Dependence on the Motif YIPP for the Physical Association of Jak2 Kinase with the Intracellular Carboxyl Tail of the Angiotensin II AT1 Receptor*

Angiotensin II is the effector molecule of the renin-angiotensin system. Virtually all of its biochemical actions are mediated through a single class of cell-surface receptors called AT1. These receptors contain the structural features of the seven-transmembrane, G-protein-coupled receptor superfamily. Angiotensin II, acting through the AT1 receptor, also stimulates the Jak/STAT pathway by inducing ligand-dependent Jak2 tyrosine phosphorylation and activation. Here, we show that a glutathione S-transferase fusion protein containing the carboxyl-terminal 54 amino acids of the rat AT1A receptor physically binds to Jak2 in an angiotensin II-dependent manner. Deletional analysis, using both in vitro protocols and cell transfection analysis, showed that this association is dependent on the AT1Areceptor motif YIPP (amino acids 319–322). The wild-type AT1A receptor can induce Jak2 tyrosine phosphorylation. In contrast, an AT1A receptor lacking the YIPP motif is unable to induce ligand-dependent phosphorylation of Jak2. Competition experiments with synthetic peptides suggest that each of the YIPP amino acids, including tyrosine 319, is important in Jak2 binding to the AT1A receptor. The binding of the AT1A receptor to the intracellular tyrosine kinase Jak2 supports the concept that the seven-transmembrane superfamily of receptors can physically associate with enzymatically active intracellular proteins, creating a signaling complex mechanistically similar to that observed with growth factor and cytokine receptors.

The analysis of cytokines and their receptors has implicated the intracellular Jak family of kinases as critically important for the intracellular signaling initiated in response to ligand (1)(2)(3). Cytokines induce receptor dimerization and the activation, via tyrosine phosphorylation, of the associated Jak kinases. The Jak kinases phosphorylate the cytokine receptors, leading to the binding and eventual activation of intermediate signaling molecules referred to as STAT (signal transducers and activators of transcription). The STAT proteins are a fam-ily of transcription factors that migrate to the nucleus and induce gene transcription (4). The Jak/STAT pathway was first elucidated through the study of interferon signaling, but it is now known that this pathway participates in the signaling initiated by a wide variety of cytokines and growth factors. Recently, the vasoactive peptide angiotensin II was also found to activate the Jak/STAT pathway (5).
Angiotensin II is the effector molecule of the renin-angiotensin system. It is an 8-amino acid peptide that induces several physiologic responses that act to raise blood pressure. Virtually all of its biochemical actions are mediated through a single class of cell-surface receptors called AT 1 (6). Whereas humans have a single AT 1 receptor gene, rodents possess two genes encoding highly homologous receptor isoforms termed AT 1A and AT 1B . These proteins are 95% identical and appear to bind ligand and to signal in an identical fashion (7,8). All AT 1 receptors contain the structural features of the seven-transmembrane, G-protein-coupled receptor superfamily and are structurally quite different from cytokine receptors. However, studies by our group (5) and by Baker and co-workers (9,10) have independently demonstrated that angiotensin II, acting through the AT 1 receptor, also stimulates the Jak/STAT pathway. In rat aortic smooth muscle (RASM) 1 cells, angiotensin II leads to the rapid tyrosine phosphorylation and activation of Jak2 (5). Angiotensin II also induces the physical association of Jak2 with the AT 1 receptor. The AT 1 receptor contains no intrinsic kinase activity. However, it is now known that ligand occupancy of this receptor stimulates several different intracellular signaling cascades in which tyrosine phosphorylation plays an important role (11). At present, the structural features of the AT 1 receptor necessary for intracellular tyrosine kinase activation are not understood. In this study, we show that the carboxyl-terminal 54 amino acids of the rat AT 1A receptor physically bind to Jak2 in an angiotensin II-dependent manner. Both in vitro and in vivo analyses show that this association is dependent on the AT 1A receptor motif YIPP (amino acids 319 -322). The binding of the AT 1A receptor to the intracellular tyrosine kinase Jak2 supports the concept that the seventransmembrane superfamily of receptors can physically associate with enzymatically active intracellular proteins, creating a signaling complex mechanistically similar to that observed with growth factor and cytokine receptors.

EXPERIMENTAL PROCEDURES
Cell Culture-RASM cells were cultured to near confluence at 37°C under 5% CO 2 in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal serum and supplemented with antibiotics. The cells were * This work was supported in part by National Institutes of Health Grants DK39777, DK44280, DK45215, DK51445, and HL47035. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18  Preparation of RASM Cell Lysates-Growth-arrested RASM cells were stimulated with 100 nM angiotensin II for varying times, washed two times with phosphate-buffered saline containing 1 mM Na 3 VO 4 , and lysed in 1.0 ml of lysis buffer (25 mM Tris-HCl, pH 7.6, 0.15 M NaCl, 1% Triton X-100, 10% glycerol, 50 mM NaF, 1 mM Na 3 VO 4 , 1 mM phenylmethylsulfonyl fluoride, and 1 g/ml aprotinin). Cells were scraped off the plates and gently sonicated. The lysate was cleared by centrifugation at 7500 ϫ g for 15 min and, if necessary, stored at Ϫ20°C. The protein concentration of lysates was determined by the method of Lowry et al. (12).
GST Fusion Protein Construction-A 166-base pair fragment of the Ca18b cDNA encoding the AT 1A receptor was amplified by polymerase chain reaction and cloned into the pGEX-KG vector via XbaI/HindIII restriction sites (13,14). Point mutations were created using the Bio-Rad Muta-Gene phagemid kit. Other deletion constructs were made using polymerase chain reaction. All constructs were verified by DNA sequence analysis.
Jak2 Binding Assay-GST fusion proteins were expressed in Escherichia coli DH5␣ cells and purified by affinity chromatography using immobilized glutathione-Sepharose 4B beads (Pharmacia Biotech Inc.). Glutathione-Sepharose beads containing 5 g of fusion protein or GST were incubated with 1.0 ml of RASM cell lysate (0.9 -1.0 mg of protein) for 2 h at 4°C. The beads were washed three to four times with ice-cold lysis buffer containing 1 M NaCl (deletion construct D-85 (see Fig. 2, lower panel, lanes 10 and 11) was washed in 1.5 M NaCl), and the bound proteins were eluted with SDS sample buffer. Eluted proteins were separated on a 7.5% SDS-polyacrylamide gel, transferred to nitrocellulose, and immunoblotted with Jak2 polyclonal antisera (Upstate Biotechnology, Inc., Lake Placid, NY).
For the experiments reported in Fig. 1B, GST fusion protein containing the carboxyl-terminal 54 amino acids of the AT 1A receptor was covalently linked to Affi-Gel 10 (Bio-Rad) according to the manufacturer's instructions. The column was then blocked with 1 M glycine. The Jak2 binding assay was similar to that described above except that binding was assessed in the presence of increasing concentrations of competitor protein as indicated in Fig. 1B.
Electroporation-Growth-arrested RASM cells were electroporated using an Electro Square Porator (Model T820, BTX, Inc.) and a Petri dish electrode as described previously (15). In brief, serum-starved RASM cells were equilibrated in Ca 2ϩ -and Mg 2ϩ -free Hanks' balanced salt solution for 30 min at 37°C. Cells were exposed to one pulse at 100 V for 40 ms. Fusion proteins were electroporated at a final concentration of 10 g/ml in Hanks' balanced salt solution. The cells were then allowed to recover for 30 min at 37°C (5% CO 2 ) while still adherent to the culture dish. Cells were washed twice with Hanks' balanced salt solution and then stimulated with 10 Ϫ7 M angiotensin II for 3 min. The cells were lysed as described above and incubated with glutathione-Sepharose beads for 2 h at 4°C.
Peptide Synthesis-The synthetic peptides were synthesized using an Applied Biosystems Model 420 synthesizer. The peptides were purified by high performance liquid chromatography, and the identity of each peptide was verified by mass spectrometry using a Jeol SX-102 instrument. The amino acid sequences of the synthetic peptides are as follows: peptide P1, 318 KYIPPKAK 325 ; peptide P2, IKKPAPYK (scrambled version of peptide P1); and peptide P3, 311 KYFLQLLK 318 . In addition, we synthesized mutant versions of peptide P1 designated peptide P4 (KYAPPKAK), peptide P5 (KYIAPKAK), peptide P6 (KYIPAKAK), peptide P7 (KYAAAKAK), and peptide P8 (KFIPPKAK). The change in the amino acid sequence of peptide P1 is underlined.
Cell Transfection-A rat AT 1A receptor containing a mutation of the 319 YIPP motif to FAAA was created using the Bio-Rad Muta-Gene phagemid kit. This construct, as well as the wild-type AT 1A receptor, was cloned into the XhoI site of the mammalian expression vector pZeo (Invitrogen, San Diego, CA). COS-7 cells were transfected at 60% confluence in 100-mm tissue culture dishes using Lipofectin (Life Technologies Inc.). Each dish was washed once and layered with 3 ml of DMEM containing 2.5 g of a plasmid encoding murine Jak2 (pBOSwtJk2) (16), 10 g of the appropriate AT 1A receptor expression plasmid, and 20 l of Lipofectin. After a 5-h incubation at 37°C, the Lipofectin-containing medium was aspirated, replaced with serum-containing DMEM, and incubated for 24 -28 h. Plates were then washed and incubated for 16 -20 h in 5 ml of serum-free DMEM supplemented with 0.5% (w/v) bovine serum albumin. Cells were stimulated with 100 nM angiotensin II for the indicated times, and lysates were prepared as described above. To assess association of Jak2 with the AT 1A receptor, the receptor was immunoprecipi-tated using a polyclonal antibody directed against the carboxyl terminus of the rat AT 1A receptor (Santa Cruz Biotechnology, Santa Cruz, CA). Western blots were then probed with Jak2 polyclonal antisera. To measure the tyrosine phosphorylation of Jak2, the protein was immunoprecipitated using a polyclonal anti-Jak2 antibody (Santa Cruz Biotechnology) and immunoblotted with a mixture of monoclonal antiphosphotyrosine antibodies, clone 4G10 (Upstate Biotechnology, Inc.) and PY20 (Transduction Laboratories, Lexington, KY). Immunoprecipitates were collected by the addition of 20 l of Protein A/G Plus (Santa Cruz Biotechnology, Inc.). Precipitates were washed three times with radioimmune precipitation assay lysis buffer (RIPA), and proteins were eluted by boiling in SDS sample buffer. Proteins were separated on an 8% SDS-polyacrylamide gel, transferred to nitrocellulose, and immunoblotted as described above.
Binding Assays-COS-7 cells were transfected as described above. Approximately 30 -32 h after transfection, cells were trypsinized and seeded at a density of 1.5 ϫ 10 5 cells/well using 24-well tissue culture plates. The following day, transfected cells were tested for their ability to bind [ 125 I-Sar 1 ,Ile 8 ]angiotensin II (DuPont). Cells were incubated for 1 h at 25°C in binding buffer (10 mM HEPES, pH 7.4, 120 mM NaCl, 5 mM KCl, 1.2 mM MgSO 4 , 10 mM NaHCO 3 , 1.2 mM KH 2 PO 4 , 1 mM CaCl 2 , and 0.25% (w/v) bovine serum albumin) to remove endogenous ligand. After aspiration, 200 l of fresh binding buffer was added to each well, and binding was initiated by the addition of [ 125 I-Sar 1 ,Ile 8 ]angiotensin II. Saturating concentrations of [ 125 I-Sar 1 ,Ile 8 ]angiotensin II were set at 1 nM, and serial dilutions were made from this point. All samples were done in duplicate. Cells were incubated for 1 h at 25°C, and reactions were terminated by placing the plates on ice. Cells were washed four times with ice-cold binding buffer without bovine serum albumin. Cells were lysed with 250 l of 2 N NaOH for 30 min at 25°C, and 150 l was counted on a Beckman ␥-counter. Protein concentrations were determined using the Bio-Rad D c protein assay with the remaining lysates. Nonspecific binding was defined as binding in the presence of 1.0 M angiotensin II.

RESULTS AND DISCUSSION
A GST fusion protein containing the carboxyl-terminal 54 amino acids of the rat AT 1A receptor was purified to homogeneity. As a control, equally pure GST protein was made. Cultured RASM cells were treated with 100 nM angiotensin II for time points of 0.5-10 min. A cell lysate was prepared, and 1 mg of cell protein was mixed with 5 g of the GST-AT 1A fusion protein bound to glutathione-agarose beads. After a 2-h incubation, the beads were washed, and any remaining bound proteins were eluted by boiling. Jak2 was detected by Western blot analysis using a polyclonal anti-Jak2 antibody. As shown in Fig. 1A, there was both a ligand-and a time-dependent binding of Jak2 to the GST-AT 1A fusion protein. In the absence of angiotensin II (lane 1), Jak2 did not bind to the fusion protein, an observation that was confirmed in Ͼ10 separate experiments. Maximal Jak2 binding was observed 3 min after the addition of angiotensin II (lane 4). Treating RASM cells with angiotensin II for longer periods of time (lanes 5 and 6) resulted in less Jak2 binding to the GST-AT 1A fusion protein. An equivalent experiment was performed using GST (lanes 7 and 8); no Jak2 binding to GST was detected.
We have also used an identical protocol to investigate the binding of Jak1 and Tyk2. In contrast to Jak2, angiotensin II induced no detectable complex formation of Jak1 or Tyk2 with the GST-AT 1A fusion protein (data not shown).
To investigate the specificity of the binding assay, we covalently linked the GST-AT 1A fusion protein to an agarose matrix. This preparation allowed us to compete for the binding of Jak2 to the GST fusion protein matrix with increasing concentrations of free GST-AT 1A fusion protein, GST, or an irrelevant protein such as albumin (Fig. 1B). Lane 1 shows that in the absence of a competitor, there was abundant binding of Jak2 to the GST-AT 1A fusion protein matrix. Increasing concentrations of free GST-AT 1A fusion protein competed with this binding (lanes 2-4). In contrast, free GST or bovine serum albumin showed no competition with the binding of Jak2 to the fusion protein matrix (lanes 5-8).
To identify specific regions within the AT 1A receptor carboxyl tail important for the association with Jak2, we created a series of GST fusion proteins containing overlapping portions of the AT 1A receptor tail (Fig. 2, upper panel). Each of these proteins was purified and individually tested for the ability to bind to Jak2 kinase. As before, Jak2 association was quantitated by Western blot analysis. We observed that fusion proteins containing the motif YIPP (Tyr-Ile-Pro-Pro, amino acids 319 -322) bound Jak2 in a fashion similar to the full-length construct (Fig. 2, lower panel, lanes 1-4, 7, and 10). Receptor fusion proteins lacking this motif showed either markedly reduced or no binding of Jak2 (lanes 5, 6, 8, 9, and 11).
The carboxyl-terminal 54 amino acids of the AT 1A receptor contain tyrosine residues at positions 319 and 339. While Jak2 association with cytokine receptors is not thought to be dependent on tyrosines, the presence of a tyrosine in the 319 YIPP motif convinced us to make GST-AT 1A fusion proteins in which these individual tyrosine residues were converted to phenylalanine. When the conversion at position 319 was made, the GST fusion protein bound less Jak2 than the parent protein (Fig. 3, lower  panel, lane 2). This was consistently observed in five separate experiments. In contrast, the Y339F change had no effect on Jak2 binding (lane 3).
To further investigate the specificity of the YIPP motif, we created an 8-amino acid peptide (KYIPPKAK) corresponding to positions 318 -325 of the AT 1A receptor. As controls, we also synthesized this peptide in a scrambled configuration (IKKPA-PYK) and a peptide corresponding to positions 311-318 of the AT 1A receptor (KYFLQLLK). Increasing amounts of the synthetic peptides were used to compete for the angiotensin II-dependent binding of Jak2 to a GST-AT 1A fusion protein containing the carboxyl-terminal 54 amino acids of the receptor (Fig.  4A). Peptide P1 demonstrated a dose-dependent inhibition of Jak2 binding to the AT 1A receptor fusion protein (lanes 2-4).
No inhibition was observed using either the scrambled peptide (P2) or the peptide encompassing amino acids 311-318 (P3) (lanes 5-10). Even when 50 g of competitor peptide P2 or P3 was used, no competition was observed (data not shown). On a molar basis, peptide P1 is a less effective inhibitor than the entire GST-AT 1A fusion protein (Fig. 1B), a finding probably related to the small size of the peptide.
The specificity of individual amino acids within the YIPP motif was tested by synthesizing peptides P4 -P8 and testing the ability of each peptide to inhibit Jak2 binding to the GST-AT 1A fusion protein (Fig. 4B). Competition assays were performed using 10 g of competitor peptide. These data demonstrate that peptides containing a single change in the IPP portion of peptide P1 were no longer able to compete (Peptides P4 -P6). Peptide P7, containing a 3-amino acid substitution, was also unable to compete. Peptide P8, containing FIPP in place of YIPP, showed a partial ability to inhibit Jak2 binding to the GST-AT 1A fusion protein. These results are consistent with data in Fig. 3, showing that a GST-AT 1A fusion protein with a Y319F mutation bound Jak2, but with reduced efficiency.
To investigate the behavior of the AT 1A receptor carboxyl tail in vivo, RASM cells were electroporated with the GST-AT 1A fusion protein; deletion construct D-35, D-55, or D-85; or GST ( Fig. 5). We have previously shown that electroporation is an effective method of inserting proteins into RASM cells (15). After electroporation, the cells were incubated for 30 min at 37°C and then washed extensively. Angiotensin II was added for 3 min, and the cells were lysed and incubated with glutathione-Sepharose beads at 4°C. The beads were washed with buffer containing 1 M NaCl and eluted with SDS sample buffer. Jak2 was then assessed by Western blot analysis. In the absence of angiotensin II, no Jak2 associated with the GST-AT 1A fusion protein (data not shown). However, after angiotensin II addition, both the GST-AT 1A fusion protein and deletion construct D-35 bound Jak2 (Fig. 5, lanes 1 and 2). In contrast, deletion constructs D-55 and D-85, both of which lack the YIPP motif, bound very much less Jak2 (lanes 3 and 4). No binding of Jak2 was observed with GST (lane 5). These data recapitulate the in vitro studies and indicate the importance of the YIPP sequence.
We have also used a cell transfection approach to evaluate the importance of the YIPP motif. These experiments used the mammalian expression vector pZeo containing either a fulllength, wild-type AT 1A receptor (called pZeo/WT) or a construct in which the YIPP motif was converted to FAAA (called pZeo/ FAAA). Transient transfection was performed using COS-7 cells, which contain little endogenous AT 1 receptor or Jak2 (data not shown). Initial experiments used standard procedures to measure the binding of [ 125 I-Sar 1 ,Ile 8 ]angiotensin II to the wild-type receptor or the FAAA mutant expressed in these cells. Scatchard analysis indicated K d values of 0.172 nM for the wild-type receptor and 0.225 nM for the FAAA mutant. Thus, the conversion of the YIPP motif to FAAA does not markedly  1-6, respectively). 1 mg of cell protein was incubated for 2 h with 5 g of GST-AT 1A fusion protein bound to glutathione-Sepharose. Protein complexes were washed in buffer containing 1 M NaCl, and 130-kDa Jak2 was detected by Western blot analysis. In the absence of ligand (lane 1), Jak2 did not bind to the fusion protein. In response to angiotensin II, Jak2 bound to the fusion protein in a time-dependent fashion, with maximal binding present 3 min after ligand addition (lane 4). Using a similar protocol, Jak2 showed no binding to GST (lanes 7 and 8). B, the GST-AT 1A receptor fusion protein was covalently attached to agarose. RASM cells were treated with angiotensin II for 3 min, and a cell lysate was prepared. Lane 1 shows that Jak2 binds to the immobilized GST-AT 1A receptor fusion protein. In lanes 2-8, increasing concentrations of soluble competitor proteins were added to the cell lysate as indicated. Free AT 1A receptor fusion protein showed a dose-dependent inhibition of Jak2 binding (lanes 2-4). In contrast, free GST or bovine serum albumin (BSA) had no effect on the binding of Jak2 to the immobilized GST-AT 1A receptor fusion protein (lanes 5-8).
To measure the physical association of the AT 1A receptor with Jak2, constructs encoding both these proteins were transfected into COS-7 cells (Fig. 6). Two days after transfection, the cells were treated with angiotensin II for 0, 3, 6, or 15 min. Cells were lysed, and proteins were immunoprecipitated using a rabbit polyclonal anti-rat AT 1A receptor antibody and Protein A/G. The precipitated proteins were washed, and associated Jak2 was measured by Western blot analysis using polyclonal anti-Jak2 antibody. Controls for this protocol included transfection of the vector pZeo lacking any angiotensin II receptor insert (lanes 1-4). When pZeo/WT was cotransfected with a plasmid encoding Jak2 (lanes 5-8), treatment of cells with angiotensin II for 3 min induced the physical association of the AT 1A receptor with Jak2 (lane 6). This experiment was performed four times, and association was typically strongest after 3 min of ligand treatment. However, other repetitions of the experiment indicated association above the background levels at the 6-min point (data not shown). Equivalent cotransfection experiments were also performed with pZeo/FAAA (lanes 9 -12). In contrast to the wild type, an AT 1A receptor bearing FAAA in place of the YIPP motif never showed ligand-dependent association of the receptor with Jak2.
In smooth muscle cells, angiotensin II stimulates the tyrosine phosphorylation of Jak2 (5). We have analyzed this using cotransfection of the AT 1A receptor and Jak2 into COS-7 cells. Two days after transfection, cells were treated with angiotensin II, lysed, and immunoprecipitated with polyclonal anti-Jak2 antibody. Proteins were collected, washed, and analyzed by Western blotting using monoclonal anti-phosphotyrosine antibodies (Fig. 7A). The blot was then stripped and reprobed with anti-Jak2 antibody to verify equivalent loading of protein (Fig. 7B). In the absence of ligand, there was a basal level of Jak2 phosphorylation. In response to ligand, the wild-type receptor induced increased tyrosine phosphorylation of Jak2 (Fig. 7A, lanes 5-8). In contrast, no such increase was ever seen with the AT 1A receptor containing the FAAA mutation (lanes 9 -12). In the absence of transfected Jak2 (lane 13), no endogenous Jak2 was identified with this protocol.
The major observation in this study is the binding of Jak2 to FIG. 2. Sequence-dependent binding of Jak2. Upper panel, GST fusion proteins containing the indicated regions of the rat AT 1A receptor were prepared. Lower panel, RASM cells were treated with angiotensin II for 3 min, and a cell lysate was prepared. Each fusion protein was tested for its ability to bind Jak2 as described under "Experimental Procedures." Fusion proteins containing the AT 1A receptor motif YIPP (amino acids 319 -322) bound Jak2 (lanes 1-4, 7, and 10); fusion proteins lacking this motif showed either no or very much reduced binding of Jak2 (lanes 5, 6, 8, 9, and 11). W.T., wild type.

FIG. 3.
Upper panel, GST-AT 1A receptor fusion proteins were prepared containing Tyr-to-Phe point mutations at amino acid 319 or 339. Lower panel, each protein was tested for its ability to bind Jak2 from a RASM cell lysate prepared from cells treated with angiotensin II for 3 min. While the mutation of position 339 had no effect on binding (lane 3), the Y319F conversion reduced Jak2 binding (lane 2). W.T., wild type. a GST fusion protein containing the carboxyl-terminal portion of the rat AT 1A receptor. This association appears to be dependent on the receptor motif YIPP and is stable to washing in 1.5 M NaCl (Fig. 2, lower panel, lane 10). Transient cellular expression of either the wild-type AT 1A receptor or the receptor containing a mutation of the YIPP motif confirmed the importance of this sequence in the association of Jak2 with the seventransmembrane AT 1A receptor. The YIPP motif also appears to be important in angiotensin II-dependent tyrosine phosphorylation of Jak2. These data are consistent with previously published observations showing the coprecipitation of the AT 1A receptor and Jak2 from RASM cells (5). In that study, the association of Jak2 with the AT 1A receptor was dependent on the addition of angiotensin II to the RASM cells. This dependence on ligand for binding Jak2 was recapitulated in both the in vitro and in vivo studies reported here. At present, the precise changes induced by angiotensin II are not known. Presumably, ligand binding to the AT 1A receptors within cells changes the chemistry of these cells such that the Jak2 present in a cell lysate now binds to the GST-receptor fusion proteins. Whether our protocol is stripping activated Jak2 from endogenous receptors or whether activated Jak2 is naturally shuttling onto and off of AT 1 receptors is not known. Indeed, it is not known whether the association of Jak2 with the GST-AT 1A fusion protein is a bimolecular event or whether additional linker molecules participate. Finally, we are not certain if the Jak2-AT 1A association that occurs within cells is a precedent or a consequence of Jak2 activation.
We have considered the possibility that the interaction of Jak2 with the GST-AT 1A fusion protein is the result of the YIPP sequence acting as a substrate-binding site for Jak2. This seems unlikely given that Jak2 still shows affinity for a receptor fusion protein lacking Tyr 319 (Fig. 3). A peptide lacking this same tyrosine also showed some ability to inhibit the interaction of Jak2 with the GST-AT 1A fusion protein (Fig. 4B). To  -4), inhibited the binding of Jak2 in a dose-dependent fashion. Peptide P2 (IKKPAPYK) contains the same amino acids as peptide P1, but in a scrambled configuration. This peptide showed no inhibition of Jak2 binding (lanes 5-7). Peptide P3 (KYFLQLLK), containing amino acids 311-318 of the AT 1A receptor, also did not inhibit the binding of Jak2 (lanes 8 -10). All experiments were performed with a RASM cell lysate prepared from cells treated with angiotensin II for 3 min. B, inhibition studies similar to those described in A were performed with 10 g of peptides P1 and P4 -P8. Peptides P4 -P6 contain single alanine substitutions for the sequence IPP (amino acids 320 -322), but were unable to inhibit the binding of Jak2 to the GST-AT 1A receptor fusion protein. Peptide P8, containing phenylalanine in place of tyrosine 319, has a reduced capacity to inhibit the binding of Jak2.  Fig. 2) using a protocol that has previously been shown to be effective in inserting proteins into these adherent cells (15). The cells were then cultured for 30 min and washed. Angiotensin II was added for 3 min, and the cells were lysed. Fusion proteins were collected by affinity for glutathione and washed with buffer containing 1 M NaCl. Associated Jak2 was determined by Western blot analysis. Fusion proteins containing the AT 1A receptor YIPP motif bound Jak2 (lanes 1 and 2), whereas proteins lacking this motif bound very little or no Jak2 (lanes [3][4][5]. WT, wild type. FIG. 6. Cotransfection of the AT 1A receptor and Jak2. Using Lipofectin, COS-7 cells were cotransfected with plasmid pBOSwtJk2 encoding murine Jak2 (16) and the expression vector pZeo (lanes 1-4), pZeo encoding the wild-type rat AT 1A receptor (pZeo/WT) (lanes 5-8), or pZeo containing an AT 1A construct in which the YIPP motif was converted to FAAA (pZeo/FAAA) (lanes 9 -12). Two days after transfection, the cells were treated with angiotensin II for 0, 3, 6, or 15 min. Cells were lysed, and proteins were precipitated with polyclonal anti-AT 1A antisera. Associated Jak2 was measured by Western blot analysis. In response to angiotensin II, the wild-type AT 1A receptor formed a complex with Jak2 that peaked at 3 min (lane 6) and often remained above background levels at 6 min. In four separate experiments, no such ligand-dependent complex was observed with the pZeo/FAAA construct. directly address this issue, we measured Jak2 binding to the GST-AT 1A fusion protein in the presence of increasing concentrations of AG-490, a selective inhibitor of Jak2 kinase activity (Fig. 8) (17). This compound was added to a RASM cell lysate before the addition of the GST-AT 1A fusion protein. Even in the presence of 50 M AG-490, no reduction in Jak2 binding to the GST-AT 1A fusion protein was observed.
Previous studies of Jak2 binding to cytokine receptors have demonstrated the importance of the "box 1" motif (18 -22). This is a region of 8 -11 amino acids containing the consensus sequence PXXPXP, but such a sequence is not found in the AT 1A receptor. To our knowledge, the YIPP motif has not been associated with Jak2 binding to cytokine or other receptor types. Whether this sequence acts alone to define the Jak2-binding site or acts in combination with other regions of the AT 1 receptor awaits further analysis. In a sense, it is not surprising that the 319 YIPP motif is functionally important. It is the only region of the AT 1A receptor in which 2 prolines are adjacent or separated by 1 amino acid. Previously, we (23) and others (24) have noted that this region is analogous to the motif YIIP found within the platelet-derived growth factor receptor and the motif YLPP found within the epidermal growth factor receptor. In these growth factor receptors, the sequences have been shown to be SH2 target sequences when the tyrosine is phosphorylated (25). For instance, phospholipase C-␥1 contains an SH2 domain that interacts with the indicated SH2 target sequences of the platelet-derived growth factor and epidermal growth factor receptors (26,27). In contrast to phospholipase C, the study of cytokine receptors has suggested that Jak2 binding is not dependent on tyrosines and is consistent with the lack of an SH2 domain in these kinases (2, 3). Whether our data, showing an effect of Tyr 319 on Jak2 binding, can be explained by a nonspecific steric effect or whether these data indicate that tyrosine (and perhaps a linker protein) may play a specific role in Jak2-AT 1A receptor association is an issue that still must be addressed.
The study of growth factor receptors has also demonstrated that ligand binding induces receptor dimerization and the assembly of a signaling complex. This complex consists of the receptor, linker proteins, and enzymatically active molecules (such as Jak2) capable of transducing the binding of ligand into an intracellular signaling cascade (27,28). The equivalent concept does not exist for seven-transmembrane receptors. These receptors can be modified by intracellular enzymes such as ␤-adrenergic receptor kinase, but they are not typically portrayed as the assembly point for a signaling complex containing enzymatically active intracellular enzymes. Our previously published study (5) and the work presented in this article support such a concept for the seven-transmembrane AT 1A receptor. The association of Jak2 with the AT 1A receptor provides a pathway in which signals can be transduced from the cell surface into the cell nucleus. While the role of heterotrimeric G-proteins in the pathway remains unclear, the association of Jak2 with the AT 1A receptor suggests that the paradigm of a receptor-based signaling complex applies to seven-transmembrane receptors as well as to those for growth factors and cytokines. FIG. 8. Effect of the Jak2 inhibitor AG-490. RASM cells were treated with angiotensin II for 3 min, and a cell lysate was prepared. AG-490 was added to a final concentration of 0, 10, or 50 M, with all samples receiving an equivalent amount of the vehicle Me 2 SO. The lysate was incubated for 30 min at room temperature, and Jak2 binding to the GST-AT 1A fusion protein was then measured as described under "Experimental Procedures." AG-490 had no effect on the association of Jak2 with the GST-AT 1A fusion protein.  1-4), pZeo/WT (lanes 5-8), or pZeo/FAAA (lanes 9 -13) as described in the legend to Fig. 6. After the addition of angiotensin II, the level of Jak2 tyrosine phosphorylation was measured by immunoprecipitating cellular proteins with anti-Jak2 antibody, followed by Western blot analysis of the precipitated proteins with anti-phosphotyrosine antibody. The wild-type AT 1A receptor induced increased Jak2 tyrosine phosphorylation in response to angiotensin II (lanes [5][6][7][8]. No such increase was observed with the AT 1A receptor lacking the YIPP motif (lanes 9 -12). In the absence of transfected Jak2 (lane 13), no Jak2 was identified using this protocol. B, the Western blots from A were stripped and reprobed with anti-Jak2 antibody to verify equivalent loading of protein within each group.