Up-regulation of the Angiotensin II Type 1 Receptor by the MAS Proto-oncogene Is Due to Constitutive Activation of Gq/G11 by MAS*

Coexpression of the MAS proto-oncogene with the angiotensin II type 1 (AT1) receptor in CHO-K1 cells has been reported to increase the number of [3H]angiotensin II-binding sites, although MAS does not bind [3H]angiotensin II. In HEK293 cells stably expressing AT1 receptor-cyan fluorescent protein (CFP), MAS-yellow fluorescent protein (YFP) expression from an inducible locus caused strong up-regulation of AT1 receptor-CFP amounts and [3H]angiotensin II binding levels. The time course of AT1 receptor-CFP up-regulation was also markedly slower than that of induction of MAS expression. These effects were not mimicked by induced expression of I138D MAS-YFP, a mutant unable to cause constitutive loading of [35S]guanosine 5′-O-(thiotriphosphate) onto the phospholipase Cβ-linked G protein Gα11. Protein kinase C (PKC) inhibitors and the selective Gαq/Gα11 inhibitor YM-254890 fully blocked MAS-induced up-regulation of AT1 receptor-CFP amounts, whereas the PKC activator phorbol 12-myristate 13-acetate produced strong up-regulation of AT1 receptor-CFP without induction of MAS-YFP expression and in the presence of I138D MAS-YFP. The C-terminal tail of the AT1 receptor is a known target for PKC-mediated phosphorylation. In cells stably expressing a C-terminally truncated version of the AT receptor, induction of MAS expression did not up-regulate the truncated construct levels. These data demonstrate that the ability of MAS to up-regulate AT1 receptor levels reflects the constitutive capacity of MAS to activate Gαq/Gα11 and hence stimulate PKC-dependent phosphorylation of the AT1 receptor.

The octapeptide hormone angiotensin (Ang) 2 II is one of the key components of the renin-angiotensin system and, as such, plays a major role in the regulation of blood pressure and cardiovascular homeostasis (1). Ang II exerts its effects through at least two subtypes of G proteincoupled receptors (GPCRs), the angiotensin II type 1 (AT 1 ) and 2 (AT 2 ) receptors. Whereas the functional role of the AT 2 receptor is not fully understood (2,3), important biological functions such as vasoconstric-tion, salt/water reabsorption, and stimulation of aldosterone release are mediated through AT 1 receptor activation (4). However, in a number of situations, the AT 1 receptor is modulated by other coexpressed GPCRs. For example, interactions with the AT 2 receptor have been demonstrated in which the AT 2 receptor acts as a functional antagonist of the AT 1 receptor (5). AT 1 and bradykinin B 2 receptor interactions have also been shown, and these result in enhanced signaling of ligands at each receptor (6). Furthermore, in addition to interactions with GPCRs, agonist-induced functional interactions between the AT 1 receptor and the single transmembrane-spanning tyrosine kinase epidermal growth factor receptor have also been reported (7,8).
The MAS proto-oncogene was first detected in vivo by its tumorigenic activity (9) and later identified as a member of the rhodopsin-like class A GPCR subfamily. However, although suggested in early studies to be a potential candidate as an Ang II receptor (10,11), it remained an "orphan" until recently, when it was demonstrated that the Ang II metabolite Ang-(1-7) is an endogenous agonist of MAS (12). In the last decade, there has been emerging evidence that, although not able to bind or respond directly to Ang II, MAS has a physiological role in modulating the functions of Ang II in both the neuronal (13) and cardiovascular (14) systems. In MAS knock-out mice, AT 1 receptor signaling is altered in the amygdala (13); and, recently, Castro et al. (15) reported functional interactions between MAS and both the AT 1 and AT 2 receptors in mouse heart. In a previous study, we showed that MAS and the AT 1 receptor interact in heterologous expression systems and that MAS acts as an functional antagonist of the AT 1 receptor both in cotransfected CHO-K1 cells and in mesenteric microvessels of mice because greater levels of Ang II-mediated contraction were observed in vessels from MAS knock-out mice compared with wild-type controls (14). However, although Ang II produced lower levels of inositol phosphate accumulation and reduced increases in intracellular [Ca 2ϩ ] in CHO-K1 cells coexpressing MAS and the AT 1 receptor compared with those expressing the AT 1 receptor alone, we also observed that MAS coexpression with the AT 1 receptor resulted in an increase in [ 3 H]Ang II binding capacity (14). The mechanism by which MAS increases [ 3 H]Ang II binding has remained elusive. In this study, we demonstrate that it is due to the constitutive capacity of MAS to stimulate the G proteins G␣ q /G␣ 11 and hence the activity of protein kinase C (PKC) and that this effect is cell type-independent.
Site-directed Mutagenesis-To introduce amino acid substitutions into the primary structure of the human MAS receptor, site-directed mutagenesis of the encoding nucleotide sequence was performed using the QuikChange II site-directed mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer's instructions. The following primers were designed for the production of the I138D mutation (with the bases underlined): GTCCTTTACCCCGACTGGTACCGATGC (forward) and GCATCGGTACCAGTCGGGGTAAAGGAC (reverse).
Flp-In Constructs-To create MAS receptor constructs N-terminally tagged with the vesicular stomatitis virus G (VSV-G) epitope sequence and fused at the C terminus with yellow fluorescent protein (YFP), the human MAS receptor and I138D MAS were used as PCR templates. An oligonucleotide encoding a BamHI restriction site, the VSV-G epitope sequence, and the first 21 bases of the MAS receptor sequence was used as the forward primer (CGGGATCCATGTACACCGACATCGA-AATGACCCGCCTTGGTAAGGATGGGTCAAACGTGACATCA), and an oligonucleotide containing the last 21 bases of the MAS sequence without its stop codon and a NotI restriction site was employed as the reverse primer (TTTTCCTTTTGCGGCCGCTGACGACAGTCTC-AACTGTGACCGT). The PCR product was then inserted into vector pcDNA5/FRT/TO (Invitrogen) already containing YFP and previously digested with BamHI and NotI.
c-Myc-AT 1 receptor (AT 1 R)-cyan fluorescent protein (CFP) was obtained using the same strategy as described above with the human AT 1 receptor as a template; a forward primer encoding a HindIII restriction site, the c-Myc epitope sequence, and the first 21 bases of the receptor sequence (CCCAAGCTTATGGAACAAAAACTTATTTCT-GAAGAAGATCTGATTCTCAACTCTTCTACTGAAGATTGG); and a reverse primer containing the last 21 bases of the AT 1 receptor without its stop codon and a KpnI restriction site (CGGGGTACCCTCAACCT-CAAAACATGGTGC). The PCR product was then inserted in pcDNA3.1 already containing CFP and previously digested with HindIII and KpnI. c-Myc-AT 1 R-YFP in pcDNA5/FRT/TO was subcloned by digesting c-Myc-AT 1 R-CFP with HindIII and KpnI and inserting it into pcDNA5/FRT/TO already containing YFP and previously digested with the same restriction enzymes. The truncated form of the AT 1 receptor, c-Myc-⌬ 325 AT 1 R-CFP, was obtained using the same strategy as described above with the human AT 1 receptor as a template. The same primer encoding a HindIII restriction site, the c-Myc epitope sequence, and the first 21 bases of the receptor sequence was used as the forward primer, and the reverse primer contained the last 21 bases up to amino acid 325 of the AT 1 receptor without its stop codon and a KpnI restriction site (CGGGGTAC-CTTTTGGGGGAATATATTT). The PCR product was then inserted in pcDNA3.1 already containing CFP and previously digested with HindIII and KpnI.
Cell Culture and Transfection-HEK293 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 0.292 g/liter L-glutamine and 10% (v/v) newborn calf serum at 37°C in a 5% CO 2 humidified atmosphere. Cells were grown to 60 -80% confluence before transient transfection. Transfection was performed using Effectene transfection reagent (Qiagen Inc., West Sussex, UK) according to the manufacturer's instructions.
CHO-K1 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 2 mmol/liter L-glutamine. Cells at 50 -80% confluence were transfected transiently with the indicated cDNAs using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer's instructions.
Generation of Stable Flp-In TM T-REx TM HEK293 Cells-To generate Flp-In T-REx HEK293 cells able to inducibly express the receptors of interest, the cells were transfected with a mixture containing the desired receptor cDNA in the pcDNA5/FRT/TO vector and pOG44 vectors (1:9) using Effectene according to the manufacturer's instructions. Cell maintenance and selection were as described (16). Resistant clones were screened for receptor expression by both fluorescence and Western blotting. To induce expression of receptors cloned into the Flp-In locus, cells were treated with 0.1 g/ml doxycycline for varying periods of time. PKC activation and inhibition, as well as G␣ q /G␣ 11 inhibition treatments, were performed 6 h after inducing the receptors for an overnight period.
Live Cell Epifluorescence Microscopy-Cells expressing the appropriate receptors tagged with CFP or YFP were grown on poly-D-lysinetreated coverslips. The coverslips were placed into a microscope chamber containing physiological saline solution (130 mM NaCl, 5 mM KCl, 1 mM CaCl 2 , 1 mM MgCl 2 , 20 mM HEPES, and 10 mM D-glucose, pH 7.4). Fluorescent images of the cells were acquired using a Nikon TE2000-E inverted microscope (Nikon Instruments, Melville, NY) equipped with a ϫ40 (numerical aperture ϭ 1.3) oil immersion Plan Fluor lens and a cooled digital CoolSNAP HQ charge-coupled device camera (Photometrics, Tucson, AZ) (see Ref. 17 for details).
Visualization of the Plasma Membrane-To fluorescently visualize the plasma membrane in live HEK293 cells expressing CFP-or YFPfused receptors, cells were treated (as specified by the manufacturer) with the reagents in the Image-iT plasma membrane and nuclear labeling kit (Invitrogen), in which the plasma membrane is specifically labeled with wheat germ agglutinin-Alexa Fluor 594, and nuclei are stained simultaneously with Hoechst 33342. CFP and YFP were excited as described above, and Alexa Fluor 594 was excited at 575/12 nm and imaged using the following filter set: dichroic, Q595LP; and emitter, HQ645/75m. Using these filters, no bleed-through was observed, and the resultant sequential 12-bit images were overlaid using MetaMorph software (Version 6.3.5, Universal Imaging Corp., Downingtown, PA). For three-dimensional imaging, stacks of images (2 ϫ 2 binning, 150 -200-ms exposure/image) with a 0.339-m Z step (ϳ20 -25 frames/ stack) were sequentially acquired for each fluorescent dye. Reactions were incubated for 30 min at 30°C and terminated by the addition of 0.5 ml of ice-cold buffer containing 20 mM HEPES, pH 7.4, 3 mM MgCl 2 , 100 mM NaCl, and 0.2 mM ascorbic acid. The samples were centrifuged at 16,000 ϫ g for 10 min at 4°C, and the resulting pellets were resuspended in solubilization buffer (100 mM Tris, 200 mM NaCl, 1 mM EDTA, and 1.25% Nonidet P-40) plus 0.2% SDS. Samples were precleared with Pansorbin (Calbiochem, Nottingham, UK), followed by immunoprecipitation with antiserum CQ (18). Finally, the immunocomplexes were washed twice with solubilization buffer, and bound [ 35 S]GTP␥S was measured by liquid scintillation spectrometry.
Cell-surface Receptor Measurement and Enzyme-linked Immunosorbent Assay-Cells were grown in 96-well poly-D-lysine-coated plates and induced with different concentrations of doxycycline for 24 h. Afterward, cell-surface receptors were labeled with anti-VSV-G antibody (1:1000) in growth medium for 30 min at 30°C. The cells were then washed once with 20 mM HEPES and Dulbecco's modified Eagle's medium and then incubated for another 30 min at 37°C in growth medium supplemented with horseradish peroxidase-conjugated antirabbit IgG as the secondary antibody and 1 M Hoechst nuclear stain (Sigma) to determine the number of cells in each well. The cells were washed twice with phosphate-buffered saline, and the Hoechst fluorescence was measured. Finally, the cells were incubated with SureBlue (KPL, Inc., Gaithersburg, MD) for 5 min in the dark at room temperature, and the absorbance was read at 620 nm using a VICTOR 2 plate reader (PerkinElmer Life Sciences).
Endoglycosidase Treatment-Endoglycosidase treatment was carried out overnight at 32°C using peptide N-glycosidase F (Roche Diagnostics, Mannheim, Germany) at a final concentration of 1 unit/l.
After samples were heated at 65°C for 15 min, cell lysates were subjected to SDS-PAGE analysis using 4 -12% BisTris gels (NuPAGE, Invitrogen) and MOPS buffer. After electrophoresis, proteins were transferred onto nitrocellulose membranes that were incubated in a solution of 5% nonfat milk and 0.1% Tween 20 in Tris-buffered saline at room temperature on a rotating shaker for 2 h to block nonspecific binding sites. The membrane was incubated overnight with anti-c-Myc polyclonal antibody (Cell Signaling, Hertfordshire, UK) or anti-VSV-G antiserum and detected using horseradish peroxidase-linked anti-rabbit IgG secondary antiserum (Amersham Biosciences, Buckinghamshire, UK). Immunoblots were developed by application of enhanced chemiluminescence solution (Pierce).
Cell-surface Biotinylation Experiments-For cell-surface biotinylation, cells were grown in 6-well plates coated with poly-D-lysine and induced as described. Confluent cells were washed with ice-cold borate buffer (10 mM boric acid, 154 mM NaCl, 7.2 mM KCl, and 1.8 CaCl 2 , pH 9.0) and incubated on ice with 1 ml of 0.8 mM EZ-Link sulfosuccinimidyl 2-(biotinamido)ethyl-1,3-dithiopropionate (Pierce) in borate buffer for 15 min. The cells were then rinsed with a solution of 0.192 M glycine and 25 mM Tris, pH 8.3, to quench the excess biotin and lysed with radioimmune precipitation assay buffer. Lysates were centrifuged for 30 min at 14,000 ϫ g, and the supernatant was recovered. An aliquot of the lysates was saved for Western blotting. Cell-surface biotinylated proteins were isolated using 100 l of ImmunoPure immobilized streptavidin (Pierce). After 1 h of incubation at 4°C with constant rotation, samples were spun, and the streptavidin beads were washed three times with radioimmune precipitation assay buffer. Finally, the biotinylated proteins were eluted with 100 l of SDS sample buffer for 1 h at 37°C, and SDS-PAGE and Western blotting were performed as described above.
Intact Cell Radioligand Binding Assays-Flp-In T-REx HEK293 cells expressing the wild-type or I138D MAS receptor with the constitutively expressed AT 1 receptor were subcultured in 6-well plates coated with poly-D-lysine and grown to confluence. Each well was washed twice with prewarmed Krebs-Ringer buffer (145 mM NaCl, 20 mM HEPES, 1.3 mM MgCl 2 , 1.2 mM NaPO 4 , 5 mM KCl, 1.3 mM CaCl 2 , and 10 mM glucose, pH 7.4). Then, the Krebs-Ringer buffer was replaced with 0.8 ml of prewarmed [ 3 H]Ang II (Amersham Biosciences) in Krebs-Ringer buffer supplemented with 0.25% (w/v) bovine serum albumin. Nonspecific binding was determined in the presence of 10 M unlabeled Ang II. After 30 min at 37°C (5% CO 2 and 95% air), each well was washed twice with ice-cold Krebs-Ringer buffer, and the cells were solubilized by the addition of 1 ml of 0.1 M NaOH and 2% (w/v) SDS and incubated overnight at room temperature (20 -25°C). 1 ml of 0.1 M HCl was added to each well to neutralize the NaOH. The samples were mixed, and 1.6 ml was mixed with scintillation mixture and counted (Packard 2000 CA scintillation counter) to measure bound [ 3 H]Ang II. The remaining cell lysates from each well were pooled and used for protein determination. A radioligand concentration of 10 nM [ 3 H]Ang II was used for single point studies.

RESULTS
We recently reported that coexpression of the GPCR MAS with the AT 1 receptor in CHO-K1 cells results in a decreased capacity of Ang II to increase intracellular [Ca 2ϩ ] and inositol phosphate accumulation (14). Paradoxically, however, this is associated with an increase in [ 3 H]Ang II-binding sites, although MAS does not bind this ligand (14). We resolved to explore the molecular basis for this up-regulation of Ang II binding.
Generation of Flp-In T-REx HEK293 Cell Lines-A Flp-In T-REx HEK293 clonal cell line was established in which a form of the human AT 1 receptor C-terminally tagged with CFP and N-terminally tagged with the c-Myc epitope sequence (c-Myc-AT 1 R-CFP) was expressed stably and constitutively. These cells also harbored, at the Flp-In locus, a form of human MAS C-terminally tagged with YFP and N-terminally tagged with the VSV-G epitope sequence (VSV-G-MAS-YFP). The Flp-In locus ensures a single defined site of chromosomal integration; and in the T-REx form of these cells, expression from this locus is controlled in a Tet-on-inducible fashion by the addition of either tetracycline or the related antibiotic doxycycline (16). In the absence of doxycycline, cell imaging demonstrated expression of c-Myc-AT 1 R-CFP predominantly in punctate intracellular vesicles, likely to represent recycling endosomes, and a lack of expression of VSV-G-MAS-YFP (Fig. 1A). The addition of doxycycline (0.1 g/ml, 24 h) resulted in induction of VSV-G-MAS-YFP expression. This receptor construct had an unusual, widespread, cellular distribution, with at least some of the YFP signal overlapping with the nucleus (Fig. 1B). With expression of VSV-G-MAS-YFP, the fluorescent signal corresponding to c-Myc- AT 1 R-CFP was markedly increased (Fig. 1B), with much of the signal apparently inside the cell.
MAS Constitutively Activates G␣ 11 -Transient transfection of MAS into HEK293 cells along with the phospholipase C␤-linked G protein G␣ 11 resulted in loading of ϳ4-fold greater amounts of [ 35 S]GTP␥S onto the G protein than observed in the absence of MAS, consistent with the substantial ligand-independent, constitutive activity of this GPCR (Fig. 2). We have demonstrated previously that mutation of key hydrophobic residues in the second intracellular loop of many rhodopsin-like class A GPCRs ablates their capacity to activate cognate G proteins (18,19). Conversion of Ile 138 to Asp in MAS (I138D MAS) and expression of this form of the receptor with G␣ 11 failed to enhance [ 35 S]GTP␥S binding to the G protein (Fig. 2).
I138D MAS Is Delivered to the Cell Surface but Does Not Up-regulate the AT 1 Receptor-Based on the ability of the I138D mutation to eliminate constitutive MAS-induced loading of [ 35 S]GTP␥S onto G␣ 11 , we generated additional Flp-In T-REx HEK293 cell lines that constitutively expressed c-Myc-AT 1 R-CFP but also harbored VSV-G-I138D MAS-YFP at the Flp-in locus (Fig. 3A). Induction of VSV-G-I138D MAS-YFP expression by treatment with doxycycline resulted in a completely different distribution pattern compared   and ii) or VSV-G-I138D MAS-YFP (panels iii and iv), were left untreated (panels i and iii) or were treated with doxycycline (0.1 g/ml) for 24 h (panels ii and iv) and stained with Image-iT membrane dye (red).
with VSV-G-MAS-YFP. The I138D MAS construct appeared to be located largely at the plasma membrane (Fig. 3B). Expression of VSV-G-I138D MAS-YFP neither up-regulated c-Myc-AT 1 R-CFP nor markedly altered its cellular distribution (Fig. 3B). To confirm substantially more effective plasma membrane localization of VSV-G-I138D MAS-YFP, we performed a series of intact cell anti-VSV-G enzyme-linked immunosorbent assays. Although induction of VSV-G-MAS-YFP expression with increasing concentrations of doxycycline resulted in little increase in anti-VSV-G cell-surface immunoreactivity (Fig. 3C), a clear, doxycycline concentration-dependent increase in signal was obtained when VSV-G-I138D MAS-YFP expression was induced (Fig. 3C). Furthermore, staining and image overlay of cells induced to express either VSV-G-MAS-YFP or VSV-G-I138D MAS-YFP with Image-iT membrane dye confirmed effective plasma membrane delivery of VSV-G-I138D MAS-YFP, but not VSV-G-MAS-YFP (Fig. 3D). Cell-surface biotinylation studies also confirmed cell-surface delivery of VSV-G-I138D MAS-YFP, but not VSV-G-MAS-YFP (Fig. 4A).
Only the Core Glycosylated Form of the AT 1 Receptor Is Delivered to the Cell Surface-Many GPCRs are produced as immature forms that require final core glycosylation prior to effective plasma membrane delivery and insertion. Membranes of cells induced to coexpress VSV-G-MAS-YFP and c-Myc-AT 1 R-CFP were treated with or without peptide N-glycosidase F, resolved by SDS-PAGE, and immunoblotted with anti-c-Myc antibody to identify forms of c-Myc-AT 1 R-CFP (Fig. 4B). In the untreated samples, polypeptides with apparent masses of 60 and 90 kDa were detected as well as those with higher apparent molecular mass/lower mobility, which may represent dimeric or oligomeric complexes. Treatment with peptide N-glycosidase F resulted in the appearance of a predominant band at an apparent molecular mass of 50 kDa with substantially lower amounts of bands at ϳ60 kDa. These results suggest that both the 60-and 90-kDa forms are N-glycosylated and that the 90-kDa polypeptide is likely to represent the mature, core glycosylated receptor monomer. To confirm the concept that the higher molecular mass species was the mature form, we performed cell-surface biotinylation assays. In cells expressing the c-Myc-AT 1 R-CFP construct, such cell-surface biotinylation assays identified only the 90-kDa polypeptide, although immunoblots of total cell lysates indicated that the 60-kDa species was present at similar levels (Fig. 4C). Having identified the different forms of c-Myc-AT 1 R-CFP and to further validate the imaging studies, we treated cells constitutively expressing c-Myc-AT 1 R-CFP and harboring either VSV-G-MAS-YFP or VSV-G-I138D MAS-YFP with or without doxycycline. Cell lysates from these cells were then immunoblotted with anti-c-Myc antibody (Fig. 4D). These results confirmed the up-regulation of c-Myc-AT 1 R-CFP upon coexpression of VSV-G-MAS-YFP, but not VSV-G-I138D MAS-YFP. Another Flp-In T-REx HEK293 cell line in which VSV-G-MAS-YFP expression could be induced but which did not express c-Myc-AT 1 R-CFP confirmed that the immunodetected polypeptides truly represent c-Myc-AT 1 R-CFP (Fig. 4D).

MAS Expression Precedes AT 1 Receptor Up-regulation-
If the presence of active MAS were required to cause up-regulation of the AT 1 receptor construct, we reasoned that the time course of AT 1 receptor up-regulation would be slower than that of induction of MAS expres- Regulation of PKC Activity Modulates AT 1 Receptor Levels-Because MAS is able to constitutively activate G q /G 11 (Fig. 2) and hence presumably activate PKC, we added the PKC inhibitor Ro 31-8220 to cells constitutively expressing c-Myc-AT 1 R-CFP with and without doxycycline to induce expression of VSV-G-MAS-YFP. Although without effect on [ 3 H]Ang II binding in cells not induced to express VSV-G-MAS-YFP, Ro 31-8220 fully inhibited the increase in [ 3 H]Ang II binding produced by expression of MAS (Fig. 5A). Ro 31-8220 was also without effect on [ 3 H]Ang II binding in cells induced to express VSV-G-I138D MAS-YFP (Fig. 5A). Parallel immunoblots confirmed the effect of Ro 31-8220 (Fig. 5B), whereas equivalent immunoblots of total ERK1 and ERK2 MAPKs amounts confirmed equal sample loading. A second PKC inhibitor, GF 109203X, produced similar results (Fig. 5C). We reasoned that activation of PKC in the absence of MAS induction should also up-regulate c-Myc-AT 1 R-CFP levels. Treatment of cells harboring VSV-G-MAS-YFP with PMA produced a high level of c-Myc-AT 1 R-CFP up-regulation without induction of VSV-G-MAS-YFP expression (Fig. 5C). The effect of PMA was also blocked by the co-addition of Ro 31-8220 and was not increased further by induction of VSV-G-MAS-YFP expression (Fig. 5C). Simple quantitation of the levels of CFP fluorescence in living cells (Fig. 5D) confirmed the immunoblot results. As anticipated, PMA treatment of cells expressing c-Myc-AT 1 R-CFP and induced to express VSV-G-I138D MAS-YFP also resulted in marked up-regulation of c-Myc-AT 1 R-CFP, which was again blocked by the co-addition of Ro 31-8220 (Fig. 6).
A Novel G q /G 11 Inhibitor Prevents MAS-induced Up-regulation of the AT 1 Receptor-G q and G 11 are upstream of PKC and, as shown in Fig. 2, are constitutively activated by MAS, but not I138D MAS. Recently, YM-254890 has been described as a selective G q /G 11 inhibitor (21). We initially tested the ability of YM-254890 to inhibit receptor activation of G q /G 11 by its capacity to prevent phenylephrine-mediated [ 35 S]GTP␥S binding to ␣ 1b -adrenoreceptor-G␣ q and ␣ 1b -adrenoreceptor-G␣ 11 fusion proteins (19,22). In membranes of HEK293 cells expressing these fusion proteins, YM-254890 inhibited agonist-mediated loading of [ 35 S]GTP␥S with EC 50 ϭ 4 nM. This inhibition was highly selective. 100 nM YM-254890 had no ability to inhibit agonist-mediated activation of G␣ s via a corticotropin-releasing factor-1 receptor-G␣ s fusion protein, G␣ i3 via a GPR41-G␣ i3 fusion protein, or G␣ o1 via an ␣ 2A -adrenoreceptor-G␣ o1 fusion protein (Fig. 7). At 100 nM, YM-254890 also blocked the up-regulation of c-Myc-AT 1 R-CFP produced following induction of VSV-G-MAS-YFP expression (Fig. 8). Because PKC is downstream of G q /G 11 , we predicted that YM-254890 would not be able to block PMAmediated c-Myc-AT 1 R-CFP up-regulation, and this was confirmed (Fig. 8).  (Fig. 9).

The AT 1 Receptor Lacking C-terminal PKC Phosphorylation Sites Is Not Up-regulated by Coexpression of MAS-
The AT 1 receptor has a group of three potential PKC phosphorylation sites in the C-terminal tail, and phosphorylation of these residues in response to both Ang II and PKC activation has been explored previously (23)(24)(25). We generated a variant of c-Myc-AT 1 R-CFP (c-Myc-⌬ 325 AT 1 R-CFP) that was truncated after amino acid 325 of the receptor and hence lacks the PKC phosphorylation sites and generated additional Flp-In T-REx HEK293 cell lines with VSV-G-MAS-YFP at the Flp-In locus and c-Myc-⌬ 325 AT 1 R-CFP expressed constitutively. In the absence of VSV-G-MAS-YFP, the distribution pattern of c-Myc-⌬ 325 AT 1 R-CFP was not different from that of c-Myc-AT 1 R-CFP (Fig. 10), but expression of VSV-G-MAS-YFP failed to substantially up-regulate c-Myc-⌬ 325 AT 1 R-CFP levels judged by either CFP fluorescence or c-Myc immunoreactivity (Fig. 10).
To confirm that the PKC-based mechanism of MAS-induced up-regulation of the AT 1 receptor is not produced only in HEK293 cells, CHO-K1 cells were transiently transfected to express c-Myc-AT 1 R-CFP with or without coexpression of VSV-G-MAS-YFP. These cells were subsequently treated with combinations of PMA to activate PKC and Ro 31-8220 to inhibit this activity. As in the Flp-In T-REx HEK293 cell lines, treatment with PMA in the absence of MAS resulted in strong up-regulation of c-Myc-AT 1 R-CFP levels detected in anti-c-Myc immunoblots, and this was blocked by co-administration of Ro 31-8220 (Fig. 11). MAS-induced up-regulation of c-Myc-AT 1 R-CFP immunoreactivity was largely blocked by treatment with Ro 31-8220 (Fig. 11). As an additional control for the effects of MAS in the Flp-In T-REx HEK293 system, which requires the addition of doxycycline to cause expression of MAS, we transiently transfected HEK293 cells with c-Myc-AT 1 R-CFP and confirmed that the addition of doxycycline did not result in c-Myc-AT 1 R-CFP up-regulation, whereas treatment with PMA produced strong up-regulation (Fig. 11).

DISCUSSION
The AT 1 receptor and the MAS proto-oncogene are often coexpressed in, for example, vascular smooth muscle cells. After the cloning of MAS cDNA (9), some early experiments suggested that it might be a receptor for angiotensin peptides (10), but it is now well appreciated that MAS does not bind Ang II. However, Ang-(1-7), the product of ACE2 activity (26), has recently been described as an  endogenous agonist of MAS (12). Furthermore, because Ang-(1-7) counters many of the regulatory actions of Ang II, there has been considerable interest in the interplay between MAS and the AT 1 receptor. Following expression of the AT 1 receptor in CHO-K1 cells, Ang II produces a strong, concentration-dependent increase in intracellular [Ca 2ϩ ] (14). However, with coexpression of MAS, the effect of a maximally effective concentration of Ang II is substantially reduced, an effect also observed when inositol phosphate accumulation is measured (14). Despite these effects on Ang II-generated signals, coexpression with MAS actually results in higher levels of [ 3 H]Ang II binding (14). The interplay between MAS and the AT 1 receptor is further underlined when measuring Ang II-mediated contraction of mouse mesenteric microvessels. Ang II produces greater contraction in vessels from MAS knock-out animals than in vessels from wild-type controls (14). It was concluded that MAS forms a complex with the AT 1 receptor that is inhibitory to AT 1 receptor function, as had been described previously for AT 2 /AT 1 receptor interactions (5).
To confirm MAS-induced increases in [ 3 H]Ang II binding when coexpressed with the AT 1 receptor in a separate cell system and to explore the molecular basis of this effect required a means to control MAS expression in the face of AT 1 receptor expression. We thus employed Flp-In T-REx HEK293 cells. Introduction of a construct at the single defined Flp-In locus results in induction of expression only when the cells are exposed to tetracycline or the related antibiotic doxycycline (16). These cells were also transfected to stably and constitutively express the AT 1 receptor. By employing receptor constructs tagged at the C terminus with CFP and YFP, we could also monitor their cellular distribution. In the absence of MAS, AT 1 R-CFP was predominantly intracellular with a distribution pattern reminiscent of the endocytic recycling pathway. With induction of MAS expression, a marked up-regulation of AT 1 R-CFP could be easily visualized, although a significant fraction appeared trapped within the cells. Although MAS is a GPCR, little of the MAS-YFP construct was present at the plasma membrane; and indeed, the distribution apparently overlapped, at least in part, with the nucleus. Interestingly, following expression in HEK293 cells, a number of GPCRs that appear to have a nuclear localization sequence in their C-terminal tails have been reported to show nuclear localization (27). Although not tested directly, Lee et al. (27) did note a similar, potential nuclear localization sequence in MAS. MAS has also been suggested to show high levels of constitutive activity in a number of deorphanization/ligand screening programs, and we demonstrated the ability of MAS to load [ 35 S]GTP␥S onto the G protein G␣ 11 in a ligandindependent fashion. Because we have shown that mutation of key hydrophobic residues in the second intracellular loop of many GPCRs eliminates both constitutive and agonist-dependent G protein activation (19,20), we generated I138D MAS. I138D MAS had no ability to activate G␣ 11 ; and when expressed as I138D MAS-YFP from the Flp-In locus of Flp-In T-REx HEK293 cells, this construct was plasma membrane-delineated and failed to cause up-regulation of AT 1 R-CFP. It is unclear if it is the constitutive activity of MAS that results in the unusual cellular distribution of MAS-YFP, but certainly I138D is not close (at least in the primary sequence) to the identified nuclear localization sequence in the C-terminal tail (27) and would not inherently be anticipated to alter this. However, it did seem possible that the constitutive activity of MAS could be responsible for the up-regulation of AT 1 R-CFP. A series of inhibitor studies confirmed this conclusion. The most direct was via inhibition of G q /G 11 using YM-254890 (21). This compound fully blocks the MAS-induced effect and is both a remarkably selective and potent G q /G 11 inhibitor.
The MAS-induced up-regulation of the AT 1 receptor monitored in cell imaging studies showed substantial accumulation in an intracellular compartment that may be the Golgi. It is well established that effective core glycosylation is an important quality control check prior to cellsurface delivery of many GPCRs (28,29) and that a substantial amount of expressed protein fails this control and is routed to the proteasome for destruction. Immunoblot studies showed strong up-regulation of the AT 1 receptor by both MAS and activation of protein kinase C and also demonstrated a mixture of highly and less well glycosylated forms. Cell-surface biotinylation studies indicated that only the fully glycosylated form was delivered to the cell surface. Thus, because the imaging studies cannot discriminate between these forms, the combination of cell imaging, immunoblot, ligand binding, and cell-surface biotinylation studies offers the best means to understand the molecular diversity of the forms and cellular distribution of the AT 1 receptor construct. Because PKC lies downstream of G q /G 11 , we reasoned that PKC inhibitors should also block MAS-induced up-regulation of AT 1 R-CFP and that PKC activators should do so without induction of constitutively active MAS. Both these expectations were fully met. Many analyses have shown roles for agonist-and PKC-mediated phosphorylation of the C-terminal tail of the AT 1 receptor (23)(24)(25). Although key previous studies have been performed with the rat receptor, the C-terminal tail of the AT 1 receptor is well conserved between human and rat with three clear potential sites for PKC-mediated phosphorylation. Truncation to amino acid 325 removes these three sites. As such, it was satisfying that MAS-YFP was unable to cause significant up-regulation of c-Myc-⌬ 325 AT 1 R-CFP.
Although our study does not directly address the mechanistic basis for enhanced Ang II-mediated contraction in mesenteric vessels of MAS knock-out mice, it is likely that it may also reflect a loss of constitutive MAS-induced activation of PKC and phosphorylation of the AT 1 receptor. PKC-mediated phosphorylation of the AT 1 receptor is associated with decreased function, whereas truncation to eliminate PKC phosphorylation sites, as in ⌬ 325 AT 1 R, is associated with an increased capacity of Ang II to stimulate inositol phosphate production because of reduced desensitization. Elimination of constitutive MAS-induced PKC activation and hence AT 1 receptor phosphorylation in vessels of MAS knock-out animals is therefore also consistent with the enhanced function of Ang II in producing contraction of mesenteric microvessels from MAS knock-out mice (14); and of course, heterologous desensitization of a coexpressed receptor by either constitutive or agonist-induced activation of a GPCR is a commonly employed regulatory strategy (30). It still remains to be established if the MAS-G q /G 11 -PKC-mediated upregulation of the AT 1 receptor reflects enhanced stability of the protein and hence slower turnover. Future studies will assess this issue.