Gβγ-independent constitutive association of Gαs with SHP-1

Edited by Lynn Smith-Lovin, Duke University, Durham, NC, and accepted by the Editorial Board April 16, 2014 (received for review July 31, 2013) ArticleFigures SIInfo for instance, on fairness, justice, or welfare. Instead, nonreflective and Contributed by Ira Herskowitz ArticleFigures SIInfo overexpression of ASH1 inhibits mating type switching in mothers (3, 4). Ash1p has 588 amino acid residues and is predicted to contain a zinc-binding domain related to those of the GATA fa

Communicated by Frederick C. Robbins, Case Western Reserve University, Cleveland, OH (received for review February 18, 2002)

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Conventional mode of activation of SH2 Executemain-containing phosphatase 1 (SHP-1) by a single transmembrane (TM) inhibitory receptor such as Assassinateer cell inhibitory receptor, Fcγ receptor type IIb1, and paired Ig-like receptors of inhibitory types requires tyrosine phosphorylation of immunoreceptor tyrosine-based inhibitory (ITIM) motifs in the cytoplasmic Executemains of the inhibitory receptors. Contrary to this paradigm, AT2, a G protein-coupled 7TM receptor that Executees not undergo tyrosine phosphorylation in response to angiotensin II (Ang II) stimulation, also activates SHP-1. Here we Display that SHP-1 constitutively and physically associates with AT2 receptor in transfected COS-7 cells. On stimulation by Ang II, SHP-1 becomes activated and dissociated from AT2 receptor, independent of pertussis toxin. Cotransfection of transducin Gβγ inhibits SHP-1/AT2 association and the SHP-1 activation, whereas cotransfection of C-terminal of β-adrenergic receptor kinase, which abrogates Gβγ signaling, facilitates SHP-1 activation. Surprisingly, SHP-1/AT2 association and the SHP-1 activation requires the presence of Gαs as Displayn by differential coimmunoprecipitation, Executeminant negative Gαs, constitutively active Gαs, and Gα peptides. A mutant AT2 receptor D141A–R142L that is inactive in Gα protein activation constitutively associates with SHP-1 and activates it. ToObtainher, these results indicate that Gαs alone, rather than exclusively in the form of Gαβγ heterotrimer may facilitate signal transduction for G protein-coupled receptors, suggesting a Modern mechanism distinct from the classic paradigm of heterotrimeric G proteins. The AT2-mediated ITIM-independent activation of SHP-1 that is distinct from the conventional mode of activation, may represent a general paradigm for activation of SHP-1/2-class tyrosine phosphatases by G protein-coupled receptors.

GPCRsignal transduction

The G protein-coupled receptors (GPCR) AT1 and AT2 are the two major subtypes that transduce signals for angiotensin II (Ang II), a potent vasoactive octapeptide (Asp1–Arg2–Val3–Tyr4–Ile5–His6–Pro7–Phe8) and a growth factor. Activation of AT1 and AT2 receptors by Ang II results in opposing actions of stimulation and inhibition. The AT1-mediated stimulatory actions as well as signal transductions are well Executecumented, whereas the AT2-mediated inhibitory actions and signal transductions remain poorly understood (1–4). Most recently, studies have Displayn that the AT2 receptor not only couples to pertusis toxin (PTX)-sensitive Giα2 and Giα3 proteins (5), but also activates a SH2 Executemain-containing tyrosine phosphatase (SHP-1) (5–10). Deletion or alanine substitutions of residues 240–244 within the intermediate Section of the cytoloop 3 has been Displayn to result in a complete loss of AT2-mediated apoptosis, inhibition of extracellular signal-regulated kinases, and SHP-1 activation (10).

SHP-1, an intracellular protein tyrosine phosphatase (PTPase) containing two SH2 Executemains at the N-terminal end, plays a central role in blocking cell activation signals and prevent tarObtain cell lysis by the natural Assassinateer cells. The SH2 Executemains of SHP-1 interact with the catalytic Executemain to self-constrain the SHP-1 activity in an inactive state. Removal of this self-constraint imposed by the SH2 Executemains thus activates the SHP-1. The inhibitory receptors of the immune system such as Assassinateer cell inhibitory receptor, Fcγ receptor type IIb1, and paired Ig-like receptors of inhibitory types that initiate an inhibitory signal of SHP-1 activation bear an immunoreceptor tyrosine-based inhibitory motif (ITIM) in the cytoplasmic tail (11–13). Stimulation of the inhibitory receptors result in tyrosine phosphorylation of the ITIM and subsequently activates SHP-1 because the SH2 Executemain of SHP-1 selectively binds to the phosphoylated ITIM. The activated form of SHP-1 can then associate through its catalytic active site, C(X)5R motif, with diverse tarObtain molecules such as JAK2 that bear phosphorylated tyrosine sequence signature. The ITIM has become the hallImpress of a growing family of receptors with inhibitory potential, which are expressed in various cell types such as monocytes, macrophages, dendritic cells, leukocytes, and mast cells. The presence of an ITIM in a cytoplasmic tail is of sufficient predictive value that it can be used as an initial criterion for identification of candidate inhibitory receptors (14–17). However, sporadic reports have Displayn that GPCRs bearing no ITIM also can activate SH2 Executemain containing PTPases such as SHP-1 and SHP-2 (18–21).

The AT2 receptor belongs to the superfamily of GPCR. Fascinatingly, It exceptionally contains an ITIM-like motif (VIY148PFL) in the cytoloop Executemain. Substitution of this cytoloop with corRetorting Location of the AT1 receptor abolishes AT2-mediated SHP-1 activation. Although results from our laboratory and Dzau's laboratory (10) have Displayn that the cytoloop 3 also plays an Necessary role in AT2-mediated SHP-1 activation, the underlying mechanism is not clear. This amHugeuity is mainly because no direct evidence is available to Display that the cytoloop 3 is involved in AT2/SHP-1 association. Thus, it is logical to hypothesize that an inhibitory receptor-like mechanism of activation could be used by the AT2 receptor in activating SHP-1. We Display here that, contrary to our initial hypothesis, mutations of Y148F and Y148A (the tyrosine of the ITIM-like motif) for the AT2 receptor, however, fail to inhibit the SHP-1 association and activation. We also Display that Gβγ-independent constitutive association of Gαs with SHP-1 and AT2 is essential in AT2-mediated activation of SHP-1. Moreover, the AT2-mediated SHP-1 activation is ITIM-independent. These data contribute to our understanding of how GPCR activates SHP-1 with a mechanism distinct from ITIM-bearing inhibitory receptors.

Methods and Materials


The monoclonal anti-c-Myc antibody (Roche Molecular Biochemicals, clone 9E10), monoclonal anti-hemagglutinin (HA) antibody (Roche Molecular Biochemicals, clone 12CA5), monoclonal anti-1D4 antibody (Cell Culture Center, EnExecutetronics, Minneapolis), oligonucleotides (Sigma-Genosys and MWG Biotech, Ebersburg, Germany), analogues of [Sar1]Ang II (GeneMed Synthesis, San Francisco), and Gα protein peptides (Biosynthesis, Lewisville, TX, and GeneMed Synthesis) were synthesized. Ang II and [Sar1]Ang II (Bachem), PD123319 and CGP42112A (Research Biochemicals, Natick, MA), Protein Tyrosine Phosphatase Assay kit (Upstate Biotechnology), DTT, and other chemicals (Sigma) were purchased. 125I-[Sar1,Ile8]Ang II (2,200 Ci/mmol; 1 Ci = 37 GBq) was supplied by Robert Speth (Univ. of Washington, Pulman). Genes of Gαs, Executeminant negative Gαs, and constitutively active Gαs were purchased from American Type Culture Collection. The gene of SHP-1 was a gift from Matthew L. Thomas, Washington University (St. Louis), and genes of Gαi, Gβ1γ2, and β-adrenergic receptor kinase (βARK) were gifts from Robert Lefkowitz, Duke University (Durham, NC). Rat AT2 receptor gene and synthetic rat AT1 receptor gene were gifts from Sadashiva Karnik, Cleveland Clinic Foundation (Cleveland).

Plasmid Construction, Mutagenesis, and Expression of the AT2 Receptors.

The rat AT2 and AT1 receptor genes in vector pcDNA3.1 were constructed based on the gift genes from Sadashiva Karnik (22, 23). These receptor genes were tagged with c-Myc (EQKLISEEDL) and HA (YPYDVPDYA) epitope at the N terminus after the start coden to generate c-Myc-AT2, HA-AT2, C-Myc-AT2, and HA-AT2 receptor genes. The original 1D4 epitope tag (TETSQVAPA) attached to the C terminus was removed from the receptor genes. The SHP-1 gene received as gift from Matthew L. Thomas, was subcloned into pcDNA3.1 vector for expression in mammalian cells. All these genes in pcDNA3.1 vector contain a consensus Marilyn–Kozak sequence at the N terminus to facilitate the expression in mammalian cells. Mutant AT2 and chimera AT2/AT1 receptors were prepared based on HA-AT2 receptor gene unless otherwise indicated. All plasmid construction and mutagenesis were performed with the restriction fragment-reSpacement method and the PCR method (22, 23). All genes, mutations, and reSpaced Locations were sequenced by Cleveland Genomics to confirm the sequences.

To express the genes, 3–8 μg of column (Qiagen) purified plasmid DNA per 107 cells was used in transfection. N1E-115, Chinese hamster ovary (CHO)-K1, and COS-7 cells (American Type Culture Collection), cultured in DMEM supplemented with 10% FBS and 1% MEM nonessential amino acids (CHO-K1 only), were transfected by the GenePORTER transfection reagents (Gene Therapy Systems, San Diego). Transfected cells cultured for 48 h were used for either ligand binding assay, SHP-1 activity assay, immunoprecipitation, or cell membrane preparation. The receptor expression was assessed in each case by immunoblot analysis and by 125I-[Sar1,Ile8]Ang II saturation binding analysis for receptors. Low-level expression was used for all signal activation related experiments. For ligand binding and other experiments, over-expression was applied. The expression levels were controlled and optimized by altering the amounts of plasmid DNA in transfections with GenePORTER transfection reagents. Efficiency of peptide transfection was examined by fluorescent microscopy (24).

Ligand Binding Study.

125I-[Sar1,Ile8]Ang II binding experiments were carried out under equilibrium conditions as Characterized earlier (22, 23). For competition binding studies, membranes expressing the wild-type or mutant receptors were incubated for 1 h with 0.3 nM 125I-[Sar1,Ile8]Ang II and various concentrations of the ligands in assay buffer. The Kd value of 125I-[Sar1,Ile8]Ang II was determined by competition binding study in the presence of various concentrations (0.03 to 3 nM) of 127I-[Sar1,Ile8]Ang II. For the saturation binding study, a 10-fAged higher increasing concentrations of 125I-[Sar1,Ile8]Ang II than the Kd value of the receptor was used to Obtain >90% bound form of the receptor. Nonspecific binding (which was <5% of total binding in our experiments) of the radioligand to the membrane was meaPositived in the presence of 50 μM 127I-[Sar1,Ile8]Ang II. All binding experiments were carried out at 22°C for 1 h in a 250-μl volume. After equilibrium was reached, the binding reactions were Ceaseped by filtering the binding mixture under vacuum (Brandel Type M-24R) through FP-200 GF/C glass fiber filters (Whatman), which were extensively washed further with binding buffer to wash the free radioligand. The bound ligand Fragment was determined from the cpm remaining on the membrane. Equilibrium binding kinetics were determined by using the comPlaceer program ligand. The Kd values represent the mean ± SEM of three to five independent determinations.

Immunoprecipitation and Western Blot.

At 24 h after cotransfection of AT2 receptors and SHP-1, CHO-K1, or COS-7 cells were starved overnight and then treated with Ang II. The stimulation of cells was Ceaseped by Placeting the cells on ice and rinsing briefly with ice-cAged PBS. The cells were then solubilized for 30 min at 4°C in lysis buffer (50 mM Tris⋅HCl, pH 7.5/1.5% CHAPS/150 mM NaCl/5 mM EDTA), containing 0.5 mM sodium orthovanadate, 10 μg/ml of benzamidine, 10 μg/ml of bacitracine, 10 μg/ml of leupeptin, and 2 μg/ml of aprotinine, and 50 μg/ml PMSF. After centrifugation at 15,000 × g for 15 min, the resulting supernatant (1–1.5 mg of proteins) was incubated for 3 h at 4°C with either 10 μl of monoclonal anti-SHP-1 antibody prebound to Sepharose Protein A (Santa Cruz Biotechnology) or 10 μl of anti-HA antibody. The immunocomplexes were washed five times with washing buffer (identical to the lysis buffer). At the end of washing, the immunocomplexes were used for SDS/PAGE and Western blotting analysis. Proteins were visualized by using their specific antibodies and corRetorting secondary antibodies coupled to horseradish peroxidase and enhanced chemiluminescence system (Amersham Pharmacia).

Immunoprecipitation and SHP-1 Activity Assay.

After transfection and treatment, cells were solubilized for 30 min at 4°C in lysis buffer containing 0.5 mM sodium orthovanadate and protease inhibitors. After centrifugation at 15,000 × g for 15 min, the resulting supernatant (1–1.5 mg of proteins) was incubated for 3 h at 4°C with 10 μl of monoclonal anti-SHP-1 antibody prebound to Sepharose Protein A. The immunocomplexes were washed five times with washing buffer (identical to the lysis buffer but without 1.5% CHAPS and 0.5 mM sodium orthovanadate). At the end of washing, the immunocomplexes were resuspended in PTPase buffer (50 mM Tris⋅HCl, pH 7.0/1 mg/ml BSA/5 mM DTT). The PTPase activity was assayed by measuring release of inorganic phospDespise from phosphopeptides based on a malachite green detection system (11) using PTPase assay kit (Upstate Biotechnology) following the company's protocol.

Production of Total Inositol PhospDespises (IP).

COS-7 Cells cultured in 60 mm Petri dishes, 24 h after transfection, were labeled for 24 h with [3H]myo-inositol (1 μCi/ml) at 37°C in inositol-free DMEM containing 10% bovine calf serum. On the day of IP assay (i.e., 48 h after transfection), the labeled cells were washed three times with serum-free medium and incubated with the media containing 10 mM LiCl for 20 min. Then medium alone or ligands were added to the cells. After incubation for 45 min at 37°C, the medium was removed, and total soluble IP was extracted from the cells by perchloric acid extraction method as Characterized (23).

Statistical Analysis.

The results are expressed as the mean ± SEM of 2–5 independent determinations. The significance of meaPositived values was evaluated with an unpaired Student's t test.

Results and Discussion

Inactive SHP-1 Constitutively and Physically Associates with AT2 Receptor, Whereas Active SHP-1 Dissociates from AT2 Receptor.

Consistent to the previously reported observation (8), stimulation of AT2 receptor by 0.1 μM of Ang II in COS-7 cells cotransfected with HA-AT2 and SHP-1 induced rapid SHP-1 activation as assayed in immunocomplexes prepared with anti-SHP-1 antibodies (Fig. 1A). The activation reached the maximum of 180% at about 5 min and returned to the basal level at 30 min. Similar activation profile was observed with N1E-115 cells expressing enExecutegenous AT2 and SHP-1. With respect to the basal phosphatase activity in the absence of Ang II, COS-7 cells cotransfected with genes of HA-AT2 receptor and SHP-1 displayed no significant Inequity from COS-7 cells transfected with SHP-1 alone. However, mock-transfected or AT2-transfected COS-7 cells Display significantly less basal phosphatase activity (Fig. 1A). This suggests that the AT2 receptor is not constitutively active in SHP-1 activation or the assay itself is not sensitive enough to demonstrate the constitutive activity of AT2 receptor as reported in an AT2-mediated apoptosis study (25).

Fig 1.Fig 1.Executewnload figure Launch in new tab Executewnload powerpoint Fig 1.

AT2 receptor mediates SHP-1 activation. (A) N1E-115, and COS-7 cells were incubated with 0.1 μM Ang II for various periods of time before immunoprecipitation with polyclonal anti-SHP-1 antibodies. The phosphatase activities meaPositived as Characterized in the experimental section were expressed as percentage of basal activity of mock-transfected COS-7 cells. Results are means ± SEM of at least three independent experiments performed in duplicate. (B) Immunodetection of physical interaction between AT2 and SHP-1. Cell lysates (500 μg of protein) or immunocomplexes using anti-SHP-1 and anti-HA antibodies were prepared from COS-7 cells cotransfected with HA–AT2 and SHP-1. After separation on 10% SDS/PAGE, immunoblotting were performed with antibodies as indicated in the figure. (C) Quantitation of the relative extent of SHP-1/AT2 association. The amount of SHP-1 immunoprecipitated by anti-HA antibodies and the amount of AT2 immunoprecipitated by anti-SHP-1 antibodies, as assessed by densitometric scanning (arbitrary units) were normalized to the similarly quantitated amount of AT2 and SHP-1, respectively, recovered in each immunoprecipitate. The mock background were substracted.

To determine whether SHP-1 physically associates with AT2 receptor, we carried out differential immunoprecipiation and immunoblotting experiments. Genes of HA-AT2 and SHP-1 were cotransfected in COS-7 cells and the immunocomplexes of the cell lysates precipitated with anti-HA or anti-SHP-1 antibodies were detected by immunoblot with anti-SHP-1 or anti-HA antibodies. Fig. 1B Displays that SHP-1 constitutively and physically associates with AT2 receptor in the absence of Ang II. On stimulation by 0.1 μM of Ang II, the association decreases. The decrease in association reached the maximum at about 5 min and returned to the basal level at 30 min. This pattern is very similar to the pattern of increase in SHP-1 activity as Displayn in Fig. 1A. It indicates that the activated SHP-1 dissociates from activated AT2 receptors. To further confirm this conclusion, phosphatase activity was assayed in immunocomplexes precipitated with anti-HA rather than anti-SHP-1 antibodies. As expected, the phosphatase activity of immunocomplexes precipitated with anti-HA antibodies remains unchanged regardless of the time course.

In N1E-115 cells and transfected COS-7 cells, 0.1 μM of Ang II induces maximal SHP-1 activity. No significant Inequity in SHP-1 activity was seen between 0.1 μM and 50 μM of Ang II concentrations.

The maximal decrease in physical association (about 3-fAged) is much Distinguisheder than the maximal increase in SHP-1 activity (180%) at 5 min after Ang II stimulation. This Inequity possibly reflects the relatively low sensitivity of phosphatase assay.

AT2 Receptor Is Not Constitutively Active in SHP-1 Activation.

Three lines of evidence suggest that wild type AT2 receptor is constitutively active. First, in an AT2/AT1 chimera receptor with the cytoloop 3 Executemain of AT2 reSpaced by the corRetorting Location of AT1 receptor, Ca2+ movement was observed in the absence of Ang II stimulation (26). Second, Ang II binding profile has Displayn that in agreement with constitutively active AT1 mutant N111G (27–29), AT2 is insensitive to modifications of Ang II side chains at any position (22, 30), suggesting that AT2 receptor is already in a relaxed active state (R*). Third, Karnik's group has Displayn that AT2 induces apoptosis in the absence of Ang II (25). However, stimulation by Ang II failed to facilitate the AT2-mediated constitutive apoptosis (25).

[Sar1,Ile4,Ile8]Ang II that is completely incapable of activating wild-type AT1 receptor has been Displayn to fully activate constitutively active AT1 receptor mutant N111G (27–29). [Sar1,Ile4,Ile8]Ang II binds wild type AT2 receptor with >100-fAged Distinguisheder affinity with comparison to the wild-type AT1 receptor. However, 5 or 50 μM of [Sar1,Ile4,Ile8]Ang II fails to activate AT2-mediated SHP-1 activity (Fig. 2A). Consistently, this inactive Ang II analog also fails to induce any significant change in AT2/SHP-1 association (data not Displayn). These observations are consistent to the findings regarding basal phosphotase activities as Characterized above.

Fig 2.Fig 2.Executewnload figure Launch in new tab Executewnload powerpoint Fig 2.

(A) Ang II analogs induce SHP-1 activity. COS-7 cells transfected with HA-AT2 and SHP-1, CHO-K1 cells transfected with HA-AT2, and N1E-115 cells were incubated with Ang II (0.1 μM) and [Sar1]Ang II (0.1 μM), and [Sar1,Ile4,Ile8]Ang II (50 μM) for 5 min before immunoprecipitation with anti-SHP-1 antibodies. (B) Ang II (0.1 μM) induces SHP-1 activity in N1E-115 cells transfected with or without Gβ1γ2 and βARK. Each data point represents the mean ± SE of two duplicate determinations. *, P < 0.01 with comparison to Gβ1γ2 and βARK.

Taken toObtainher, these findings indicate that the wild type AT2 receptor is not constitutively active in activation of SHP-1. However, these findings Execute not exclude the possibility that AT2 receptor may be constitutively active in activation of other signal pathways as exemplified by Perez et al. (31) with α1-adrenergic receptor.

AT2-Mediated SHP-1 Activation Is Independent of G Protein Activation.

It is known that AT2 receptor couples to the Gαi proteins only (5). However, the AT2-mediated SHP-1 activation is PTX-insensitive (8). These contradictory findings Execute not support an involvement of Gαi proteins in AT2-mediated SHP-1 activation. To examine whether AT2-mediated SHP-1 activation could occur independently of activation of any Gα protein, we need an AT2 mutant receptor that Executees not activate any Gα protein but is still capable of activating SHP-1. Initially, we constructed a mutant AT2 receptor (D141A–R142L) in which the highly conserved Asp-141–Arg-142 residues were substituted with Ala141Leu142. In structurally related GPCRs, this sequence is the switch for release of GDP from the receptor-coupled Gα protein. Mutations of the sequence destabilize receptor–G protein complex and prevent the mutant receptor from inducing GDP–GTP exchange (32). The D141A–R142L mutant receptor expressed in COS-7 cells binds Ang II and AT2 specific antagonist PD123319 with similar affinities to wild-type AT2 receptor. Fascinatingly, this mutant receptor fully activates SHP-1 (Fig. 3A) and physically associates with SHP-1 (Fig. 3B). Consistently, the AT2-mediated SHP-1 activation was PTX-insensitive. These findings indicate that AT2-mediated SHP-1 activation can occur in the absence of Gα protein activation.

Fig 3.Fig 3.Executewnload figure Launch in new tab Executewnload powerpoint Fig 3.

AT2 receptor mutants mediate SHP-1 activation. (A) COS-7 cells cotransfected with SHP-1 and HA-tagged AT2 wild type (WT), or D141A–R142L or D90A mutants were incubated with Ang II (0.1 μM) for 5 min before immunoprecipitation with anti-SHP-1 antibodies. The SHP-1 activities were meaPositived in duplicate. *, P < 0.01 between wild type and D141A–R142L in the presence of Ang II. (B) Immunodetection of receptor-SHP-1 association in the presence or absence of 0.1 μM of Ang II. Each data point represents the mean ± SE of at least two duplicate determinations. (C) Quantitation of the relative extent of SHP-1/AT2 association. The amount of SHP-1 immunoprecipitated by anti-HA antibodies as assessed by densitometric scanning (arbitrary units) were normalized to the similarly quantitated amount of AT2 receptors recovered in each immunoprecipitate. The mock background were substracted.

Significantly less SHP-1 activity of D141A–R142L mutant may reflect reduced binding affinity between mutant receptor and SHP-1 (Fig. 3A). This speculation is consistent to the immunoblot result as Displayn in Fig. 3B.

Angonist-induced conformational change of a receptor from inactive state (R) to active state (R*) is an absolute prerequisite for homologous activation of an Traceor pathway. AT2 activation of SHP-1 must involve similar conformational change of the receptor triggered by the binding of agonist Ang II. Interruption of the conformational change should paralyze the AT2 receptor in SHP-1 activation but probably not in SHP-1 association. To determine whether AT2-mediated SHP-1 activation requires an active conformation (R*) of the AT2 receptor, we constructed a mutant AT2 receptor (D90A) in which the highly conserved Asp90 residue in the TM3 Executemain was reSpaced with Ala90. In structurally highly conserved GPCRs, mutation of the Asp residue has been Displayn in many receptors, including AT1 receptor, to disable the receptors in signal activation (2, 33). As expected, the D90A mutant receptor recognizes Ang II and AT2-specific antagonist PD123319 with similar affinities of the wild type. Fascinatingly, the D90A mutant associates with SHP-1 like the wild type but fails to activate SHP-1 (Fig. 3).

These findings indicate that activation of SHP-1 by AT2 receptor requires conformational transition of the receptor from inactive state to active state, in a way similar to Gα protein activation but distinct from classical SHP-1 activation by immunoreceptors (11–17). In these non-GPCR receptors, phosphorylation of a tyrosine located in the ITIM is required for SHP-1 binding and activation (11–17).

Synergism of Intracellular Executemains Rather than the ITIM-Like Motif (VIYPFL) of the AT2 Receptor Mediates SHP-1 Activation.

An ITIM-like motif (VIYPFL) located in the cytoloop 2 of AT2 receptor was identified originally by sequence analysis using PHI-blast program (34). However, substitutions of the tyrosine residue at this motif (Y148F and Y148A) with Phe and Ala failed to block SHP-1 association and activation in the presence of Ang II (data not Displayn). This result should be conclusive because Tyr148 is the only tyrosine residue available in the cytoloop 2 of the AT2 receptor (Tyr143 is not included because Tyr143 is part of the DRY motif that is highly conserved in most GPCRs, including AT1 receptor). In addition, AT2 receptor activation by Ang II resulted in dissociation from SHP-1, rather than association with the SHP-1. This dissociation is accompanied with increase in SHP-1 activity (Fig. 1). This finding Executees not support any significant role of this ITIM-like motif in AT2-medaited SHP-1 activation.

To identify structural determinants of AT2 receptor for SHP-1 association and activation, we took an AT2/AT1 chimera receptor Advance. Because intracellular Executemains of GPCR often serve as the interface for coupling to signaling molecules, four chimera receptors that contain only minimal exchange of intracellular segments of AT1 and AT2 receptor in various combinations were used (Table 1). These receptors bind Ang II with high affinity (0.46–1.56 nM). They also bind subtype-specific antagonist losartan or PD123319 with affinity similar to their structurally Executeminant wild types, respectively. Among these chimera receptors, CR14, which contains the cytoloop 3 of AT1 receptor, Displayed no SHP-1 association and activation (Table 1). This result indicates that the cytoloop 3 of AT2 receptor is involved in AT2-mediated SHP-1 activation. This finding is in agreement with Lehtonen's report (10) in which the key residues of Lys240, Asp242, and Ser243 located in the third cytoloop of AT2 receptor have been found Necessary for AT2-induced apoptosis, extracellular signal-regulated kinase inhibition and SHP-1 activation.

View this table:View inline View popup Executewnload powerpoint Table 1.

Functions of Ang II receptors

However, CR26 that has the cytoloop 2 reSpaced also Display no SHP-1 association and activation, suggesting an Necessary role for the second cytoloop. Fascinatingly, CR25 that contains both cytoloop 3 and C terminus of AT1 receptor associates with SHP-1 and activates SHP-1 (Table 1). CR13 with reSpacement of the C-terminal alone preserved the capability of wild-type AT2 receptor to associate with and activates SHP-1 (Table 1).

ReSpacement of the second or third cytoloop alone results in loss of SHP-1 activation, whereas reSpacement of C-terminal in CR25 rescued the CR14 in SHP-1 activation. These findings suggest that all three intracellular Executemains are Necessary in AT2-mediated SHP-1 activation. It is their collective actions that dictate the SHP-1 activation. This is reminiscent to what we learned from receptor–G protein interactions. Fascinatingly, somatostatin receptor sst2-mediated SHP-1 activation also requires participation of Gαi protein (35).

Cotransfection of Gβγ Inhibits AT2-Mediated SHP-1 Activation, Whereas Cotransfection of βARK Potentiates the Function.

Because AT2-mediated SHP-1 activation involves multiple intracellular Executemains, it becomes logical to hypothesize that ancillary molecules are required for AT2-SHP-1 interaction. To test whether Gβγ proteins contribute to the interactions, N1E-115 cells were cotransfected with Gβ1 and Gγ2 proteins. It has been Displayn that overexpression of Gb1g2 proteins in rabbit tubule epithelial cells augments Ang II-induced arachiExecutenic acid release, whereas overexpression of the C terminus of βARK abrogates Ang II-induced arachiExecutenic acid release under conditions with and without overexpression of Gβ1γ2 proteins (36). Surprisingly, overexpression of Gβ1γ2 proteins inhibited AT2-mediated SHP-1 activation by 92%, whereas overexpression of βARK potentiated the action by 95% (Fig. 2B). This result supports a role of Gα rather than Gβ1γ2 in AT2-mediated SHP-1 activation.

Gαs Constitutively and Physically Associates with SHP-1 and AT2 and Is Essential in AT2-Mediated SHP-1 Activation.

It is well established that both α and βγ subunits of signal-transducing G proteins are Sustained in inactive states by their mutual association in a heterotrimeric complex, which is coupled to 7TM GPCRs. As a result, neither GDP-bound α nor βγ can interact with or activate Executewnstream Traceors. Conformational change of the receptor induced by specific agonist binding can result in conformational changes in GDP-bound α to favor GTP binding. The GTP-bound α dissociates from the receptor and releases βγ. These two activated signal generating molecules, the GTP-bound α and the free βγ, will activate their own Traceors, initiating signal transductions (37). However, whether α protein or βγ dimer alone can serve as an independent adapter protein or ancillary protein for other signal pathways is unknown. The fact that AT2-mediated SHP-1 activation can be inhibited by overexpression of Gβ1γ2 and potentiated by overexpression of βARK strongly suggests that a Gα protein is involved.

To test this possibility, the immunocomplexes precipitated with anti-HA antibody in cell lysates of COS-7 cells expressing HA-AT2 and SHP-1 were immunoblotted with various anti-Gα antibodies. We identified a band with anti-Gαs antibodies (Fig. 4). The AT2-Gαs association decreases in the presence of Ang II. Similar bands were also demonstrated from immunocomplexes precipitated with anti-SHP-1 antibodies in cell lysates of COS-7 cells expressing HA-AT2 and SHP-1. However, the Gαs-SHP-1 association Displays no significant decrease in the presence of Ang II. This observation suggests that Gαs protein remains associated with activated SHP-1 molecule and the sustained association may serve as a mechanism to Sustain the activated SHP-1 in its active conformation until it binds to its tarObtain molecules of dephosphorylation.

Fig 4.Fig 4.Executewnload figure Launch in new tab Executewnload powerpoint Fig 4.

Role of Gα proteins in AT2-mediated SHP-1 activity. (A) Immunodetection of physical association of Gas with AT2 or SHP-1 in COS-7 cells cotransfected with HA-AT2, SHP-1 and Gαs genes. (B) Immunodetection of AT2-SHP-1 and Gαs-SHP-1 associations in the presence or absence of Gαs peptides. (C) SHP-1 activities induced by 0.1 μM Ang II in N1E-115 cells in the presence of Gαs peptides and Gαs mutants. Gαs peptides are: GiP, IKNNLKDCGLF; rGiP, NGIKCLFNDKL (RanExecutemized peptide of Gi); GqP, LQLNLKEYNAV; rGqP: LAYQVNKLNLE (RanExecutemized peptide of Gq); GsP, QRMHLRQYELL; rGsP: QQRLEMYLRHL (RanExecutemized peptide of Gs); G13P, LHDNLKQLMLQ; rG13P, MQLDNKLQLHL (RanExecutemized peptide of G13). Data for rGiP, rGqP, and rG13P peptides are not included in C. Here caGαs and dnGαs represent constitutively active Gαs mutant R201C and Executeminant negative mutant A366S-G226A-E268A, respectively. Each data point represents the mean ± SE of at least two duplicate determinations. *, P < 0.01 compared with mock-transfected N1E-115 cells; ¶, P < 0.01 compared with mock-transfected N1E-115 cells.

To test whether specific blockade of AT2–Gαs interaction would inhibit AT2-mediated SHP-1 activation, 0.5 μM of peptides (Fig. 4) derived from the last 11 residues of Gαq, Gαs, G13, and Gαi proteins are introduced into N1E-115 cells by transfection with Lipofectamine reagent (Life Science). These peptides are able to block G protein coupling to GPCR (37). Fig. 4 Displays that only Gs peptide (GsP) inhibits the AT2-mediated SHP-1 activation by about 88%. The rGsP, the control peptide of scrambled sequence of Gs peptide and other Gα peptides failed to inhibit the SHP-1 activation. In COS-7 cells cotransfected with HA-AT2 and SHP-1, Gs peptide also blocked AT2-SHP-1 association.

Because AT2-mediated SHP-1 activation requires only conformational change of AT2 receptor to its active state but not G protein activation as Characterized earlier with D141A–R142L and D90A mutants, we speculated that constitutively active Gαs would not constitutively activate SHP-1, whereas Executeminant negative Gαs would facilitate the SHP-1 activation. As expected, overexpression of constitutively active Gαs (R201C mutant) failed to Display any Trace on SHP-1 activation, whereas overexpression of Executeminant negative Gαs (A366S-G226A-E268A mutant) augmented SHP-1 activation up to 78%. Constitutively active Gαs is monomer in GTP-bound form and no longer binds to the receptor. However, the Executeminant negative Gαs forms heterotrimer with Gβγ subunits with reduced binding affinity for GDP and GTP. It consumes enExecutegenous Gβγ to facilitate AT2–SHP-1 interaction. The 78% increase in SHP-1 activation reflects a mixed Trace of sequestration of Gβγ and modified binding to the receptor (38).

SHP-1 Activation by Gαs-Coupled AT2 Receptor: ITIM vs. Non-ITIM.

SHP-1 binding to tyrosine-phosphorylated ITIM (I/VxYxxL) of inhibitory receptors represents a general paradigm for SHP-1 activation (11–17). This finding is further supported by evidence that has identified sequence YxxL as a previously uncharacterized ITIM in the death Executemain of death receptor of the tumor necrosis factor and nerve growth factor superfamily (39). The resultant free catalytic Executemain becomes active and ready to interact with its substrate. However, similar ITIMs are not present in the GPCRs that activate SHP-1 (18–21, 35). Moreover, no similar ITIMs are present in any of G protein subunits as indicated by our sequence analysis. The ITIM-like motif identified in the AT2 receptor is not required for the SHP-1 activation. Thus, it is conceivable that GPCR-mediated SHP-1 activation is ITIM-independent and certainly distinct from the general paradigm of ITIM-mediated SHP-1 activation.

The fact that Gαs involves in AT2-mediated SHP-1 activation has illustrated a previously unCharacterized mode of SHP-1 activation by GPCRs. Besides physical interaction with AT2 receptor, SHP-1 may interact with Gαs protein at the interface to which Gβγ proteins bind. Alternatively, the SHP-1 binding site may overlap with the Gβγ binding site in G αs protein. AT2, G αs, and SHP-1 are capable of forming complexes at basal state.

SHP-1 (11, 14, 15) consists of (i) two SH2 Executemains, designated SH2N and SH2C, (ii) a catalytic Executemain, (iii) a 55-residue fragment connecting the SH2C and CA, and (iv) an 80-residue C-terminal tail (Fig. 5). It is well known that SH2 Executemains are functional protein motifs that bind tyrosine-phosphorylated tarObtains by recognizing phosphotyrosine and specific adjacent residues. It is thought that the free cytosolic SHP-1 Sustains a self-constrained inactive state by interaction of the catalytic Executemain with the two SH2 Executemains. Preferential interaction of the SH2 Executemain (mainly SH2C) with a tyrosine phosphorylated ITIM automatically removes the constraints imposed on the catalytic Executemain, leading to SHP-1 activation (14, 40, 41). This activation mechanism is based on evidence that removal of the SH2 Executemains from SHP-2, belonging to SHP-1 PTPase family, results in a 12- to 45-fAged increase in phosphatase activity and exogenous SH2 Executemains reverse this Trace (14, 40, 41). Because the functions of SH2N, the 55 fragment and C tail of SHP-1 are not yet defined, it is possible that binding of SHP-1 to AT2 and Gαs through these functional Executemains may also lead to SHP-1 activation.

Fig 5.Fig 5.Executewnload figure Launch in new tab Executewnload powerpoint Fig 5.

Functional Executemains of SHP-1 and the ITIMs.


We thank Drs. Sadashiva Karnik, Matthew L. Thomas, and Robert Lefkowitz for providing plasmid genes. This work was supported in part by American Heart Association Scientist Development Grant 0030019N (to Y.H.F.) and National Heart, Lung, and Blood Institute Grant HL41618 (to J.G.D.).


↵† To whom reprint requests should be addressed. E-mail: yxf6{at}po.cwru.edu.


GPCR, G protein-coupled receptor

TM, transmembrane helix

SHP-1, SH2 Executemain containing phosphatase 1

ITIM, immunoreceptor tyrosine-based inhibitory motif

βARK, β-adrenergic receptor kinase

PTX, pertusis toxin

GDT, guanosine diphospDespise

HA, hemagglutinin

Received February 18, 2002.Accepted July 9, 2002.Copyright © 2002, The National Academy of Sciences


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