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HDL normally transports about 50–70% of plasma sphingosine 1-phosphate (S1P), and the S1P in HDL reportedly mediates several HDL-associated biological effects and signaling pathways. The HDL receptor, SR-BI, as well as the cell surface receptors for S1P (S1PRs) may be involved partially and/or completely in these HDL-induced processes. Here we investigate the nature of the HDL-stimulated interaction between the HDL receptor, SR-BI, and S1PR1 using a protein-fragment complementation assay and confocal microscopy. In both primary rat aortic vascular smooth muscle cells and HEK293 cells, the S1P content in HDL particles increased intracellular calcium concentration, which was mediated by S1PR1. Mechanistic studies performed in HEK293 cells showed that incubation of cells with HDL led to an increase in the physical interaction between the SR-BI and S1PR1 receptors that mainly occurred on the plasma membrane. Model recombinant HDL (rHDL) particles formed in vitro with S1P incorporated into the particle initiated the internalization of S1PR1, whereas rHDL without supplemented S1P did not, suggesting that S1P transported in HDL can selectively activate S1PR1. In conclusion, these data suggest that S1P in HDL stimulates the transient interaction between SR-BI and S1PRs that can activate S1PRs and induce an elevation in intracellular calcium concentration.

Bone remodeling involves the coordinated actions of osteoclasts, which resorb the calcified bony matrix, and osteoblasts, which refill erosion pits created by osteoclasts to restore skeletal integrity and adapt to changes in mechanical load. Osteoblasts are derived from pluripotent mesenchymal stem cell precursors, which undergo differentiation under the influence of a host of local and environmental cues. To characterize the autocrine/paracrine signaling networks associated with osteoblast maturation and function, we performed gene network analysis using complementary “agnostic” DNA microarray and “targeted” NanoString™ nCounter datasets derived from murine MC3T3-E1 cells induced to undergo synchronized osteoblastic differentiation in vitro. Pairwise datasets representing changes in gene expression associated with growth arrest (day 2 to 5 in culture), differentiation (day 5 to 10 in culture), and osteoblast maturation (day 10 to 28 in culture) were analyzed using Ingenuity Systems™ Pathways Analysis to generate predictions about signaling pathway activity based on the temporal sequence of changes in target gene expression. Our data indicate that some pathways known to be involved in osteoblast differentiation, e.g. Wnt/β-catenin signaling, are most active early in the process, while others, e.g. TGFβ/BMP, cytokine/JAK-STAT and TNFα/RANKL signaling, increase in activity as differentiation progresses. Collectively, these pathways contribute to the sequential expression of genes involved in the synthesis and mineralization of extracellular matrix. These results provide insight into the temporal coordination and complex interplay between signaling networks controlling gene expression during osteoblast differentiation. A more complete understanding of these processes may aid the discovery of novel methods to promote osteoblast development for the trea™ent of conditions characterized by low bone mineral density.

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, [Sar(1),Ile(4),Ile(8)]-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 [Sar(1),Ile(4), Ile(8)]-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 [Sar(1),Ile(4),Ile(8)]-AngII blunts B2 receptor activation of Gq/11-dependent intracellular calcium influx and Gi/o-dependent inhibition of adenylyl cyclase. In contrast, [Sar(1),Ile(4),Ile(8)]-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 [Sar(1),Ile(4),Ile(8)]-AngII promotes internalization of AT1-B2 heterodimers. Thus, [Sar(1),Ile(4),Ile(8)]-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.

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.

We recently reported that IGF-II binding to the IGF-II/mannose-6-phosphate (M6P) receptor activates the ERK1/2 cascade by triggering sphingosine kinase 1 (SK1)-dependent transactivation of G protein-coupled sphingosine 1-phosphate (S1P) receptors. Here, we investigated the mechanism of IGF-II/M6P receptor-dependent sphingosine kinase 1 (SK1) activation in human embryonic kidney 293 cells. Pretreating cells with protein kinase C (PKC) inhibitor, bisindolylmaleimide-I, abolished IGF-II-stimulated translocation of green fluorescent protein (GFP)-tagged SK1 to the plasma membrane and activation of endogenous SK1, implicating PKC as an upstream regulator of SK1. Using confocal microscopy to examine membrane translocation of GFP-tagged PKCα, β1, β2, δ, and ζ, we found that IGF-II induced rapid, transient, and isoform-specific translocation of GFP-PKCβ2 to the plasma membrane. Immunoblotting of endogenous PKC phosphorylation confirmed PKCβ2 activation in response to IGF-II. Similarly, IGF-II stimulation caused persistent membrane translocation of the kinase-deficient GFP-PKCβ2 (K371R) mutant, which does not dissociate from the membrane after translocation. IGF-II stimulation increased diacylglycerol (DAG) levels, the established activator of classical PKC. Interestingly, the polyunsaturated fraction of DAG was increased, indicating involvement of phosphatidyl inositol/phospholipase C (PLC). Pretreating cells with the PLC inhibitor, U73122, attenuated IGF-II-dependent DAG production and PKCβ2 phosphorylation, blocked membrane translocation of the kinase-deficient GFP-PKCβ2 (K371R) mutant, and reduced sphingosine 1-phosphate production, suggesting that PLC/PKCβ2 are upstream regulators of SK1 in the pathway. Taken together, these data provide evidence that activation of PLC and PKCβ2 by the IGF-II/M6P receptor are required for the activation of SK1.

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. © 2011 by The American Society of Hematology., National Institutes of Health (NIH); US Army; NIH, National Center for Research Resources (C06 RR015455)

The kallikrein-kinin system, along with the interlocking renin-angiotensin system, is a key regulator of vascular contractility and injury response. The principal effectors of the kallikrein-kinin system are plasma and tissue kallikreins, proteases that cleave high molecular weight kininogen to produce bradykinin. Most of the cellular actions of kallikrein (KK) are thought to be mediated by bradykinin, which acts via G protein-coupled B1 and B2 bradykinin receptors on VSMCs and endothelial cells. Here, we find that primary aortic vascular smooth muscle but not endothelial cells possess the ability to activate plasma prekallikrein. Surprisingly, exposing VSMCs to prekallikrein leads to activation of the ERK1/2 mitogen-activated protein kinase cascade via a mechanism that requires kallikrein activity but does not involve bradykinin receptors. In transfected HEK293 cells, we find that plasma kallikrein directly activates G protein-coupled protease-activated receptors (PARs) 1 and 2, which possess consensus kallikrein cleavage sites, but not PAR4. In vascular smooth muscles, KK stimulates ADAM (a disintegrin and metalloprotease) 17 activity via a PAR1/2 receptor-dependent mechanism, leading sequentially to release of the endogenous ADAM17 substrates, amphiregulin and tumor necrosis factor-α, metalloprotease-dependent transactivation of epidermal growth factor receptors, and metalloprotease and epidermal growth factor receptor-dependent ERK1/2 activation. These results suggest a novel mechanism of bradykinin-independent kallikrein action that may contribute to the regulation of vascular responses in pathophysiologic states, such as diabetes mellitus.

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 and AdipoR2, 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 and AdipoR2 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 attenutated 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. Additional results suggest that adiponectin can exert proliferative effects by activating Ras signaling pathways.

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 G(q/11) activation leads to a rapid and transient rise in ERK1/2 activity, whereas beta-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 beta-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 beta-arrestin1 and beta-arrestin2 upon angiotensin binding and internalized with the receptor. In contrast, only beta-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 beta-arrestin1 or beta-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 beta-arrestin2-dependent ERK1/2 activation. These results indicate that the transcriptional response to AT1a receptor activation primarily reflects heterotrimeric G protein activation. Although beta-arrestin1 and beta-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.

Angiotensin II (AngII) plays a critical role in the regulation of vascular tone and blood pressure mainly via regulation of Ca2+ mobilization. Several reports have implicated sphingosine kinase 1 (SK1)/sphingosine 1-phosphate (S1P) in the mobilization of intracellular Ca2+ through a yet-undefined mechanism. Here we demonstrate that AngII-induces biphasic calcium entry in vascular smooth muscle cells, consisting of an immediate peak due to inositol tris-phosphate-dependent release of intracellular calcium, followed by a sustained transmembrane Ca2+ influx through store-operated calcium channels (SOCs). Inhibition of SK1 attenuates the second phase of transmembrane Ca2+ influx, suggesting a role for SK1 in AngII-dependent activation of SOC. Intracellular S1P triggers SOC-dependent Ca2+ influx independent of S1P receptors, whereas external application of S1P stimulated S1P receptor-dependent Ca2+ influx that is insensitive to inhibitors of SOCs, suggesting that the SK1/S1P axis regulates store-operated calcium entry via intracellular rather than extracellular actions. Genetic deletion of SK1 significantly inhibits both the acute hypertensive response to AngII in anaesthetized SK1 knockout mice and the sustained hypertensive response to continuous infusion of AngII in conscious animals. Collectively these data implicate SK1 as the missing link that connects the angiotensin AT1A receptor to transmembrane Ca2+ influx and identify SOCs as a potential intracellular target for SK1.

Angiotensin II plays a critical role in the regulation of vascular tone and blood pressure mainly via regulation of Ca2+ mobilization. Several reports have implicated sphingosine kinase 1 (SK1)/sphingosine 1-phosphate (S1P) in the mobilization of intracellular Ca2+ through a yet undefined mechanism. Here we demonstrate that angiotensin II induces biphasic calcium entry in vascular smooth muscle cells, consisting of an immediate peak due to inositol tris-phosphate-dependent release of intracellular calcium, followed by a sustained transmembrane Ca2+ influx through store operated calcium channels (SOC). Inhibition of SK1 attenuates the second phase of transmembrane Ca2+ influx, suggesting a role for SK1 in angiotensin II-dependent activation of SOC. Intracellular S1P triggers SOC-dependent Ca2+ influx independent of S1P receptors, while external application of S1P stimulated S1P receptor-dependent Ca2+ influx that is insensitive to inhibitors of SOC, suggesting that the SK1/S1P axis regulates SOC entry via ...

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 Galpha(q/11)-(305-359) peptide that inhibits G(q/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.

The beta-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 beta-arrestin-mediated ERK1/2 activation, we compared ERK1/2 activation by the wild-type neurokinin 1 (NK1) receptor with a chimeric NK1 receptor having beta-arrestin1 fused to the receptor C terminus (NK1-betaArr1). The NK1 receptor couples to both G(s) and G(q/11), resides on the plasma membrane, and mediates rapid ERK1/2 activation and nuclear translocation in response to neurokinin A. In contrast, NK1-betaArr1 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-betaArr1 expression caused robust constitutive activation of cytosolic ERK1/2 and that endogenous Raf, MEK1/2, and ERK1/2 coprecipitated in a complex with NK1-betaArr1. While agonist-dependent ERK1/2 activation by the NK1 receptor was independent of protein kinase A (PKA) or PKC activity, NK1-betaArr1-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-betaArr1-expressing cells, suggesting that beta-arrestin-bound ERK1/2 is protected from mitogen-activated protein kinase phosphatase activity. These data suggest that beta-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 angiotensin II peptide analog [Sar(1),Ile(4),Ile(8)]AngII (SII) is a biased AT(1A) 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 AT(1A) receptor signaling, we performed a gel-based phosphoproteomic analysis of AngII and SII-induced signaling in HEK cells stably expressing AT(1A) 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 E(2) synthesis. These findings suggest that AT(1A) 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.

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 kinase-dependent "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.

The beta-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 beta-arrestin-mediated ERK1/2 activation, we compared ERK1/2 activation by the wild-type neurokinin 1 (NK1) receptor with a chimeric NK1 receptor having beta-arrestin1 fused to the receptor C terminus (NK1-betaArr1). The NK1 receptor couples to both G(s) and G(q/11), resides on the plasma membrane, and mediates rapid ERK1/2 activation and nuclear translocation in response to neurokinin A. In contrast, NK1-betaArr1 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-betaArr1 expression caused robust constitutive activation of cytosolic ERK1/2 and that endogenous Raf, MEK1/2, and ERK1/2 coprecipitated in a complex with NK1-betaArr1. While agonist-dependent ERK1/2 activation by the NK1 receptor was independent of protein kinase A (PKA) or PKC activity, NK1-betaArr1-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-betaArr1-expressing cells, suggesting that beta-arrestin-bound ERK1/2 is protected from mitogen-activated protein kinase phosphatase activity. These data suggest that beta-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.

Diverse extracellular stimuli activate the ERK1/2 MAPK cascade by transactivating epidermal growth factor (EGF) receptors. Here, we have examined the role of EGF receptors in IGF-I-stimulated ERK1/2 activation in several cultured cell lines. In human embryonic kidney 293 cells, IGF-I triggered proteolysis of heparin binding (HB)-EGF, increased tyrosine autophosphorylation of EGF receptors, stimulated EGF receptor inhibitor (AG1478)-sensitive ERK1/2 phosphorylation, and promoted EGF receptor endocytosis. In a mixed culture system that employed IGF-I receptor null murine embryo fibroblasts (MEFs) (R(-) cells) to detect paracrine signals produced by MEFs expressing the human IGF-I receptor (R(+) cells), stimulation of R(+) cells provoked rapid activation of green fluorescent protein-tagged ERK2 in cocultured R(-) cells. The R(-) cell response was abolished by either the broad-spectrum matrix metalloprotease inhibitor batimastat or by AG1478, indicating that it resulted from the proteolytic generation of an EGF receptor ligand from adjacent R(+) cells. These data suggest that the paracrine production of EGF receptor ligands leading to EGF receptor transactivation is a general property of IGF-I receptor signaling. In contrast, the contribution of transactivated EGF receptors to IGF-I-stimulated downstream events, such as ERK1/2 activation, varies in a cell type-dependent manner.

The present study was performed on the basis of the hypothesis that the low molecular weight (MW) compounds, DMSO and glycerol, permeate the cell and interact hydrophobically with intracellular proteins, thereby perturbing the cytoskeletal architecture of frozen cells and diminishing islet cell integrity and function. Isolated rat islets were cultured overnight (18-24 h) at 37 degrees C in RPMI medium supplemented with 10% fetal calf serum and 1% mixture of penicillin/streptomycin. Using a programmable temperature controller, samples of precounted islets were then frozen under liquid nitrogen, in the presence of either 2 M DMSO (MW = 0.078 kDa), 3 M glycerol (MW = 0.092 kDa), 5% polyethylene glycol (PEG, MW = 20 kDa), or 10% polyvinylpyrrolidone (PVP, MW = 40 kDa), and stored at -80 degrees C for 1 week. Following thawing and overnight (18-24 h) culture, intact islet recovery was determined by islet counting after dithizone staining. Islet function was assessed by determination of glucose-stimulated insulin secretion in perifusion experiments with Krebs-Ringer bicarbonate buffer, pH 7.4, containing either basal (3.3 mM) or high (16.7 mM) glucose concentrations. The assessment of islet recovery and function of all cryopreserved samples was performed only after thawing and overnight culture (18-24 h) of islets. The mean +/- SEM percent intact islet recovery was higher with PVP compared with DMSO (82 +/- 4.6 vs. 62.7 +/- 3.1%, respectively, p < 0.005, n = 9). Furthermore, the glucose stimulation index of insulin secretion by islets taken from samples frozen with PEG and PVP, after thawing and overnight culture, was comparable to that of freshly isolated islets, in contrast to DMSO and glycerol. There was no significant difference in intact islet recovery and function between samples frozen with PVP and those frozen with PEG. Samples frozen with DMSO and glycerol had similar results in islet recovery and function. These data show that PVP is a new and potent cryoprotectant for islet cell freezing.