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Direct protein–protein interaction between PLCγ1 and the bradykinin B2 receptor—Importance of growth conditions
BBRC Biochemical and Biophysical Research Communications 326 (2005) 894–900 www.elsevier.com/locate/ybbrc
Direct protein–protein interaction between PLCc1 and the bradykinin B2 receptor—Importance of growth conditions Johan Duchenea, Sharmila D. Chauhana, Fre´de´ric Lopezb, Christiane Pechera, Jean-Pierre Este`veb, Jean-Pierre Girolamia, Jean-Loup Bascandsa, Joost P. Schanstraa,* b
a Inserm U388, IFR 31, Hopital Rangueil, TSA 50032, 31059 Toulouse Cedex 9, France Functional Proteomics Facility, IFR 31, Hopital Rangueil, TSA 50032, 31059 Toulouse Cedex 9, France
Received 17 November 2004
Abstract Recently, we have described a novel protein–protein interaction between the G-protein coupled bradykinin B2 receptor and tyrosine phosphatase SHP-2 via an immunoreceptor tyrosine-based inhibition motif (ITIM) sequence located in the C-terminal part of the B2 receptor and the Src homology (SH2) domains of SHP-2. Here we show that phospholipase C (PLC)c1, another SH2 domain containing protein, can also interact with this ITIM sequence. Using surface plasmon resonance analysis, we observed that PLCc1 interacted with a peptide containing the phosphorylated form of the bradykinin B2 receptor ITIM sequence. In CHO cells expressing the wild-type B2 receptor, bradykinin-induced transient recruitment and activation of PLCc1. Interestingly, this interaction was only observed in quiescent and not in proliferating cells. Mutation of the key ITIM residue abolished this interaction with and activation of PLCc1. Finally we also identified bradykinin-induced PLCc1 recruitment and activation in primary culture renal mesangial cells. 2004 Elsevier Inc. All rights reserved. Keywords: G-protein coupled receptor; Bradykinin B2 receptor; Protein–protein interaction; Phospholipase Cc1; Tyrosine phosphatase SHP-2; Src homology 2 domain; Surface plasmon resonance; Mesangial cells; Quiescent and proliferating conditions
G-protein coupled receptor (GPCR) signaling pathways involve activation of heterotrimeric G proteins which can regulate a large panel of downstream effectors. Stimulation of GPCRs by ligands promotes the interaction between the third intracellular-loop of the receptor and the G protein. This induces the exchange of GDP by GTP on the G protein a subunit and the dissociation of the bc complex. Both a and bc subunits can initiate intracellular signaling by activation or inhibition of different effectors such as adenylate cyclase, phospholipases, ion channels or kinases. This GPCR signaling is now considered as ‘‘classical’’ GPCR signaling [1]. The bradykinin B2 receptor belongs to the GPCR superfam*
Corresponding author. Fax: +33 5 62 17 25 54. E-mail address: [email protected] (J.P. Schanstra).
0006-291X/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.11.126
ily and is coupled to a Gq/11 protein [2] which activates phospholipase beta (PLC-b) [3], protein kinase C (PKC) [4], and the mitogen-activated protein kinase (MAPK) cascade [4]. The bradykinin B2 receptor and other GPCRs have also been shown to induce ‘‘alternative’’ (G-independent) pathways. Indeed, such direct protein–protein interactions of intracellular proteins with GPCRs have been observed: (i) between endothelial nitric-oxide (NOS) synthase and the B2, angiotensin AT1 and endothelin receptors [5], (ii) between neuronal NOS and the B2 receptor [6], (iii) between c-Src and the b3-adrenergic receptor [7], and (iv) between SHP-2 and phospholipase Cc1 (PLCc1) with the B2 and AT1 receptors [8–11]. Although a direct interaction between an intracellular protein and a GPCR was found in all studies, few of them have
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identified the actual sequence motif involved in this interaction. It has been shown however, that proline rich motifs (PxxP) present in the third intracellular loop of the b3 adrenergic receptor allow the direct interaction with c-Src [7]. Another example is the YIPP motif in the C-terminal domain of the AT1 receptor which has been identified to directly interact with both PLCc1 and SHP-2 [10,11]. This single motif can bind two different downstream effector proteins (PLCc1 and SHP-2) when the key amino-acid (Y309) is phosphorylated. The bradykinin B2 receptor does not possess a YIPP motif, but recently we have described that SHP-2 (via its SH2 domain) can be recruited by the immunoreceptor tyrosine-based inhibitor motif (ITIM) of the B2 receptor [8]. This B2 receptor ITIM is located at the interface of the C-terminal part of the 7th transmembrane region and the C-terminal intracellular tail of the bradykinin B2 receptor and is 100% conserved in human, mouse, rabbit, rat, and pig. Under proliferating conditions, and upon bradykinin-treatment-induced phosphorylation of the key tyrosine residue (Y307) of the B2 receptor ITIM sequence, the bradykinin B2 receptor recruits, via a direct protein–protein interaction, tyrosine phosphatase SHP-2. This interaction is involved in the anti-mitogenic properties of the B2 receptor. In analogy with the AT1 receptor, it is possible that the B2 receptor ITIM sequence can recruit both SHP-2 and PLCc1. In agreement with this, Venema et al. [9], showed that in vascular endothelial cells expressing the human B2 receptor, PLCc1 could associate with the B2 receptor, but the involvement of the B2 receptor ITIM sequence was not studied. In this study the extreme C-terminal end of the receptor which does contain the ITIM sequence was used for the identification of regions of the B2 receptor involved in the interaction (see Fig. 1). We therefore explored the possibility that, in addition to recruiting SHP-2, the ITIM sequence also plays a role in PLCc1 recruitment. Using a combination of surface plasmon resonance, Western blot, and mutational analyses, we found in non-proliferating cells that PLCc1 interacts with the B2 receptor via the phosphorylated ITIM.
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Materials and methods Materials. Primary culture mesangial cells were obtained and cultivated in DMEM as previously described [12]. The culture media DMEM, aMEM, streptomycin, penicillin, and fetal calf serum (FCS) were from GIBCO. Bradykinin and chemical products were from Sigma. Monoclonal anti-PLCc1, anti-phosphotyrosine (PY20), SHP-2, and anti-B2 receptor antibodies were from Transduction Laboratories. The recombinant PLCc1 was from Santa Cruz. Surface plasmon resonance analysis. Surface plasmon resonance analysis of real time PLCc1 binding to B2 receptor ITIM peptides was performed on a BIAcore 3000 instrument (BIAcore AB). Synthetic biotinylated peptides containing the bradykinin B2 receptor ITIM sequence (SCLNPLVYVIVGKRF: ITIM residues in bold, which is 100% conserved among species) tyrosyl-phosphorylated or non-tyrosyl-phosphorylated were immobilized on separate channels of a streptavidin-coated carboxymethylated dextran chips (BIAcore AB) at 60 resonance units (RU). Different concentrations (1–200 nM) of purified recombinant PLCc1 (530–850 amino acids) were passed over the immobilized peptide surface at a flow rate of 30 ll/min at 25 C during 300 s in HBS buffer (10 mM Hepes, pH 7.4, 150 mM NaCl, 3 mM EDTA, and 0.005% polysorbate). After each binding assay, sensor chips were regenerated by two pulses of 5 ll of 0.01% SDS. The level of phosphotyrosine residues on phosphorylated peptides was verified using an anti-phosphotyrosine antibody (4G10, Upstate Biotechnology). The association and dissociation curves (sensorgrams) corresponding to the binding of PLCc1 to the ITIM peptides were used to determine association, dissociation, and affinity constants with the software BIAevaluation 3.1 (BIAcore AB). CHO cells expressing wild-type and ITIM mutated rat B2 receptor. As previously described [8], two different clonal cell lines overexpressing the rat wild-type and ITIM mutated B2 receptor (Y307F) were used. These cell lines express functional B2 receptors as previously shown [8]. Lysates. Semiconfluent cells were washed twice with phosphatebuffered saline and treated with bradykinin (100 nM in medium) or with medium only at 37 C. Cells were collected and lysed with RIPA buffer (10 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 2 mM Na3VO4, 0.1% SDS, 1% NP40, 1% sodium deoxycholate, 0.36 lg/ ml PMSF, 0.01% SBTI, 0.01% leupeptin, and 0.01% aprotinin). The mixture was gently agitated for 30 min at 4 C and centrifuged at 15,000g during 15 min at 4 C followed by collection of the supernatant. Immunoprecipitation. Five hundred micrograms of soluble proteins were incubated with 1.5 lg of antibody for 30 min at 4 C. The mixture was then incubated 2 h with Sepharose–protein A beads prewashed with RIPA buffer. The mixture was then washed three times with RIPA buffer and resuspended in 32.5 ll of Laemmli buffer for immunoblotting.
Fig. 1. Comparison of the rat and human bradykinin B2 receptor and outline of peptides used in this study and the study of Venema et al. [9]. Both the human and rat B2 receptor contain an ITIM sequence (in bold) which is 100% conserved among species. Both the rat (Y320) and human (Y322) possess an additional tyrosine downstream of the ITIM sequence, possibly capable of interacting with SH2 domains. Peptides used in the different studies are positioned in the boxed sections.
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Immunoblotting. Solubilized or immunoprecipitated proteins in Laemmli buffer, incubated 6 min at 100 C, were loaded on a 10% SDS–polyacrylamide gel, transferred to a nitrocellulose membrane, and immunoblotted with anti-PLCc1, anti-B2 receptor or anti-phosphotyrosine antibodies. Immunoreactive proteins were visualized by ECL immunodetection (Amersham) and quantified by densitometric analysis.
Results A peptide containing the phosphorylated rat B2 receptor ITIM sequence interacts with PLCc1 in vitro To verify the hypothesis that the bradykinin B2 receptor ITIM sequence can, besides with SHP-2, also interact with PLCc1 we performed surface plasmon resonance experiments using a synthetic peptide containing the B2 receptor ITIM sequence which is 100% conserved in human, mouse, rabbit, rat, and pig [8] (see Fig. 1 for a comparison between the rat and human B2 receptor). This sequence does not include the sequence used by Venema et al. [9], which also studied the interaction between the B2 receptor and PLCc1. Synthetic peptides containing the bradykinin B2 receptor ITIM sequence tyrosyl-phosphorylated or non-tyrosyl-phosphorylated were immobilized on a sensorchip. Recombinant PLCc1 (530–850 amino acids; containing two SH2 domains and one SH3 domain) was passed over the immobilized peptides, and the interactions analyzed in real-time. Using this technique, we observed a clear interaction between the phosphorylated ITIM peptide and recombinant PLCc1. In contrast, PLCc1 did not bind to the nonphoshorylated ITIM peptide (Fig. 2A). Binding of PLCc1 was dose-dependent (Fig. 2B) and the affinity constant calculated from sensorgrams showed a high affinity of PLCc1 for the phosphorylated ITIM peptide (KD = 1.3 nM; Kon = 6.88 · 105 M 1 s 1 and Koff = 8.94 · 10 4 s 1) which is four times higher than SHP-2 with the same ITIM sequence (KD = 5.5 nM) [8]. Thus, in vitro, recombinant PLCc1 is able to directly bind to the tyrosine-phosphorylated ITIM sequence of the bradykinin B2 receptor. Stimulation of the B2 receptor with bradykinin induces recruitment and activation of PLCc1 via the ITIM sequence in quiescent but not in proliferating cells To verify whether the interaction between PLCc1 and the B2 receptor exists in a cellular context, we performed immunoprecipitation experiments using an anti-PLCc1 antibody on cell lysates of quiescent (0% FCS) CHO cells transfected with the wild-type rat B2 receptor. This was followed by Western blotting analysis using an antiB2 receptor antibody on the immunoprecipitates. In untreated cells, no interaction was observed between PLCc1 and the bradykinin B2 receptor. However, stim-
Fig. 2. Surface plasmon resonance analysis of PLCc1 binding to B2 receptor ITIM peptides. (A) Interaction of PLCc1 (100 nM) with phosphorylated and non-phosphorylated B2 receptor ITIM peptide. (B) Dose-dependent (1–200 nM) binding of PLCc1 to phosphorylated B2 receptor ITIM peptide. RU, resonance units.
ulation with bradykinin induced a transient interaction at 2 min after bradykinin addition (Fig. 3A, left panel). This interaction was confirmed by immunoprecipitation using an anti-B2 receptor antibody and immunoblotting of the immunoprecipitate by anti-PLCc1 antibodies (data not shown). Stripping and reblotting of the membrane with anti-PLCc1 antibodies showed that the amount of immunoprecipitated PLCc1 was comparable between the samples (Fig. 3A, left panel). B2 receptor stimulation thus induces PLCc1 recruitment. To correlate the increased B2 receptor-PLCc1 binding observed after bradykinin treatment with an activation of PLCc1, tyrosine phosphorylation of PLCc1 was investigated. The level of PLCc1 tyrosine phosphorylation being a measure for PLCc1 activation [13]. After bradykinin treatment, CHO cells transfected with the wild-type rat B2 receptor were immunoprecipitated using PLCc1 antibodies followed by immunoblotting with phosphotyrosine antibodies. Thirty seconds after bradykinin addition, PLCc1 phosphorylation was observed (142 ± 14% tyrosine phosphorylated PLCc1 expressed as a percentage of basal phosphorylation, Fig. 3B, left panel, lane 2) this reaction peaked at 2 min
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Fig. 3. Bradykinin leads to recruitment and activation of PLCc1 and involves the ITIM sequence in quiescent cells only. (A,B) Quiescent or (C,D) proliferating cells were incubated with 100 nM bradykinin for the indicated time, lysates were immunoprecipitated (IP) with anti-PLCc1 antibodies and subjected to Western blotting (blot) with anti-B2 receptor antibodies (A,C) or with anti-phosphotyrosine antibodies (B,D). The same filters were stripped and reprobed with anti-PLCc1 antibodies. (E) PLCc1, SHP-2 and b-actin expression in whole cell lysates (30 lg) of proliferating (+) and non-proliferating ( ) CHO cells transfected with the wild-type B2 receptor. Blots are representative of at least three independent experiments and results are expressed as the means ± SE of three independent experiments. **Significantly different from control values (non-stimulated cells), p < 0.05, StudentÕs t test. WT, cells transfected with an expression plasmid containing the rat wild-type B2 receptor; Y307F, cells transfected with an expression plasmid containing the rat B2 receptor carrying the ITIM mutation Y307F. Tyr-phospho, tyrosine phosphorylation of PLCc1.
(194 ± 11% tyrosine phosphorylated PLCc1, lane 3) and then decreased at 10 min (168 ± 6% tyrosine phosphorylated PLCc1, lane 4). The same results were observed in immunoprecipitates obtained using phosphotyrosine antibodies followed by immunoblotting with PLCc1 antibodies (data not shown). To verify the involvement of the ITIM sequence in the interaction between the bradykinin B2 receptor and PLCc1 and also the effect of this ITIM-dependent protein–protein interaction on the activation PLCc1, the same experiments (as described above) were undertaken with the rat B2 receptor mutated in the key residue (tyrosine) of the ITIM sequence (Y307F) [8]. This mutation still results in a functional B2 receptor as measured by bradykinin-induced PGE2 release and MAPK activation [8]. This ITIM mutation abolished bradykinin-induced recruitment and subsequent activation of PLCc1 (Figs. 3A and B, right panel). Since we previously described an interaction of the B2 receptor ITIM sequence with tyrosine phosphatase SHP-2 under proliferating conditions [8], we also investigated the possibility of an interaction between this receptor and PLCc1 in proliferating cells. As observed in Figs. 3C and D, we were not able to detect an interaction between the B2 receptor and PLCc1 or PLCc1 activation under proliferating conditions (10% of SVF)
(in CHO cells transfected with the wild-type B2 receptor). This was not due to the absence of PLCc1 in these cells since expression as analyzed by Western blot of PLCc1 under these conditions was 5.9 higher than in quiescent cells while, for comparison, SHP-2 expression was similar between proliferating and quiescent cells (Fig. 3E). In conclusion, these experiments show that the bradykinin B2 receptor ITIM sequence is necessary for the protein–protein interaction between the B2 receptor and PLCc1, for PLCc1 activation and exists in quiescent cells only. The stimulated bradykinin B2 receptor interacts with and activates PLCc1 in quiescent primary culture rat mesangial cells To verify whether the interaction between PLCc1 and the B2 receptor was not caused by the artificial expression of the B2 receptor in CHO cells, we analyzed the existence of the interaction in primary culture quiescent rat renal mesangial cells. We observed co-immunoprecipitation of PLCc1 and the bradykinin B2 receptor in non stimulated quiescent (0.5% FCS) primary culture mesangial cells (Figs. 4A and B, lane 1). This interaction was further confirmed by immunoprecipitation using an
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anti-B2 receptor antibody and immunoblotting of the immunoprecipitate by anti-PLCc1 antibodies (data not shown). This basal interaction between the B2 receptor and PLCc1 was modified by bradykinin treatment which induced an increase in PLCc1–B2 receptor complex formation. This interaction reached a maximum 2 min after bradykinin addition (243 ± 17% interaction compared with basal interaction, Figs. 4A and B, lane 3), which returned to basal levels at 10 min after stimulation (103 ± 8%, Figs. 4A and B, lane 4). Stripping and reblotting of the membrane with anti-PLCc1 antibodies showed that the amount of immunoprecipitated PLCc1 was again comparable between the samples (Fig. 4A). The basal interaction between the B2 receptor and PLCc1 is thus dynamically modified by B2 receptor stimulation in quiescent primary culture mesangial cells. This bradykinin-induced recruitment of PLCc1 was accompanied by PLCc1 activation. Indeed, stimulation with bradykinin (after 30 s) increased levels of PLCc1 phosphorylation (124 ± 7% tyrosine phosphorylated PLCc1 compared to basal phosphorylation, Fig. 4C, lane 2), which reached a maximum at 2 min (148 ± 4% tyrosine phosphorylated PLCc1, lane 3) and then decreased at 10 min (102 ± 2% tyrosine phosphorylated PLCc1, lane 4). Stripping and reblotting of the membrane with anti-PLCc1 antibodies showed that the amount of immunoprecipitated PLCc1 was comparable between the samples (Fig. 4A). Similar results were observed with phosphotyrosine antibodies immunoprecipitates followed by immunoblotting with PLCc1 antibodies (data not shown). Taken together, these results clearly indicate an interaction between the bradykinin B2 receptor and PLCc1 in primary culture quiescent mesangial cells. Stimulation by bradykinin induces a rapid increase of the number of PLCc1 molecules recruited to the B2 receptor which in turn is accompanied by PLCc1 activation.
Discussion
Fig. 4. Bradykinin recruits and activates PLCc1 in primary culture mesangial cells. (A) Quiescent mesangial cells were incubated with 100 nM of bradykinin for the indicated time followed by immunoprecipitation (IP) of 500 lg of cell lysate with anti-PLCc1 antibodies. These immunoprecipitates were subjected to Western blotting (blot) with anti-B2 receptor antibodies (A,B) or anti-phosphotyrosine antibodies (C). The same filters were stripped and reprobed with anti-PLCc1 antibodies. (B,C) Immunoblots were analyzed by densitometric analysis. Data are expressed as the percentage of the interaction between the B2 receptor and PLCc1 (B), or as the percentage of the PLCc1 phosphorylation (C), observed in non-treated cells (t = 0). Results are the means ± SE of three independent experiments. **Significantly different from control values (basal), p < 0.05, StudentÕs t test. BK, bradykinin.
GPCRs are now known to interact directly with other proteins than G proteins [5–11]. The bradykinin B2 receptor belongs to the GPCR family and has shown to directly interact with endothelial- and neuronal-nitric oxide synthase [5,6], although the protein–protein interactions involved have not been fully investigated. Recently, our laboratory has demonstrated that the bradykinin B2 receptor can directly interact with tyrosine phosphatase SHP-2 [8]. This interaction is mediated through a protein–protein interaction between the SH2 domains (of SHP-2) and the ITIM sequence located on C-terminal tail of the bradykinin B2 receptor. In this current paper we demonstrate that the same ITIM sequence can also interact with another protein containing SH2 domains: PLCc1.
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This interaction has been previously demonstrated by Venema et al. The authors of this study showed that in vascular endothelial cells, PLCc1 associates with the B2 receptor [9]. However, in contrast with our results, this interaction appeared to be independent of the ITIM sequence. In the study of Venema et al., an in vitro binding assay with a GST-B2 receptor fusion protein containing the 55 C-terminal amino acids (residues 310–364) of the human B2 receptor which does not contain the ITIM sequence (Fig. 1) was used to show the interaction. PLCc1 contains two SH2 domains and one SH3 domain, which interact with phosphorylated tyrosine and proline-rich region (pPro), respectively. Residues 310–364 of the human B2 receptor contain only one tyrosine residue (Y320) and neither possess a pPro region or ITIM sequence. Therefore it is likely that the interaction observed in this study is mediated by tyrosine 320. Thus, although this study shows an interaction between B2 and PLCc1, this was mediated through a different tyrosine residue and no investigation of the ITIM sequence could be made. In addition, this study was conducted entirely in vitro and thus the implication of these findings in vivo (in a cellular context) is unknown. Moreover, investigation of the involvement of the ITIM and PLCc1 was warranted in light of its previously shown interaction with another SH2 domain containing protein (SHP-2, [8]). Indeed there is a definite protein–protein interaction between the tyrosine located in ITIM sequence and PLCc1; BIAcore binding experiments using a sequence from residues 300 to 314, which contains the ITIM of the rat B2 receptor (Fig. 1) with tyrosine Y307 (equivalent of tyrosine Y305 in human) but without tyrosine Y322 (equivalent of Y320 in human) showed distinct binding to PLCc1. This interaction could also be confirmed in cells. In line with our findings in vitro, cells expressing the wild-type rat B2 receptor showed an increase in PLCc1 recruitment and phosphorylation when stimulated with bradykinin. However, bradykinin had no effect on these factors in cells with an ITIM mutated B2 receptor. Using the whole receptor, our study suggests that both the tyrosine inside and outside the ITIM sequence (as shown by Venema et al.) participate in the recruitment of PLCc1. The respective contribution of these two tyrosines is still not known. A similar scenario exists between PLCc1 and the epidermal growth factor (EGF) receptor, which is also believed to have two binding sites for PLCc1. The N-terminal SH2 domain of PLCc1 is involved in the primary association while the C-terminal SH2 domain is necessary for maximal association [14]. It is possible that a similar relationship exists between B2 and PLCc1, but more experiments are necessary (to determine which tyrosine interacts with which SH2 domain). Using surface plasmon resonance, we found that the B2 receptor ITIM peptide bound PLCc1 with an affinity
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four times higher affinity than for SHP-2 [8]. As the bradykinin B2 receptor and thus the ITIM sequence used in both experiments is identical, the difference in affinity is likely to originate within the different SH2 domains of SHP-2 and PLCc1. Based on these affinities PLCc1 should be the protein preferentially recruited by the B2 receptor. However, this difference in in vitro affinity is not sufficient to determine the binding partner of the B2 receptor in vivo, since we observed that growth conditions play an important role in the recruitment of PLCc1 and SHP-2 as well. In quiescent cells, stimulation of the B2 receptor induces recruitment of PLCc1 while in proliferating cells, SHP-2 is the preferential binding partner despite of an significant increase in PLCc1 expression in proliferating cells. The origin of this growth condition dependent binding of the different partners remains to be elucidated. This growth condition dependence suggests that the recruitment of PLCc1 to the B2 receptor may have important implications in a pathophysiological context. Especially since our findings could be extrapolated to a primary cell line, of quiescent cultured mesangial cells, where it had identical effects to the former experiments (Fig. 3). The role of PLCc1 in renal physiology has been investigated using PLCc1 / chimeric mice (deficient mice are not viable). These mice show an alteration in renal development and hematopoiesis [15]. However, at present the role of bradykinin in the renal development seems limited. Bradykinin B2-deficient (B2 / ) mice are viable and the kidney seems to develop normally [16]. Nevertheless B2 / mice have a decreased renal nitric oxide excretion and a reduced glomerular tuft area [17]. A recent study on the involvement of the B2 receptor in diabetic nephropathy, which used Akita diabetic mice (a dominant mutation in the insulin 2 gene that leads to maturity onset diabetes) lacking the bradykinin B2 receptor, suggested that bradykinin can strongly reduce diabetic nephropathy and most particularly decrease glomerular mesangial sclerosis [18]. In the present study, we describe that the direct protein–protein interaction between PLCc1 and the bradykinin B2 receptor is found in the renal mesangial cell present in glomerulus. Furthermore, we previously described that the direct protein–protein interaction between SHP-2 and the bradykinin B2 receptor is involved in the antiproliferative effect of the bradykinin on mesangial cells [8]. Taken together, these results suggest that these two interactions may be important during renal development or in a pathological context. In conclusion, we show for the first time that an ITIM sequence situated in a GPCR can recruit two different proteins containing SH2 domains and that recruitment is growth condition dependant. A similar mechanism, without growth condition dependence, has been demonstrated for the angiotensin AT1 receptor which, via a YIPP sequence, can recruit both SHP-2
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[10] and PLCc1 [11]. It is interesting to point out that whilst the bradykinin B2 receptor does not possess a YIPP motif, the AT1 receptor possesses both. The implications of these have yet to be studied.
Acknowledgments J. Duchene received a grant from the Ministe`re de lÕEducation Nationale de la Recherche et de la Technologie. S.D. Chauhan was supported by an ‘‘Entente Cordiale’’ grant.
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Direct protein–protein interaction between PLCc1 and the bradykinin B2 receptor—Importance of growth conditions Johan Duchenea, Sharmila D. Chauhana, Fre´de´ric Lopezb, Christiane Pechera, Jean-Pierre Este`veb, Jean-Pierre Girolamia, Jean-Loup Bascandsa, Joost P. Schanstraa,* b
a Inserm U388, IFR 31, Hopital Rangueil, TSA 50032, 31059 Toulouse Cedex 9, France Functional Proteomics Facility, IFR 31, Hopital Rangueil, TSA 50032, 31059 Toulouse Cedex 9, France
Received 17 November 2004
Abstract Recently, we have described a novel protein–protein interaction between the G-protein coupled bradykinin B2 receptor and tyrosine phosphatase SHP-2 via an immunoreceptor tyrosine-based inhibition motif (ITIM) sequence located in the C-terminal part of the B2 receptor and the Src homology (SH2) domains of SHP-2. Here we show that phospholipase C (PLC)c1, another SH2 domain containing protein, can also interact with this ITIM sequence. Using surface plasmon resonance analysis, we observed that PLCc1 interacted with a peptide containing the phosphorylated form of the bradykinin B2 receptor ITIM sequence. In CHO cells expressing the wild-type B2 receptor, bradykinin-induced transient recruitment and activation of PLCc1. Interestingly, this interaction was only observed in quiescent and not in proliferating cells. Mutation of the key ITIM residue abolished this interaction with and activation of PLCc1. Finally we also identified bradykinin-induced PLCc1 recruitment and activation in primary culture renal mesangial cells. 2004 Elsevier Inc. All rights reserved. Keywords: G-protein coupled receptor; Bradykinin B2 receptor; Protein–protein interaction; Phospholipase Cc1; Tyrosine phosphatase SHP-2; Src homology 2 domain; Surface plasmon resonance; Mesangial cells; Quiescent and proliferating conditions
G-protein coupled receptor (GPCR) signaling pathways involve activation of heterotrimeric G proteins which can regulate a large panel of downstream effectors. Stimulation of GPCRs by ligands promotes the interaction between the third intracellular-loop of the receptor and the G protein. This induces the exchange of GDP by GTP on the G protein a subunit and the dissociation of the bc complex. Both a and bc subunits can initiate intracellular signaling by activation or inhibition of different effectors such as adenylate cyclase, phospholipases, ion channels or kinases. This GPCR signaling is now considered as ‘‘classical’’ GPCR signaling [1]. The bradykinin B2 receptor belongs to the GPCR superfam*
Corresponding author. Fax: +33 5 62 17 25 54. E-mail address: [email protected] (J.P. Schanstra).
0006-291X/$ - see front matter 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.11.126
ily and is coupled to a Gq/11 protein [2] which activates phospholipase beta (PLC-b) [3], protein kinase C (PKC) [4], and the mitogen-activated protein kinase (MAPK) cascade [4]. The bradykinin B2 receptor and other GPCRs have also been shown to induce ‘‘alternative’’ (G-independent) pathways. Indeed, such direct protein–protein interactions of intracellular proteins with GPCRs have been observed: (i) between endothelial nitric-oxide (NOS) synthase and the B2, angiotensin AT1 and endothelin receptors [5], (ii) between neuronal NOS and the B2 receptor [6], (iii) between c-Src and the b3-adrenergic receptor [7], and (iv) between SHP-2 and phospholipase Cc1 (PLCc1) with the B2 and AT1 receptors [8–11]. Although a direct interaction between an intracellular protein and a GPCR was found in all studies, few of them have
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identified the actual sequence motif involved in this interaction. It has been shown however, that proline rich motifs (PxxP) present in the third intracellular loop of the b3 adrenergic receptor allow the direct interaction with c-Src [7]. Another example is the YIPP motif in the C-terminal domain of the AT1 receptor which has been identified to directly interact with both PLCc1 and SHP-2 [10,11]. This single motif can bind two different downstream effector proteins (PLCc1 and SHP-2) when the key amino-acid (Y309) is phosphorylated. The bradykinin B2 receptor does not possess a YIPP motif, but recently we have described that SHP-2 (via its SH2 domain) can be recruited by the immunoreceptor tyrosine-based inhibitor motif (ITIM) of the B2 receptor [8]. This B2 receptor ITIM is located at the interface of the C-terminal part of the 7th transmembrane region and the C-terminal intracellular tail of the bradykinin B2 receptor and is 100% conserved in human, mouse, rabbit, rat, and pig. Under proliferating conditions, and upon bradykinin-treatment-induced phosphorylation of the key tyrosine residue (Y307) of the B2 receptor ITIM sequence, the bradykinin B2 receptor recruits, via a direct protein–protein interaction, tyrosine phosphatase SHP-2. This interaction is involved in the anti-mitogenic properties of the B2 receptor. In analogy with the AT1 receptor, it is possible that the B2 receptor ITIM sequence can recruit both SHP-2 and PLCc1. In agreement with this, Venema et al. [9], showed that in vascular endothelial cells expressing the human B2 receptor, PLCc1 could associate with the B2 receptor, but the involvement of the B2 receptor ITIM sequence was not studied. In this study the extreme C-terminal end of the receptor which does contain the ITIM sequence was used for the identification of regions of the B2 receptor involved in the interaction (see Fig. 1). We therefore explored the possibility that, in addition to recruiting SHP-2, the ITIM sequence also plays a role in PLCc1 recruitment. Using a combination of surface plasmon resonance, Western blot, and mutational analyses, we found in non-proliferating cells that PLCc1 interacts with the B2 receptor via the phosphorylated ITIM.
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Materials and methods Materials. Primary culture mesangial cells were obtained and cultivated in DMEM as previously described [12]. The culture media DMEM, aMEM, streptomycin, penicillin, and fetal calf serum (FCS) were from GIBCO. Bradykinin and chemical products were from Sigma. Monoclonal anti-PLCc1, anti-phosphotyrosine (PY20), SHP-2, and anti-B2 receptor antibodies were from Transduction Laboratories. The recombinant PLCc1 was from Santa Cruz. Surface plasmon resonance analysis. Surface plasmon resonance analysis of real time PLCc1 binding to B2 receptor ITIM peptides was performed on a BIAcore 3000 instrument (BIAcore AB). Synthetic biotinylated peptides containing the bradykinin B2 receptor ITIM sequence (SCLNPLVYVIVGKRF: ITIM residues in bold, which is 100% conserved among species) tyrosyl-phosphorylated or non-tyrosyl-phosphorylated were immobilized on separate channels of a streptavidin-coated carboxymethylated dextran chips (BIAcore AB) at 60 resonance units (RU). Different concentrations (1–200 nM) of purified recombinant PLCc1 (530–850 amino acids) were passed over the immobilized peptide surface at a flow rate of 30 ll/min at 25 C during 300 s in HBS buffer (10 mM Hepes, pH 7.4, 150 mM NaCl, 3 mM EDTA, and 0.005% polysorbate). After each binding assay, sensor chips were regenerated by two pulses of 5 ll of 0.01% SDS. The level of phosphotyrosine residues on phosphorylated peptides was verified using an anti-phosphotyrosine antibody (4G10, Upstate Biotechnology). The association and dissociation curves (sensorgrams) corresponding to the binding of PLCc1 to the ITIM peptides were used to determine association, dissociation, and affinity constants with the software BIAevaluation 3.1 (BIAcore AB). CHO cells expressing wild-type and ITIM mutated rat B2 receptor. As previously described [8], two different clonal cell lines overexpressing the rat wild-type and ITIM mutated B2 receptor (Y307F) were used. These cell lines express functional B2 receptors as previously shown [8]. Lysates. Semiconfluent cells were washed twice with phosphatebuffered saline and treated with bradykinin (100 nM in medium) or with medium only at 37 C. Cells were collected and lysed with RIPA buffer (10 mM Tris–HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 2 mM Na3VO4, 0.1% SDS, 1% NP40, 1% sodium deoxycholate, 0.36 lg/ ml PMSF, 0.01% SBTI, 0.01% leupeptin, and 0.01% aprotinin). The mixture was gently agitated for 30 min at 4 C and centrifuged at 15,000g during 15 min at 4 C followed by collection of the supernatant. Immunoprecipitation. Five hundred micrograms of soluble proteins were incubated with 1.5 lg of antibody for 30 min at 4 C. The mixture was then incubated 2 h with Sepharose–protein A beads prewashed with RIPA buffer. The mixture was then washed three times with RIPA buffer and resuspended in 32.5 ll of Laemmli buffer for immunoblotting.
Fig. 1. Comparison of the rat and human bradykinin B2 receptor and outline of peptides used in this study and the study of Venema et al. [9]. Both the human and rat B2 receptor contain an ITIM sequence (in bold) which is 100% conserved among species. Both the rat (Y320) and human (Y322) possess an additional tyrosine downstream of the ITIM sequence, possibly capable of interacting with SH2 domains. Peptides used in the different studies are positioned in the boxed sections.
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Immunoblotting. Solubilized or immunoprecipitated proteins in Laemmli buffer, incubated 6 min at 100 C, were loaded on a 10% SDS–polyacrylamide gel, transferred to a nitrocellulose membrane, and immunoblotted with anti-PLCc1, anti-B2 receptor or anti-phosphotyrosine antibodies. Immunoreactive proteins were visualized by ECL immunodetection (Amersham) and quantified by densitometric analysis.
Results A peptide containing the phosphorylated rat B2 receptor ITIM sequence interacts with PLCc1 in vitro To verify the hypothesis that the bradykinin B2 receptor ITIM sequence can, besides with SHP-2, also interact with PLCc1 we performed surface plasmon resonance experiments using a synthetic peptide containing the B2 receptor ITIM sequence which is 100% conserved in human, mouse, rabbit, rat, and pig [8] (see Fig. 1 for a comparison between the rat and human B2 receptor). This sequence does not include the sequence used by Venema et al. [9], which also studied the interaction between the B2 receptor and PLCc1. Synthetic peptides containing the bradykinin B2 receptor ITIM sequence tyrosyl-phosphorylated or non-tyrosyl-phosphorylated were immobilized on a sensorchip. Recombinant PLCc1 (530–850 amino acids; containing two SH2 domains and one SH3 domain) was passed over the immobilized peptides, and the interactions analyzed in real-time. Using this technique, we observed a clear interaction between the phosphorylated ITIM peptide and recombinant PLCc1. In contrast, PLCc1 did not bind to the nonphoshorylated ITIM peptide (Fig. 2A). Binding of PLCc1 was dose-dependent (Fig. 2B) and the affinity constant calculated from sensorgrams showed a high affinity of PLCc1 for the phosphorylated ITIM peptide (KD = 1.3 nM; Kon = 6.88 · 105 M 1 s 1 and Koff = 8.94 · 10 4 s 1) which is four times higher than SHP-2 with the same ITIM sequence (KD = 5.5 nM) [8]. Thus, in vitro, recombinant PLCc1 is able to directly bind to the tyrosine-phosphorylated ITIM sequence of the bradykinin B2 receptor. Stimulation of the B2 receptor with bradykinin induces recruitment and activation of PLCc1 via the ITIM sequence in quiescent but not in proliferating cells To verify whether the interaction between PLCc1 and the B2 receptor exists in a cellular context, we performed immunoprecipitation experiments using an anti-PLCc1 antibody on cell lysates of quiescent (0% FCS) CHO cells transfected with the wild-type rat B2 receptor. This was followed by Western blotting analysis using an antiB2 receptor antibody on the immunoprecipitates. In untreated cells, no interaction was observed between PLCc1 and the bradykinin B2 receptor. However, stim-
Fig. 2. Surface plasmon resonance analysis of PLCc1 binding to B2 receptor ITIM peptides. (A) Interaction of PLCc1 (100 nM) with phosphorylated and non-phosphorylated B2 receptor ITIM peptide. (B) Dose-dependent (1–200 nM) binding of PLCc1 to phosphorylated B2 receptor ITIM peptide. RU, resonance units.
ulation with bradykinin induced a transient interaction at 2 min after bradykinin addition (Fig. 3A, left panel). This interaction was confirmed by immunoprecipitation using an anti-B2 receptor antibody and immunoblotting of the immunoprecipitate by anti-PLCc1 antibodies (data not shown). Stripping and reblotting of the membrane with anti-PLCc1 antibodies showed that the amount of immunoprecipitated PLCc1 was comparable between the samples (Fig. 3A, left panel). B2 receptor stimulation thus induces PLCc1 recruitment. To correlate the increased B2 receptor-PLCc1 binding observed after bradykinin treatment with an activation of PLCc1, tyrosine phosphorylation of PLCc1 was investigated. The level of PLCc1 tyrosine phosphorylation being a measure for PLCc1 activation [13]. After bradykinin treatment, CHO cells transfected with the wild-type rat B2 receptor were immunoprecipitated using PLCc1 antibodies followed by immunoblotting with phosphotyrosine antibodies. Thirty seconds after bradykinin addition, PLCc1 phosphorylation was observed (142 ± 14% tyrosine phosphorylated PLCc1 expressed as a percentage of basal phosphorylation, Fig. 3B, left panel, lane 2) this reaction peaked at 2 min
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Fig. 3. Bradykinin leads to recruitment and activation of PLCc1 and involves the ITIM sequence in quiescent cells only. (A,B) Quiescent or (C,D) proliferating cells were incubated with 100 nM bradykinin for the indicated time, lysates were immunoprecipitated (IP) with anti-PLCc1 antibodies and subjected to Western blotting (blot) with anti-B2 receptor antibodies (A,C) or with anti-phosphotyrosine antibodies (B,D). The same filters were stripped and reprobed with anti-PLCc1 antibodies. (E) PLCc1, SHP-2 and b-actin expression in whole cell lysates (30 lg) of proliferating (+) and non-proliferating ( ) CHO cells transfected with the wild-type B2 receptor. Blots are representative of at least three independent experiments and results are expressed as the means ± SE of three independent experiments. **Significantly different from control values (non-stimulated cells), p < 0.05, StudentÕs t test. WT, cells transfected with an expression plasmid containing the rat wild-type B2 receptor; Y307F, cells transfected with an expression plasmid containing the rat B2 receptor carrying the ITIM mutation Y307F. Tyr-phospho, tyrosine phosphorylation of PLCc1.
(194 ± 11% tyrosine phosphorylated PLCc1, lane 3) and then decreased at 10 min (168 ± 6% tyrosine phosphorylated PLCc1, lane 4). The same results were observed in immunoprecipitates obtained using phosphotyrosine antibodies followed by immunoblotting with PLCc1 antibodies (data not shown). To verify the involvement of the ITIM sequence in the interaction between the bradykinin B2 receptor and PLCc1 and also the effect of this ITIM-dependent protein–protein interaction on the activation PLCc1, the same experiments (as described above) were undertaken with the rat B2 receptor mutated in the key residue (tyrosine) of the ITIM sequence (Y307F) [8]. This mutation still results in a functional B2 receptor as measured by bradykinin-induced PGE2 release and MAPK activation [8]. This ITIM mutation abolished bradykinin-induced recruitment and subsequent activation of PLCc1 (Figs. 3A and B, right panel). Since we previously described an interaction of the B2 receptor ITIM sequence with tyrosine phosphatase SHP-2 under proliferating conditions [8], we also investigated the possibility of an interaction between this receptor and PLCc1 in proliferating cells. As observed in Figs. 3C and D, we were not able to detect an interaction between the B2 receptor and PLCc1 or PLCc1 activation under proliferating conditions (10% of SVF)
(in CHO cells transfected with the wild-type B2 receptor). This was not due to the absence of PLCc1 in these cells since expression as analyzed by Western blot of PLCc1 under these conditions was 5.9 higher than in quiescent cells while, for comparison, SHP-2 expression was similar between proliferating and quiescent cells (Fig. 3E). In conclusion, these experiments show that the bradykinin B2 receptor ITIM sequence is necessary for the protein–protein interaction between the B2 receptor and PLCc1, for PLCc1 activation and exists in quiescent cells only. The stimulated bradykinin B2 receptor interacts with and activates PLCc1 in quiescent primary culture rat mesangial cells To verify whether the interaction between PLCc1 and the B2 receptor was not caused by the artificial expression of the B2 receptor in CHO cells, we analyzed the existence of the interaction in primary culture quiescent rat renal mesangial cells. We observed co-immunoprecipitation of PLCc1 and the bradykinin B2 receptor in non stimulated quiescent (0.5% FCS) primary culture mesangial cells (Figs. 4A and B, lane 1). This interaction was further confirmed by immunoprecipitation using an
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anti-B2 receptor antibody and immunoblotting of the immunoprecipitate by anti-PLCc1 antibodies (data not shown). This basal interaction between the B2 receptor and PLCc1 was modified by bradykinin treatment which induced an increase in PLCc1–B2 receptor complex formation. This interaction reached a maximum 2 min after bradykinin addition (243 ± 17% interaction compared with basal interaction, Figs. 4A and B, lane 3), which returned to basal levels at 10 min after stimulation (103 ± 8%, Figs. 4A and B, lane 4). Stripping and reblotting of the membrane with anti-PLCc1 antibodies showed that the amount of immunoprecipitated PLCc1 was again comparable between the samples (Fig. 4A). The basal interaction between the B2 receptor and PLCc1 is thus dynamically modified by B2 receptor stimulation in quiescent primary culture mesangial cells. This bradykinin-induced recruitment of PLCc1 was accompanied by PLCc1 activation. Indeed, stimulation with bradykinin (after 30 s) increased levels of PLCc1 phosphorylation (124 ± 7% tyrosine phosphorylated PLCc1 compared to basal phosphorylation, Fig. 4C, lane 2), which reached a maximum at 2 min (148 ± 4% tyrosine phosphorylated PLCc1, lane 3) and then decreased at 10 min (102 ± 2% tyrosine phosphorylated PLCc1, lane 4). Stripping and reblotting of the membrane with anti-PLCc1 antibodies showed that the amount of immunoprecipitated PLCc1 was comparable between the samples (Fig. 4A). Similar results were observed with phosphotyrosine antibodies immunoprecipitates followed by immunoblotting with PLCc1 antibodies (data not shown). Taken together, these results clearly indicate an interaction between the bradykinin B2 receptor and PLCc1 in primary culture quiescent mesangial cells. Stimulation by bradykinin induces a rapid increase of the number of PLCc1 molecules recruited to the B2 receptor which in turn is accompanied by PLCc1 activation.
Discussion
Fig. 4. Bradykinin recruits and activates PLCc1 in primary culture mesangial cells. (A) Quiescent mesangial cells were incubated with 100 nM of bradykinin for the indicated time followed by immunoprecipitation (IP) of 500 lg of cell lysate with anti-PLCc1 antibodies. These immunoprecipitates were subjected to Western blotting (blot) with anti-B2 receptor antibodies (A,B) or anti-phosphotyrosine antibodies (C). The same filters were stripped and reprobed with anti-PLCc1 antibodies. (B,C) Immunoblots were analyzed by densitometric analysis. Data are expressed as the percentage of the interaction between the B2 receptor and PLCc1 (B), or as the percentage of the PLCc1 phosphorylation (C), observed in non-treated cells (t = 0). Results are the means ± SE of three independent experiments. **Significantly different from control values (basal), p < 0.05, StudentÕs t test. BK, bradykinin.
GPCRs are now known to interact directly with other proteins than G proteins [5–11]. The bradykinin B2 receptor belongs to the GPCR family and has shown to directly interact with endothelial- and neuronal-nitric oxide synthase [5,6], although the protein–protein interactions involved have not been fully investigated. Recently, our laboratory has demonstrated that the bradykinin B2 receptor can directly interact with tyrosine phosphatase SHP-2 [8]. This interaction is mediated through a protein–protein interaction between the SH2 domains (of SHP-2) and the ITIM sequence located on C-terminal tail of the bradykinin B2 receptor. In this current paper we demonstrate that the same ITIM sequence can also interact with another protein containing SH2 domains: PLCc1.
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This interaction has been previously demonstrated by Venema et al. The authors of this study showed that in vascular endothelial cells, PLCc1 associates with the B2 receptor [9]. However, in contrast with our results, this interaction appeared to be independent of the ITIM sequence. In the study of Venema et al., an in vitro binding assay with a GST-B2 receptor fusion protein containing the 55 C-terminal amino acids (residues 310–364) of the human B2 receptor which does not contain the ITIM sequence (Fig. 1) was used to show the interaction. PLCc1 contains two SH2 domains and one SH3 domain, which interact with phosphorylated tyrosine and proline-rich region (pPro), respectively. Residues 310–364 of the human B2 receptor contain only one tyrosine residue (Y320) and neither possess a pPro region or ITIM sequence. Therefore it is likely that the interaction observed in this study is mediated by tyrosine 320. Thus, although this study shows an interaction between B2 and PLCc1, this was mediated through a different tyrosine residue and no investigation of the ITIM sequence could be made. In addition, this study was conducted entirely in vitro and thus the implication of these findings in vivo (in a cellular context) is unknown. Moreover, investigation of the involvement of the ITIM and PLCc1 was warranted in light of its previously shown interaction with another SH2 domain containing protein (SHP-2, [8]). Indeed there is a definite protein–protein interaction between the tyrosine located in ITIM sequence and PLCc1; BIAcore binding experiments using a sequence from residues 300 to 314, which contains the ITIM of the rat B2 receptor (Fig. 1) with tyrosine Y307 (equivalent of tyrosine Y305 in human) but without tyrosine Y322 (equivalent of Y320 in human) showed distinct binding to PLCc1. This interaction could also be confirmed in cells. In line with our findings in vitro, cells expressing the wild-type rat B2 receptor showed an increase in PLCc1 recruitment and phosphorylation when stimulated with bradykinin. However, bradykinin had no effect on these factors in cells with an ITIM mutated B2 receptor. Using the whole receptor, our study suggests that both the tyrosine inside and outside the ITIM sequence (as shown by Venema et al.) participate in the recruitment of PLCc1. The respective contribution of these two tyrosines is still not known. A similar scenario exists between PLCc1 and the epidermal growth factor (EGF) receptor, which is also believed to have two binding sites for PLCc1. The N-terminal SH2 domain of PLCc1 is involved in the primary association while the C-terminal SH2 domain is necessary for maximal association [14]. It is possible that a similar relationship exists between B2 and PLCc1, but more experiments are necessary (to determine which tyrosine interacts with which SH2 domain). Using surface plasmon resonance, we found that the B2 receptor ITIM peptide bound PLCc1 with an affinity
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four times higher affinity than for SHP-2 [8]. As the bradykinin B2 receptor and thus the ITIM sequence used in both experiments is identical, the difference in affinity is likely to originate within the different SH2 domains of SHP-2 and PLCc1. Based on these affinities PLCc1 should be the protein preferentially recruited by the B2 receptor. However, this difference in in vitro affinity is not sufficient to determine the binding partner of the B2 receptor in vivo, since we observed that growth conditions play an important role in the recruitment of PLCc1 and SHP-2 as well. In quiescent cells, stimulation of the B2 receptor induces recruitment of PLCc1 while in proliferating cells, SHP-2 is the preferential binding partner despite of an significant increase in PLCc1 expression in proliferating cells. The origin of this growth condition dependent binding of the different partners remains to be elucidated. This growth condition dependence suggests that the recruitment of PLCc1 to the B2 receptor may have important implications in a pathophysiological context. Especially since our findings could be extrapolated to a primary cell line, of quiescent cultured mesangial cells, where it had identical effects to the former experiments (Fig. 3). The role of PLCc1 in renal physiology has been investigated using PLCc1 / chimeric mice (deficient mice are not viable). These mice show an alteration in renal development and hematopoiesis [15]. However, at present the role of bradykinin in the renal development seems limited. Bradykinin B2-deficient (B2 / ) mice are viable and the kidney seems to develop normally [16]. Nevertheless B2 / mice have a decreased renal nitric oxide excretion and a reduced glomerular tuft area [17]. A recent study on the involvement of the B2 receptor in diabetic nephropathy, which used Akita diabetic mice (a dominant mutation in the insulin 2 gene that leads to maturity onset diabetes) lacking the bradykinin B2 receptor, suggested that bradykinin can strongly reduce diabetic nephropathy and most particularly decrease glomerular mesangial sclerosis [18]. In the present study, we describe that the direct protein–protein interaction between PLCc1 and the bradykinin B2 receptor is found in the renal mesangial cell present in glomerulus. Furthermore, we previously described that the direct protein–protein interaction between SHP-2 and the bradykinin B2 receptor is involved in the antiproliferative effect of the bradykinin on mesangial cells [8]. Taken together, these results suggest that these two interactions may be important during renal development or in a pathological context. In conclusion, we show for the first time that an ITIM sequence situated in a GPCR can recruit two different proteins containing SH2 domains and that recruitment is growth condition dependant. A similar mechanism, without growth condition dependence, has been demonstrated for the angiotensin AT1 receptor which, via a YIPP sequence, can recruit both SHP-2
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[10] and PLCc1 [11]. It is interesting to point out that whilst the bradykinin B2 receptor does not possess a YIPP motif, the AT1 receptor possesses both. The implications of these have yet to be studied.
Acknowledgments J. Duchene received a grant from the Ministe`re de lÕEducation Nationale de la Recherche et de la Technologie. S.D. Chauhan was supported by an ‘‘Entente Cordiale’’ grant.
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