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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 Accepted: October 20, 2011