Estrogen α and β and β-2-adrenergic Drug Receptor Similarities
Across Species and Environmental Implication
Elizabeth Cates and Shen Chen
April 21, 2010
Georgia Institute of Technology
School of Biology
310 Ferst Dr.
Atlanta, Georgia 30332
(770)605-4683
ecates3@gatech.edu
Abstract
Unabsorbed pharmaceuticals enter the waste water and are released back into
the environment, where they wreak havoc on the non-humans species. Estrogens
contained in oral contraceptives and beta blockers have been detected at low levels in
water sources and have been to have anatomical and physiological impacts on the
surrounding species including altered reproduction and behavior. By comparing the
estrogen receptor α, estrogen receptor β and β-2-adrenergic receptor binding amino
acid across four different species, it was possible to determine that designing drugs that
would only target humans would be impossible. The amino acids in the binding site of
estrogen receptor α were conserved across all four species (human, mouse, chicken,
and fish). Estrogen receptor β and β-2-adrenergic receptor varied in fish by single
amino acids that would be unlikely to prevent binding of human intended drugs. The
conservation of the hypothalamic-gonadal axis and the receptors and hormones
involved allow for human intended drugs to affect other species. Efforts should
concentrate on removing the pharmaceutical from the water before the water is
released back in the environment.
2
Introduction
As the use of pharmaceuticals has increased, more drug metabolites and
unabsorbed drugs have been released into the environment [1]. Human intended
pharmaceuticals have been detected within the environment and have been shown to
affect other species [2]. One of the primary routes of pharmaceutical or drug metabolite
exposure occurs when medication enters the waste water after excretion or disposal.
Current waste cleaning methods fail to sufficiently remove the pharmaceuticals from the
water before releasing the water back in the environment. The pharmaceuticals
suspended in the water are absorbed by the organisms in the surrounding environment.
Two of these pharmaceuticals are estrogens, contained in oral contraceptives, and beta
blockers, which are used to treat various conditions including high blood pressure.
These two are contained with the category of endocrine active compounds, as they
affect the neuro-endrocine system and have been shown to have effects on nonhumans species [2,3].
Estrogens are a category of sex hormones that help regulate reproduction [4].
Along with other hormones, estrogen is one of communicators involved in the
hypothalamic-pituitary-gonadal axis (HPG axis) [5]. One of the primary estrogens in
humans is estradiol [4]. Estrodiol, which is produced mainly during the fertile years, is
produced by the follicles in the ovaries, the corpus luteum, and the placenta to regulate
the ovarian cycle. This system are highly conserved within the vertebrates and is
thought to have emerged prior to or during the differentiation of the ancestral agnathans
3
[6]. Although the effects of the sex steroids vary among the species, the communicators
are the same.
There are two main estrogen receptors, α and β. Estrogen receptor α is coded by
the ERS1 gene and concentrated in the hypothalamus [7]. Estrogen receptor β is coded
by ERS2 and is distributed throughout the body. When estrogen binds to these
receptors the transcription rate of certain genes is up-regulated leading eventually to
overall physiological and anatomical changes. Oral contraceptives, which contain
synthetic estrogens and progesterone, function by mimicking the body’s natural
estrogen [4]. Estrogen inhibits the release of the gonadatropin releasing hormones
(GnRH) of the hypothalamus, which prevent luteinizing and follicle stimulating hormone
(LH and FSH respectively) from being secreted from the pituitary. LH and FSH stimulate
the development of eggs and create ovulation. Progesterone is normally produced
primary by the corpus luteum signaling pregnancy to the rest of the body. Together
estrogen and progesterone stop the ovarian cycle and simulate pregnancy hormones.
Estrogen causes various peripheral effects via estrogen receptors β [8]. Studies have
shown overexposure to estrogen in animals can have equally detrimental effects.
Overexposure to estrogen cause song disruption, increased egg shell breakage, and
decreased male fertility in zebra finches [9].Exposure to estrogen in a fish population
below a water treatment facility resulted in male fish developing follicles within their
seminal vesicles, disruption of reproduction, and abnormal sex ratios [10].
Beta blockers are drugs that antagonize β-adrenergic receptors, opposing the
stimulatory effects of epinephrine and norepinephrine [2]. β-2-adrenergic receptors are
4
found throughout the body including the heart, lungs, and reproductive systems [3].
During the activation of the sympathetic branch of the autonomic nervous system,
epinephrine and norepinephrine are released and bind to β adrenergic receptors [2] The
receptors stimulate blood vessels to constrict, the bronchioles to dilate, heart rate to
increase, and digestive processes to arrest [3]. Beta blockers act by inhibiting the
activation of these receptors causing the opposite effects of epinephrine and
norepinephrine. One of the effects is blood vessel dilation, which decreases blood
pressure. For this reason beta blockers are commonly used to treat high blood
pressure. Beta blockers have been detected in waste water and have been shown to
have effects on various species [2]. Chronic low level exposure in fish caused
decreased egg production, reduced growth, and decreased heart rate. Because β-2adrenergic receptors are found in all vertebrates, there is the possibly that all the
species will be effect if they absorb beta blockers.
The purpose of the study was to determine if conservation of the amino acid in
the binding site of the receptors across the species exists and might explain how a
diverse number of species are affected by waste water pharmaceuticals intended for
human use. Using data provided by other experiments along with analysis and visual
tools, the conservation of the drug binding site across the species will be determined.
Methods
The protein structure of receptors was found in the Protein Data Bank (PDB)
along with the amino acid sequences for the corresponding protein (estrogen receptor α
– 3DT3, estrogen receptor β – 2NV7, β-2-adrenergic receptor – 2RH1). The files were
5
visualized using PyMOL. To find the amino acid residues involved in binding the ligand,
the ligand explorer software provided by the PDB was used. This program shows the
interactions between the ligand and amino acids. To find the sequences of other
species, the human receptor sequence was used to search in a Basic Alignment Search
Tool protein (BLASTp) search. Based on the search results, four species sequences
were chosen for each of the receptors. The human sequences were then compared to
the sequences of the other species using ClustalW2. Based on the amino acid found
using ligand viewer, the ClustalW2 results provided data on the conservation of the
amino acids contained within the binding site.
Results
Estrogen Receptor α
The PDB entry for estrogen receptor α contained two receptors bound together
each binding an estradiol (Figure 1a). The structure of estrogen receptor α consists of
twelve α helices and two anti-parallel β sheets. Based on the information from the
Ligand Explorer, each amino acids for the human binding site were recorded (Table 1).
To highlight the areas responsible for binding estradiol, the surfaces of the involved
amino acids were highlighted in orange (Figure 1b). The specific amino acids involved
in binding are shown in orange in stick mode and are labeled (Figure 1c).
The sequences for the other animals were then aligned with the human
sequences, and then the binding amino acids for the human were compared to the
aligned amino acids in the other species (Table 1). The amino acids at the binding site
for estrogen receptor α are conserved in all four species (350-alanine, 353-glutamic
6
acid, 387-leucine, 388-methionine, 394-argenine, 404-phenylalanine, 424-isoleucine,
524-histadine, 525-leucine) The binding site conservation means that a molecule
designed to bind to human estrogen receptor α will also bind to the receptors of other
species.
Estrogen Receptor β
The binding site of estrogen on estrogen receptor β was found in the same
manner as for estrogen receptor α (Figure 2). Like estrogen receptor α, estrogen
receptor β was also crystallized a dimer. Each receptor consists of four α helices. The
channel to binding site appeared to go inward and then turn (Figure 2a). The specific
amino acids involve in binding are labeled and shown in orange (Figure 2a).
The sequences for the species were aligned, and the amino acids in the human
binding site were compared with the aligned amino acids of the other species (Table 2).
The human, mouse, and chicken amino acids of the binding site were found to be
conserved (307-leucine, 305-glutamic acid, 339-leucine, 346-argenine, 356phenylalanine, 373-isoleucine, 376-isoleucine, 377-phenylalanine, 475-histadine, and
476-leucine). The single variance was in the zebra fish 373 where the isoleucine was
replaced with valine. All the species except for the zebra fish would bind estrogens with
the same strength
β-2-adrengeric receptor
The β-2-adrengeric receptor consists of nine helices and was shown bound to Gcoupled protein receptor in the PDB entry (Figure 3a). Because the G-couple protein
7
was not of interest in this study, it was removed. The binding site for beta blockers on β2-adrenergic was found using ligand explorer software provided by the PDB. The
binding site and the amino acid involved in binding were labeled in orange (Figure 3b,c)
The amino acids for the human binding site were recorded (Table 3).
The sequences for the other animals were then aligned with the human
sequences, and then the binding amino acids for the human were compared to the
aligned amino acids in the other species (Table 3). The binding site amino acids for
humans, mice, and domestic dogs are the same (113-aspartic acid, 114-valine, 193phenylalanine, 199-tryptophan, 203-serine, 204-serine, 289-phenylalanine, 290phenylalanine, 293-aspartic acid, 312-aspartic acid). Aspartic acid 113 and valine 114
were found in positions 122 and 123 when the sequences were aligned using
ClustalW2. Only one of the amino acids in the binding site was altered in the rainbow
trout. In the rainbow trout, Val 114 of the other animals was altered to a leucine, which
differs from valine by one methyl group.
Discussion
Estrogens
Both estrogen receptors show strong conservation of the amino acid residues
involved in binding estradiol. The conservation of the binding site of estradiol on
estrogen receptor α implies that the binding for all four species would be the same. The
variance in the estrogen receptor β zebra fish binding site was a single amino acid.
Although the difference in one amino acid might alter the binding affinity, it would be
8
unlikely to totally prevent the estrogen from binding. A study of human single
polymorphisms for the estrogen receptor β showed that differences the single amino
acid has a minimal effect on the binding affinity [9].
The similarity in the receptors across species explains the mechanisms of the
observed environmental phenomenon. When estrogens are not absorbed in the human
digestive tracts, they pass into the waste water system. Once released into the
environment, animals absorb the estrogens from the water. Because the system and
molecular conservation of the hormone reproductive control, the estrogens are able to
bind to the receptors of the non-human species. The binding, dimerization, and
complexing with other factors change the transcription of particular gene that change
sex characteristics and behavior. These changes can disrupt the reproduction of the
species and potential contributed to the extinction of a species. Although the effects on
each species may differ due to difference in sex characteristics, the mechanism for
causing alterations remains the same. Studies should examine how altering the amino
acids of the binding site could alter the binding affinities of estrogens.
Beta blockers
The difference of one amino acid for rainbow trout in the β-2-adrenergic receptor
differed would be unlikely to significantly affect the binding affinity for beta blockers.
Arakawa, Yanamala, Upadhyaya, Halayko, Klein-Seetharaman, and Chelikani modified
the binding site strategically substituting different amino acids in the binding site [10].
When substitutions occurred with a similar amino acid, the binding affinity was reduced
9
but binding still occurred. Based on this, the variance of Val 114 to Leu 114 would be
unlikely to greatly decrease the binding affinity for beta blockers.
Because the variances in the amino acid causes little significant variance in the
binding affinity, beta blockers would affect all four species. β-2-adrenergic receptors are
found in all vertebrates, which means potentially any of these vertebrates could be
affected an exposure to beta blockers. One study showed that invertebrates were also
affected by beta blocker exposure, but invertebrates do not have any β adrenergic
receptors [11]. Research should look for alternate mechanism to explain how
invertebrates would be affected by beta blockers. Impairing the physiology of organism
could result in early death and eventually decimate the populations. As prescriptions for
beta blockers increase, efforts should be made to prevent or minimize any
environmental impacts.
Possibilities of the future
Future research should look for other receptor-drug combinations
environmentally active compounds where the binding site may vary more across
species and determine if it would be possible to design drugs that would only bind to the
human receptors. Also the different environments and species should be further
examined to determine how many ecosystems and species are being affected by
environmentally active compounds. It would be possible that other humans are being
affected by the compounds when they consume water although sufficient
concentrations would be necessary. Studies should be done to examine if humans are
being affected by the low levels of pharmaceuticals in the water. Research should
10
continue to look for methods of preventing the pharmaceutical exposure from animals
possibly through increasing the amount of pharmaceuticals metabolized and destroyed
during waste water treatment process.
Conclusions
Binding site similarities do exist and explain why non-human animals are affected by
human pharmaceuticals. No other study has examined the receptors across species or
linked the conservation of the binding site to the cause of the environmentally observed
phenomena. It would be improbable to design drugs that would only bind to human
estrogen receptors α and β and β-2-adregenergic receptors. Future research should
focus on how to prevent animals from being exposed to human pharmaceuticals and
search for any possible humans exposure with effects on individuals.
Acknowledgements
Shen Chen
Professor Ingenborg Schmidt-Krey Ph.D.
Tanawadee Preprem
11
References
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E, Pettersson K, Warner M, & Gustafsson J (2001) Mechanisms of Estrogen
Action. Physiological Reviews 81(4), 1535-1565.
[7] Vaja A., Barber L., Gray J, Lopez E, Wooding J, Norris D (2008) Reproductive
Disruption in Fish Downstream from an Estorgenic Wastewater Effleunt.
Environmental Science Technology 42, 3407-3414.
[8] Rochester J., Heiblum R, Rozenboim I. & Milam J. (2008) Post-hatch Oral
Estrogen Exposure Reduces Oviduct and Egg Mass and Alters Net-building
Behavior in Adult Zebra Finches (Taeinopygia guttata). Physiology &
Behavior 95, 370-380.
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Q., et al. (2004). Identification of a functional variant of estrogen receptor beta
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[10]
Arakawa, M., Yanamala, N., Upadhyaya, J., Halayko, A., Klein-Seetharaman,
J., & Chelikani, P. (2010). The importance of valine 114 in ligand binding in
beta(2)-adrenergic receptor. Protein Science, 19(1), 85-93.
[11]
Stanley, J. K., Ramirez, A. J., Mottaleb, M., Chambliss, C. K., & Brooks, B. W.
(2006). Enantiospecific toxicity of the beta-blocker propranolol to Daphnia
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25(7), 1780-1786.
12
(a)
(b
(c)
(C)
Figure 1. Estrogen Receptor α Structure and Binding Site
(a) The crystallization process left two estrogen receptors α bound together each with its own
estradiol (yellow) bound in the binding region. It is shown in cartoon mode using PyMol. (b)
The binding pocket is shown in surface mode with orange highlighting the areas involve in
binding the ligand. (c) Shown in cartoon-stick mode in PyMol, the amino acids involved in
binding estradiol are highlighted in orange and labeled.
13
(a)
(b)
(c)
Figure 2. Estrogen Receptor β Structure and Binding Site
(a) The crystallization process left two estrogen receptors β bound together each with its own
estradiol (yellow) bound in the binding region. It is shown in cartoon mode using PyMol. (b)
The binding pocket is shown in surface mode with
14orange highlighting the areas involve in
binding the ligand. (c) Shown in cartoon-stick mode in PyMol, the amino acids involved in
binding estradiol are highlighted in orange and labeled.
(a)
(b)
(c)
Figure 3. β-2-adrengeric Receptor Structure and Binding Site
(a) The structure for β-adrenergic receptor is shown in cartoon mode using PyMol, and
carazolol, a common beta blocker, is the ligand
15 colored yellow. (b) The binding pocket is
shown in surface mode with orange highlighting the areas involve in binding the ligand. (c)
Shown in cartoon-stick mode in PyMol, the amino acids involved in binding carazolol is
highlighted in orange and labeled.
Table 1. Estrogen Receptor α Binding Site Amino Acid Conservation
Human
Mouse
Chicken
Zebrafish
(Homo sapiens)
(Mus musculus)
(Gallus gallus)
(Danio rerio)
ALA 350
C
C
C
GLU 353
C
C
C
LEU 387
C
C
C
MET 388
C
C
C
LEU 391
C
C
C
ARG 394
C
C
C
PHE 404
C
C
C
ILE 424
C
C
C
HIS 524
C
C
C
LEU 525
C
C
C
Each amino acid using in binding the estradiol in humans is compared with the
corresponding amino acid from the Clustal W2 alignement. C indicates the amino acid
conserved from humans to the other species. U means the corresponding amino acid
differed from the human amino acid, and the difference is noted.
Table 2. Estrogen Receptor β Binding Site Amino Acid Conservation
Human
Mouse
Chicken
Zebrafish
(Homo sapiens)
(Mus musculus)
(Gallus gallus)
(Danio rerio)
LEU 301
C
C
C
GLU 305
C
C
C
LEU 339
C
C
C
ARG 346
C
C
C
PHE 356
C
C
C
ILE 373
C
C
U ( Val)
ILE 376
C
C
C
PHE 377
C
C
C
HIS 475
C
C
C
LEU 476
C
C
C
Each amino acid using in binding the estradiol in humans is compared with the
corresponding amino acid from the Clustal W2 alignement. C indicates the amino acid
conserved from humans to the other species. U means the corresponding amino acid
differed from the human amino acid, and the difference is noted.
16
Table 3. β-2-adrenergic Receptor Binding Site Amino Acid Conservation
Mouse
Rainbow trout
Human
Chicken
(Mus
(Oncorhynchus
(Homo sapiens)
(Gallus gallus)
musculus)
mykiss)
Asp 113 (122)
C
C
C
Val 114 (123)
C
C
U (Lue)
Phe 193
C
C
C
Tyr 199
C
C
C
Ser 203
C
C
C
Ser 204
C
C
C
Phe 289
C
C
C
Phe 290
C
C
C
Asn 293
C
C
C
Asn 312
C
C
C
Each amino acid using in binding the carazolol in humans is compared with the
corresponding amino acid from the Clustal W2 alignement. C indicates the amino acid
conserved from humans to the other species. U means the corresponding amino acid
differed from the human amino acid, and the difference is noted.
17