Hesham M. El-Shewy

The adiponectin receptors AdipoR1 and AdipoR2 activate ERK1/2 through a Src/Ras-dependent pathway and stimulate cell growth †

Adiponectin is an adipocyte-derived cytokine that has attracted much attention because of its insulin-sensitizing effects in liver and skeletal muscle. Two adiponectin receptors, AdipoR1/R2, have been cloned, but relatively little is known about their intracellular signaling mechanisms. We found that full-length adiponectin rapidly and robustly activates the ERK1/2 mitogen-activated protein kinase pathway in primary vascular smooth muscle, vascular endothelial cells, and hepatocytes. In a HEK293 cell model, we found that downregulating AdipoR1/R2 simultaneously, but not individually, by RNA interference attenuated adiponectin-induced ERK1/2 activation, suggesting that either receptor was sufficient to mediate the response. Downregulation of T-cadherin, another adiponectin binding protein, enhanced the response. Downregulation of APPL1, an adapter protein and putative mediator of AdipoR1/R2 signaling, impaired adiponectin-stimulated ERK1/2 activation. Inhibiting PKA modestly attenuated ERK1/2 activation, while inhibition of Src family tyrosine kinases with PP2 abolished the response. The small GTPase inhibitor Clostridium difficile toxin B also produced complete inhibition. Adiponectin caused rapid, PP2-sensitive activation of Ras, but not the cAMP-regulated small GTPase, Rap1, suggesting that Src-dependent Ras activation is the dominant mechanism of adiponectin-stimulated ERK1/2 activation. To test whether Ras-ERK1/2 signaling by adiponectin was physiologically relevant, we determined the effects of overexpressing AdipoR1, adiponectin, or both on the rate of HEK293 cell growth. Overexpression of adiponectin alone, but not AdipoR1 alone, supported growth under serum-free conditions, while simultaneous expression of both led to further enhancement. These results suggest that adiponectin can exert proliferative effects by activating Ras signaling pathways.

Sphingosine kinase-1 and sphingosine 1-phosphate receptor 2 mediate Bcr-Abl1 stability and drug resistance by modulation of protein phosphatase 2A

The mechanisms by which sphingosine kinase-1 (SK-1)/sphingosine 1-phosphate (S1P) activation contributes to imatinib resistance in chronic myeloid leukemia (CML) are unknown. We show herein that increased SK-1/S1P enhances Bcr-Abl1 protein stability, through inhibition of its proteasomal degradation in imatinib-resistant K562/IMA-3 and LAMA-4/IMA human CML cells. In fact, Bcr-Abl1 stability was enhanced by ectopic SK-1 expression. Conversely, siRNA-mediated SK-1 knockdown in K562/IMA-3 cells, or its genetic loss in SK-1(-/-) MEFs, significantly reduced Bcr-Abl1 stability. Regulation of Bcr-Abl1 by SK-1/S1P was dependent on S1P receptor 2 (S1P2) signaling, which prevented Bcr-Abl1 dephosphorylation, and degradation via inhibition of PP2A. Molecular or pharmacologic interference with SK-1/S1P2 restored PP2A-dependent Bcr-Abl1 dephosphorylation, and enhanced imatinib- or nilotinib-induced growth inhibition in primary CD34(+) mononuclear cells obtained from chronic phase and blast crisis CML patients, K562/IMA-3 or LAMA4/IMA cells, and 32Dcl3 murine progenitor cells, expressing the wild-type or mutant (Y253H or T315I) Bcr-Abl1 in situ. Accordingly, impaired SK-1/S1P2 signaling enhanced the growth-inhibitory effects of nilotinib against 32D/T315I-Bcr-Abl1-derived mouse allografts. Since SK-1/S1P/S1P2 signaling regulates Bcr-Abl1 stability via modulation of PP2A, inhibition of SK-1/S1P2 axis represents a novel approach to target wild-type- or mutant-Bcr-Abl1 thereby overcoming drug resistance.

Insulin-like Growth Factors Mediate Heterotrimeric G Protein-dependent ERK1/2 Activation by Transactivating Sphingosine 1-Phosphate Receptors

Although several studies have shown that a subset of insulin-like growth factor (IGF) signals require the activation of heterotrimeric G proteins, the molecular mechanisms underlying IGF-stimulated G protein signaling remain poorly understood. Here, we have studied the mechanism by which endogenous IGF receptors activate the ERK1/2 mitogen-activated protein kinase cascade in HEK293 cells. In these cells, treatment with pertussis toxin and expression of a Gαq/11-(305–359) peptide that inhibits Gq/11 signaling additively inhibited IGF-stimulated ERK1/2 activation, indicating that the signal was almost completely G protein-dependent. Treatment with IGF-1 or IGF-2 promoted translocation of green fluorescent protein (GFP)-tagged sphingosine kinase (SK) 1 from the cytosol to the plasma membrane, increased endogenous SK activity within 30 s of stimulation, and caused a statistically significant increase in intracellular and extracellular sphingosine 1-phosphate (S1P) concentration. Using a GFP-tagged S1P1 receptor as a biological sensor for the generation of physiologically relevant S1P levels, we found that IGF-1 and IGF-2 induced GFP-S1P receptor internalization and that the effect was blocked by pretreatment with the SK inhibitor, dimethylsphingosine. Treating cells with dimethylsphingosine, silencing SK1 expression by RNA interference, and blocking endogenous S1P receptors with the competitive antagonist VPC23019 all significantly inhibited IGF-stimulated ERK1/2 activation, suggesting that IGFs elicit G protein-dependent ERK1/2 activation by stimulating SK1-dependent transactivation of S1P receptors. Given the ubiquity of SK and S1P receptor expression, S1P receptor transactivation may represent a general mechanism for G protein-dependent signaling by non-G protein-coupled receptors.

Role of β-Arrestin-mediated Desensitization and Signaling in the Control of Angiotensin AT1a Receptor-stimulated Transcription

Heptahelical G protein-coupled receptors employ several mechanisms to activate the ERK1/2 cascade and control gene transcription. Previous work with the angiotensin AT1a receptor has shown that Gq/11 activation leads to a rapid and transient rise in ERK1/2 activity, whereas β-arrestin binding supports sustained ERK1/2 activation by scaffolding a Raf·MEK·ERK complex associated with the internalized receptor. In this study, we compared the role of the two β-arrestin isoforms in AT1a receptor desensitization, ERK1/2 activation and transcription using selective RNA interference. In HEK293 cells, both the native AT1a receptor and a G protein-coupling deficient DRY/AAY mutant recruited β-arrestin1 and β-arrestin2 upon angiotensin binding and internalized with the receptor. In contrast, only β-arrestin2 supported protein kinase C-independent ERK1/2 activation by both the AT1a and DRY/AAY receptors. Using focused gene expression filter arrays to screen for endogenous transcriptional responses, we found that silencing β-arrestin1 or β-arrestin2 individually did not alter the response pattern but that silencing both caused a marked increase in the number of transcripts that were significantly up-regulated in response to AT1a receptor activation. The DRY/AAY receptor failed to elicit any detectable transcriptional response despite its ability to stimulate β-arrestin2-dependent ERK1/2 activation. These results indicate that the transcriptional response to AT1a receptor activation primarily reflects heterotrimeric G protein activation. Although β-arrestin1 and β-arrestin2 are functionally specialized with respect to supporting G protein-independent ERK1/2 activation, their common effect is to dampen the transcriptional response by promoting receptor desensitization.

Insulin-like growth factor-2/mannose-6 phosphate receptors.

The insulin-like growth factor type 2/mannose-6-phosphate (IGF-2/M6P) receptor is a multifunctional single transmembrane glycoprotein that is known to regulate diverse biological functions. It is composed of a large extracytoplasmic domain, a single transmembrane region and a short cytoplasmic tail that lacks intrinsic catalytic activity. The receptor cycles continuously between intracellular compartments and the plasma membrane, and at steady state is predominantly localized in the trans-Golgi network and endosomal compartments, and to a lesser extent on the cell surface. The receptor binds IGF-2 with higher affinity than IGF-1 and does not bind insulin. It interacts, via distinct sites, with lysosomal enzymes and a variety of other M6P-containing ligands. IGF-2/M6P receptors perform diverse cellular functions related to lysosome biogenesis and the regulation of growth and development. It regulates extracellular IGF-2 concentrations, modulating signaling through the growth-stimulatory IGF-1 receptor pathway. It appears to mediate the uptake and processing of M6P-containing cytokines and peptide hormones, such as transforming growth factor-beta, leukemia inhibitory factor, and proliferin. Some data suggest that the IGF-2/M6P receptor also functions in signal transduction by transactivating G protein-coupled sphingosine 1-phosphate receptors. Genetic evidence clearly supports a role for IGF-2/M6P receptors in organ development and growth, and recent data indicate that it may play an important role in tumor progression.

The β-Arrestin Pathway-selective Type 1A Angiotensin Receptor (AT1A) Agonist [Sar1,Ile4,Ile8]Angiotensin II Regulates a Robust G Protein-independent Signaling Network

The angiotensin II peptide analog [Sar1,Ile4,Ile8]AngII (SII) is a biased AT1A receptor agonist that stimulates receptor phosphorylation, β-arrestin recruitment, receptor internalization, and β-arrestin-dependent ERK1/2 activation without activating heterotrimeric G-proteins. To determine the scope of G-protein-independent AT1A receptor signaling, we performed a gel-based phosphoproteomic analysis of AngII and SII-induced signaling in HEK cells stably expressing AT1A receptors. A total of 34 differentially phosphorylated proteins were detected, of which 16 were unique to SII and eight to AngII stimulation. MALDI-TOF/TOF mass fingerprinting was employed to identify 24 SII-sensitive phosphoprotein spots, of which three (two peptide inhibitors of protein phosphatase 2A (I1PP2A and I2PP2A) and prostaglandin E synthase 3 (PGES3)) were selected for validation and further study. We found that phosphorylation of I2PP2A was associated with rapid and transient inhibition of a β-arrestin 2-associated pool of protein phosphatase 2A, leading to activation of Akt and increased phosphorylation of glycogen synthase kinase 3β in an arrestin signalsome complex. SII-stimulated PGES3 phosphorylation coincided with an increase in β-arrestin 1-associated PGES3 and an arrestin-dependent increase in cyclooxygenase 1-dependent prostaglandin E2 synthesis. These findings suggest that AT1A receptors regulate a robust G protein-independent signaling network that affects protein phosphorylation and autocrine/paracrine prostaglandin production and that these pathways can be selectively modulated by biased ligands that antagonize G protein activation.

The Insulin-like Growth Factor Type 1 and Insulin-like Growth Factor Type 2/Mannose-6-phosphate Receptors Independently Regulate ERK1/2 Activity in HEK293 Cells

Insulin-like growth factor types 1 and 2 (IGF-1; IGF-2) and insulin-like peptides are all members of the insulin superfamily of peptide hormones but bind to several distinct classes of membrane receptor. Like the insulin receptor, the IGF-1 receptor is a heterotetrameric receptor tyrosine kinase, whereas the IGF-2/ mannose 6-phosphate receptor is a single transmembrane domain protein that is thought to function primarily as clearance receptors. We recently reported that IGF-1 and IGF-2 stimulate the ERK1/2 cascade by triggering sphingosine kinasedependent “transactivation” of G protein-coupled sphingosine-1-phosphate receptors. To determine which IGF receptors mediate this effect, we tested seven insulin family peptides, IGF-1, IGF-2, insulin, and insulin-like peptides 3, 4, 6, and 7, for the ability to activate ERK1/2 in HEK293 cells. Only IGF-1 and IGF-2 potently activated ERK1/2. Although IGF-2 was predictably less potent than IGF-1 in activating the IGF-1 receptor, they were equipotent stimulators of ERK1/2. Knockdown of IGF-1 receptor expression by RNA interference reduced the IGF-1 response to a greater extent than the IGF-2 response, suggesting that IGF-2 did not signal exclusively via the IGF-1 receptor. In contrast, IGF-2 receptor knockdown markedly reduced IGF-2-stimulated ERK1/2 phosphorylation, with no effect on the IGF-1 response. As observed previously, both the IGF-1 and the IGF-2 responses were sensitive to pertussis toxin and the sphingosine kinase inhibitor, dimethylsphingosine. These data indicate that endogenous IGF-1 and IGF-2 receptors can independently initiate ERK1/2 signaling and point to a potential physiologic role for IGF-2 receptors in the cellular response to IGF-2.

Constitutive ERK1/2 Activation by a Chimeric Neurokinin 1 Receptor-β-Arrestin1 Fusion Protein PROBING THE COMPOSITION AND FUNCTION OF THE G PROTEIN-COUPLED RECEPTOR “SIGNALSOME”

The β-arrestins, a small family of G protein-coupled receptor (GPCR)-binding proteins involved in receptor desensitization, have been shown to bind extracellular signal-regulated kinases 1 and 2 (ERK1/2) and function as scaffolds for GPCR-stimulated ERK1/2 activation. To better understand the mechanism of β-arrestin-mediated ERK1/2 activation, we compared ERK1/2 activation by the wild-type neurokinin 1 (NK1) receptor with a chimeric NK1 receptor having β-arrestin1 fused to the receptor C terminus (NK1-βArr1). The NK1 receptor couples to both Gs and Gq/11, resides on the plasma membrane, and mediates rapid ERK1/2 activation and nuclear translocation in response to neurokinin A. In contrast, NK1-βArr1 is a G protein-uncoupled “constitutively desensitized” receptor that resides almost entirely in an intracellular endosomal compartment. Despite its inability to respond to neurokinin A, we found that NK1-βArr1 expression caused robust constitutive activation of cytosolic ERK1/2 and that endogenous Raf, MEK1/2, and ERK1/2 coprecipitated in a complex with NK1-βArr1. While agonist-dependent ERK1/2 activation by the NK1 receptor was independent of protein kinase A (PKA) or PKC activity, NK1-βArr1-mediated ERK1/2 activation was completely inhibited when basal PKA and PKC activity were blocked. In addition, the rate of ERK1/2 dephosphorylation was slowed in NK1-βArr1-expressing cells, suggesting that β-arrestin-bound ERK1/2 is protected from mitogen-activated protein kinase phosphatase activity. These data suggest that β-arrestin binding to GPCRs nucleates the formation of a stable “signalsome” that functions as a passive scaffold for the ERK1/2 cascade while confining ERK1/2 activity to an extranuclear compartment.

The arrestin-selective angiotensin AT1 receptor agonist [Sar1,Ile4,Ile8]-AngII negatively regulates bradykinin B2 receptor signaling via AT1-B2 receptor heterodimers

Background: Hemodynamic regulation involves extensive cross-talk between the renin-angiotensin and kallikrein-kinin systems. Results: In vascular smooth muscle, “biased” AT1 agonists inhibit both AT1 and B2 signaling by internalizing AT1-B2 heterodimers. Conclusion: AT1 antagonists and arrestin-selective biased AT1 agonists have opposing effects on B2 signaling. Significance: Negative allosteric modulation of B2 signaling by biased AT1 agonists may impact their clinical utility. The renin-angiotensin and kallikrein-kinin systems are key regulators of vascular tone and inflammation. Angiotensin II, the principal effector of the renin-angiotensin system, promotes vasoconstriction by activating angiotensin AT1 receptors. The opposing effects of the kallikrein-kinin system are mediated by bradykinin acting on B1 and B2 bradykinin receptors. The renin-angiotensin and kallikrein-kinin systems engage in cross-talk at multiple levels, including the formation of AT1-B2 receptor heterodimers. In primary vascular smooth muscle cells, we find that the arrestin pathway-selective AT1 agonist, [Sar1,Ile4,Ile8]-AngII, but not the neutral AT1 antagonist, losartan, inhibits endogenous B2 receptor signaling. In a transfected HEK293 cell model that recapitulates this effect, we find that the actions of [Sar1,Ile4, Ile8]-AngII require the AT1 receptor and result from arrestin-dependent co-internalization of AT1-B2 heterodimers. BRET50 measurements indicate that AT1 and B2 receptors efficiently heterodimerize. In cells expressing both receptors, pretreatment with [Sar1,Ile4,Ile8]-AngII blunts B2 receptor activation of Gq/11-dependent intracellular calcium influx and Gi/o-dependent inhibition of adenylyl cyclase. In contrast, [Sar1,Ile4,Ile8]-AngII has no effect on B2 receptor ligand affinity or bradykinin-induced arrestin3 recruitment. Both radioligand binding assays and quantitative microscopy-based analysis demonstrate that [Sar1,Ile4,Ile8]-AngII promotes internalization of AT1-B2 heterodimers. Thus, [Sar1,Ile4,Ile8]-AngII exerts lateral allosteric modulation of B2 receptor signaling by binding to the orthosteric ligand binding site of the AT1 receptor and promoting co-sequestration of AT1-B2 heterodimers. Given the opposing roles of the renin-angiotensin and kallikrein-kinin systems in vivo, the distinct properties of arrestin pathway-selective and neutral AT1 receptor ligands may translate into different pharmacologic actions.

Low-Density Lipoprotein Induced Expression of Connective Tissue Growth Factor via Transactivation of Sphingosine 1-Phosphate Receptors in Mesangial Cells

The pro-fibrotic connective tissue growth factor (CTGF) has been linked to the development and progression of diabetic vascular and renal disease. We recently reported that low-density lipoproteins (LDL) induced expression of CTGF in aortic endothelial cells. However, the molecular mechanisms are not fully defined. Here, we have studied the mechanism by which LDL regulates CTGF expression in renal mesangial cells. In these cells, treatment with pertussis toxin abolished LDL-stimulated activation of ERK1/2 and c-Jun N-terminal kinase (JNK), indicating the involvement of heterotrimeric G proteins in LDL signaling. Treatment with LDL promoted activation and translocation of endogenous sphingosine kinase 1 (SK1) from the cytosol to the plasma membrane concomitant with production of sphingosine-1-phosphate (S1P). Pretreating cells with SK inhibitor, dimethylsphinogsine or down-regulation of SK1 and SK2 revealed that LDL-dependent activation of ERK1/2 and JNK is mediated by SK1. Using a green fluorescent protein-tagged S1P₁ receptor as a biological sensor for the generation of physiologically relevant S1P levels, we found that LDL induced S1P receptor activation. Pretreating cells with S1P₁/S1P₃ receptor antagonist VPC23019 significantly inhibited activation of ERK1/2 and JNK by LDL, suggesting that LDL elicits G protein-dependent activation of ERK1/2 and JNK by stimulating SK1-dependent transactivation of S1P receptors. Furthermore, S1P stimulation induced expression of CTGF in a dose-dependent manner that was markedly inhibited by blocking the ERK1/2 and JNK signaling pathways. LDL-induced CTGF expression was pertussis toxin sensitive and inhibited by dimethylsphinogsine down-regulation of SK1 and VPC23019 treatment. Our data suggest that SK1-dependent S1P receptor transactivation is upstream of ERK1/2 and JNK and that all three steps are required for LDL-regulated expression of CTGF in mesangial cells.