More interestingly, whereas inhibition of IGFR by AG1024 completely abolished ERK1/2 activation in response to AVP (Fig. activation, indicating that a pool of -arrestins distinct from those -arrestins recruited to the V2R acts downstream of the receptor tyrosine kinase to activate ERK1/2. Such a dual site of action for -arrestins helps explain the pleiotropic actions of this scaffolding protein. Given the role that V2R-stimulated ERK1/2 plays in kidney cell proliferation, this transactivation mechanism may have important implications for renal pathophysiology. Still, the role of -arrestins downstream of a transactivation event is not limited to the V2R, because we observed a similar involvement for an unrelated GPCR (the platelet-activating factor receptor), indicating that it may be a general mechanism shared among GPCRs. using the antiCIGF-1R 1H7 antibody followed by Western blot (WB) detection of IGF-1R tyrosine autophosphorylation with antiCIGF-1R pY1131 antibody. Total IGF-1R population was detected with an antiCIGF-1R. Typical immunoblots representative of three independent experiments are shown. ** 0.01; *** 0.001. To further explore the mechanism linking V2R stimulation to IGFR transactivation, the potential role of metalloproteinases was investigated. As shown in Fig. 2 0.01; *** 0.001. Metalloproteinase-Dependent Transactivation of IGFR Requires Src Activity. Given that Src has been shown to promote metalloproteinase-dependent MAPK activation by several GPCRs (29) but is also known to regulate ERK1/2 activity in response to many growth factor receptor ligands (30, 31), notably the IGFR (32, 33), we examined the role of Src in the signaling cascade uncovered in our study. To determine whether Src is involved in the transactivation of IGFR (acting upstream) or contributes to the activation of ERK1/2 downstream of IGFR, we took advantage of a phosphospecific antibody recognizing the activated form of Src phosphorylated at tyrosine 416. Fig. S3 shows that AVP can promote a transient time-dependent increase in Src phosphorylation at tyrosine 416 that peaked at 5 min. Consistent with our previous finding that V2R-promoted ERK1/2 activation is Gs-independent (3), down-regulating Gs with long-term cholera toxin treatment did not inhibit the AVP-stimulated Src phosphorylation (Fig. S4). More interestingly, whereas inhibition of IGFR by AG1024 completely abolished ERK1/2 activation in response to AVP (Fig. 3and 0.05; ** 0.01; *** 0.001. -Arrestins Are Required Downstream of IGFR Transactivation for V2R-Mediated ERK1/2 Stimulation. -Arrestins have been shown to play a role in ERK1/2 Ticagrelor (AZD6140) activation in response to the stimulation of many GPCRs (34), including V2R (3). To explore the role of -arrestins in the IGFR transactivation mechanism, we used a C-tail truncated form of -arrestins-1 (arr 318C419), which has been shown to act as a dominant negative (3, 35). Coexpression of this -arrestin dominant negative mutant with V2R significantly blunted ERK1/2 activation in response to AVP stimulation (Fig. 4 0.05; ** 0.01; *** 0.001. -Arrestin Action Downstream of GPCR-Promoted Transactivation Ticagrelor (AZD6140) Is Not Limited to V2R. To assess whether a -arrestin action downstream of a transactivation event could also be involved in the ERK1/2 activation by another GPCR, a supernatant transfer assay was performed using donor cells expressing the platelet-activating factor receptor (PAFR). As shown in Fig. 5 and and 0.05; ** 0.01. V2R Activation Can Promote -Arrestin-1 Association to the Transactivated IGFR. Given that V2R-null HEK293 cells do not express any endogenous V2R (Fig. 2and Fig. S2), the role of -arrestins downstream of IGFR transactivation in these cells after supernatant transfer implicates the existence of a distinct stimulatory signal triggering -arrestin engagement. The recently appreciated ability of some RTK to recruit -arrestins in response to their cognate ligands (21) makes IGFR an attractive candidate as the membrane receptor initiating -arrestin translocation and activation. Indeed,.ERK phosphorylation was normalized according to the loading of proteins by expressing the data as a percentage of P-ERK over total ERK (or P-ERK2-GFP over total ERK2-GFP) of the level observed in the agonist-stimulated condition. IGFR transactivation. Unexpectedly, the engagement of -arrestins by the IGFR but not by the V2R was needed to promote the vasopressin-stimulated ERK1/2 activation, indicating that a pool of -arrestins distinct from those -arrestins recruited to the V2R acts downstream of the receptor tyrosine kinase to activate ERK1/2. Such a dual site of action for -arrestins helps explain the pleiotropic actions Ticagrelor (AZD6140) of this scaffolding protein. Given the role that V2R-stimulated ERK1/2 plays in kidney cell proliferation, this transactivation mechanism may have important implications for renal pathophysiology. Still, the role of -arrestins downstream of a transactivation event is not limited to the V2R, because we observed a similar involvement for an unrelated GPCR (the platelet-activating factor receptor), indicating that it may be a general mechanism shared among GPCRs. using the antiCIGF-1R 1H7 antibody followed by Western blot (WB) detection of IGF-1R tyrosine autophosphorylation with antiCIGF-1R pY1131 antibody. Total IGF-1R population was detected with an antiCIGF-1R. Typical immunoblots representative of three independent experiments are shown. ** 0.01; *** 0.001. To further explore the mechanism linking V2R stimulation to IGFR transactivation, the potential role of metalloproteinases was investigated. As demonstrated in Fig. 2 0.01; *** 0.001. Metalloproteinase-Dependent Transactivation of IGFR Requires Src Activity. Given that Src offers been shown to promote metalloproteinase-dependent MAPK activation by several GPCRs (29) but is also known to regulate ERK1/2 activity in response to many growth element receptor ligands (30, 31), notably the IGFR (32, 33), we examined the part of Src in the signaling cascade uncovered in our study. To determine whether Src is definitely involved in the transactivation of IGFR (acting upstream) or contributes to the activation of ERK1/2 downstream of IGFR, we required advantage of a phosphospecific antibody realizing the activated form of Src phosphorylated at tyrosine 416. Fig. S3 demonstrates AVP can promote a transient time-dependent increase in Src phosphorylation at tyrosine 416 that peaked at 5 min. Consistent with our earlier finding that V2R-promoted ERK1/2 activation is definitely Gs-independent (3), down-regulating Gs with long-term cholera toxin treatment did not inhibit the AVP-stimulated Src phosphorylation (Fig. S4). More interestingly, whereas inhibition of IGFR by AG1024 completely abolished ERK1/2 activation in response to AVP (Fig. 3and 0.05; ** 0.01; *** 0.001. -Arrestins Are Needed Downstream of IGFR Transactivation for V2R-Mediated ERK1/2 Stimulation. -Arrestins have been shown to play a role in ERK1/2 activation in response to the stimulation of many GPCRs (34), including V2R (3). To explore the part of -arrestins in the IGFR transactivation mechanism, we used a C-tail truncated form of -arrestins-1 (arr 318C419), which has been shown to act as a dominating bad (3, 35). Coexpression of this -arrestin dominating bad mutant with V2R significantly blunted ERK1/2 activation in response to AVP activation (Fig. 4 0.05; ** 0.01; *** 0.001. -Arrestin Action Downstream of GPCR-Promoted Transactivation Is Not Limited to V2R. To assess whether a -arrestin action downstream of a transactivation event could also be involved in the ERK1/2 activation by another GPCR, a supernatant transfer assay was performed using donor cells expressing the platelet-activating element receptor (PAFR). As demonstrated in Fig. 5 and and 0.05; ** 0.01. V2R Activation Can Promote -Arrestin-1 Association to the Transactivated IGFR. Given that V2R-null HEK293 cells do not communicate any endogenous V2R (Fig. 2and Fig. S2), the part of -arrestins downstream of IGFR transactivation in these cells after supernatant transfer implicates the living of a distinct stimulatory signal triggering -arrestin engagement. The recently appreciated ability of some RTK to recruit -arrestins in response to their cognate ligands (21) makes IGFR a good Rabbit Polyclonal to HOXA11/D11 candidate as the membrane receptor initiating -arrestin translocation and activation. Indeed, IGF1 offers been shown to promote -arrestin-1 translocation to the IGFR (20, 23, 36). We therefore hypothesized that IGFR could possibly preserve this signaling capacity in the context of a transactivation event when stimulated by a GPCR ligand. Assisting this hypothesis, coimmunoprecipitation experiments exposed that AVP Ticagrelor (AZD6140) as well as IGF1 can promote -arrestin-1 association to the endogenously indicated IGFR (Fig. 6 and and and and and and 0.001. Conversation Our results lead us to propose a unique model for the activation of ERK1/2 by a GPCR. This model entails the.