REVIEWS
STEROID-HORMONE RAPID ACTIONS,
MEMBRANE RECEPTORS AND A CONFORMATIONAL ENSEMBLE MODEL
Anthony W. Norman*,‡, Mathew T. Mizwicki* and Derek P. G. Norman*
Steroid hormones can act as chemical messengers in a wide range of species and target tissues
to produce both slow genomic responses, and rapid non-genomic responses. Although it is
clear that genomic responses to steroid hormones are mediated by the formation of a complex
of the hormone and its cognate steroid-hormone nuclear receptor, new evidence indicates that
rapid responses are mediated by a variety of receptor types associated with the plasma
membrane or its caveolae components, potentially including a membrane-associated nuclear
receptor. This review summarizes our current knowledge of membrane-associated steroid
receptors, as well as details of structure–function relationships between steroid hormones and
the ligand-binding domains of their nuclear and membrane-associated receptors. Furthermore,
a new receptor conformational ensemble model is presented that suggests how the same
receptor could produce both rapid and genomic responses. It is apparent that there is a
cornucopia of new drug development opportunities in these areas.
Department of
*Biochemistry &
‡
Division of Biomedical
Sciences, University of
California, Riverside,
California 92521, USA.
Correspondence to A.W.N.
e-mail:
[email protected]
doi:10.1038/nrd1283
At the level of the organism, steroid hormones are
widely recognized for their generation of a wide array
of essential cellular and physiological responses,
resulting from their endocrine production and subsequent functioning as chemical messengers throughout the domains of their cognate endocrine systems1.
However, a detailed understanding of the actions of
steroid hormones can be achieved only by experimentation at the cellular and molecular levels. Such
work has resulted in the elucidation of complex signalling networks. In this regard, the key parameters in
these networks are the chemical structure of the
steroid hormones and their partner receptors. It is now
becoming accepted that in addition to the ‘traditional’
actions of steroid-hormone–receptor complexes —
functioning in the cell nucleus as transcription factors
to selectively modulate gene expression (that is, to
generate genomic responses) — steroid hormones
also show an essential type of action referred to as
‘rapid actions’ (also called non-genomic or nongenotropic actions).
NATURE REVIEWS | DRUG DISCOVERY
In this context, the term ‘rapid actions’ describes
the general rate of appearance of specific biological
responses that result from a steroid hormone acting
through a receptor; that is, the steroid hormone forms
a ligand–receptor complex that is linked by some
mechanism to the production of a rapid biological
response. The rapidity, of course, is system-dependent
and can vary from seconds (for example, the opening
of ion channels) to an hour or so (for example, the
inhibition of apoptosis; see TABLE 1). This contrasts
with genomic responses, which generally take a few
hours to days to manifest fully, and which can be
blocked by inhibitors of transcription and translation.
Steroid hormones can initiate biologically useful
rapid responses in the absence of a functional nucleus
(for example, in spermatozoa2,3, or in bone-cartilage
matrix vesicles4), indicating that a classic nuclear
receptor need not necessarily be involved. Virtually all
classes of steroid hormones have been shown repeatedly, and by many laboratories, to be able to induce
rapid responses (TABLE 1).
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Table 1 | Examples of rapid responses for steroid hormones and related compounds*
Steroid system Rapid response(s)
Oestradiol
Tissue
Increase intracellular Ca2+; opening of maxi-K channels
Activation of PI3 kinase linked to cardiovascular
protective effects
Plasma-membrane receptors signal to block apoptosis
Androgens
2+
Cellular Ca influx
Inhibition of apoptosis
Triggers S-phase entry via activation of SRC and
PI3 kinase
Testosterone or oestradiol activate the AR or ER to
interact with SRC and activate MAP kinase to promote
cell proliferation
Stimulation of intracellular Ca2+ release and MAP kinase
Progesterone
1α, 25(OH)2vitamin D3
Cellular maturation
Activation of PI3 kinase by nuclear progesterone receptor
Liganded PR forms a heterodimer with the ER which
in turn forms a ternary complex with c-SRC to activate
the MAP kinase pathway.
Ca2+ influx linked to the acrosome reaction
2+
–
Opening of voltage-gated Ca and Cl channels
Activation of PKC and PI3 kinase
Stimulation of insulin secretion
Activation of MAP kinase linked to cell differentiation.
References
Endometrial
Endothelial
Endothelial
68
69
70
Breast cancer cells
71
Splenic T cells,
osteoblasts
Osteoblasts, HeLa cells
NIH3T3 cells
72–74
24
42
LNCaP prostate cells
75
Skeletal muscle myotubes
76
Xenopus oocytes
Xenopus oocytes
T47D breast cancer cells
Spermatozoa
Osteoblasts
Cartilage
Endothelial
Pancreas
Leukemia cells
31
77
78,79
2,3
80,81
4,82
83
84,85
86,87
Glucocorticoids
Inhibition of nicotine-induced Ca2+ influx through a
G-protein–PKC pathway
Stimulation of mating response in male newts
Newt salamanders
89
Mineralocorticoids
Rapid effects of aldosterone
Working rat heart;
positive inotropic action
36
Thyroid
hormones
Activation of MAP kinase by a G-protein-coupled receptor
Shortening of action potentials
Stimulates oxygen consumption
Activation of PKA and PKC are linked to interferon-γinduced antiviral activity
HeLa cells
Rat ventricular myocytes
Rat ventricular myocytes
HeLa cells
90
91,92
PPAR
None yet clearly identified
Retinoids
None yet clearly identified
Brassinosteroids Stimulation of H2O2 production in 30 min
PC-12 cells
Arabidopsis
88
90
30
AR, androgen receptor; ER, oestrogen receptor; PKA, protein kinase A; PKC, protein kinase C; PR, progestin receptor; PPAR, peroxisome
proliferator-activated receptor. *This table is not intended to provide a comprehensive enumeration of all rapid responses known to be
mediated by steroid hormones and related compounds; such information can be found in REFS 93–98.
The study of steroid-mediated rapid responses has
been active for at least 40 years5–7, which is similar to the
time frame for the study of genomic responses mediated
by steroid nuclear receptors8,9. However, it is only in the
past ten years or so that rapid responses to steroid
hormones have achieved as much prominence or
received similar acceptance as genomic responses. This
article will review the present knowledge of the rapid
actions of steroid hormones from a structure–function
standpoint and relate this to the potential opportunities
such knowledge generates for drug discovery.
Steroid hormone biological responses
A scheme describing the present understanding of the
two major pathways by which natural steroid hormones
produce biological responses is shown in FIG. 1. The
figure also illustrates how analogues (some of which
could be drugs) might act as surrogates for the natural
hormones in functioning as agonists of the natural hormone receptors (see discussion below). Both animals
and plants that use steroids as chemical messengers
28
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(TABLES 1 and 2) possess complex cellular signalling networks to generate both genomic and rapid responses. For
example, in the plant Arabidopsis, a leucine-rich-repeat
receptor kinase is involved in rapid signal transduction
by brassinosteroid10.
The prerequisite for the generation of a final outcome
(a biological response) is the formation of a stable
ligand–receptor complex under physiological conditions
(the ligand can be a hormone or agonist analogue) (FIG. 2).
The phrase ‘under physiological conditions’ emphasizes
that the receptor must be able to recognize and bind the
steroid hormone in question using the available freecirculating concentration of the steroid. The plasma concentrations of steroid hormones are typically in the range
of 5–500 nM; however, owing to their hydrophobic
properties, they are all bound to steroid-specific plasma
transport proteins1. Therefore, the actual ‘free’ steroid
concentration is reduced by the proportion of the
steroid bound to the transport protein; this in turn
depends on the dissociation constant, Kd, of the transport
protein for its cognate hormone(s). The ligand Kd for
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REVIEWS
most nuclear receptors is in the range of 0.05–50 nM,
which reflects the extremely strong, but non-covalent,
binding of the hormone to its cognate receptor.
The location of the ligand–receptor complex for
gene transcriptional responses is in the nucleus of the
cell (the subcellular localization of unoccupied steroid
Natural steroid
hormone
Conformationally
flexible analogues
Conformationally
restricted analogues
Receptor
Receptor
Plasma membrane
Nucleus
Ligand–receptor
complex
Ligand–receptor
complex
MAP kinase
activation
cAMP ↑
PKC ↑
Ca2+ channel
(open)
DNA
Gene expression
Crosstalk
Upregulation
Downregulation
mRNA ↑
mRNA ↓
Protein synthesis
(Minutes–hours–days)
(Seconds–minutes)
Biological
responses
Figure 1 | Pathways for generating biological responses by steroid hormones. In the
genomic pathway (left-hand side), occupancy of the nuclear receptor by the cognate steroid
hormone leads to an up- or downregulation of genes subject to hormone-receptor regulation.
In the rapid-response pathway (right-hand side), occupancy of a putative membrane receptor
(which in some instances might be a membrane-associated nuclear receptor) by the steroid
hormone can lead to the initiation of rapid responses that are coupled through appropriate
second-messenger systems, either directly to the generation of the end biological response(s) or
indirectly through modulation of genomic responses; the examples included in the figure are not
intended to be exhaustive. The figure also indicates the possibility of conformationally flexible
agonist analogues of the parent steroid hormone binding to the nuclear or proposed membrane
receptor(s). Further, it is known that conformationally restricted analogues can occupy the
membrane receptor and initiate only rapid responses. These possibilities are further discussed in
the text, FIG. 3 and FIG. 4 (examples of conformationally restricted analogues) and FIG. 6. cAMP,
cyclic AMP; MAP, mitogen-activated protein; mRNA, messenger RNA; PKC, protein kinase C.
NATURE REVIEWS | DRUG DISCOVERY
‘nuclear’ receptors varies with the particular steroid
hormone; they can be present in the cytoplasm complexed to chaperones, in the nucleus, or partitioned
between the cytoplasm and nucleus1,11). By contrast, for
most, if not all, rapid responses, the ligand–receptor
complex is believed to be associated with the plasma
membrane of the cell or in the very near vicinity (see
TABLE 2 and later discussion). So, a fundamental challenge
to researchers in the field of steroid rapid responses has
been to identify and characterize the nature of the
plasma-membrane-associated receptors that bind these
ligands with high affinity.
There is now a vast literature describing, in everincreasing detail, how steroid-hormone–receptor
complexes function as transcription factors to regulate
the processes of gene transcription and protein synthesis,
which in some instances have crucial consequences on
the proliferation and/or death of cells that affect tissue
structure and remodelling (animal development), as well
as a multitude of disease processes, such as cancer12–14.
In parallel to these developments is the increasing contribution made by the X-ray structural determination
of many cellular proteins. There are ~23, 000 protein
structures in the Protein Data Bank, which include 71
structures for human nuclear steroid-hormonereceptor ligand-binding domains (LBD) with their
cognate agonist ligands or antagonists15–18. Collectively,
these structures provide the foundation for insights into
structure–function relationships between receptors and
their partner steroid hormones, as well as agonist or
antagonist analogues, in relation to their selective cellular
or organismal biological actions.
The rapid-response-initiated biological responses are
mediated by a wide array of cellular second-messenger
systems. Steroid hormones have been shown to activate
the following signal transduction pathways: mitogenactivated protein (MAP) kinase, phosphatidylinositol-3kinase, signal transducer and activator of transcription
(STAT), tyrosine kinases and phosphatases, nitric oxide
synthase (NOS), proteinases, epidermal growth factor
receptor, SRC kinase, SHC kinase, protein kinase C
(PKC), adenylyl cyclase and GTP proteins (for details,
see the Signal Transduction Knowledge Environment
website under Further information). In general, these
signalling systems are present in the cytoplasm (sometimes near the plasma membrane) and produce outcomes in the cytoplasm/membrane (for example,
opening channels) or engage in crosstalk with the
nucleus (to modulate gene transcription).
Rapid-response and nuclear receptors
FIGURE 2a,b presents the structures of the NATURALLY OCCUR-
for the SUPERFAMILY OF NUCLEAR RECEPTORS1,19,
and that of the plant hormone brassinolide, all of which
are known to produce rapid responses (TABLE 1) in a
stereoselective manner. It is instructive to remember
that the superfamily of nuclear receptors can accommodate both relatively rigid ligands (the five classic steroid
hormones), as well as conformationally flexible ligands
(1α, 25(OH)2-vitamin D3, ecdysone and (L)-triiodothyronine). The latter three ligands individually have a
RING LIGANDS
1
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minimum of four carbon–carbon single bonds, each of
which can achieve 360° rotations, generating a vast array
of molecular shapes. In addition, the A-ring of 1α,
25(OH)2D3 can undergo the typical conformational
interchange of a cyclohexane ring (chair–chair interconversion); this dramatically changes the three-dimensional
orientation of the pair of 1α- and 3β-hydroxyls from
axial/equatorial to equatorial/axial, respectively. It is
useful to note, that from a drug discovery perspective,
the presence of significant conformational flexibility in a
ligand can be fully accommodated by the superfamily of
nuclear receptors (see below).
FIGURES 3a,b illustrate another important insight into
steroid hormone structures that can serve as specific
agonists for either, but not both, genomic and nongenomic biological responses. For the conformationally
flexible 1α, 25(OH)2-vitamin D3 molecule, one shape of
the natural hormone is an optimal full agonist for
genomic responses, whereas a different shape of the
hormone is an optimal full agonist for non-genomic/
rapid responses; however, this is not the case for
oestradiol, which is conformationally restrained. To
emphasize that shape matters in dictating hormonal
response, FIG. 3b presents space-filling representations
Table 2 | Status report for steroid receptors that initiate ligand-mediated rapid responses
Receptor
Steroid hormone studied
Novel plasma membrane
receptor: cloned
Brassinosteroid — Arabidopsis plant outer-cell-membrane receptor with
serine/threonine kinase activity and a brassinosteroid as a ligand
Progesterone — Xenopus oocytes; novel G-protein membrane receptor
cloned
10,30
Classical nuclear receptor:
associated with plasma
membrane caveolae
Oestrogen — endothelial cell caveolae ER; human breast cancer cells
membrane ruffles
1α, 25(OH)2D3: chick and mouse intestine, kidney and lung caveolae VDR
33,34
Membrane adaptor
or scaffold proteins
that interact with
nuclear receptors
ER-interacting protein that modulates non-genomic activity/crosstalk with
SRC/Erk
Adaptor protein linking ER with inner surface of caveolae membrane‡
VDR: formation of a ternary complex between VDR and 1α, 25(OH)2D3
with a Ser/Thr protein phosphatase (PP1c) and p70(S6k) is implicated
in G(1)–S cell-cycle transition
Plasma-membrane
receptor: partially purified
Glucocorticoid: membrane receptor purified from amphibian newt brain
and from lymphoma cells
1α, 25(OH)2D3: chick intestine basal lateral membrane receptor purified
× 4,000
101,102
1α, 25(OH)2D3: chick intestinal plasmalemmal receptor and rat cartilage
cell membrane receptor
Progesterone: plasma membrane receptor detected, isolated by twodimensional gel electrophoresis and analyzed by MALDI-TOF mass
spectrometry
Oestrogen: rat pituitary cells
Oestrogen: human breast cancer cells
Oestrogen: ER in mouse macrophages
Glucocorticoid: GR nuclear receptor
Testosterone: Xenopus oocytes have the AR
104,105
NATURALLY OCCURING
LIGANDS
Steroid hormones are classified
chemically on the basis of the
traditional presence of a fourmembered ring structure, A, B,
C, D, which is related to
cyclopentanoperhydrophenanthrene; see as an
example the structure of
17β-oestradiol in figure 2b.
1α, 25(OH)2-vitamin D3 is
technically a seco-steroid, which
indicates that one of its rings is
‘broken’; in the case of 1α,
25(OH)2-vitamin D3, the 9, 10
carbon bond is broken, which
creates a conformationally
flexible molecule; see figure 2a.
The brassinolide steroid shown
in figure 2b has an expanded
seven-membered B ring.
SUPERFAMILY OF NUCLEAR
HORMONE RECEPTORS
Originally, the family of steroid
hormones included only
oestradiol, testosterone,
progesterone, cortisol,
aldosterone and ecdysone.
However, with the discovery
of a nuclear receptor for the
primary metabolite of vitamin
D3, 1α, 25(OH)2-vitamin D3 in
1969, it became classified as a
steroid hormone. As a
consequence of cloning the
protein receptors, the classes
of ligands for the superfamily of
nuclear receptors was further
expanded beyond steroids to
include thyroid hormone
(3, 5, 3′-L-triodothyronine) and
all trans-retinoic acid.
30
Plasma membrane
receptor detected:
immunochemistry studies
References
Plasma membrane
receptor detected:
ligand-binding studies
Oestradiol: endometrial cells
1α, 25(OH)2D3: chick intestine
Cortisol: salamander synaptic membranes
Aldosterone: porcine kidney plasma membrane
Testosterone: LNCap prostate cells plasma membrane
Plasma membrane
receptor: linked to
production of rapid
responses
Oestrogens and androgens: attenuation of apoptosis by the LBD of either
the ER or AR in mouse osteoblasts, human fibroblasts or HeLa cells
Estradiol, progesterone, testosterone, cortisol or thyroxine: decreased
vascular leukocyte accumulation after ischaemia and reperfusion injury
in endothelial cells via the ER, PR, AR, GR or TR binding to the p85
regulatory subunit of PI3-kinase
Estradiol: antibodies to ER stimulate prolactin release from pituitary cells
Estradiol: plasma membrane ER-α stimulates eNOS via coupling to Gα(i)
Testosterone: testosterone stimulates via a G-protein-coupled receptor
intracellular Ca2+ and MAP kinase
Testosterone: testosterone stimulates the secretion of PSA in LNCaP
prostate cells
Plasma transport
proteins linked
to membrane ‘effects’
Sex-hormone-binding globulin
Vitamin-D-binding protein
PKC binds steroids
Aldosterone: colonic epithelial PKC activation
1α, 25(OH)2D3: recombinant PKC activation in vitro
31
35,37
99
100
103
106
107
34
108
109
110
35,37
89,111
112
113
24
70
114
40
76
113
43
44,115
45
46,47
AR, androgen receptor; eNOS, endothelial nitric oxide synthase; ER, oestrogen receptor; GR, glucocorticoid; LBD, ligand-binding
domain; MALDI-TOF, matrix-assisted laser desorption ionization time-of-flight; MAP, mitogen-activated protein; PKC, protein kinase C;
PR, progestin receptor; PSA, prostate-specific antigen; TR, thyroid hormone receptor; VDR, vitamin D3 receptor. *This table presents
selected examples for each topic and is not intended to be comprehensive. Additional information is available in REFS 93–98.
‡
R. H. Karas and M. E, personal communication.
| JANUARY 2004 | VOLUME 3
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REVIEWS
a
H
C
D
OH
OH
H
C
A
D
H
H
HO
A
HO
B
OH
O
OH
1α, 25(OH)2-vitamin D3
17β-oestradiol
Testosterone
OH
OH
O
HO
O
HO
O
O
OH
H
H
H
H
O
O
O
Progesterone
Cortisol
Aldosterone
OH
I
COOH
I
HO
OH
HO
O
NH2
I
HOOC
3,5,3′-L-triiodothyronine
All-trans retinoic acid
b
OH
HO
H
O
α-Ecdysone
OH
HO
HO
HO
O
H
O
Brassinolide
Figure 2 | Structures of naturally occurring hormones. a | Structures of compounds that function as hormones or analogues
of hormones via binding to the ligand-binding domain of one of the members of the superfamily of nuclear receptors16 or to
appropriate cell-membrane receptors. These include 1α, 25(OH)2-vitamin D3, oestradiol, testosterone, progesterone, cortisol,
aldosterone, all-trans-retinoic acid, 3, 5, 3′, -L-triiodothyronine (T3), and the insect steroid hormone ecdysone. b | Brassinolide,
a plant steroid hormone that binds to a plant-cell-membrane receptor.
CONNOLLY REPRESENTATIONS
An illustration of the threedimensional shape of the
collective electron orbits of a
molecule (ligand). Accordingly,
a Connolly shape also defines
the volume of receptor space
minimally required to
accommodate (or ‘accept’) that
molecule as a bound ligand.
(known as CONNOLLY REPRESENTATIONS) of the natural
steroid hormone (FIG. 3b; top row — these function as
agonists for both genomic and non-genomic responses)
and the separate genomic (FIG. 3b; middle row) and nongenomic (FIG. 3b; bottom row) agonists. (In general, the
natural ligands of nuclear receptors have conserved
volumes with only limited variation (318 ± 53 Å3.) It has
been suggested that this conservation is due to evolutionary selection, and it has also been pointed out that it
serves as a “useful criterion in the design of high-affinity
analogues” — that is, new drugs20. The importance of
how creative chemistry is used in designing ligand
shapes that can satisfy the requirements of the receptor’s
LBD (for genomic or rapid responses) is illustrated
briefly in the following three case studies.
Case study I. Structure–function studies of the vitamin
D/vitamin D receptor (VDR) system led to the conclusion that the shape of a 6-s-cis-shaped analogue, such
as 1α, 25(OH)2-lumisterol (FIG. 3a), is a full agonist for
NATURE REVIEWS | DRUG DISCOVERY
rapid responses, but is only a poor genomic agonist. By
contrast, the shape of a 6-s-trans bowl-shaped analogue
(FIG. 2a; FIG. 3b, middle row) is a full agonist for genomic
responses, but is only a poor non-genomic agonist.
Furthermore, the conformationally flexible natural
hormone 1α, 25(OH)2-vitamin D3, which can achieve
both the 6-s-cis and 6-s-trans shapes, is a full agonist
for both rapid and genomic responses21,22.
Case study II. Structure–function studies in the
oestradiol/oestrogen receptor (ER) system have led to
the conclusion that the shape of an oestradiol analogue,
such as oestren (FIG. 3a; FIG. 3b, bottom row), is a full
agonist for rapid responses, but is a poor genomic agonist. By contrast, the analogue 1, 3, 5-tris(4-hydroxyhenyl-4-propyl-1H-pyrazole (FIG. 3a; FIG. 3b, middle
row) is a full genomic agonist, but is a poor nongenomic agonist. 17β-oestradiol can satisfy the
requirements of being both a full genomic and nongenomic agonist23,24.
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a
VDR ligands
b
ER-α ligands
VDR ligands
ER-α ligands
OH
OH
OH
H
H
H
Natural ligand X-ray
(genomic and nongenomic agonist)
HO
HO
Non-genomic
Non-genomic
1α, 25(OH)2-lumisterol
Oestren
Natural ligand X-ray
(genomic and nongenomic agonist)
O
OH
HO
O
O
N
N
Synthetic non-steroidal
ligand (genomic agonist)
O
Synthetic non-steroidal
ligand (genomic agonist)
OH
Non-steroidal genomic
Non-steroidal genomic
3-[4-(2-oxo-3,3-dimethyl
butoxy)-3-methylphenyl)]-3[4-(4′-acetyl-benzyloxy)-3methylphenyl]pentane
1,3,5-tris(4-hydroxyphenyl4-propyl-1H-pyrazole
Synthetic ligand
(non-genomic agonist)
Synthetic ligand
(non-genomic agonist)
Figure 3 | Structural diversity of ligand shapes accommodated by steroid-hormone receptors. a | Comparison of agonist
ligand structural diversity for the nuclear vitamin D (VDR) and the estrogen (ER-α) receptors that are known to mediate both nongenomic and genomic responses. One example each is shown for a non-genomic steroid ligand and a non-steroidal genomic
ligand for the VDR [1α, 25(OH)2-lumisterol-D3 and 3-[4-(2-oxo-3, 3-dimethylbutoxy)-3-methylphenyl)]-3-[4-(4′-acetylbenzyloxy)-3methylphenyl]pentane25] and the ER-α [oestren and 1, 3, 5-tris(4-hydroxyphenyl-4-propyl-1H-pyrazole26] receptors. b | Space-filling
(Connolly) shapes of the natural steroid ligands that mediate both genomic and non-genomic responses (top row), genomic nonsteroidal agonists (middle row) and non-genomic agonists (bottom row) for the VDR and ER-α receptors. The natural agonists for
VDR and ER-α are 1α, 25(OH)2-vitamin D3 and 17β-oestradiol, respectively; for the synthetic non-steroidal genomic agonists, the
ligands are [3-[4-(2-oxo-3, 3-dimethylbutoxy)-3-methylphenyl)]-3-[4-(4′-acetyl-benzyloxy)-3-methylphenyl]-pentane and 1, 3, 5tris(4-hydroxyphenyl-4-propyl-1H-pyrazole; and for the synthetic non-genomic agonists, the ligands are 1α, 25(OH)2-lumisterol D3
and oestren. The Connolly surface for each ligand was generated from computer minimized molecular models with the exception
of the non-steroidal ligands. These two ligands were oriented in a manner that was similar to their published models; they were then
each transformed into a Connolly surface25,26.
A further important observation that follows from
case studies I and II is that, in the vitamin D and
oestradiol systems, there could be two distinct receptors,
each with an LBD that can accommodate the ligands
appropriate for either, but not both, a genomic or
rapid response. Alternatively, if there is only one receptor responsible for both genomic and rapid responses,
then this receptor must somehow utilize the different
binding modes of differently shaped ligands to determine whether to initiate rapid or genomic responses.
(3-[4-(2-oxo-3, 3-dimethylbutoxy)-3-methylphenyl)]-3[4-(4′-acetylbenzyloxy)-3-methylphenyl]pentane25) and
the oestradiol/ER genomic analogue (1, 3, 5-tris(4hydroxyphenyl-4-propyl-1H-pyrazole26), are both nonsteroidal full agonists. The non-steroidal potent genomic
VDR agonist (FIG. 3a) was the best compound from a
library of 13 compounds that were the product of a computer-aided molecular design program that was used to
generate a focused library of non-steroidal analogues27.
Rapid-response receptor location
CAVEOLAE
Caveolae are small, flask-shaped
invaginations located in the
plasma membranes of many
cell types; these domains have
high proportions of
sphingolipids and cholesterol as
well as characteristic integral
membrane protein, either
caveolin-1, -2 or -3 (22 kDa).
32
Case study III. Structure–function studies illustrate one
more observation crucial to the drug discovery process.
In FIG. 3a, it is apparent that the two non-genomic analogues (1α, 25(OH)2-lumisterol and oestren) are clearly
steroids; they each have the full ‘A, B, C, D complement’
of rings that are a hallmark of steroids. It is not surprising
that these steroid analogues can effectively mimic the
non-genomic actions of their parent steroid hormones.
By contrast, the two genomic agonist analogues selected
illustrate that molecules which violate the A, B, C, D
steroid paradigm (that is, non-steroidal analogues) can
still be fully potent agonists. In this example, for genomic
responses, the vitamin D/VDR genomic analogue
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A major focus during the past decade for researchers in
the steroid-hormone rapid-responses field has been to
identify, characterize and determine the subcellular
localization of receptors linked to rapid responses; TABLE 2
presents a summary of the current status of these
studies. So far, only two unique receptors have been
cloned, fully characterized and shown to be capable of
using specific hormones to produce rapid responses.
A schematic diagram of a steroid hormone interacting with four classes of membrane receptor is
shown in FIG. 4. Membrane receptors can either be
present in the general plasma membrane or associated
with CAVEOLAE (much evidence indicates that caveolae
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act as a platform for the accumulation of signaltransduction molecules; see REFS 28,29). This schematic
will serve as a point of comparison for the findings
summarized in TABLE 2.
The first breakthrough in determining the identity of
rapid-response receptors came in 1997 with the plant
Arabidopsis thaliana, when an outer-cell-membrane
receptor with serine/threonine kinase activity that uses a
brassinosteroid (FIG. 2b) as its ligand was cloned30.
Brassinosteroids are a class of growth-promoting regulators responsible for plant growth and development. In
the absence of the hormone brassinolide, Arabidopsis
does not respond to fluctuations in ambient light. The
cloned receptor (1,196 amino acids) consisted of a signal
peptide followed by an extracellular domain that contains
25 tandem copies of a 24-amino-acid leucine-rich repeat
(LRR), followed by a transmembrane domain (amino
acids 793–814), and an intracellular domain (382
amino-acid residues) that has 12 sub-domains that
are characteristic of almost all eukaryotic protein
kinases. The receptor corresponds to the receptor configuration shown in FIG. 4C: a transmembrane receptor
with intrinsic kinase activity. In a follow-up report, the
extracellular domain of the Arabidopsis membrane
receptor was definitively shown to be the LBD for the
hormone brassinosteroid30. This Arabidopsis LBD has
no known amino-acid sequence similarity with the
LBDs of the superfamily of steroid receptors; as yet, an
X-ray crystallographic structure is not available. The
physiological role of this membrane receptor is to initiate
a rapid oxidative peroxide burst (60 min), followed by
defensive gene activation and cell death. This plasma
membrane steroid receptor therefore functions to signal
to the nucleus using crosstalk to modulate gene transcription. The Kd for the binding of the steroid ligand to
the receptor has not been reported.
The second breakthrough occurred in 2003 with
the cloning, expression and characterization of a
membrane progestin receptor isolated from spotted
seatrout ovaries31. The cloned protein (352 amino
acids, 40.5 kDa) has seven transmembrane domains,
which is characteristic of a G-protein-coupled receptor
(FIG. 4B). Using progestins as the ligand, the expressed
protein exhibits saturable binding with high affinity
(Kd = 30 nM). Transfection of the receptor into MDAMB-231 breast cancer cells allowed evaluation of its
ability to initiate rapid responses: within 5 minutes,
there was a reduction in the production of cyclic AMP
C
B
D
SH
SH
SH
SH
Plasma membrane
DAG
PKC
DAG
PIP3
PLC
α
γ
Caveola
PKC
IP3
β
Ras-GTP
GTP
c-Raf
Src
P
Shc
P
P
Pi
Pi
Aa
AKT
SH
AC
P
c-Raf
Shc
PKA
cAMP
P
Grb2
Sos
PI-3,4,5 P
P85
PI-3-kinase
Src
P110
Scaffold
SH
Ac
SH
Ab
Signal transduction systems
PKA
PKC
PLC
PI-3-kinase
Ras/MAP-kinase
e-NOS
EGFR/matrix metalloproteinases
Rapid response outcomes
Ion channels
Transcription
Translation
Protein kinase/phosphatases
Structural proteins
Signalling enzymes
Feedback regulation
SH
Steroid hormone
Figure 4 | Schematic diagram of a steroid hormone interacting with four classes of membrane receptors to generate second messengers linking to
variety of signal-transduction systems. A | Three classes of membrane receptor are shown illustrating the classic nuclear steroid-hormone receptor associated
with a caveola. Aa | The receptor is technically outside the cell and is associated with the outer surface of the plasma membrane in the flask of the caveola.
Ab | The receptor is tethered by a scaffolding protein to the plasma membrane on the inner surface of a caveola. Ac | The receptor is tethered to the caveolae by a
palmitic acid molecule that is esterified to a receptor Ser or Thr with the fatty-acid side chain ‘inserted’ into the membrane (palmitoylation). B | A G-protein-coupled
receptor with its ligand-binding domain on the outside of the cell and a seven-membrane spanning peptide transition followed by an intracellular peptide domain that
can bind Gα, β and γ proteins. C | A single-spanning membrane receptor with intrinsic kinase activity that might be functional as a monomer. D | Same as C except
a homodimer. Caveolae are flask-shaped membrane invaginations present in the outer cell membrane of many cells; they are believed to serve as a ‘platform’ to
accumulate or ‘dock’ signal-transduction-related molecules.The signal-transduction systems are listed as candidates for mediating rapid responses to steroid
hormones and are based on published data93–98. The details remain to be defined on the basis of careful experimentation. The two ovals with Ras-GTP and c-Raf
‘touching’ are to suggest that c-Raf was recruited to the complex. AC, adenylyl cyclase; DAG, diacylglycerol; EGFR, epidermal growth factor receptor;
e-NOS, endothelial nitric oxide synthase; IP3, inositol triphosphate; MAP, mitogen-activated protein; PI3K, phosphatidylinositol 3-kinase; PIP3, phosphatidylinositol
triphosphate; PKA, protein kinase A; PKC, protein kinase C; PLC, phospholipase C.
NATURE REVIEWS | DRUG DISCOVERY
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REVIEWS
after addition of a progestin that could be blocked by
pertussis toxin, suggesting that the receptor is specific for
Gi/o proteins. In addition, MAP kinase-3 (also known as
extracellular regulated kinase-3) and MAP kinase-1 (also
known as extracellular regulated kinase-2) were activated
within 5 minutes of progesterone treatment in transfected cells. Collectively, the data support the idea that
this fish protein is a membrane progestin receptor that
is linked to the non-classical actions of progesteronemediated oocyte maturation in fish31. A companion
paper reported the startling discovery that the fish membrane progesterone receptor was one of 14 members of a
new family of steroid membrane receptors present in
man, mouse, pig, Xenopus, zebrafish and puffer fish32.
Computer analyses indicate that this new family of
membrane receptors is unrelated to the nuclear superfamily of steroid receptors. It will be intriguing to learn
the identity of the natural ligands for these steroid membrane receptors, as well as their biological roles.
There is emerging biochemical (radioactive-ligand
binding and immunological studies with antibodies to
the nuclear receptors) and confocal microscopic evidence that ~5% of the classical nuclear receptor can
be associated with the plasma-membrane caveolae
(TABLE 2; FIG. 4A). Strong evidence supports the idea
that ER-α and ER-β are localized in caveolae of
endothelial33 and human breast cancer cells 34, and
emerging evidence suggests that the VDR is also
found in association with caveolae of intestine, kidney
and lung cells35–37. However, it should be stated as a
cautionary note that no absolute proof has been presented as to the precise identity of the membraneassociated ‘nuclear’ receptors. Such identification will
require the determination of a significant portion of
the amino-acid sequence of the membrane receptor.
This will complicate drug discovery efforts, because
until unequivocal evidence as to the biochemical
properties of the membrane steroid receptors is available, it will not be known whether the LBD is identical
to that of the superfamily of nuclear receptor proteins
or just a close variant.
Several uncertainties exist regarding the precise position of the caveolae membrane-associated hormone
receptor. It is not yet clear on which side of the caveolae
membrane the nuclear receptor is localized; FIG. 4A illustrates three possibilities. The model shown in FIG. 4Aa
proposes that the receptor is ‘outside’ the cell but inside
the flask of a caveola interacting with the outer plasma
membrane; although this is a possibility, it is not clear
what cellular mechanism(s) would be needed to move
the receptor through the membrane. The model shown
in FIG. 4Ab illustrates how an adaptor/scaffold protein
might form a heterodimer with a nuclear receptor to
tether it to or near the caveolae (see also TABLE 2). It is
known that in SRC tyrosine kinase, Gα subunits and
H-RAS each engage in protein–protein interactions with
caveolin, the integral membrane protein of caveolae38.
Last, the model shown in FIG. 4Ac illustrates how palmitoylation could tether the receptor to the inner surface of
the caveola; some evidence has been presented supporting such a mechanism for caveolae-ER-α association39.
34
| JANUARY 2004 | VOLUME 3
Clear and convincing evidence has been presented
that caveolae-associated ER-α can, for example, couple
to Gαi and stimulate endothelial NOS40 or, as another
example, couple sequentially to G-proteins that activate
matrix metalloproteases linked to membrane heparinbinding epidermal growth factor, and then to epidermal
growth factor (EGF) that finally activates the MAPkinase signalling pathway41.
The next categories of TABLE 2 hint of possible future
developments regarding the identification and characterization of membrane receptors for steroid hormones.
The potential existence of novel plasma membrane
receptors implies that there will be additional receptor
LBD targets to consider.
Another interesting variation on the properties of
membrane-related steroid receptors, which relates to their
ligand concentration, has been described by Auricchio’s
group. In this model (studied in NIH3T3 cells), the outcome of signal transduction can depend on ligand concentration: high ligand concentrations (10 nM) inhibited
DNA synthesis and entry into the cell cycle, whereas low
concentrations (1 pM) stimulated association of the
androgen receptor (AR) with both SRC and phosphatidylinositol 3-kinase, leading to S-phase entry42.
Two other tantalizing pieces of data are the binding
of steroids to PKC and extracellular plasma transport
proteins, which might interact with the membrane to
initiate signal-transduction events. For the plasma
transport protein for oestrogens and androgens, there
is preliminary evidence that the sex-hormone-binding
globulin (SHBG), when liganded, has the ability to
selectively interact with the plasma membrane of target cells and to transduce its signal via a G-protein that
stimulates adenylyl cyclase, leading to the production
of cAMP43. For the vitamin-D-binding protein (DBP),
the ‘membrane effects’ alluded to in TABLE 2 are mediated by the interaction of DBP with a target cell membrane to alter the chemotactic activity of the cell (in
this case, a neutrophil)44; these effects are not known to
be dependent on the DBP being fully liganded. The
suggestions that PKC possibly has an LBD for the
steroids aldosterone45 and 1α, 25(OH)2-vitamin D346,47
are intriguing, but in need of further investigation. In
principle, it would be possible for these proteins to
have a unique LBD specific for these steroids. This
would represent an attractive drug development consideration, because it would seem unlikely that these
LBDs have any structural similarity with the corresponding nuclear receptors.
Receptor and plasma transport protein structures
A dramatic advance in the understanding of steroidhormone receptors has occurred during the past eight
years with the determination of X-ray crystal structures
of the LBDs of seven steroid-hormone-related receptors:
oestrogen receptor (ER); progestin receptor (PR); glucocorticoid receptor (GR); AR; mineralocorticoid receptor
(MR); VDR; and the ecdysone receptor (ECR). In addition, three ‘other’ receptors with hormones as ligands
(retinoic acid receptor (RAR), retinoid-X-receptor
(RXR), and the thyroid hormone receptor (TR)) have
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REVIEWS
a
b
Domain II
Domain III
Domain I
Figure 5 | Ligand-binding domain structures. a | Overlay of the X-ray ribbon structures of the ligand-binding domain (LBD) of
the VDR116 (red) and ER-α52 (blue) receptors. For the VDR and ER-α, respectively, only the LBD portion of the structure was
crystallized; this represents aa118-427; ∆165–215 (VDR) and aa305–538 (ER-α). b | Space-filling views of the vitamin-D-binding
protein (DBP, 458 amino acids117) complexed to 1α, 25(OH)-vitamin D3 and the sex-hormone-binding globulin (SHBG, 373
amino acids55) complexed to 5α-dihydrotestosterone. For DBP, a surface crevice on domain I houses the ligand-binding site,
whereas for SHBG the LBD is located in an interior hydrophobic pocket within a β-sheet barrel. The DBP binds the parent
vitamin D3 and all its metabolites with high affinity (1–100 nM) and is found only in the serum compartment. SHBG binds both
androgens and oestrogens with high affinity and is responsible for their transport in the plasma compartment. ER-α, oestrogen
receptor-α; VDR, vitamin D3 receptor.
ORPHAN RECEPTOR
In this case, a receptor whose
structure makes it a member of
the superfamily of steroid
receptors, but for which no
ligand has yet been identified.
had their structures determined. Along with significant
primary amino-acid sequence similarity, these receptors have the same overall three-dimensional fold16,48,49.
FIGURE 5a presents the canonical ribbon structures of the
LBD of ER and VDR. A surprising revelation was that
not only are the ER-α and VDR LBD backbones
nearly superimposable (FIG. 5a), but all the members of
the superfamily seem to contain a structurally homologous protein fold, indicating a remarkable spatial conservation of the secondary and tertiary structure. Each of
these receptors contains a three-stranded β-sheet and 12
α-helices that are arranged to create a three-layer sandwich, which creates an LBD that completely engulfs the
cognate ligand in a hydrophobic core (FIG. 6a). So, a
challenge to the drug discovery process is to understand
the dynamic molecular detail of the interior surface of the
LBDs, which are composed of hydrophobic and hydrophilic amino-acid side-chains that make themselves
available to close in on, and interact with, the cognate
ligand or drugs. Although all the steroid-hormone
receptors have the same 12-helical three-dimensional
organization, each receptor has a unique ligand-binding
pocket shape that determines the shape of the hormone
that can bind with a high affinity.
NATURE REVIEWS | DRUG DISCOVERY
On the basis of an evaluation of the human genome
database, it is known that there are 48 members of the
nuclear receptor superfamily50. In this superfamily, 28
receptors have an LBD which binds a small ligand molecule; of these, eight are classical hormone receptors and
twenty are ORPHAN RECEPTORS50,51.
When the classical hormone receptors bind their
cognate ligands, a new conformation of the receptor
becomes energetically preferred, in which helix-12
closes over the mouth of the ligand-binding pocket.
An X-ray structure is available for the unoccupied
RXR48, providing the crucial insight that the position
of helix-12 in an unoccupied receptor is distinct from
that of a ligand-bound receptor. Further along these
lines, the structure of ER bound to raloxifene, which
can act as an antagonist of the transcriptional activation
function of the nuclear receptor52, results in helix-12
assuming an altogether different position on the surface
of the receptor (compared with hormone-bound or
apo receptor). In the case of raloxifene-bound ER, this
conformation renders the receptor incapable of stimulating gene transcription. In this manner, the position of
helix-12 sensitively modulates the biological activity
of the hormone receptor.
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REVIEWS
a
ER-α
VDR
b
ER-α
VDR
H1
H1
H3
H5
H12
H5
H3
H5
R394
R274
H2
Figure 6 | Details of the ligand-binding domain in the ribbon structures of the VDR and ER-α. a | Differences in the volume
and shape of the interior of the receptor’s ligand-binding domain (LBD; light blue) with the structure of their natural ligands 1α,
25(OH)2D3 and oestradiol shown in wire-frame. The Connolly surface displayed was generated by unmerging the ligand from the X-ray
assembly and selecting atoms that were within 5.0 Å of the ligand. b | Illustration for the VDR and ER-α of the location of the classic
LBD (red: 1α, 25(OH)2-vitamin D3 and 17β-oestradiol; genomic agonists, respectively) and a putative alternative LBD (blue: 1,
25(OH)2-lumisterol and oestren; non-genomic agonists, respectively) as studied by molecular modelling (helix-12 (H12) is coloured
brown and shown in the closed conformation in VDR (Protein Data Bank (PDB) code: 1db1) and in the opened conformation in ER-α
(PDB code: 1a52)). The conserved C-terminal H5 Arg residue (R274, VDR; R394, ER-α) is shown in CPK COLOURS. This residue forms
hydrogen bonds with ligands docked in either the putative alternative pocket or the genomic pocket. The proposed alternative pocket
portal lies between the C terminus of H1 and N terminus of H3. In both VDR and ER-α this region contains a large amount of loop
character; however, the VDR does contain a small two-turn helix labelled H2. The models were produced using Insight II (Discover_3,
cff91 force-field) and suggest that specific non-genomic agonists form favourable complexes with both the genomic and alternative
pockets; however, the interaction energies, calculated between the ligand and residues forming both ligand-binding pockets, indicate
that the specific non-genomic agonists oestren and 1, 25(OH)2-lumisterol have, respectively, better and equivalent interaction
energies, with the putative alternative pocket and weaker interaction energies with the genomic pocket when compared with the
natural hormones 17β-estradiol and 1α, 25(OH)2D3 docked in both pockets36,57. These models as yet lack functional validation, but it
is known that both the VDR22,118 and ER-α23,24 elicit non-genomic rapid responses. This indicates that an alternative pocket is
probably present within their LBDs that stereospecifically bind agonists unique for mediating some rapid or non-genomic responses.
ER, oestrogen receptor; VDR, vitamin D receptor.
CPK COLOURING
The CPK colour scheme for
elements is based on the colours
of the popular plastic spacefilling models developed by
Corey, Pauling and Kultun, and
is conventionally used by
chemists. In this scheme, carbon
is represented in light grey,
oxygen in red, nitrogen in blue
and sulphur in yellow.
PLASMA TRANSPORT PROTEINS
In addition to the vitamin-Dbinding protein and the sexhormone-binding globulin,
there are plasma transport
proteins for the following
hormones: retinoids (retinolbinding proteins), the thyroid
hormones (thyroxine-binding
globulin), and glucocorticoids
and progesterone
(corticosteroid-binding
globulin). Each plasma transport
protein binds its ligands with
high affinity; the Kd values fall in
the range 5–500 nM. In general,
for any given hormone, the Kd of
the hormone for its target organ
receptor is 10–100 times
stronger than for its plasma
transport protein, for example,
0.05–50 nM.
36
FIGURE 5b presents a space-filling view of the X-ray
structures of two steroid plasma transport proteins,
DBP and SHBG. Unlike the nuclear superfamily of
receptors, the PLASMA TRANSPORT PROTEINS for steroids and
related molecules are not evolutionarily related. The
three-domain structure of DBP (458 amino acids) is
apparent; domains I, II and III have been postulated to
have evolved from a progenitor that arose from the
triple repeat of a 192-amino-acid sequence53; however,
domain III is significantly truncated at the C terminus.
The most intriguing structural feature of DBP is the
ligand-binding cleft on the surface of the protein.
The presence of the 1α, 25(OH)2-vitamin D3 ligand in
domain I is clearly visible, and one surface of the ligand
is completely exposed to solvent. The protein’s affinity
for this ligand is still quite strong (Kd = 60 nM), yet, at
the same time, conveniently lower than the Kd (0.5–1
nM) for the VDR, thereby facilitating movement of
ligand from DBP to the VDR. It is well known in the
vitamin D field that the shape and chemical properties
of the ligand required for optimal occupancy of the
DBP cleft differ from those required for initiating
genomic and non-genomic signalling, a fact which must
be taken into consideration during drug discovery54.
The X-ray structure of SHBG is radically different
from that of DBP. Human SHBG is a homodimer of
| JANUARY 2004 | VOLUME 3
373-amino-acid monomers, each of which contains two
laminin G-like domains in a tandem repeat55. Only the
N-terminal G-domain residues of SHBG (1–194) are
required for ligand binding. The G domains comprise
strands of multiple β-pleated sheets forming a β-sandwich or jellyroll. Each monomer of SHBG has a binding
domain in which the steroid, 5α-dihydrotestosterone
(5α-DHT), intercalates into the interior hydrophobic
pocket within the β-sheet sandwich. A loop segment
(residues 130–135) covers the steroid-binding site and
could possibly regulate the access of ligands into the
binding pocket. Intriguingly, crystallographic studies
indicate that C18 (oestrogenic) and C19 (androgenic)
steroids are bound in opposite orientations within the
same SHBG steroid-binding site56. Again, here is a
hurdle for drug discovery. Given the subtle difference
in steroid ring structures for oestrogenic and androgenic
steroids, how can these be manipulated so as to control
their affinity for SHBG?
Possible alternative LBD in ER-α and VDR
As discussed earlier and summarized in TABLE 2, it is
becoming increasingly clear that some members of the
superfamily of steroid receptors (for example, ER-α, ER-β
and VDR) are in close association with the outer plasma
membrane of the cell, and more particularly caveolae
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REVIEWS
(FIG. 4A). This suggests that these receptor proteins can
mediate both genomic as well as rapid responses. This
then poses the conundrum as to how a receptor with one
LBD can bind ligands of quite different shapes to generate
two quite different biological outcomes.
Using the atomic coordinates of the X-ray structures
of the ER-α and VDR, and computer modelling (see FIG.
6b and its legend), we have been able to identify the
presence of a putative alternative LBD in each receptor
that can accommodate, via computer ‘docking’, either
the appropriate natural hormone or analogues that are
known to be agonists only for rapid responses (FIG. 3a,b;
1α, 25(OH)2-lumisterol for the VDR and oestren for the
ER-α)57. FIGURE 6b illustrates for the ER-α and VDR
both the classical nuclear ligand pocket (red) and the
proposed alternative ligand pocket (blue). Each ligand
pocket is envisioned to have separate portals. Entry to
the nuclear pocket requires that helix-12 is in an ‘open’
configuration (see brown H12 for ER-α; FIG. 6b) so that
the hormone/ligand can enter into the ligand-binding
pocket and gain access to integral H-bonding residues
(particularly the conserved C-terminal H5 Arg residue),
thereby fully occupying the genomic pocket. Next,
helix-12 moves to ‘re-close’ the portal (see brown H12
for VDR; FIG. 6b) shown for the VDR. The portal to the
alternative pocket is proposed to exist between a flexible
and variable region between H1 and H3 and the β-sheet
loop (FIG. 6b). Interestingly, this region had been proposed previously to be the portal to the nuclear pocket
and the conserved H5 Arg in TR-β58.
Receptor ensemble model
We propose a receptor ensemble model that can describe
how a classic steroid (nuclear) receptor could accommodate differently shaped ligands so as to result in the initiation of either rapid or genomic responses (FIG. 7). There
has been, over time, an evolution of the understanding
of how a substrate or ligand can interact with its cognate
enzyme or receptor, starting with the original ‘LOCK AND
59
KEY’ MODEL (first formulated by Fischer in 1898), which
was followed by the ‘INDUCED FIT’ MODELS (initially proposed by Koshland60), and finally leading to the current
protein ensemble model61. This model posits that
unbound receptor macromolecules exist in the cytoplasm as multiple receptor conformations in equilibrium
that follow the laws associated with standard statistical
b
k2
c
k–2
kon
koff
k–1
k3
Kinetically favourable
ligand–receptor complex
mediating rapid responses
Natural ligand able
to sample the X-ray,
genomic pocket
Thermodynamically
favourable ligand–receptor
complex mediating
genomic responses
k1
kon
k–3
Natural ligand able
to sample the
alternative pocket
koff
a
LOCK AND KEY MODEL
In the ‘lock and key’ model of
ligand binding, ligand-binding
sites of proteins are rigid and
complementary in shape to
their ligand.
INDUCED FIT MODEL
Koshland’s ‘induced fit’
hypothesis proposes that
a flexible interaction between a
ligand and the protein induces
a conformational change in
the protein, resulting in its
increased ligand-binding affinity.
Figure 7 | Schematic of a receptor ensemble model to describe how a classic steroid receptor could accommodate
differently shaped ligands that result in the initiation of either rapid responses or genomic responses. Three different
conformers of the ER-α are illustrated by different positions of helix-12 (H12; coloured brown). The ensemble model proposes that
there is a population of different unoccupied receptor species (a, b and c) that are in rapid equilibrium with one another; each
receptor conformer species might preferentially bind differently shaped ligands61. In this example, occupancy of the alternative
pocket by a ligand would lead to initiation of rapid responses, whereas occupancy of the classical pocket by a ligand would lead to
activation of genomic responses; both of these pockets are illustrated for the VDR and ER-α in FIG. 6b. The alternative pocket is
accessible in all three H12 conformers (a, b and c) due to the accessibility of the conserved H5 Arg residue from the right of H3.
Occupancy of the alternative pocket is assumed to be favourable before a steady-state is achieved in the genomic pocket by the
natural ligand. It is possible that the alternative pocket provides weaker van der Waals stabilization of the ligand than is achieved in
the genomic pocket. The receptor conformer (a) is the only conformer able to accept ligands that can bind to the classical ligandbinding site and lead to genomic responses, because in conformers b and c H12 blocks ligand accessibility to the conserved H5
Arg residue located at the innermost part of the classic ligand-binding pocket. An interesting consequence of the receptor ensemble
concept could be that association of the nuclear receptor with caveolae or cell membranes might change the ligand-binding
preferences to favour the alternative pocket (see FIG. 4Ab and the text). Ligand occupancy of this genomic pocket is favoured under
steady-state conditions because of the increase in thermodynamic stability derived from hydrophobic interactions. The designation
of ‘ligand occupancy of the alternative pocket affecting only rapid responses’ and the ‘genomic pocket affecting only genomic
responses’ is for simplicity. In theory, some non-genomic responses could be due to ligand occupancy of the genomic pocket and
the closed H12 conformation. In addition, it is expected that drug analogues, especially those related to the natural hormone(s),
might have differential fractional occupancies of the two pockets, thereby affecting the efficiency of the cellular signalling pathways
mediated by either pocket.
NATURE REVIEWS | DRUG DISCOVERY
VOLUME 3 | JANUARY 2004 | 3 7
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Table 3 | Proposed rapid-response drug development targets
Rapid-response hormone Disease target
Oestrogens
Cardiovascular protection against coronary heart disease
Androgens
Male osteoporosis; modulation of T splenic cells
Progestins
Modulation of sperm acrosome reaction
1α, 25(OH)2-vitamin D3
Plaque psoriasis, leukaemia, prostate cancer, immunosuppression (type 1 diabetes; organ transplantation)
Glucocorticoids
Anti-inflammation, plaque psoriasis
Mineralocorticoids
Hypertension, coronary heart disease
Thyroid hormone
Cardiac systems
distributions61. A steroid hormone or drug ligand would
therefore sample the existing population of receptor conformations available and form a receptor–hormone
complex with the receptor species that formed the
best complementary fit between the two molecules; this
would shift the equilibrium amongst the receptor species
so as to favour the energetically most stable hormonebound receptor conformation. It should be noted that
1α, 25(OH)2D3 should be able to change its conformation much more quickly than the receptor protein, so
essentially the whole ensemble of 1α, 25(OH)2D3 conformations can sample each of the individual protein
ensemble conformations. Implicit in this model is that
the receptor ‘sampling’ by the ligand involves gaining
entrance and exploring the interior surface of the LBD to
determine whether a complementary ‘fit’ can be
achieved. A related model to describe ligand/receptorinduced dissociation of rapid from genomic responses
was included in the comprehensive analysis of nongenomic, sex-nonspecific signalling by the ER and AR24.
In the ensemble model (FIG.7), the ER and VDR are
proposed to have two ligand-binding pockets, which
when occupied by the appropriately shaped ligand will
lead to the onset of either rapid or genomic responses.
In solution, for this model, there are three different ER or
VDR conformer species, which are illustrated by the
different positions of helix-12; all of these species are in
equilibrium with one another. When using the example
of the VDR, when the conformationally flexible, natural
seco steroid hormone 1α, 25(OH)2-vitamin D3 is present,
the 6-s-cis shape will sample (that is, attempt to dock
with) all three receptor species, but will preferentially
occupy the alternative ligand pocket (FIG. 7b) before the
system reaches the steady state. Simultaneously, the 6-strans-shaped hormone will also sample all three receptor
species, preferentially occupying the genomic pocket
(FIG. 7a) under steady-state conditions. The alternative
pocket, during the period of active ‘sampling’ of the conformational ensemble, should display greater ligand
accessibility than the genomic pocket before the steady
state, due to the proposed differences in the physical
properties of their respective portals. As stated previously,
the genomic pocket portal is controlled by the position of
helix-12, and a finite time interval will be required for
the helix to move to an ‘open’ position; therefore entrance
to the genomic pocket is gated (an introduction to the
field of biological gating or how proteins can have their
38
| JANUARY 2004 | VOLUME 3
functions switched ‘on’ or ‘off’ can be found in REFS 62–64).
Although details of access to the alternative pocket are not
yet available, it is proposed — given the increased loop
character of the amino-acid residues forming the lid
of the alternative pocket — that the energy barrier
between the opened and closed states is far less then that
required for helix-12 repositioning. This effectively
makes the ligand more accessible to the alternative
pocket than the nuclear pocket. This mechanism might
be analogous to the ‘flickering gate’ properties of certain
membrane channels that open and close rapidly65,66 (the
‘flickering gate’ concept has been proposed to mechanistically explain how membrane channels can be either
open or closed65; in this system, there is evidence that
inwardly rectifying potassium channels are regulated by
Gβγ). By contrast, over a long time-frame (steady-state
equilibrium), occupancy of the genomic pocket is significantly favoured over the alternative pocket in the
case of the natural ligand, because the increase in
hydrophobic stability prolongs the half-life of the ligand
in the genomic pocket, thereby requiring a large enthalpy
of activation for the re-opening of helix-12.
The existence of the two proposed pockets in the
receptor ensemble model raises an interesting possibility.
Specifically, it suggests that when the nuclear receptor
is associated with caveolae membranes or scaffolding
protein (see models in FIG. 4Aab), rather than the
nucleus, a conformation of the receptor favouring ligand
sampling of the alternative pocket might be enhanced by
intermolecular interactions that do not occur when the
receptor is in the nucleus. This could result in enhancement of steroid-hormone binding to the alternate
pocket, with a concomitant loss or significant reduction
in binding to the nuclear pocket. This might occur if
helix-12 of the receptor could not open as a consequence
of heterodimer interaction with a scaffold protein or a
caveolae membrane protein. Such a consequence would
effectively decrease the concentration of ‘classical’ receptor
from the family of ensemble members.
Receptor LBD and partner ligand shapes
What are the consequences of a match or mismatch? It is
instructive to compare carefully the Connolly surface
shape of a receptor LBD with their cognate steroid
hormone, or proposed analogue or drug. The natural
partners (receptor and its steroid hormone) should have
surface shapes that are reasonably complementary.
FIGURE 6a schematically illustrates, for the VDR and ER-α,
differences in the volume and shape of the interior
surface of the receptor’s LBD (shown in a Connolly presentation) with the fit of the resident natural steroid
hormone (shown in wire-frame presentation). A better
view of the Connolly natural ligand shape is provided in
the top row of FIG. 3b; the complementarity of the fit
between receptor and ligand is striking. Then, using the
Connolly shapes of the non-steroidal genomic ligands
(FIG. 3b, middle row) and the synthetic non-genomic
ligands (FIG. 3b, bottom row) one can explore how well
each ligand shape fits in the nuclear pocket or the alternative pocket of the VDR and ER-α of FIG. 6b. Clearly,
the synthetic non-steroidal ligands fit nicely into the
www.nature.com/reviews/drugdisc
REVIEWS
genomic pockets, but are a bad mismatch for the alternative pocket. From a drug discovery perspective, it is
imperative to evaluate the proposed new drug candidates to select those that might produce only genomic
responses, only rapid responses, or, if possible, both
genomic and rapid responses.
Drug development targets
Target selection in drug discovery is a complex topic67.
In the case of steroid-hormone receptors, it is important to have an in-depth understanding of the structural
and biochemical properties of the receptor and to
identify the logical population of receptor conformations available to bind the hormone or drug. Then, as
discussed, computer modelling should be used to
compare the interior shape(s) of the LBD of the receptor (when available) with the surface shape(s) of the
ligand. It might be possible to identify a small family of
structurally related potential drugs to be combinatorially
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NATURE REVIEWS | DRUG DISCOVERY
synthesized and biologically evaluated. Some proposed examples of possible drug development disease
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that these are suggestions, and it should not be
inferred that the absence of rapid responses specifically causes these diseases. The disease process could
be much more complex, and it remains to be determined how the interplay between steroid-mediated
genomic and rapid responses might be altered by the
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resulting implications for hormone or drug interaction
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Acknowledgements
A.W.N. and M.T.M. contributed equally to the preparation of this
review. Work in the laboratory of A.W.N. was supported by NIH
grant DK-09012. The authors thank H. Henry for her critical review
of the manuscript, and D. Keidel for his help in the initial modelling
experiments and helpful conversation.
Competing interests statement
The authors declare that they have no competing financial interests.
Online links
DATABASES
The following terms in this article are linked online to:
LocusLink: http://www.ncbi.nlm.nih.gov/LocusLink/
AR | DBP | epidermal growth factor receptor | ER | MAP kinase-1 |
MAP kinase-3 | MR | SHBG | SRC kinase | VDR
FURTHER INFORMATION
Signal Transduction Knowledge Environment website:
http://stke.sciencemag.org/cgi/content/full/sigtrans;2002/138/re9
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