KINA: Wqucncc, cxprcssion and homology IO v-crb-A. NiIIurc Xl), 1.34. Gubkr, Ll. and B.J. !!~~ffman~ IOH3. A simple and wry cfficicnt method fur gcncr;ltltlg
cl)NA
lilrriu’lcs.
GCW 1.5. X3.
lnnw. 51.A.. K.8. hl>amh>. D.H. Grlfaud and MAD. Brown. 1988. DN.4 zequrncing with 77r~rrnrr.rorlrctrrrc-rcs DNA poiymerase and direct scqurncing of pirlymerasr chain reaction-amplified DNA. Proc. Natl. Acad. Sci. USA 85, Vf3h. Kcrikc. S.. M. Saka~ and M. Muramatsu. IYli7. Molecular cloning and ch.uacterr~atron of ran estrogen receptor cDNA. Nucleic Acids Res. 15. 1-w. Krw. A.. S. Grrn. P. Argus. V. Kumar. P. Walter. J.-M. Bornert and P. Chambon. 19X6. The chicken estrogen receptor sequence: homology with v-erh-A and the human estrogen and glucocorticoid receptors. EMBO J. 5. 891.
Kumar V.. S. Green. A. Stau and P. Chamhon. 198~5,Localization of oestriol hinding and putattve DNA hinding of the human oestrogen receptor, EMBO J. 5. 2231. O’Malley, B.. 1990. Steroid receptor superfamily: more excitement predicted ior the future. Mol. Endncrinol. 4, 363. White. R.. J.A. Lees, J. Ham and M. Parker, 1987, Structural organization and expression of the mouse estrogen receptor. Mol. Endocrinol. I. 735.
-
Molecular
Pharmacology
Swriotr. 227 ( 1992) 353-356
Elsevier Science Publishers B.V. All rights reserved 0922-410~/92/$OS.O0
EJPMOL X0095
Short communication
kcleotide
sequence of estrogen receptor cDNA from Sprague-Dawley rat
Elena Spreafico, Ezio Bettini, Giuseppe Pollio and Adriana Milana
Mokcular
Pharmacology
Loboraroty,
Inslimfe of Phannarolo~ical
Sciences, litritwsity
Maggi of Milan, Milatr, Ita!,
Received 24 July 1992. accepted 1 September 1992
The cDNA of tbc Sprague-Dawlcy rat estrogen receptor was scqucnccd. With rcspcct to the published Wistar rat estrogen receptor sequence, 3 single amino acid difference (tryptophan instead of asparagine) was found in the hormone binding site. Since tryptophan was found at the same position in chicken, human and mouse cslrogcn receptors, it is proposed that the Wistar rat could represent an interesting natural mutant for estrogen rcccptor studies. Estrogen rcccptors; Polymerax chain reaction; (Sprague-Dawley rat)
I. Introduction In the past few years the comprehension of the mechanism of action of steroid receptors has been facilitated by the availability of recombinant DNA technologies. To date, most of the intracellular receptors have been sequenced and a series of studies were conducted in order to clarify structural-functional relationships. A schematic view of some of the functional domains identifiable in the members of the steroid receptors family indicates that the hormone binding site is generally located at the COOH terminal, whereas the DNA binding domain is in the central position and the NH,-terminal portion is important for a number of functions among which is to be cited the interaction with transcription factors (O’Malley, 1990). With regard to the estrogen receptor, the sequences of human (Green et al., 19863, chicken (Krust et al., 1986), mouse (White et al., 1987) and Wistar rat (Koike et al., 1987) cDNA were reported recently. In the present study the sequence of the SpragueDawley rat was determined. A difference between the Wistar and Sprague-Dawley estrogen receptor sequences was found. The interest of the present study resides in the fact that the discrepancy concerns one single amino acid present in the hormone binding site
Corrcspondcncc10: Adriana Muggi. Institute of Phnrmacoloyical Sciences.Via BulzareUi 9, 2(!!2Y Milan, Italy. Tel. 3Y-2-2il4HH375: Fi~x OY-2.2Y404961,
which is very well conserved among all the species analyzed to the present time.
2. Materials and methods
2.1. Animals Female Sprague-Dawley rats (Charles River, Calco, Italy) were kept in an environmentally controlled room (ligli on from 6:00 am. to 8:00 p.m., temperature 20°C) and provided with food and drinking water ad libitum. 2.2. Total RNA preparation
and
PCR
Total cytoplasmic RNA was purified from hypothalamus or uterus of female rats according to Chirgwin ct al. (1979). Single-strand cDNA was synthssizcd from 10 pg of total RNA using oligo-dT,,_,, as described by Gubler and Hoffman (1983). The cDNA amplification was performed in a volume of 100 ~1 in the prcscncc of: 1X buffer (SO mM KCI, 10 mM Tris-KCI, 1.5 mM MgCl,, 0.01% Triton X-100 pH 9.0 a: 25°C) (Promega. Madison, WI, USA), 2.5 units of Taq polymerase (Promega, Madison, WI, USA), 200 PM of each deoxynucleotide. The oligonucleotides were added at a final concentration of 50 pmo1/100 ~1. On the basis of the published sequence (Koikc ct al., 1987), two sets of oligonucleotides wcrc synthcsizcd: \i: in the first set, the 2lmcrs were complcmentaty to the coding strand: the first Zlmcr corresponded to the region (- 179)-
a
i
0
A
C
,A 2
[3H]-ESTl?ADIOL
4
6
(nM) P
000 BOUND (fmol/mgprot) Fig. 2. [‘H]Estradiol binding in cytosol of rat hypothalamus. A: Different concentrations of [“H]E, were used in the absence (total binding, q ) or presence (non-specific binding, A 1 of cold @-estradiol. Specific binding (e) was calculated as the difference between total and non-specific binding. B: Scatchard plot of specifically bound [“HI&. B,,,, = 8.4 fmol/mg protein; K, = 0.37 nM.
amino acid, however, did not affect the hydrophilicity profile of the entire receptor protein. Since the difference found occurs in the hormone binding site (Kumar et al., 19861, a series of saturation analyses were performed in order to establish whether this mutation had any functional consequence. The results obtained are shown in fig. 2; in the Sprague-Dawley rat the estrogen receptor bound [‘HIP-estradiol with a Kd = 0.37 nM, the same affinity described for human (Green et al., 1986) and chicken (Krust et al., 1986) receptors.
4. Discussion The present study demonstrates a difference of two nucleotides between the estrogen receptor sequence of the Sprague-Dawley rat and the sequence published for the Wistar rat. The first difference occurs in the 5’ flanking sequence and should not bear any functional consequence since: this region has never been associated with any regulatory function. The second, howcvcr, occurs in the hormone binding region and could bear functional consequences since tryptophan and asparaginc may diffcrcntially influence the activity of this segment of rcccptor. Interestingly, when an intcrspccics comparison was made, it was found that in chicken (Krust ct al., 1986), mouse (White c.t al., 19871
and man (Green et al., 19863 the amino acid 488 of estrogen receptor is very well conserved and is invariably tryptophan. On the basis of this finding, it is not surprising to observe that the affinity of the SpragucDawlcy estrogen receptor is identical to the affinity reported for this receptor in other species (Green et al., 1986). Therefore, the Wistar rat seems to be the on!y one showing asparaginc at this position. The Sprague-Dawley strain originates from a male of unknown origin and a female of Wistar descent, the Sprague-Dawley stocks in existence today are practically continued as outbred population (Baker et al., 1.979). Therefore, it is unlikely that the presence of tryptophan in position 488 is due to any retromutation; it simply must have been inherited from the male ancestor. In spite of the fact that the hydrophilicity pattern of the hormone binding site is the same in the presence of both amino acids, it would still be very interesting to study in more detail the effect of the mutation present in the Wistar rat. In fact, previous studies done in the Wistar rat have reported a lower affinity (1.5 nM) of estrogen receptor for 6.‘HIPestradiol (Gesell and Roth, 1981), suggesting that the mutation described could somehow influence the estrogen receptor hormone binding activity. On the basis of the results presented, the Wistar rat could represent an interesting natural mutant for studies of the relationships between estrogen receptor structure and function.
Acknowledgements We are grateful to Simona Frigerio for her technical support. This work was partially scpported by CNR Target Projects ‘Genetic Enpinecring’ and ‘Biotechnology and Bioinstrumenlalion’.
References Baker, H.J.. J.R. Lindsey and S.H. Wcisbruth. 1’170. The Lahl~raI1)ry Rat, Vol. I (Academic Press. New York) p. 2Y. Bettini E. and A. Maggi. 1901. A rapid method for the quanritaticm of estrogen receptors in small amounts of tissue. 9. Immunol. Methods 144, X7. Bradford, H.H., lY7h, A rapid and sensitive method for quantirication of microgram quantities of prolcin ulilizinp the principle :tf protein-dye binding, Anal. Biochem. 72, 24X. Chirgwig, J.M., A.E. Przyhyla. R.J. MacDlmald and W.J. RU(hx. IY7Y. Isolation of biologically active ribimuclcic acid flom sourcec enriched in rihonucleasc, Biochemistry 18. S204. &sell, S.M. and G.S. Roth, 19x1, Decrease in rat Ulerine cslroecn receptors during aging: physic and immunochcmical properties. Endocrinology l(9Y. 1502. Green, S.. P. Waher. V. Kumar. A. Krust. J.-M. Borne& P. Argos and I’. Cltamhon. I‘)#(>. Human ocs~ro~gen rcccptor KINA: Wqucncc, cxprcssion and homology IO v-crb-A. NiIIurc Xl), 1.34. Gubkr, Ll. and B.J. !!~~ffman~ IOH3. A simple and wry cfficicnt method fur gcncr;ltltlg
cl)NA
lilrriu’lcs.
GCW 1.5. X3.
lnnw. 51.A.. K.8. hl>amh>. D.H. Grlfaud and MAD. Brown. 1988. DN.4 zequrncing with 77r~rrnrr.rorlrctrrrc-rcs DNA poiymerase and direct scqurncing of pirlymerasr chain reaction-amplified DNA. Proc. Natl. Acad. Sci. USA 85, Vf3h. Kcrikc. S.. M. Saka~ and M. Muramatsu. IYli7. Molecular cloning and ch.uacterr~atron of ran estrogen receptor cDNA. Nucleic Acids Res. 15. 1-w. Krw. A.. S. Grrn. P. Argus. V. Kumar. P. Walter. J.-M. Bornert and P. Chambon. 19X6. The chicken estrogen receptor sequence: homology with v-erh-A and the human estrogen and glucocorticoid receptors. EMBO J. 5. 891.
Kumar V.. S. Green. A. Stau and P. Chamhon. 198~5,Localization of oestriol hinding and putattve DNA hinding of the human oestrogen receptor, EMBO J. 5. 2231. O’Malley, B.. 1990. Steroid receptor superfamily: more excitement predicted ior the future. Mol. Endncrinol. 4, 363. White. R.. J.A. Lees, J. Ham and M. Parker, 1987, Structural organization and expression of the mouse estrogen receptor. Mol. Endocrinol. I. 735.
