Download Receptor Fragments: Intracellular Signaling and

60
Current Hypertension Reviews, 2012, 8, 60-70
Receptor Fragments: Intracellular Signaling and Novel Therapeutic Targets
Julia L. Cook*
Laboratory of Molecular Genetics, Ochsner Clinic Foundation, Biomedical Research Building, 1514 Jefferson
Highway, New Orleans, LA 70121, USA
Abstract: Many conventional GPCRs such as those associated with apelin, endothelin, prostaglandin E2, and angiotensin
have also been localized to the intracellular space, principally the nucleus. These observations have involved a broad
range of tissues, isolated primary cells, and cell lines and a variety of techniques including confocal microscopy,
immunohistochemistry, immunocytochemistry, and western blotting. Some receptors are transported to nucleus as
holoreceptors while other receptors have been shown to be cleaved with only a portion of the receptor trafficking to
nucleus. Several studies from many different laboratories indicate that, depending on the cell type, the angiotensin II type
1 receptor can exist in nuclear membrane or nucleosol and that nuclear accumulation can be induced by ligand-treatment.
Moreover, a population of the angiotensin receptor is cleaved in response to angiotensin II and the cytoplasmic carboxyterminal fragment trafficks to nucleus and is a potent apoptotic reagent. In this review, we discuss AT 1R cleavage in light
of several other receptor cleavage events which similarly produce apoptotic fragments; functionally active intracellular
cleavage fragments represent novel targets for drug development.
Keywords: Angiotensin II, intracellular, intracrine, nuclear AT1 receptor, receptor cleavage.
NUCLEAR GPCRs
A number of transmembrane receptors, including several
receptor tyrosine kinases, (e.g., receptors for epidermal
growth factor (EGF), insulin, fibroblast growth factor (FGF),
nerve growth factor (NGF), interleukin (IL-1), ErbB-4 and
Her22/neu) [1] have been reported to localize to the nucleus,
either as holoproteins or protein cleavage fragments (Fig. 1).
In fact, the most innovative and exciting studies at the
receptor forefront converge on the idea that many
conventional plasma membrane receptors also accumulate
within cell nuclei (within the nuclear membrane and/or
nucleosol) and that others undergo “regulated intramembrane
proteolysis” (also known as RIP) to produce receptor
fragments that can continue to function within (or outside)
cells to mediate biologically relevant events [2-5]. This
principal has been demonstrated for many receptor tyrosine
kinases and other single-pass (cross the membrane only
once) membrane receptors.
Many G protein-coupled receptors (GPCRs) are also
known to associate with nuclei (nuclear membrane or
nucleosol) and several of these are known to undergo
regulated proteolysis, usually at the extracellular domain [68]. However, from studies of most GPCRs, it is unclear
whether an intracellular fragment (as compared to an
ectodomain fragment) is also generated during proteolysis,
often because the appropriate assays have not yet been
*Address correspondence to this author at the Laboratory of Molecular
Genetics, Ochsner Clinic Foundation, Biomedical Research Building, 1514
Jefferson Highway, New Orleans, LA 70121, USA; Tel: 504-842-3316;
E-mail: [email protected]
17-/12 $58.00+.00
performed. Nuclear GPCRs, including those for acetylcholine,
angiotensin II, apelin, dynorphin B, endothelin 1, and
prostaglandin E2, have been identified, often using multiple
different approaches [9, 10] (Fig. 1). Confocal microscopy
studies indicate that the endothelin B (ETB) receptor, a
classic rhodopsin-like Class A GPCR (as is the angiotensin
AT1 receptor), is located primarily in nuclei of rat ventricular
cardiac myocytes. Western blot analyses of purified nuclei
show this receptor to copurify with nucleoporin 62, and
ligand-binding and antagonist studies of isolated myocyte
nuclei confirm the association [11]. These studies were
conducted using C-terminal-specific antibodies that do not
differentiate between the presence of holoreceptor and
cleaved receptor C-terminal fragment.
GABAB receptors are Class C GPCRs. Functional
GABAB receptors consist of a heterodimer of GABABR1
and GABABR2. Using electron microscopic analyses of
immunoperoxidase- and immunogold-labeled tissues,
Burkhalter and associates found both subunits to be present
not only on the plasma membrane but in cellular nuclei of
the visual cortex as well [12]. Since the antibodies used in
that study were reactive to the receptor subunit carboxytermini, it is not clear whether the holoreceptors or only the
cytosolic domains are present in nuclei. Yeast two-hybrid
screens have also revealed that both subunits associate with
CREB2 [activating transcription factor 4 (ATF4)] and ATFx
and, in theory, may, through this association, modulate gene
transcription [13]. White and colleagues further show that
CREB2 binds to the cytoplasmic carboxy-terminus of the
heterodimer, translocates to the nucleus in response to
receptor activation, and directly upregulates the Gadd153
promoter [14]. It is possible, therefore, that the GABA B
© 2012 Bentham Science Publishers
Angiotensin Receptor Fragments
Current Hypertension Reviews, 2012, Vol. 8, No. 1
Multipass
TM Receptors
ETA, ETB
Kappa Opioid
Apelin
AT1, AT2
mAChR
PGE2
βAR
Neurokinin
Bradykinin B2
PTHrP
61
Signal Transduction
Transcription
Replication
Singlepass
TM Receptors
EGF
Insulin
FGFR1, FGFR2
Trk-A
IL-1
ErbB-2, ErbB-4
Fig. (1). Representative conventional plasma membrane receptors that have also mapped to the nucleus (nucleosol, matrix, or nuclear
membrane) either as holoreceptors or cleaved products [9, 18, 105, 118-142].
holoreceptor or C-terminal fragment(s)
transport CREB2 into the nucleus.
may
directly
The 7-transmembrane angiotensin AT1 receptor (AT1 R)
has been localized to nuclei by several different independent
studies using techniques which include radioligand binding
and chromatin solubilization assays of rat liver nuclei,
immunohistochemistry of rat brain, electrophysiology assays
of rat cardiac myocytes, Ang II microinjection and calcium
assays, immunocytochemistry and western blot of rat brain
neurons, and immunocytochemistry and western blot of
human VSMCs [10, 15-17]. In these nuclear association
studies, assays have not generally been designed to
differentiate between cleaved receptor fragments and
holoreceptors. In contrast, our studies as described below,
have been specifically designed to address cleavage and
trafficking. We find that a population of the AT1R undergoes
cleavage with trafficking of the C-terminus into the nucleus.
That being said, intact receptor is also clearly present within
nuclear membranes and retains biologically important
functions perhaps through interaction with intranuclear Ang
II [18-25].
Since the G protein-coupled receptors (GPCRs)
constitute the largest family of cell surface proteins involved
in signal transduction and, indeed, are the target of more
than 50% of current marketed therapeutic agents [26], the
identification of new functions for these proteins, and the
corresponding opportunities for new drug design are exciting.
This review will discuss AT1 R cleavage from the perspective
of cleavage of other receptors from divergent families.
CLEAVAGE OF MULTI-PASS MEMBRANE RECEPTORS
A number of GPCRs, in addition to the AT1 R, are
reported to undergo regulated limited proteolysis to produce
peptides with bioactivity. The ETB GPCR was perhaps the
first multispanning membrane protein reported to be cleaved
by a metalloprotease but to retain functional activity and
overall structure [6]. ETB possesses a cleavable 26 amino
acid signal peptide. In addition to removal of the signal
peptide, the N-terminus (38 amino acids) of the mature
protein undergoes proteolytic cleavage in a ligand-mediated
fashion; the ETA subtype is not cleaved [6]. Moreover,
batimastat [inhibitor of TNFα converting-enzyme (TACE)]
and metal chelators (EDTA and phenanthroline) block the
cleavage indicating involvement of a metalloprotease. The
functional importance of the processing is unknown.
However, the receptor subtypes demonstrate disparate spatial
distributions in cardiac myocytes [10, 11]. Endothelin A
(ETA) receptors primarily localize to plasma membrane
while ETB receptors localize primarily to nuclei; receptors in
both compartments retain functionality. Note that these
studies were conducted using amino-terminal ETA receptorspecific antibodies and carboxy-terminal ETB receptorspecific antibodies which may have influenced the outcome
[11]. In any case, the fate of the plasma membraneassociated ETB receptor following cleavage has not been
reported but it is clear that this receptor, wholly or in part,
localizes to the nucleus.
The β1-adrenergic GPCR (β1AR), the predominant βAR
in the heart has also been found to undergo limited
proteolysis, both constitutive and regulated, to produce an Nterminal cleavage product [7]. Agonist enhances cleavage in
both a time and concentration dependent manner; cleavage
occurs in vivo via metalloproteases. Moreover, cleavage
occurs at the plasma membrane rather than internal
compartments and in a regulated manner. Since mutation
of the cleavage site stabilizes the mature receptor, the
investigators suggest that N-terminal cleavage represents a
62 Current Hypertension Reviews, 2012, Vol. 8, No. 1
novel mechanism for regulation of cell surface receptor
accumulation. Once again, the fate of the remaining portion
of the receptor is unknown.
Recent studies suggest that the extracellular domain of
the β2-adrenergic receptor is also cleaved [8]. Spontaneously
hypertensive rats (SHR) appear to have enhanced levels of
matrix metalloproteases (MMPs) which lead to reduced
density of the β2AR extracellular domain on aortic
endothelial cells and cardiac microvessels of SHR compared
to WKY or Wistar rats. Moreover, treatment of the aorta and
the heart of control Wistar rats with plasma from SHR
reduced the extracellular but not intracellular domain of
β2AR in these tissues, a process that was prevented by MMP
inhibitors. In related studies, the NFκB transcription factor
has been shown to be upregulated in SHR and to augment
MMP activity which in turn increases cleavage of β2AR [27,
28]. Treatment of SHR with the NFκB inhibitor, pyrrolidine
dithiocarbamate (a metal chelator), reduces NFκB, MMP-2,
MMP-9 and systolic pressure of SHR. Collectively, these
studies suggest that MMPs contribute to cleavage of the
extracellular domain of β2AR, to inactivation of the
vasodilatory B2AR, and to increased arteriolar tone in SHRs.
Vasopressin (AVP), the antidiuretic hormone, is a cyclic
nonpeptide that is involved in the regulation of body fluid
osmolality [29-31]. AVP mediates its effects through a
family of G-protein coupled receptors, the vasopressin
receptors type V1a, V2 and V3 (also designated V1b). The
human vasopressin receptor V2 gene maps to chromosome
Xq28 and is expressed in lung and kidney [32, 33].
Mutations in the V2 receptor result in nephrogenic diabetes
insipidus (NDI), a rare X-linked disorder characterized by
the inability of the kidney to concentrate urine in response to
AVP [33, 34]. The vasopressin receptor V2 activates the G s
protein and the cyclic AMP second messenger system [34].
V2 GPCR has been shown to undergo ligand-mediated
metalloprotease cleavage at the second transmembrane helix
close to the extracellular agonist binding site [35]. While no
stable intracellular domain has been identified, other yet
unknown fragments may be generated in a regulated fashion.
The protease-activated GPCR, PAR1, is angiogenic and
has a role in vascular development [36]. The N-terminal 41
amino acid cleavage fragment, parstatin, can be cleaved
by MMP-1, thrombin or activated protein C. It is antiangiogenic and pro-apoptotic. Interestingly, exogenous
parstatin rapidly localizes to the cell surface, penetrates the
cell membrane and accumulates in the intracellular space; no
extracellular receptor has been identified but the uptake
seems to be dependent upon the hydrophobic N-terminus of
parstatin [37]. It is not clear at this point to what extent
parstatin serves to regulate and counterbalance signaling
from the parent molecule but it is being investigated for
pharmacologic value.
Polycystin-1 (PC1) is an atypical 11-transmembrane
GPCR which is involved in autosomal dominant polycystic
kidney disease (ADPKD) [38]. PC1 is cleaved at the N-
Julia L. Cook
terminus and C-terminus; the C-terminus trafficks and
accumulates in nucleus preferentially in the absence of
mechanical stimuli (i.e., low blood flow). This C-terminal
tail interacts with the transcription factor STAT6 and the
coactivator P100; all of these are upregulated in nuclei
of cyst-lining cells in ADPKD [39]. The C-terminal tail
apparently binds to both STAT6 and P100 and may be
involved in shuttling them to the nucleoplasm where they are
involved in regulating several pathways including wnt [40] ,
AP-1 [38, 41-43], calcium signaling [44, 45] and activation
of STAT1 [46] all of which may influence kidney disease
progression.
An intracellular fragment is also produced from the
GPCR, D-frizzled 2, a post-synaptic protein which interacts
with the presynaptic protein, “wingless”. Following endosome
internalization, the cytoplasmic domain is cleaved and
translocated to the nucleus where it is involved in transcriptional events that support synapse development [47, 48].
Interference with D-frizzled 2 cleavage reduces proliferation
of synaptic boutons and formation of pre-and postsynaptic
specializations in many boutons.
Collectively, these studies indicate that cleavage of
receptors including GPCRs and other multi-membrane
spanning cell surface proteins, release of extracellular
peptides and accumulation of stable intracellular products
can be regulated processes which serve, perhaps, to further
amplify or enhance effects of ligand:receptor signal
transduction events which initiate at the plasma membrane.
Alternatively such products may directly, as in the case of
parstatin, counterbalance downstream effects from the initial
ligand:receptor interaction events. As such, cleavage
products may act in a homeostatic manner to control the
transduced signal magnitude or duration.
AT1 RECEPTOR CLEAVAGE
The existence of an intracellular renin-angiotensin
system (iRAS) implies that components of the RAS
are made locally and result in biologically functional
intracellular angiotensins, renin and/or receptor. Studies
show that measurable levels of angiotensin II (Ang II) exist
within some cells and that Ang II may be released from
certain cell types (e.g., cardiac myocytes and mesangial
cells) following stretching [49-55]. Existing intracellular
Ang II may be internalized from the circulation or extracellular fluid, or alternatively, produced intracellularly.
Consistent with intracellular Ang II function, a number of
studies independently support the existence of intracellular
Ang II binding sites. Early studies by Re and colleagues
showed (1) that isolated rat liver and spleen nuclei
specifically bind 125I-labeled Ang II with high affinity, (2)
that 125I-Ang II binds to solubilized rat liver chromatin
fragments and the existence of discrete Ang II-binding
nucleoprotein particles, and (3) direct effects of nuclear
angiotensin on transcription [21, 22, 56]. Their studies
extended earlier investigations which suggested that labeled
Ang II localized to nuclear and mitochondrial regions of
myocardium, brain and smooth muscle cells [23, 57, 58].
Angiotensin Receptor Fragments
Baker and colleagues [57] further characterized the kinetics
of binding of Ang II (versus competitors) to nuclei and
nuclear envelopes, and showed that inhibitors of AT1 R-Ang
II binding (losartan) also inhibited Ang II-nuclear receptor
interactions. Dzau and colleagues [25] contributed to this
research area by characterizing the Ang II nuclear receptor
as being AT1-like (similar in size, losartan-inhibited) but
distinct with respect to several physicochemical properties.
Baker and colleagues have further shown that in vivo
expression from a plasmid encoding intracellular Ang II,
through introduction into mouse tail vein as a liposome
complex, results in biventricular cardiac hypertrophy [59].
Many other laboratories have, in various ways, contributed to
our understanding of the nature of intracellular components of
the RAS [20, 24, 60-71].
Our published studies have shown that intracellular
expression in a rat vascular smooth muscle cell line, of Ang
II with the AT1 R, results in nuclear accumulation of the
AT1 R, activation of p38MAPK and CREB pathways, and
enhanced cell proliferation [72, 73]. In those studies, Ang II
was expressed as ECFP/AngII (fused at the amino-terminus
to enhanced cyan fluorescent protein) and AT1 R was
expressed as AT1R/EYFP (fused at the carboxy-terminus to
enhanced yellow fluorescent protein). Our studies using
fluorescent colocalization markers, show that AT 1R/EYFP
accumulates in the endoplasmic reticulum, Golgi, vesicles,
and plasma membrane when expressed exclusively, but that
the distribution changes upon co-expression with ECFP/AII.
While less than 1% of rat A10 vascular smooth muscle cells
transfected with pAT1R/EYFP show yellow nuclear
fluorescence, 48% of cells that express both ECFP/AII and
AT1 R/EYFP show nuclear yellow fluorescence indicating
nuclear transport of the protein [72]. Therefore, nuclear
transport of the receptor is temporally linked to several
quantifiable cellular changes (e.g., proliferation and
activation of signaling pathways).
These studies [72, 73], were not designed to differentiate
between transport of the AT1 R holoprotein versus transport
of cleaved intracellular fragments into the nucleus. We
sought, therefore, to expand those studies and address that
particular issue. We designed and employed an expression
plasmid (pECFP/AT1R/EYFP) that encodes a fusion protein
in which the AT1 receptor is labeled at the extracellular
amino-terminus with ECFP and at the intracellular carboxyterminus with EYFP [15]. In principle, cleavage within the
AT1 R leads to dissociation of the two fluorescent moieties
and prospect for spatial separation within the cell or at the
plasma membrane. We investigated this construct and related
control plasmids using 3D deconvolution microscopy and
western blot analyses and showed that a population of
the AT1 R undergoes cleavage at approximately the 7th
transmembrane domain/cytoplasmic domain junction. We
showed that the AT1 R cytoplasmic domain is essential for
the processing event; YFP is not cleaved from the fusion
protein when the encoded protein is deleted with respect
to AT1 R amino acid residues 306-359. We further
demonstrated that the amino-terminal extracellular domain
Current Hypertension Reviews, 2012, Vol. 8, No. 1
63
also undergoes cleavage and can be recovered from the
tissue culture media. This is consistent with the idea that a
population of the AT1 R undergoes cleavage at the plasma
membrane releasing the extracellular and intracellular
domains. The intracellular domain accumulates in cytoplasm
and cell nuclei. We have corroborated the processing events
using alternate tags (short amino acid sequences, Flag
upstream and myc downstream). We have further confirmed,
using immunoblotting and specific inhibitors, that the
cleavage occurs in native protein as well as in genetically
tagged proteins, releasing from native AT1 R a stable 6 kDa
protein within cells [15].
Native AT1 receptor is internalized from the plasma
membrane and undergoes extensive recycling, accumulating
in endosomes of the short recycling pathway as well as the
long-recycling perinuclear compartment (PNRC) [74]. The
function of the latter compartment is unknown but
materialization in the PNRC appears to slow the return of
receptor to the plasma membrane. At the start of our studies
designed to characterize intracellular receptor, we speculated
that AT1 R derived from intracellular endosomes (in
particular, those of the PNRC) might function in an
intracellular fashion to stimulate cell proliferation and
hypertrophy. Indeed, functionally active intracellular and
intranuclear holoreceptor might yet be derived from
endosome-associated intracellular pools of AT1 R, but our
published studies indicate that at least some AT 1 R subunits
undergo cleavage at the plasma membrane to produce a
carboxy-terminal fragment population that traffics to cell
nuclei, and that these events are accompanied by measurable
biological changes.
Our most recent published investigations indicate that the
cleavage site lies between Leu(305) and Gly(306) at the
junction of the 7th transmembrane domain and cytoplasmic
C-terminus [75]. Our studies also indicate that overexpression of the C-terminal fragment independent of the
holoreceptor dramatically increases apoptosis as measured
by morphological and nuclear changes, plasma membrane
phosphotidylserine displacement, caspase activation, TUNEL
DNA labeling, and DNA laddering [75].
CLEAVAGE OF SINGLE-PASS TRANSMEMBRANE
PROTEINS
The principal of regulated intramembrane proteolysis
(RIP) has been demonstrated for many receptor tyrosine
kinases and other single-pass (cross the membrane only
once) membrane receptors [2-5]. The ErbB-4 receptor, a
member of the EGF receptor family of tyrosine kinases is
arguably the prototype receptor for RIP. When induced by
activators such as TPA (tissue plasminogen activator), the
TACE metalloprotease cleaves the extracellular domain
of ErbB-4 after which the enzyme complex, γ-secretase,
cleaves within the transmembrane domain to generate an
intracellular cleavage fragment which accumulates in the
nucleus. The intracellular domain contributes to ErbB-4dependent differentiation of mammary epithelial cells
through activation of the STAT5A transcription factor [76].
64 Current Hypertension Reviews, 2012, Vol. 8, No. 1
Non-receptor tyrosine kinase membrane proteins undergo
RIP as well. The Notch receptors, for example, are a family
of single-pass transmembrane proteins activated by cell-cell
contact through ligands which are usually also transmembrane proteins. This permits cell contact-driven polarity
and spatial information exchange. Upon ligand stimulation,
Notch family members, undergo sequential cleavage by
TACE and γ-secretase. The intracellular domain traffics to
the nucleus where it activates, as a ternary complex with the
CSL (Suppressor of Hairless/LAG-1) transcription factor
and Mastermind coactivator, transcription of specific
target genes including those involved in myogenesis and
myopathies [77, 78]. The newborn mouse heart has a
population of cardiac stem cells (CSCs) that are selfrenewing and multipotent. These CSCs express the Notch1
receptor and show nuclear localization of the intracellular
domain; overexpression of the intracellular domain expands
the CSC population while blockage of the intracellular
domain accumulation using a gamma-secretase inhibitor
leads to a reduction in myocyte number and to dilated
myopathy and high mortality rates [79]. The intracellular
domain is clearly related to myogenesis.
An important clinical target that is subject to the RIP
process is the amyloid precursor protein (APP). The normal
functions of the APP are still under investigation but they
include links to neuronal outgrowth and maintenance [80].
Cleavage of APP by γ-secretase leads to accumulation of the
hydrophobic amyloid β peptide (Aβ) in the extracellular
space, and aggregation or clustering of the peptide produces
the plaques and fibrils characteristic of Alzheimer’s Disease
(AD). A number of γ-secretase inhibitors have been found to
significantly reduce Aβ deposition in animal models [81]
and ongoing clinical trials are directed towards inhibiting γsecretase activity in patients. BMS-708163, for example, is
now in Phase II clinical testing. Phase I trials showed it to
decrease cerebrospinal fluid Aβ levels by approximately 30
percent at a daily dose of 100 mg and by 60 percent at a
daily dose of 150 mg (28 days of treatment). It also appears
to be about 190-fold more selective for APP than Notch
suggesting that it may have reduced side-effects. Despite
these encouraging results, the disease appears to be complex
and reduction of the Aβ fragment alone may not be sufficient
to rescue patients. Generation of the Aβ peptides are
obligatorily coupled to that of a second APP cleavage
product, the amyloid intracellular domain (AICD), which
also contributes significantly to the disease pathogenesis [82,
83]. One mouse study has shown that a single point mutation
in the AICD (D664A) is enough to rescue mice from an AD
phenotype despite a high load of Aβ deposits and significant
plaque formation [84]. In these mutant mice, synaptic loss,
astrogliosis, neural atrophy, and behavioral abnormalities
were completely prevented suggesting that accumulation of
the intracellular fragment contributes to disease progression.
The AICD is derived from APP by cleavage via a series of
α-, β-, and γ-secretases and is increased in Hirano bodies of
degenerating neurons from AD patients. AICD translocates
Julia L. Cook
to nucleus and interacts with several proteins including the
adaptor protein Fe65 and the histone acetyltransferase tatinteractive protein (Tip60). This complex is believed to turn
on target genes such as RAB3B, IGFBP3 and MICAL2 [85,
86]. GSK-3β is another potential target of the AICD. GSK3β is a serine/threonine kinase initially identified in glycogen
metabolism. It is highly expressed in the central nervous
system and phosphorylates, among other proteins, the tau
protein. Tau, in its hyperphosphorylated state becomes the
main component of neurofibrillary tangles. AICD overexpressing transgenic mice show activation of GSK-3β at 12 months of age, increased phosphorylation of tau at 3-4
months of age and tau aggregation at 7-8 months of age [83].
Working memory is compromised by 7-8 months of ages.
These changes correspond to neurodegenerative alterations
seen in human AD brains [87]. Contributing to the
complexity of the AICD-generating system, the pathway by
which it is generated may affect its efficacy in the cell.
AICD may be generated through sequential cleavage by βsecretase then γ-secretase, or α-secretase then γ-secretase.
In the latter pathway, α-secretase cleaves within the Aβ
domain, precluding generation of toxic Aβ products. The
minor cleavage pathway (amyloidogenic pathway) which
involves β-secretase followed by γ-secretase cleavage is
probably that primarily involved in generating stable, active
nuclear AICD. This pathway appears to occur primarily
within endosomes following internalization of APP [88].
It is estimated by the national Alzheimer’s Association
that 5.3 million Americans are living with Alzheimer’s, with
one new development occurring every 70 seconds;
Alzheimer’s is the seventh leading cause of death in the
United States (http://www.alzforum.org/dis/tre/drc/detail.
asp?id=124). A more detailed understanding of the cellular
biology and biochemistry of the RIP of APP will be required
to develop the most effective Alzheimer’s prevention drugs
[5].
Interestingly, many of these intracellular and intranuclear
cleavage products resulting from RIP or alternative cleavage
mechanisms, correlate with cell death and apoptosis,
consistent with the action of AT1 R cleavage fragment. For
example, the receptor for advanced glycation endproducts
(RAGE) has been linked to several chronic diseases thought
to result from vascular damage, including atherosclerosis,
peripheral vascular disease, Alzheimer’s disease and
congestive heart failure. RAGE is targeted by RIP,
producing both an extracellular soluble fragment (sRAGE)
as well as an intracellular domain; the intracellular protein is
detected in both cytoplasm and nucleus [89]. Transfected
HEK293 cells that exhibit accumulation of this product in
nucleus also show nuclear condensation and cell shrinkage.
This is accompanied by a 16% and 38% reduction in cell
viability at 16 and 40 h post-transfection, respectively, and
also in an increase in TUNEL positive cells at 16 h posttransfection.
RIP is also involved in the pathogenesis of Alzheimer’s
disease through a pathway distinct from RAGE. As
Angiotensin Receptor Fragments
discussed above, the transmembrane amyloid precursor
protein (APP) gives rise to the Aβ peptide cleavage product
which is found in plaque fibrils and tangles [90] and also to
the intracellular domain (AICD) which translocates to the
nucleus and appears to contribute to the pathogenesis of
Alzheimer’s perhaps by regulating nuclear signaling [91].
Recent studies have shown that overexpression of the AICD
in neurons induces cell death as determined by TUNEL
assays and DNA laddering [92], possibly in collaboration
with Fe65 and p53. Another example of cleavage fragmentinduced apoptosis occurs in a family of receptors that are
involved both in internalization of ligands and also in signal
transduction and neurotransmission. Cleavage of both the
low density lipoprotein receptor-related protein (LRP) as
well as the related LRP1 contributes to apoptosis. LRP
undergoes RIP in response to ischemia in neurons with
nuclear translocation of the intracellular domain. The latter
induces caspase-3 cleavage, TUNEL positivity and significant
cell death [93].
Clearly then, other receptor cleavage fragments, like the
AT1 R cleavage fragment, have been associated with nuclear
transport and apoptosis. An underlying homology in the
sequences of the cleaved peptides, however, is not readily
apparent. Nor is there any clear reason why regulated
proteolysis of these particular diverse receptors might be
linked to cell death. Further investigation of the caspase
pathways activated by the AT1 R cleavage fragment may be
helpful in forming a hypothesis.
NUCLEAR MEMBRANE-ASSOCIATED RECEPTORS
In addition to downstream cellular effects of fragments
RIPed from cell surface receptors, it is also clear that some
prototypical receptors, including GPCRs, exist, as
holoproteins, in the nuclear membrane and possess nuclear
functions. The Type I LPA (lysophosphatidic acid) GPCR
(LPA1) associated with hepatocytes and endothelial cells has
been found in nuclear as well as plasma membrane cell
fractions [9]. Isolated nuclei respond to LPA with increased
Ca2+ accumulation and induction of iNOS (inducible nitric
oxide synthase) both of which are prevented by inhibitors of
LPA1 . LPA treatment of endothelial cells also induces LPA 1
nuclear translocation and upregulates iNOS and Cox-2
(cyclooxygenase 2) [10].
Several studies have shown directly that the AT1 R and
ETB (but not ETA) are present in both nuclear membranes
and nucleosol [18, 94, 95] and are directly activated to
increase nuclear free calcium suggesting that they are
functional receptors. The fact that the corresponding
ligands can be found within the nucleus as well, suggests
that ligand:receptor interactions which recapitulate those
found at the plasma membrane may exist at the nuclear
membrane:nucleosol interface. Chappell and colleagues have
characterized AT1 nuclear membrane receptors in rat and in
sheep kidney [64, 65]. They find that Ang II upregulates
reactive oxygen species in isolated renal nuclei through AT 1
receptors and that nuclear AT2 receptors are functionally
linked to nitric oxide production. In both fetal and adult
Current Hypertension Reviews, 2012, Vol. 8, No. 1
65
sheep, the majority of cortical nuclear and plasma membrane
sites are AT2 receptor-like while the majority of medullary
nuclear and plasma membrane sites correspond to AT 1
receptors. While they observe only one AT1 R band by
kidney cortex nuclear immunoblot (full-length, ~52 kDa),
the Ab is made to the amino-terminus. Since our studies
suggest that the amino-terminal cleavage precedes Cterminal cleavage in regulated processing of the AT1 R, this
antibody would not be expected to detect cleaved receptor on
a western blot. The receptor that they identify biochemically
and functionally corresponds to nuclear membrane-associated
full-length AT1 R.
Many plasma membrane receptors can be found within
the nuclear membrane in addition to the AT 1 R. Since the
nuclear double-membrane is continuous with the endoplasmic
reticulum (ER), receptors can flow freely in between the
two compartments. The diffusion-retention model for
nuclear trafficking predicts that transmembrane or integral
membrane proteins in the ER can diffuse laterally in a
retrograde direction from the ER to the outer nuclear
membrane and then through the phospholipid bilayer
flanking the nuclear pores and into the inner nuclear
membrane [96]. This model further predicts that proteins
will only be retained in the inner nuclear membrane at
significant levels if the proteins bind to nucleosolic proteins,
chromatin, nuclear matrix, or other intranuclear structures
(for explanatory diagrams see [19]). Full-length functional
GPCRs like the AT1 R, therefore, can accumulate in the inner
nuclear membrane by retrograde trafficking from the ER.
Such receptors have potential to interact with ligands present
in the intranuclear membrane space and to signal events in
the nucleus through nuclear membrane signal transduction
events [10] that may recapitulate plasma membrane events.
This represents yet another emerging area of research
interest.
DISCUSSION
In addition to the many examples of single-pass
transmembrane receptors which, as holoproteins, or
processed fragments translocate to the nucleus [1, 2, 4, 97108], multi-pass seven-transmembrane GPCRs have also
been found either to be processed or to be transported to the
nucleus, or both. For example, the growth hormone-releasing
hormone (GHRH) GPCR, clearly exists in nuclei from
wild-type, unmanipulated tissues. Immunohistochemistry
and immunogold labeling of the GHRH receptor, which
belongs to the secretin family of GPCRs, demonstrates that it
is restricted to the somatotropes of the pituitary [109].
Moreover, it is associated with nuclear membrane and
nuclear matrix, as well as secretory granules and cytoplasmic
matrix.
Our studies indicate that expression of the singlefluorescent moiety fusion protein, AT1 R/EYFP, with
intracellular Ang II stimulates proliferation of A10 VSMCs
[15]. Moreover, the related double-fluorescent protein,
ECFP/AT1 R/EYFP, similarly stimulates cell proliferation in
Ang II-treated glial and VSMCs [15]. ECFP/AT1R/EYFP
66 Current Hypertension Reviews, 2012, Vol. 8, No. 1
also undergoes cleavage with transport of the YFP domain to
the nucleus and accumulation of a 36 kDa cleavage
fragment. Interestingly, cleavage fragment accumulates
independent of Ang II treatment but the quantity is amplified
following Ang II treatment. However, the fragment only
significantly translocates to the nucleus in Ang II-treated
cells. Therefore, Ang II seems to have a role both in
accumulation and transport of the cleavage fragment.
A number of different regulatory domains and functions
map to the carboxy-terminus of the AT1 R. Specific residues
in the carboxy-terminal tail play roles in G-protein coupling
and receptor uptake whilst phosphorylation of serine
and threonine residues by PKA and PKC may result in
uncoupling from G-proteins and receptor desensitization.
Using a C-terminal deletion mutant (Δ309-359), which is
very similar to the mutant that we generated and used to
show that this region is required for cleavage the C-terminus
of AT1 R [15], Inagami and associates [110] demonstrated
that the mutant AT1 receptor shares a similar Ang II binding
affinity and maximum binding value as wild-type, but
markedly reduced G-protein interaction. This suggests that
the C-terminal cytoplasmic domain is involved in G-protein
coupling but not in cell-surface materialization or Ang II
binding. Consistent with this, our imaging studies suggest
that ECFP/AT1 RΔCT/EYFP is properly transported to the
plasma membrane upon Ang II treatment, but that the fluors
remain coincidental and there is no accumulation of nuclear
fluorescence indicating no cleavage and no nuclear transport.
Our immunoblot studies corroborate this conclusion.
Importantly, therefore, the cytoplasmic C-terminal domain is
obligatory for cleavage at the 7th transmembrane: intracellular junction. Cleavage is also sensitive to the presence
of metal-chelating metalloprotease inhibitors. Both EDTA
and OPA (1, 10-ortho-phenanthroline) inhibit accumulation
of AT1R cleavage products suggesting that metalloproteases
are involved in generating the AT1 R carboxy-terminal
fragment [15].
While our laboratory has been the first to report that the
AT1R undergoes biologically functional proteolytic cleavage,
there does exist some prior indirect supporting evidence.
Modrall and colleagues [111] postulated that receptor downregulation might occur independently of receptor endocytosis. Using endocytosis-deficient mutants, (carboxyterminal-deleted), they showed that receptors were downregulated both by measurements of 125I-Ang II endocytosis
and by radioligand binding assays for AT1 receptor binding
sites. They further demonstrated that the endocytosisdeficient mutant receptors (Δ309-359 and Δ311-359) were
fully capable of rapid down-regulation comparable to that of
the wild-type receptor. They suggest, therefore, that an
alternative pathway, that of receptor degradation, might be
responsible for loss of cell-surface receptor. Our studies,
showing nuclear accumulation of the carboxy-terminal cytoplasmic fragment of the AT1 R, accompanied by alterations
in signal transduction and cell proliferation suggest that
the “degradation” of plasma membrane receptor actually
represents a biologically-functional regulated proteolysis.
Julia L. Cook
The AT2 R, which is only 32% homologous to the AT1 R,
has recently been shown to bind, via the receptor Cterminus, to the promyelocytic zinc finger protein (PLZF)
transcription factor in a ligand-stimulated manner and to
drive its localization to the nucleus [112]. Confocal microscopy showed that Ang II induces cytosolic PLZF to
colocalize with AT2 R at the plasma membrane and then
drives the receptor and PLZF to internalize. PLZF slowly
appears in the nucleus whereas AT2R accumulates in the
perinuclear region; the AT2R, in whole or in part, does not
appear to translocate into the nucleus unlike the AT1R.
Nuclear PLZF binds to a number of genes which contribute
to protein synthesis and the authors suggest that these AT 2
receptor-mediated changes in gene regulation could, in
effect, contribute to cardiac hypertrophy. In any case, this
is an example of a receptor which, through the cytoplasmic
carboxy-terminus, serves a unique intracellular chaperone
function and, thus, contributes to alterations in gene
expression.
The existence of prototypical plasma membrane
receptors within cell nuclei and accompanying evidence
that at least some of these are involved in transcriptional
regulation of gene expression prompts us to ask why some
receptors have evolved to perform multiple functions from
more than one cellular location. Conventional wisdom
suggests that nuclear accumulation of prototypical “plasma
membrane” receptors or receptor products may (1) contribute
to amplification of a downstream response to an external
stimulus, (2) prolong a response to a stimulus, and/or (3)
increase specificity by reducing the degeneracy of signaling
pathways and nuclear responses that lie downstream of
multiple cell surface ligand:receptor relationships.
The discovery that traditional plasma membrane
receptors can also accumulate in nuclei is intriguing and has
opened new avenues of exciting research. Clear evidence
now exists for the presence of uncleaved holoreceptors in
nucleosol and nuclear membrane though the mechanism by
which proteins possessing hydrophobic domains, and
associated with lipid bilayers, may escape the membrane,
and enter nucleosol or nuclear matrix remains unclear. Moreover, many receptors have now been observed to undergo
cleavage to release soluble extracellular and cytoplasmic
domains with a variety of biological functions; these now
represent new potential therapeutic targets.
SUMMARY
GPCR-targeted drugs are generally specific for cell
surface receptors and are usually not specifically designed to
be efficiently internalized into cells [5]. Moreover, even
those drugs that are efficiently internalized will only be
effective if the original binding site (or three-dimensional
binding pocket) is intact in the internalized target membrane
protein and if it is subject to ligand regulation. For the AT 1
receptor, for example, the typical nonpeptide receptor
blockers like olmesartan, losartan, irbesartan and valsartan
bind some amino acids within the agonist binding pocket
that also interact with Ang II (e.g., Lys199 in the 5th
Angiotensin Receptor Fragments
transmembrane domain, His256 in the 6th transmembrane
domain, and Asn295 in the 7th transmembrane domain) as
well as some unique amino acids [113-115]. To the extent
that these antagonists permeate the cell membrane, they
could be effective in blocking the nuclear membraneassociated receptor but would likely not be effective against
the cytoplasmic or nucleosolic carboxy-terminal cleavage
fragment. Effective targeting of cleaved fragments or
intracellular domains generated from plasma membrane
proteins will, in most cases, require novel strategies. We
have recently reported the successful in vitro application
of decoy peptides which prevent interaction of AT1 R
with the trafficking protein, GABARAP [116, 117]. The
decoy peptides were fused to cell-penetrating peptides and
extracellular application effectively blocked trafficking of
the AT1 R to the plasma membrane and cell membrane
accumulation of AT1R. We are testing these peptides for in
vivo efficacy; reduction in plasma membrane accumulation
of AT1 R should significantly reduce blood pressure and
may be a useful anti-hypertensive approach. Cell-penetrating
peptides may, in a similar fashion, be useful in blocking
intracellular functions of some receptor fragments. The
development of new drugs targeted to atypical intracellular
receptors and receptor fragments represents a new research
sphere vital to the pharmaceutical industry.
Current Hypertension Reviews, 2012, Vol. 8, No. 1
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21]
CONFLICT OF INTEREST
Declared none.
[22]
ACKNOWLEDGMENTS
This work was supported by the Ochsner Clinic
Foundation and National Heart, Lung, and Blood Institute
Grant HL-072795.
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Revised: October 19, 2011
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