Neuropeptide Y receptor mediates activation of ERK1/2 via transactivation of the IGF receptor

Sandra Lecat a,⁎, Lazare Belemnaba b, Jean-Luc Galzi a, Bernard Bucher b

Abstract

Human embryonic kidney IGFR β-arrestin since mutant Y1 receptors unable to recruit β-arrestins, can still activate ERK signaling to the same extent as wild-type receptors. We next show that this activation of the MAPK pathway is inhibited by the MEK inhibitor U0126, is not dependent on calcium signaling at the Y1 receptor (no effect upon inhibition of phospholipase C, protein kinase C or protein kinase D) but instead dependent on Gβ/γ and associated signaling pathways that activate PI3-kinase. Although inhibition of the epidermal-growth factor receptor tyrosine kinase did not influence NPY-induced ERK1/2 activation, we show that the inhibition of insulin growth factor receptor IGFR by AG1024 completely blocks activation of ERK1/2 by the Y1 receptor. This Gβ/γ-PI3K-AG1024-sensitive pathway does not involve activation of IGFR through the release of a soluble ligand by metalloproteinases since it is not affected by the metalloproteinase inhibitor marimastat. Finally, we found that a similar pathway, sensitive to wortmannin-AG1024 but insensitive to marimastat, is implicated in activation of ERK signaling in HEK293 cells by endogenously expressed GPCRs coupled to Gq-protein (muscarinic M3 receptors) or coupled to Gs-protein (endothelin ETB receptors). Our analysis is the first to show that β-arrestin recruitment to the NPY Y1 receptor is not necessary for MAPK activation by this receptor but that transactivation of the IGFR receptor is required.
Neuropeptide Y binds to G-protein coupled receptors whose action results in inhibition of adenylyl cyclase activity. Using HEK293 cells stably expressing the native neuropeptide Y Y1 receptors, we found that the NPY agonist elicits a transient phosphorylation of the extracellular signal-regulated kinases (ERK1/2). We first show that ERK1/2 activation following Y1 receptor stimulation is dependent on heterotrimeric Gi/o since it is completely inhibited by pre-treatment with pertussis toxin. In addition, ERK1/2 activation is internalization-independent

Keywords:
Neuropeptide Y
G protein-coupled receptor
Extracellular-regulated protein kinase

1. Introduction

Neuropeptide Y (NPY) is a widely distributed peptide in the central and peripheral nervous system [1]. The physiological functions of this neurohormone are mediated by a family of five receptors which belong to the class A of the G-protein coupled receptors (GPCRs) and which are expressed in neuronal and non-neuronal tissues. Classically, signaling of GPCRs is mediated by receptor coupling to heterotrimeric G proteins that activate a variety of downstream cellular effectors. The signaling pathway of the NPY family of receptors is known to be coupled to the Gαi-protein, which leads to the inhibition of the adenylyl cyclase and inhibition of cAMP accumulation [2].
The classical paradigm for signaling via inhibition of adenylyl cyclase does not always adequately explain the full range of the effects of the activation of the NPY family receptors. Recent studies have shown that activation of the NPY receptors leads to cell proliferation, neurogenesis or gene transcription, via the phosphorylation of the extracellular signal-regulated protein kinases 1 and 2 [3–5]. GPCRs can mediate ERK1/2 activation by different mechanisms, being able to induce activation of both G protein-dependent and G protein-independent signaling pathways.
Hence, growth-promoting effects of GPCR stimulation have been shown to be mediated through transactivation of growth factor receptor tyrosine kinases (RTKs for the receptors of epidermal growth factor (EGF), insulin growth factor (IGF), platelet-derived growth factor PDGF, fibroblast growth factor FGF, vascular endothelial growth factor VEGF and TrkA) [6]. Different mechanisms of transactivation have been described. These include the extracellular release of RTK ligand by secretion or by shedding of a ligand through the processing of preligand by plasma membrane metalloproteinases. Transactivation can also occur inside the cell, for example, activation of the c-Src-kinase is a generally described mechanism. Alternatively, it has been proposed to occur via direct molecular interactions between RTK and GPCRs or via inactivation of protein-tyrosine phosphatases that repress RTK [6–9]. However, transactivation is not an essential requirement in the activation of the signaling cascade induced by GPCR activation [10,11].
During the past decade, evidence has emerged showing that a variety of GPCRs can also couple to other adaptor proteins independently of heterotrimeric G-protein [12,13]. Among these transducers are the scaffolding proteins, β-arrestin-1, and β-arrestin-2, which are cytosolic endocytic adaptors playing an important role in GPCR desensitization, internalization, and trafficking. Upon GPCR activation, they can also function as molecular mediators especially by acting on the ERK1/2 signal transduction pathway [14]. It is increasingly evident that β-arrestins can bind to MAP kinases and facilitate their activation [12].
In previous studies, we have shown that, unlike NPY Y2 receptors, NPY Y1 receptors rapidly internalize through clathrin-coated pits and recycle [15,16]. Upon stimulation by NPY, the endocytic mechanism involves activation of cytosolic β-arrestins and translocation to the plasma membrane. Deletion of the carboxyl-terminal tail of the receptor (Y1 Δ42 receptor) or substitution of key putative phosphorylated residues by alanine in the carboxyl-terminal tail of the receptor (Y1 TDST-A receptor) abolishes both internalization and β-arrestin activation. Likewise, the human NPY Y2 receptor does not internalize in our system and no significant activation of β-arrestin activation is detected [16].
Therefore, the present study was undertaken in order to better understand the involvement of G-protein- and/or β-arrestins-mediated signaling in the ERK1/2 phosphorylation pathway during activation of the NPY receptors. Different aspects of NPY-induced ERK1/2 activation were evaluated by the use of mutant receptors that do not recruit β-arrestins, and using specific inhibitors in order to sequentially investigate the downstream signaling mechanisms upon activation of the receptor.

2. Materials and methods

2.1. Cell culture

Control and transfected HEK293 cells were cultured to ~80% confluence in T-75 flasks in MEM with Earle’s salt supplemented with 10% fetal calf serum, 2 mM glutamine and 1% antibiotics (penicillin/ streptomycin) [15]. For activation of the cells, transfected HEK293 cells with the respective receptors were seeded in 6-well plates (200,000–300,000 cells/well) in order to attain 70–80% confluence after 2 days of culture. Cells were then serum starved overnight (roughly 16H) before activation with NPY with or without different inhibitors, for different times as described in the individual experiments. The stimulation was terminated by replacing on ice the culture medium by 250 μl of cold RIPA lysis buffer (50 mM Tris–HCl pH 7.4, 150 mM NaCl, 1% NP40, 0.25% Na-deoxycholate) supplemented with EDTA-free anti-protease and anti-phosphatase cocktails.
cDNA of the human NPY Y1 receptor was kindly provided by Prof. H. Herzog (Garvan Institute, Sydney, Australia). The cloning schemes for the different mutants of the native Y1 receptor used in the present study were previously described [16,17]. All constructions were verified by sequencing and stably transfected cells were established through selective antibiotic selection.

2.2. SDS page and western blot analysis

Cell lysates were sonicated three times on ice using a Branson Sonifier at constant power, output = 2.5, and continuous sonication for 1 s. After sonication, insoluble elements were cleared by centrifugation at maximum speed (~14 k) for 5 min at 4 °C. Protein concentration was determined using Bradford reagent. Homogenates of cell lysate were mixed with 4× Laemmli buffer, denatured 5 min at 95 °C, and loaded on a 12% SDS-PAGE gel (20 μg/lane). Protein samples were transferred to immobilon PVDF membranes. Membranes were blocked 1 h (using 5% non-fat dried milk in PBS, pH 7.2, Tween 0.1%), probed overnight with rabbit (polyclonal) anti-ERK1/2 [pTpY185/187] phospho-specific antibody (1:5000, Invitrogen, Camarillo, CA). The strips were thoroughly washed with PBS, Tween 0.1% then incubated 40 min with secondary antibody horseradish peroxidase-conjugated donkey anti-rabbit diluted at 1/10,000. The immune-reactive bands were visualized using Immobilon western chemoluminescent HRP substrate detection reagents (Merck-Millipore) either with hyperfilm or LAS 4000 camera, and bands were quantified using respectively either ImageJ or ImageQuant software (GE-Healthcare). The data were normalized to the maximal phospho-ERK1/2 response for each set of experiments. The statistical significance of the differences between agonist stimulated and agonist stimulated + inhibitor was assessed using one-way ANOVA (Statview). If significant differences between the samples were observed, it was followed by a post hoc Bonferroni test.

2.3. Antibodies desorption and second antibodies incubation

Antibodies were stripped from the proteins on the PVDF membrane by 3 incubations of 15 min in a desorbing solution (200 mM HCl, 3% glycine). PVDF membranes were washed thoroughly with PBS, Tween 0.1%, then blocked 1 h using 5% non-fat dried milk in PBS, pH 7.2, Tween 0.1%. PVDF membranes were incubated overnight at 4 °C with anti-ERK1/2pAb antibody (1:10,000, Enzo Life Sciences, France). The strips were thoroughly washed with PBS, Tween 0.1% then incubated 40 min with secondary antibody horseradish peroxidase-conjugated donkey anti-rabbit diluted at 1/10,000. For each western blot, we used actin as a loading control. Mouse monoclonal anti-actin clone C4 1/100,000 came from Merck-Millipore, HRP-goat anti-mouse from GEHealthcare.

2.4. Peptides and chemicals

Human NPY came from Neosystem (France). PP2 and wortmannin were obtained from Enzo Life Science (Villeurbanne, France). AG1478, AG1024, Gö6983, gallein and marimastat were from Tocris Bioscience (Bristol, UK). U73122, U0126, and pertussis toxin (PTX) were from Sigma-Aldrich (L’Isle d’Abeau Chesnes, France).

3. Results

3.1. NPY Y1 receptors mediate ERK1/2-activation via a Gi-dependent pathway

To determine the temporal pattern of ERK1/2 activation mediated by NPY, we examined the kinetics of ERK1/2 phosphorylation following stimulation by NPY in HEK293 cells expressing the Y1 receptor. For this time-course study, cells were incubated with 100 nM NPY for several time points. Kinetic analysis by western blot revealed that NPY treatment induced a rapid phosphorylation of ERK1/2 with a maximal activity at 5 min (Fig. 1A). The return to almost basal levels of phosphorylation was achieved by 20 min. NPY induced a concentrationdependent increase in levels of ERK1/2 phosphorylation in cells expressing the human Y1 receptor (Fig. 1B) with an EC50 of 300 pM. In order to assess the role of Gi/o proteins in NPY-mediated ERK1/2 phosphorylation, the effect of selective inhibition of Gi-dependent signaling in cells expressing the human Y1 receptor was examined. PTX pre-treatment inhibited ERK1/2 phosphorylation induced by 100 nM NPY in a concentration manner with an IC50 of 150 pg/ml, indicating use of Gi/o proteins in the ERK signaling pathway (Fig. 1C). In all the following experiments we used the EC50 value of NPY concentration to test which pathway mediates MAPK activation. The inhibitor of the mitogen-activated protein (MAP) kinase/ERK kinase (MEK), U0126, abolished ERK1/2 phosphorylation in response to 300 pM NPY in a concentration-dependent manner, with an IC50 of 15 nM, indicating a conventional mechanism through MEK directly upstream of ERK1/2 activation (Fig. 1D).

3.2. NPY Y1 and Y2 receptors mediate ERK1/2-activation via an internalization-independent pathway

We have previously shown that the NPY Y2 receptor and mutants in the carboxyl-terminal tail of the Y1 receptor (mutants Y1 TDST-A and Y1 Δ42 receptors) are completely unable to recruit β-arrestins and to internalize [16]. This was based on studies using confocal microscopy, Fluorescent Activated Cell Sorting and fluorescence quenching to measure the internalization kinetics of activated receptors, and through the use of Bioluminescence Resonance Energy Transfer assays to detect beta-arrestin activation and recruitment. Now, in order to test the role of β-arrestins in MAPK signaling at the level of activated NPY receptors, we have analyzed the signaling induced by these receptors.
Kinetic analysis by western blot revealed that NPY treatment induced a rapid phosphorylation of ERK1/2 by Y2 receptors with a significantly lower intensity compared to phosphorylation of ERK1/2 by wild-type Y1 receptors (Fig. 2A). It also peaked significantly later with a maximal activity at 10 min instead of 5 min (Fig. 2B). NPY exposure of cells expressing the human Y2 receptor resulted in a concentrationdependent increase in ERK1/2 phosphorylation with an EC50 value of 10 nM (Fig. 2C). PTX pre-treatment inhibited ERK1/2 phosphorylation induced by 100 nM NPY in a concentration-dependent manner with an IC50 of 110 pg/ml, indicating use of Gi/o proteins in the ERK signaling pathway (Fig. 2D).
Similarly, kinetic analysis by western blot revealed that NPY treatment induced a rapid phosphorylation of ERK1/2 with a maximal activity at 5 min for cells expressing the human Y1 TDST-A and Y1 Δ42 receptors (Fig. 3A and B respectively). Thus, Y1 mutant receptors that do not recruit β-arrestins and therefore that are not internalized trigger MAPK activation to the same extent as wild-type receptors, ruling out a mechanism of activation mediated by platforms of β-arrestins bound to Y1 receptors in the endosomes.

3.3. NPY Y1 receptors mediate ERK1/2-activation via a calcium-independent pathway

NPY is also able to stimulate intracellular Ca2+ increases via PKCdependent pathways including the upstreamsignaling molecules, phospholipase C (PLC) and phospholipase D (PLD). We evaluated the possible involvement of these signaling pathways in NPY-mediated ERK1/2 activation by Y1 receptors. Statistical analysis of the results showed that neither the PLC inhibitor, U73122, nor the PLD inhibitor, FIPI, was able to block ERK1/2 activation triggered by NPY (Fig. 4A and not shown). Likewise, the PKC inhibitor, Gö6983, was not able to block the ERK1/2 phosphorylation induced by 300 pM NPY at any concentration tested (Fig. 4B). Therefore, these results clearly indicate that calciumdependent signaling pathways do not play an important role in ERK1/ 2 activation by the human NPY Y1 receptor.

3.4. NPY Y1 receptors mediate ERK1/2-activation via a PI3K-dependent pathway

We next assessed the contribution of Gβ/γ subunits to the activation of ERK1/2 by the Y1 receptor using gallein, which has been shown to selectively inhibit the interactions between Gβ/γ and effector proteins [18]. Pre-treatment with gallein inhibited ERK1/2 activation in a concentration-dependent manner (Fig. 4C).
Earlier studies have shown that the phosphoinositide 3-kinase (PI3K) is a signaling intermediate of the βγ-subunit of Gi-mediated MAPK activation [19,20]. We have pre-treated cells expressing the human NPY Y1 receptor for 1 h with increasing concentrations of wortmannin, an inhibitor of class I, II and III PI3Ks. Phosphorylation of ERK1/2 induced by 300 pM NPY was inhibited by the PI3K-inhibitor in a concentration dependent manner (Fig. 4D) indicating that the signaling pathway conducting to ERK1/2 activation is PI3K-dependent.
It has also been demonstrated that the c-Src-tyrosine kinase is involved in ERK1/2 signaling triggered by GPCRs by mediating receptor tyrosine kinase-transactivation upstream of PI3K [6,21]. Cells expressing the human NPY Y1 receptor were pre-incubated for 1 h with increasing concentrations of PP2, a specific c-Src-inhibitor, and stimulated with 300 pM NPY. Statistical analysis of the results shows that at all concentrations tested, PP2 failed to inhibit ERK1/2 phosphorylation in response to NPY activation (Fig. 4E). Together, these results indicate that upon activation of Y1 receptors, c-Src tyrosine kinase stimulation is not implicated in ERK1/2 activation.
We next tested directly whether NPY triggers transactivation of RTKs. First, cells expressing the NPY Y1 receptor were pre-treated with AG1478, a specific EGFR inhibitor. After 1 h pre-treatment with various concentrations of AG1478, we observed no statistical difference (Fig. 4F) on ERK1/2 phosphorylation in response to 300 pM NPY, suggesting that it is unlikely that EGFR is involved in NPY-mediated activation of ERK1/2.

3.5. NPY Y1 receptors mediate ERK1/2-activation via an IGFR-dependent but metalloproteinase-independent pathway

The hypothesis of transactivation of RTKs was further explored. Cells expressing the NPY Y1 receptor were pre-treated with AG1024, a specific IGFR inhibitor. After 1 h pre-treatment with various concentrations of AG1024, we observed a dose-dependent inhibition (Fig. 5A) on ERK1/2 phosphorylation in response to 300 pM NPY with an IC50 of 115 nM. Fig. 5B and C illustrates the effect of 10 μM AG1024 pre-treatment on P-ERK1/2 compared to vehicle-treated cells, with a representative western blot (Fig. 5B) and the quantification (Fig. 5C).
The metalloproteinase inhibitor, marimastat, which has a large spectrum of action on both matrix-metalloproteinases MMPs but also on metalloproteases of the ADAM family (A Disintegrin And Metalloproteases) [22], was recently shown to inhibit the transactivation of IGFR receptors triggered by the Gs-coupled V2 vasopressin receptor and by the Go/Gq-coupled platelet-activating factor PAF receptor in HEK293 cells [23]. In order to test whether NPY induces the shedding of an IGFR ligand through the processing of preligand by plasma membrane metalloproteinases, cells expressing the NPY Y1 receptor were pre-treated for 1 h with 10 μM marimastat. We did not observe any inhibition on ERK1/2 phosphorylation in response to 300 pM NPY (Fig. 5D). In conclusion, the IGFR is transactivated by Y1 receptors in HEK293 cells via a GiGβ/γ-PI3K-dependent mechanism without the release of a soluble ligand for IGFR by metalloproteinases.

3.6. Endogenously expressed GPCRs activate ERK signaling in HEK293 cells via a PI3K- and AG1024-sensitive pathway

We have already demonstrated that ERK1/2 becomes phosphorylated upon stimulation of a number of endogenously expressed GPCRs in HEK293 cells [24] and we decided to investigate the relative universality of the pathway that leads to ERK1/2 activation by NPY Y1 receptors. We found that wortmannin and AG1024 were both inhibiting the ERK1/2 phosphorylation induced at 5 min by the Gq-coupled muscarinic M3 receptor (Fig. 6A–D and B–E respectively) and by the Gs-coupled adenosine receptor as well (Fig. 6G and H respectively). On the other hand, marimastat pre-treatment of HEK293 cells had no statistically significant effect (Fig. 6C–F for muscarinic M3 receptors and Fig. 6I for Gs-coupled adenosine receptors). This suggests that in HEK293 cells, other GPCRs lead to phosphorylation of ERK1/2 using a pathway similar to the MAPK pathway governed by NPY Y1 receptor.

4. Discussion

Activation of the wild-type NPY Y1 receptor induces a rapid and transient ERK1/2 phosphorylation in HEK293 cells stably expressing this receptor. The signaling pathway leading to this activation involves Gαi and Gβ/γ protein subunits and transactivation of the IGFR.

4.1. IGFR transactivation by GPCRs

Transactivation of endogenously expressed IGFR by GPCRs leading to MAPK activation was recently dissected using the same cell model system: HEK293 cells [23]. In this previous study, the overexpressed Gs-coupled V2 receptor was found to mediate transactivation of IGFR independently of both heterotrimeric G activation and PI3Ks (insensitive to wortmannin) [25]. On the contrary, this occurred via the release by plasma membrane metalloproteinases of an unidentified soluble agonist of IGFR and was thus inhibited by incubation of cells with 10 μM marimastat. This pathway was also dependent on c-Src activation (inhibition by PP2) upstream of IGFR. In addition, surprisingly, βarrestins were necessary downstream of the IGF receptor to trigger MAPK activation instead of being necessary downstream of the activated V2 receptor. In a more complex experimental set-up, in which EGF receptors were desensitized again using the same cell model system, HEK293 cells; overexpressed Gi-coupled-delta-opioid receptors were also found to trigger MAPK activation through transactivation of the endogenously expressed IGFR via the shedding of an IGF-1-like peptide [26].
Insulin or IGF ligand binding to the IGFR can activate MAPKs [27–29]. These observations have also been confirmed in our previous signaling study in HEK293 cells [24]. The mechanism of ligand-dependent MAPK activation of IGFR is not yet clearly determined: β-arrestins have been proposed to play a role [23]. Indeed β-arrestins can serve as platforms to recruit several effectors of the MAPK pathway [12] and β-arrestin-1 has been found necessary for MAPK activation via the IGFR [30]. Along the same lines, it has been proposed that RTKs themselves can transactivate GPCRs [31]. Therefore it is not clear whether β-arrestins are directly interacting with the RTKs or whether interaction with another GPCR is needed in this MAPK activation mechanism.

4.2. IGFR transactivation by NPY Y1 receptors

Our experiments use the same experimental frame-work as the previous studies that demonstrate the transactivation of endogenously expressed IGFR by GPCRs leading to MAPK activation: the same cells, an overexpressed GPCR, and a survey of MAPK activation [23][26]. We have confirmed the previous observation that transactivation of IGFR by several GPCRs leads to MAPK activation. We have demonstrated it using the specific IGFR inhibitor, AG1024, also used in the two previous studies. In a recent survey for selectivity, AG1024 was found among the 17 uni-specific compounds that inhibited one kinase more potently than the kinase it was intended to target [32]. Indeed, the serine/threonine kinase, RIPK2 (receptor-interacting kinase2), was identified as a much more sensitive target of AG1024. Although RIPK2 becomes phosphorylated upon activation of the Gq-coupled angiotensin AT1 receptor as described in a recent phosphoproteomic analysis [33], we instead favor the interpretation that IGFR is the target of AG1024 in our study because there is so far no indication that RIPK2 could trigger MAPK activation. Instead, RIPK2 activates the NF-KB pathway and is a component of signaling in immune responses [34].
However, in several respects, the pathway that we are describing in this paper is different to that described for MAPK activation via the vasopressin V2 receptor in HEK293 cells. MAPK activation by NPY Y1 receptor is definitely G-protein dependent, because PTX and gallein, specific inhibitors of Gαi and Gβ/γ subunits respectively, completely block ERK1/2 phosphorylation in a concentration-dependent manner. The pathway that we have dissected is also clearly insensitive to both the c-Src inhibitor PP2 and the metalloprotease inhibitor, marimastat, while it is sensitive to wortmannin.
We have not addressed the requirement of β-arrestins downstream of IGFR because the mechanism of transactivation of IGFR by the Gi-coupled NPY Y1 receptor seems to occur via a ligand-independent activation of IGFR (independent of c-Src and metalloproteases). On the other hand, we have clearly established that recruitment of β-arrestins and internalization of the NPY Y1 receptor are not necessary for ERK1/2 activation. There are two mutants of the NPY Y1 receptor that upon NPY activation are unable to bind β-arrestins, or to activate β-arrestins or to internalize into clathrin-coated pits, and these mutants were both unaltered in the amplitudes and kinetics of ERK1/2 phosphorylation that they triggered [16].
It has been observed in several studies that, in the Gi-coupled receptor and Gβ/γ-mediated MAPK signaling pathway, inhibitors of PI3Ks attenuated MAPK activation [19]. In our present study, a wortmannin-sensitive PI3K activity was required for activation of ERK1/2 upon cell stimulation with NPY. PI3K-γ isoform of the PI3K family can be directly activated by the Gβ/γ complex [35] and was especially shown to mediate signaling of chemokines and chemo-attractant receptors [36–39]. But PI3K-β has also been shown to be regulated by Gβ/γ [20]. In this particular study, the G12-coupled lysophosphatidic acid LPA receptor transactivates the EGFR to mediate activation of ERK1/2. The authors have proposed that PI3K integrates two separate signals from the activated LPA receptor, one through direct interaction with the released Gβ/γ subunit and the other, downstream of transactivated EGFR [20]. Such mechanism of action where PI3K received input of two converging pathways would contribute to an amplification of the MAPK pathway. In effect, amplification was confirmed using a phosphoproteomics approach: direct EGFR activation of the cells gave rise to weaker phosphorylations of sites compared to phosphorylated sites by transactivation of the EGFR upon LPA stimulation [40]. Convergence of two separate pathways on PI3K is probably not occurring, however, in the case of IGFR transactivation by NPY Y1 receptor since the IGFR inhibitor, AG1024, completely abolishes activation of ERK1/2 upon cell stimulation with NPY.

4.3. Towards a convergent mechanism of activation of MAPK pathway by NPY

The activation of MAPKs triggered by NPY Y1 receptor that we describe, is in agreement with previous studies: PTX and wortmannin sensitivity were observed [41,42]. Interestingly, a recent investigation has proposed that neuroproliferative effects of NPY in postnatal hippocampal precursor cells is mediated by a MAPK pathway occurring downstream of intracellular nitric oxide production, cGMP and the cGMP-dependent protein kinase PKG activation [43,44]. Further studies are required to investigate BB-2516 the relationship between transactivation of IGFR and nitric oxide production, as was studied, for example in the case of angiotensin receptor transactivation of EGFR [45] or bradykinin B2 receptor transactivation of vascular endothelial growth factor [46].

5. Conclusion

Together, our results propose for the first time, that MAPK activation by NPY Y1 receptors is an internalization-independent pathway and that this receptor can transactivate the IGFR receptor. We finally show that a similar mechanism of MAPK activation through transactivation of IGFR in a PI3K-dependent but metalloprotease-independent manner is used by endogenously expressed Gq- and Gs-coupled receptors as well.

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