Cell Membrane–Associated Mineralocorticoid Receptors?
New Evidence
Alexander W. Krug, Luminita H. Pojoga, Gordon H. Williams, Gail K. Adler
Abstract—The purpose of the present article is to provide an overview of plasma membrane steroid hormone receptors and
their implications in nongenomic signaling. We especially focus on recent evidence supporting the notion of a possible
membrane-associated aldosterone receptor, whether this receptor is different from the classic nuclear receptor, and the
possible implications of such a receptor for nongenomic and genomic aldosterone effects in physiological and
pathophysiological processes. (Hypertension. 2011;57:00-00.)
Key Words: mineralocorticoid receptor 䡲 aldosterone 䡲 nongenomic effects 䡲 plasma membrane
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ldosterone is the major physiological regulator of Na⫹
and electrolyte balance and, hence, blood pressure
homeostasis. In the kidneys, aldosterone mediates Na⫹ retention and K⫹ excretion via genomic effects through the
cytosolic mineralocorticoid receptor (MR), which acts as a
transcription factor.1 In 1984, Moura and Worcel2 described
rapid (within minutes) effects of aldosterone on Na⫹ efflux
from smooth muscle cells, and this effect could not be
blocked by the transcription inhibitor actinomycin D. Later it
was shown that aldosterone can elicit activation of Na⫹/H⫹
exchanger and signaling cascades, such as mitogen activated
protein kinases (MAPKs) or protein kinase C, through nongenomic mechanisms, and these effects may impact cellular
functions, such as growth and differentiation.3–5 Several
excellent recent reviews have extensively described the physiology and biology of aldosterone action.5–7
A
dence for the possible existence of a plasma membrane–
bound MR. For example, presumably membraneimpermeable, BSA-coupled aldosterone showed the same
rapid effects as aldosterone, supporting the idea of a plasma
membrane– bound receptor10,11; however, aldosterone-BSA
conjugates may undergo dissociation into hormone and BSA
requiring critical and careful handling,12 as shown for
estrogen-BSA.13 Additional support for a distinct membrane
MR comes from recent work by Wildling et al.14 Using
endothelial cell swelling as an index of an aldosterone effect,
it was shown that spironolactone could only block this effect
5 minutes but not 1 minute after the application of aldosterone.14,15 These investigators provided additional data supporting their hypothesis using atomic force microscopy technology. A single aldosterone molecule covalently bound to an
atomic force microscopy tip was used to detect unbinding
forces between aldosterone and plasma membrane–associated
molecules. These studies suggested the existence of an
aldosterone-binding protein in the plasma membrane. As
neither dexamethasone nor spironolactone influenced the
binding of aldosterone to its plasma membrane interaction
site, the authors concluded that the classic glucocorticoid
receptor (GR) and MR were not involved in these observations. These results, however, need to be interpreted with
caution, because the delay in spironolactone action might
relate to the kinetics of spironolactone binding to the receptor
and/or the relatively poor water solubility of spironolactone.
Moreover, the technology used cannot determine whether the
aldosterone binding sites are located in the plasma membrane
or in the several 100 nm spanning glycocalyx on the cell
surface that contains molecules that have been secreted into
the extracellular space and have then been adsorbed onto the
cell surface. Valid conclusions about the quantity of these
aldosterone-binding sites could not be made.14
Evidence for a Nonclassic Aldosterone Receptor in
the Plasma Membrane and MR- Independent
Rapid Signaling
Initially, the classic cytosolic MR protein was suggested to be
involved in mediating both nongenomic and genomic aldosterone signaling2,8; however, some studies reported that
these nongenomic effects could not be blocked by MR
antagonists such as spironolactone. In addition, although
rapid, nongenomic effects were elicited by the mineralocorticoids aldosterone, deoxycorticosterone, and 9␣fludrocortisone, these rapid effects could not be elicited by
the glucocorticoids cortisol and corticosterone.5 In analogy to
other steroid hormones, such as estrogens, Wehling et al9
hypothesized an aldosterone receptor that is distinct from the
classic MR protein, located at the membrane, and responsible
for nongenomic aldosterone effects. This view was initially
supported by several studies demonstrating functional evi-
Received July 13, 2010; first decision August 8, 2010; revision accepted March 17, 2011.
From the Brigham and Women’s Hospital/Harvard Medical School, Department of Endocrinology, Diabetes, and Hypertension, Boston, MA.
Correspondence to Alexander W. Krug, Brigham and Women’s Hospital/Harvard Medical School, Division of Endocrinology, Diabetes, and
Hypertension, 221 Longwood Ave, Boston, MA 02115. E-mail [email protected]
© 2011 American Heart Association, Inc.
Hypertension is available at http://hyper.ahajournals.org
DOI: 10.1161/HYPERTENSIONAHA.110.159459
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Functional evidence for MR-independent rapid aldosterone
effects comes from studies using MR-deficient human embryonic kidney cells. Aldosterone-induced Ca2⫹ signaling
was similar in MR-transfected and untransfected cells and
could not be prevented by MR blockade.16 Similarly, aldosterone-induced Ca2⫹ signaling in fibroblasts from MR
knockout mice is unchanged compared with the wild type,
indicating MR-independent mechanisms.17 Taken together,
results from these studies provided indirect evidence for a
unique membrane-bound MR, making conclusive and definitive statements difficult.6
However, one could speculate that the described aldosterone interaction site on the plasma membrane represents an
already known molecule with a distinct physiological function. For example, the ␣␯␤3 integrin is believed to act as a
cell surface thyroid hormone receptor mediating T4-induced
MAPK activation18 and affecting nuclear thyroid hormone
receptor and estrogen receptor (ER) function.19,20 A very
recent article by Gros et al,21 published in Hypertension,
builds on this concept, providing for the first time more direct
and convincing evidence for the existence of a plasma
membrane aldosterone receptor. At low picomolar concentrations, aldosterone increased extracellular signal–regulated
kinase (ERK) 1/2 phosphorylation in vascular smooth muscle
cells, which was inhibited both by an antagonist to the orphan
G protein– coupled receptor (GPR) 30 and the MR blocker
eplerenone. Functional data suggest that this effect seems to
be specific for aldosterone versus glucocorticoids, with an
EC50 in the low picomolar range. In rat aortic endothelial
cells with no detectable MR expression levels, aldosterone
also induced ERK1/2 activation, and GPR30 blockade as well
as adenovirus-mediated downregulation of GPR30 almost
completely prevented aldosterone-mediated ERK1/2 phosphorylation. In conclusion, both MR- and GPR30-mediated
mechanisms seem to be involved in rapid aldosterone signaling, such as ERK1/2 and phosphatidylinositol 3-kinase activation. Although this study did not include binding assays of
aldosterone and GPR30, it has established the membrane
protein GPR30 as a crucial factor in MR-independent rapid
aldosterone signaling. GPR30 has also been described as a
high-affinity/low-capacity estrogen-responsive protein and a
linker in the membrane ER signaling complex. However, it
does not seem to function as an independent ER in the
absence of classic ER protein.12 Future investigations addressing the physiological and pathophysiological significance of aldosterone-GPR30 interaction, including aldosterone-GPR30 binding studies, should further characterize the
nature of aldosterone-GPR30 interaction.
Evidence That the Classic MR Mediates Both
Nongenomic and Genomic Aldosterone Signaling
and a Small Fraction May Be Found in the
Plasma Membrane
Currently, a large body of evidence supports the view that
both nongenomic and genomic aldosterone effects are mediated via the classic cytosolic MR protein in various cell
types,22–24 and at least a small fraction of classic MR is
located in the membrane. In contrast to spironolactone, more
water-soluble, open-ring form MR blockers, such as
RU28318, inhibited the same rapid aldosterone effects,5,23
supporting a role for the classic MR in nongenomic aldosterone signaling. Moreover, a study from Alzamora et al25 has
demonstrated in strips of human vascular vessels that inhibition of 11␤-hydroxysteroid dehydrogenase, an enzyme that
metabolizes glucocorticoids to their inactive ␣-keto forms,
caused cortisol to induce similar nongenomic effects as
aldosterone. Cortisol has similar affinity to MR as aldosterone, suggesting that classic MR is involved in these nongenomic effects. Using MR-deficient human embryonic kidney cells, studies from Grossmann et al16 have shown rapid
aldosterone activation of a variety of signaling cascades,
including ERK1/2, p38, and c-Jun N-terminal kinase, only
when cells were transfected with a gene encoding the classic
human MR. Support for the idea that at least a fraction of the
classic MR protein is localized at the plasma membrane
comes from another recent study; Grossmann et al26 used
green fluorescent protein–tagged MR in a heterologous expression system, with coimmunoprecipitation and fluorescence energy transfer imaging techniques, to demonstrate that
a small fraction of MR is colocalized with epidermal growth
factor receptor (EGFR) at the plasma membrane. Similar to
other classic steroid hormone receptors,12 MR-mediated
MAPK activation depends on src-mediated EGFR transactivation.27,28 Interestingly, in cells transfected with human MR,
membrane colocalization of EGFR and MR was not dependent on the presence of MR ligand; however, the presence of
hormone seemed to be necessary to activate EGFR and to
induce ERK1/2 phosphorylation. Long-term aldosterone
stimulation resulted in the disappearance of MR-EGFR colocalization and translocation of the receptor into the nucleus.
Disruption of cholesterol-rich domains reduced MR-EGFR
interaction at the membrane, further supporting the notion of
an MR/EGFR signaling complex located in the plasma
membrane.26 In an earlier study, the group demonstrated that
the MR-green fluorescent protein construct shows similar
functional behavior with respect to nongenomic and genomic
signaling compared with the untagged human MR.16 However, it should be noted that a heterologous expression system
represents an artificial situation, and it is possible that
aldosterone binds to supramolecular complexes of MR associated with membrane-bound proteins. ER, GR, progesterone
receptor (PR), and androgen receptor (AR) all have a palmitoylation sequence, which is considered to be required for
membrane localization and rapid steroid signaling but not
nuclear localization.29,30 Palmitoylation is the reversible addition of a palmitate group to the sulfhydryl group of a
cysteine, providing a protein with weak membrane affinity
with a hydrophobic anchor.31 In contrast, MR does not seem
to have a perfect palmitoylation sequence, but it contains a
FYQLTKLL or FPAMLVEII sequence, which might serve as
a palmitoylation sequence. Additional studies are needed to
determine whether these or other MR sequences are relevant
for subcellular MR localization and possible binding of the
classic MR to the plasma membrane.26
Coimmunoprecipitation studies from our laboratory demonstrated an association between MR and caveolin (Cav) 1, as
has been noted for Cav-1 and ER.32 Cav-1 is the major
component of caveolae, which contain a variety of signaling
Krug et al
Mineralocorticoid Receptors in the Plasma Membrane
Table. Membrane-Associated Caveolin 1–Binding Proteins,
Including Steroid Hormone Receptors, Growth Factor Receptors,
and Signaling Molecules, Involved in Rapid Steroid Signaling
Protein
Reference
Receptors
Endothelin type A, B receptors
Chun et al33
Okamoto et al34
Yamaguchi et al35
Insulin receptor
Yamamoto et al36
Nystrom et al37
Epidermal growth factor receptor
Couet et al38
Steroid hormone receptors
Estrogen receptors ␣ and ␤
Schlegel et al39
Chambliss et al40
Pedram et al29
Androgen receptor
Lu et al41
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Pedram et al29
Glucocorticoid receptor
Progesterone receptors A and B
Mineralocorticoid receptor
Jain et al42
Pedram et al29
Own observations
(2010, unpublished)
Enzymes
Src
Sargiacomo et al 199343
Li et al44
Protein kinase A
Mitogen activated protein kinase kinase
Razani et al45
Engelman et al46
Phosphatidylinositol 3-kinase
Zundel et al47
Adenylyl cyclase
Fagan et al48
Ostrom et al49
Rybin et al50
Endothelial NO synthase
Garcia-Cardena et al51
Liu et al52
Shaul et al53
molecules, including steroid hormone receptors, growth factor receptors, and molecules involved in rapid steroid signaling (see Table for a selection of Cav-1– binding proteins). In
addition, Cav-1 knockout mice have decreased cardiac and
vascular MR expression.54 In mice infused with NG-nitro-Larginine methyl ester and angiotensin II, lack of Cav-1
reduces biventricular myocardial damage and reduces vascular expression of proinflammatory factors despite similar
circulating levels of aldosterone in wild-type and Cav-1
knockout mice. Loss of Cav-1 blunted the association between inflammation and aldosterone levels. Interestingly,
NG-nitro-L-arginine methyl ester/angiotensin II infusion resulted in an increase in cardiac MR expression in wild-type
but not in knockout animals, suggesting that Cav-1 is an
important factor determining cardiovascular MR expression
and signaling in vivo55; however, because Cav-1 is likely
involved in a variety of transduction pathways related to
inflammation but not associated with MR activation, these
findings need to be interpreted with caution until confirmative data are available.
3
Estrogen, Glucocorticoids, Androgens,
and Progesterone
The nature and function of plasma membrane steroid receptors have been characterized best for estrogen. Szego and
Davis56 demonstrated rapid effects of 17␤-estradiol on cAMP
and Ca2⫹ signaling in cell and animal models and identified
an estrogen-binding protein in the plasma membrane.57,58
Subsequently, both the 46-kDa and full-length 66-kDa forms
of ER␣ were found in the plasma membrane, with the 66-kD
protein probably being the functionally relevant receptor.12
Approximately 5% to 10% of the endogenous cellular ER␣
pool is found in the plasma membrane. The membrane and
nuclear ER have similar and high affinity for ligand. This
membrane-localized ER is responsible for rapid estrogen
signaling, with the ligand-binding domain of ER␣ being
necessary for both rapid signaling and membrane binding.59
Two endogenous ER␤ variants of ⬇54 kDa and 60 kDa have
also been described at the membrane, but less is known about
their function compared with that of ER␣.12,60
In endothelial and other cells, membrane-bound ER␣ and
ER␤ are found primarily in caveolae and noncaveolae rafts.
Cav-1 modifies ER and enzyme activity in vascular tissue,
facilitating rapid activation of endothelial NO synthase and
NO production.61 Recruitment of ER␣ to the plasma membrane is mediated by palmitoylation within the E domain of
the ER,30,62 which enhances physical association of the
receptor A/B domain with Cav-1, followed by ER␣ translocation to the membrane. Striatin, which was originally purified from rat brain synaptosomes, binds via its conserved
N-terminal motif to Cav-1 in a Ca2⫹-dependent way,63
promoting binding of ER␣ to the membrane. Striatin contains
a Ca2⫹-calmodulin– binding domain and a large WD
(tryptophan-aspartate)-repeat domain,64 which is commonly
found in cytoskeletal and signaling proteins and allows
multiple protein-protein interactions. For example, striatin is
found in a complex with protein phosphatase 2A, which
directly binds to and regulates ER␣ function.65 Because rapid
estrogen signaling is characterized by membrane, but not
nuclear, localization of ER,59 disruption of the ER/striatin
complex prevents nongenomic, but not genomic, estrogen
signaling; thus, this indicates a crucial role for striatin in the
regulation of nongenomic versus genomic ER signaling.66
The scaffold protein GPR30 has been described as a highaffinity/low-capacity estrogen-responsive protein, which is
likely to serve as a linker in the ER signaling complex in a
variety of cells, including endothelial and vascular smooth
muscle cells. The exact role of GPR30 in membranemediated estrogen signaling is not clear yet, but GPR30 has
been shown to collaborate with ER␣ at the membrane to
mediate rapid estrogen-mediated kinase signaling in a variety
of cells, such as endometrial and ovarian cells resulting in
proliferation.12
Estrogen stimulation induces dissociation of the Cav-1/
ER␣ complex, allowing interaction of the receptor with
signaling intermediates, such as c-src.67 In the nonactivated
state, ER␣ is bridged via its E domain to c-src by a scaffold
protein that has been named MNAR (modulator of nongenomic action of ER),68,69 which contains multiple LXXLL
protein-protein interaction motifs. LXXLL modules (where L
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The AR is even less well characterized. AR-mediated
signaling also seems to originate from caveolae located in the
plasma membrane.81 No specific membrane AR, which is
different from the classic AR protein, has been described.
Thus, both rapid and nuclear AR signaling seem to be
mediated by the same AR protein, with a fraction of AR being
located to the membrane. For example, in prostate cancer
cells, rapid AR signaling includes EGFR-mediated c-src
activation, which may involve an androgen-dependent AR/
MNAR interaction.82 Similarly, testosterone rapidly activates
src in Sertoli cells, resulting in EGFR transactivation and
MAPK signaling,83 possibly affecting androgen-dependent
and -independent prostate cell growth.
In addition to the classic PR-A/PR-B, a membrane PR
family has been described. Showing high-affinity interactions
with progestins, membrane PRs are involved in oocyte
maturation. Membrane PR also seem to be involved in
progestin-mediated myometrium regulation at the end of
pregnancy, involving activation of various kinase cascades
and cross-talk with classic PR.84 Progesterone-binding sites
have been shown on the cell surface of sperm cells. Evidence
suggests the involvement of another membrane-localized
progesterone-binding protein termed progesterone membrane
receptor component 1 in the nongenomic, transcriptionindependent acrosomal reaction in sperm,85 and in progesterone effects in granulosa and luteal cells.86 Other than genomic
signaling, classic PR-B also initiates MAPK signaling via
direct binding between the SH3 domain of src and a prolinerich sequence of the PR.87 Although it is not completely clear
whether PR-mediated src activation is initiated at the plasma
membrane, it is believed to occur outside the nucleus.88 One
ALDO
GPR30
ALDO
src
src
ALDO
cav-1
a
membrane
c
cav-1
MR
src
ALDO
src
cytosol
MAPK
NONGENOMIC
EFFECTS
MR
nucleus
MR
A LDO
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is leucine and X is any amino acid) are specific motifs
contained within a nuclear receptor coregulatory protein that
mediate the binding of the protein to this nuclear receptor.70
Other ER domains interact with additional scaffold proteins,
such as shc, and the ER/shc interaction mediates downstream
kinase activation. Depending on the cellular context and
stimulus, c-src activation initiates downstream activation of
ras, raf, MAPK, ERK1/2, or phosphatidylinositol 3-kinase,
AKT,71 resulting in different physiological cellular outcomes.
In the case of estrogens, rapid steroid signaling may persist
for several hours,72 leading to activation of protooncogenes
such as raf and ras.73
The first evidence for plasma membrane– bound GRs
(mGR) was described in neuronal cells, as well as human and
rodent leukemia cell lines.74,75 Overexpression studies of a
classic GR gene in a transfection model did not show
enhanced cell-surface GR expression, suggesting that mGR
may be a splice variant or product of posttranslational
modification of the classic GR gene.76 The exact structure and
importance of mGRs in rapid glucocorticoid signaling has not
been elucidated. Several reports suggest the involvement of
mGR in the pathogenesis of chronic inflammatory diseases.77
For example, in patients with rheumatoid arthritis, increased
numbers of mGR-positive monocytes and B-lymphocytes are
directly associated with parameters of disease activity,78 and
nongenomic glucocorticoid signaling is believed to mediate
immunosuppressive effects in T cells.79 Rapid extranuclear
glucocorticoid signaling also seems to be involved in suppression of adrenocorticotropic hormone secretion from the pituitary
gland; this effect likely involves the classic GR, because it can
be blocked by the GR antagonist mifepristone.80
MR
↑ transcription
( e.g. ENaC, Na-K ATPase, SGK1)
GENOMIC EFFECTS
Figure. Hypothetical model describing membrane-initiated rapid aldosterone signaling. Classic mineralocorticoid receptor (MR) is translocated to and associated with the membrane in a signaling complex including striatin, caveolin (Cav) 1, src, and the epidermal growth
factor receptor (EGF-R). Similar associations have been described for estrogen receptor (ER), androgen receptor (AR), and progesterone receptor (PR). On aldosterone stimulation, MR is released from the membrane-signaling complex and EGF-R is transactivated by
aldosterone, initiating c-src–mediated mitogen-activated protein kinase (MAPK) activation. In addition, G protein– coupled receptor
(GPR) 30 may function as aldosterone receptor mediating rapid MR-independent signaling.
Krug et al
Mineralocorticoid Receptors in the Plasma Membrane
study suggesting membrane localization of PR demonstrated
that the PR contains a palmitoylation sequence characteristic
of membrane proteins and a 9-amino acid membraneanchoring motif.30 Similar to estrogens, androgens, and aldosterone, rapid PR-initiated src activation mediates EGFR
transactivation, resulting in downstream MAPK activation.
Conversely, EGFR activation leads to PR-B phosphorylation
and ligand-independent accumulation of the receptor in the
nucleus.89,90 These mechanisms are believed to be involved in
breast cell proliferation and differentiation.91
Summary
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Signaling characteristics of steroid actions share similar
patterns for different hormones, such as vitamin D, androgens, progesterone, glucocorticoids, and estrogens, and
plasma membrane receptors for these hormones have been
described.32 In the case of estrogen, the plasma membrane
ER␣ is responsible for nongenomic signaling. For other
steroid hormones, such as androgens and progesterone,
plasma membrane receptors have been described, but rapid
signaling is mediated both by the cytosolic and the
membrane-associated steroid receptors.12 The evidence for
membrane-bound aldosterone receptors discussed in this
article is mainly indirect. Still, the concept of a unique
aldosterone plasma membrane receptor, which is different
from the classic cytosolic MR protein and exclusively mediates rapid aldosterone signaling, has received new support. A
recent study has shown that GPR30 mediates MRindependent rapid aldosterone effects in vascular smooth
muscle cells and endothelial cells. In addition, a large body of
evidence supports the view that both genomic and rapid
aldosterone effects are mediated via the classic MR protein. A
small fraction of classic MR protein seems to be present in the
plasma membrane and associated with Cav-1 in a signaling
complex in caveolae. This suggests that the MR is at least in
the appropriate location to initiate membrane signaling (see
Figure for a hypothetical model of plasma membrane–
initiated aldosterone signaling), but it remains to be determined in more detail whether aldosterone receptors at the
membrane play an important (patho)physiological role in
mediating rapid aldosterone effects.
Sources of Funding
This article was supported entirely by the Brigham and Women’s
Hospital, Division of Endocrinology, Diabetes, Hypertension,
Harvard Medical School.
Disclosures
None.
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Cell Membrane−Associated Mineralocorticoid Receptors?: New Evidence
Alexander W. Krug, Luminita H. Pojoga, Gordon H. Williams and Gail K. Adler
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