Raf kinase inhibitor protein (RKIP) dimer formation controls its target switch from Raf1 to G protein-coupled receptor kinase (GRK) 2

Proteins controlling cellular networks have evolved distinct mechanisms to ensure specificity in protein-protein interactions. Raf kinase inhibitor protein (RKIP) is a multifaceted kinase modulator, but it is not well understood how this small protein (21 kDa) can coordinate its diverse signaling functions. Raf1 and G protein-coupled receptor kinase (GRK) 2 are direct interaction partners of RKIP and thus provide the possibility to untangle the mechanism of its target specificity. Here, we identify RKIP dimer formation as an important mechanistic feature in the target switch from Raf1 to GRK2. Co-immunoprecipitation and cross-linking experiments revealed RKIP dimerization upon phosphorylation of RKIP at serine 153 utilizing purified proteins as well as in cells overexpressing RKIP. A functional phosphomimetic RKIP mutant had a high propensity for dimerization and reproduced the switch from Raf1 to GRK2. RKIP dimerization and GRK2 binding, but not Raf1 interaction, were prevented by a peptide comprising amino acids 127-146 of RKIP, which suggests that this region is critical for dimer formation. Furthermore, a dimeric RKIP mutant displayed a higher affinity to GRK2, but a lower affinity to Raf1. Functional analyses of phosphomimetic as well as dimeric RKIP demonstrated that enhanced dimerization of RKIP translates into decreased Raf1 and increased GRK2 inhibition. The detection of RKIP dimers in a complex with GRK2 in murine hearts implies their physiological relevance. These findings represent a novel mechanistic feature how RKIP can discriminate between its different interaction partners and thus advances our understanding how specific inhibition of kinases can be achieved.     

G protein-coupled Receptor 30 (GPR30) Forms a Plasma Membrane Complex with Membrane-associated Guanylate Kinases (MAGUKs) and Protein Kinase A-anchoring Protein 5 (AKAP5) That Constitutively Inhibits cAMP Production

GPR30, or G protein-coupled estrogen receptor, is a G protein-coupled receptor reported to bind 17β-estradiol (E₂), couple to the G proteins G_s and G_i/o, and mediate non-genomic estrogenic responses. However, controversies exist regarding the receptor pharmacological profile, effector coupling, and subcellular localization. We addressed the role of the type I PDZ motif at the receptor C terminus in receptor trafficking and coupling to cAMP production in HEK293 cells and CHO cells ectopically expressing the receptor and in Madin-Darby canine kidney cells expressing the native receptor. GPR30 was localized both intracellularly and in the plasma membrane and subject to limited basal endocytosis. E₂ and G-1, reported GPR30 agonists, neither stimulated nor inhibited cAMP production through GPR30, nor did they influence receptor localization. Instead, GPR30 constitutively inhibited cAMP production stimulated by a heterologous agonist independently of G_i/o. Moreover, siRNA knockdown of native GPR30 increased cAMP production. Deletion of the receptor PDZ motif interfered with inhibition of cAMP production and increased basal receptor endocytosis. GPR30 interacted with membrane-associated guanylate kinases, including SAP97 and PSD-95, and protein kinase A-anchoring protein (AKAP) 5 in the plasma membrane in a PDZ-dependent manner. Knockdown of AKAP5 or St-Ht31 treatment, to disrupt AKAP interaction with the PKA RIIβ regulatory subunit, decreased inhibition of cAMP production, and St-Ht31 increased basal receptor endocytosis. Therefore, GPR30 forms a plasma membrane complex with a membrane-associated guanylate kinase and AKAP5, which constitutively attenuates cAMP production in response to heterologous agonists independently of G_i/o and retains receptors in the plasma membrane.     

Promiscuous Dimerization of the Growth Hormone Secretagogue Receptor (GHS-R1a) Attenuates Ghrelin-mediated Signaling

G protein-coupled receptors (GPCRs), such as the ghrelin receptor (GHS-R1a), the melanocortin 3 receptor (MC₃), and the serotonin 2C receptor (5-HT_2C), are well known for their key role in the homeostatic control of food intake and energy balance. Ghrelin is the only known gut peptide exerting an orexigenic effect and has thus received much attention as an anti-obesity drug target. In addition, recent data have revealed a critical role for ghrelin in dopaminergic mesolimbic circuits involved in food reward signaling. This study investigates the downstream signaling consequences and ligand-mediated co-internalization following heterodimerization of the GHS-R1a receptor with the dopamine 1 receptor, as well as that of the GHS-R1a-MC₃ heterodimer. In addition, a novel heterodimer between the GHS-R1a receptor and the 5-HT_2C receptor was identified. Interestingly, dimerization of the GHS-R1a receptor with the unedited 5-HT_2C-INI receptor, but not with the partially edited 5-HT_2C-VSV isoform, significantly reduced GHS-R1a agonist-mediated calcium influx, which was completely restored following pharmacological blockade of the 5-HT_2C receptor. These results combined suggest a potential novel mechanism for fine-tuning GHS-R1a receptor-mediated activity via promiscuous dimerization of the GHS-R1a receptor with other G protein-coupled receptors involved in appetite regulation and food reward. These findings may uncover novel mechanisms of significant relevance for the future pharmacological targeting of the GHS-R1a receptor in the homeostatic regulation of energy balance and in hedonic appetite signaling, both of which play a significant role in the development of obesity.     

G Protein-coupled Receptor Kinases of the GRK4 Protein Subfamily Phosphorylate Inactive G Protein-coupled Receptors (GPCRs)

G protein-coupled receptor (GPCR) kinases (GRKs) play a key role in homologous desensitization of GPCRs. It is widely assumed that most GRKs selectively phosphorylate only active GPCRs. Here, we show that although this seems to be the case for the GRK2/3 subfamily, GRK5/6 effectively phosphorylate inactive forms of several GPCRs, including β2-adrenergic and M2 muscarinic receptors, which are commonly used as representative models for GPCRs. Agonist-independent GPCR phosphorylation cannot be explained by constitutive activity of the receptor or membrane association of the GRK, suggesting that it is an inherent ability of GRK5/6. Importantly, phosphorylation of the inactive β₂-adrenergic receptor enhanced its interactions with arrestins. Arrestin-3 was able to discriminate between phosphorylation of the same receptor by GRK2 and GRK5, demonstrating preference for the latter. Arrestin recruitment to inactive phosphorylated GPCRs suggests that not only agonist activation but also the complement of GRKs in the cell regulate formation of the arrestin-receptor complex and thereby G protein-independent signaling.     

Proteinase-activated Receptor 2 (PAR2) Decreases Apoptosis in Colonic Epithelial Cells

Mucosal biopsies from inflamed colon of inflammatory bowel disease patients exhibit elevated epithelial apoptosis compared with those from healthy individuals, disrupting mucosal homeostasis and perpetuating disease. Therapies that decrease intestinal epithelial apoptosis may, therefore, ameliorate inflammatory bowel disease, but treatments that specifically target apoptotic pathways are lacking. Proteinase-activated receptor-2 (PAR2), a G protein-coupled receptor activated by trypsin-like serine proteinases, is expressed on intestinal epithelial cells and stimulates mitogenic pathways upon activation. We sought to determine whether PAR2 activation and signaling could rescue colonic epithelial (HT-29) cells from apoptosis induced by proapoptotic cytokines that are increased during inflammatory bowel disease. The PAR2 agonists 2-furoyl-LIGRLO (2f-LI), SLIGKV and trypsin all significantly reduced cleavage of caspase-3, -8, and -9, poly(ADP-ribose) polymerase, and the externalization of phosphatidylserine after treatment of cells with IFN-γ and TNF-α. Knockdown of PAR2 with siRNA eliminated the anti-apoptotic effect of 2f-LI and increased the sensitivity of HT-29 cells to cytokine-induced apoptosis. Concurrent inhibition of both MEK1/2 and PI3K was necessary to inhibit PAR2-induced survival. 2f-LI was found to increase phosphorylation and inactivation of pro-apoptotic BAD at Ser¹¹² and Ser¹³⁶ by MEK1/2 and PI3K-dependent signaling, respectively. PAR2 activation also increased the expression of anti-apoptotic MCL-1. Simultaneous knockdown of both BAD and MCL-1 had minimal effects on PAR2-induced survival, whereas single knockdown had no effect. We conclude that PAR2 activation reduces cytokine-induced epithelial apoptosis via concurrent stimulation of MEK1/2 and PI3K but little involvement of MCL-1 and BAD. Our findings represent a novel mechanism whereby serine proteinases facilitate epithelial cell survival and may be important in the context of colonic healing.     

Platelet-derived Growth Factor Differentially Regulates the Expression and Post-translational Modification of Versican by Arterial Smooth Muscle Cells through Distinct Protein Kinase C and Extracellular Signal-regulated Kinase Pathways

The synthesis of proteoglycans involves steps that regulate both protein and glycosaminoglycan (GAG) synthesis, but it is unclear whether these two pathways are regulated by the same or different signaling pathways. We therefore investigated signaling pathways involved in platelet-derived growth factor (PDGF)-mediated increases in versican core protein and GAG chain synthesis in arterial smooth muscle cells (ASMCs). PDGF treatment of ASMCs resulted in increased versican core protein synthesis and elongation of GAG chains attached to the versican core protein. The effects of PDGF on versican mRNA were blocked by inhibiting either protein kinase C (PKC) or the ERK pathways, whereas the GAG elongation effect of PDGF was blocked by PKC inhibition but not by ERK inhibition. Interestingly, blocking protein synthesis in the presence of cycloheximide abolished the PDGF effect, but not in the presence of xyloside, indicating that GAG synthesis that results from PKC activation is independent from de novo protein synthesis. PDGF also stimulated an increase in the chondroitin-6-sulfate to chondroitin-4-sulfate ratio of GAG chains on versican, and this effect was blocked by PKC inhibitors. These data show that PKC activation is sufficient to cause GAG chain elongation, but both PKC and ERK activation are required for versican mRNA core protein expression. These results indicate that different signaling pathways control different aspects of PDGF-stimulated versican biosynthesis by ASMCs. These data will be useful in designing strategies to interfere with the synthesis of this proteoglycan in various disease states.     

Inhibition of G-protein-coupled Receptor Kinase 2 (GRK2) Triggers the Growth-promoting Mitogen-activated Protein Kinase (MAPK) Pathway

Inhibition of G-protein-coupled receptor kinase 2 (GRK2) is an emerging treatment option for heart failure. Because GRK2 is also indispensable for growth and development, we analyzed the impact of GRK2 inhibition on cell growth and proliferation. Inhibition of GRK2 by the dominant-negative GRK2-K220R did not affect the proliferation of cultured cells. In contrast, upon xenograft transplantation of cells into immunodeficient mice, the dominant-negative GRK2-K220R or a GRK2-specific peptide inhibitor increased tumor mass. The enhanced tumor growth upon GRK2 inhibition was attributed to the growth-promoting MAPK pathway because dual inhibition of the GRK2 and RAF-MAPK axis by the Raf kinase inhibitor protein (RKIP) did not increase tumor mass. The MAPK cascade contributed to the cardioprotective profile of GRK2 inhibition by preventing cardiomyocyte death, whereas dual inhibition of RAF/MAPK and GRK2 by RKIP induced cardiomyocyte apoptosis, cardiac dysfunction, and signs of heart failure. Thus, cardioprotective signaling induced by GRK2 inhibition is overlapping with tumor growth promotion.     

Role of SAP97 Protein in the Regulation of Corticotropin-releasing Factor Receptor 1 Endocytosis and Extracellular Signal-regulated Kinase 1/2 Signaling

The corticotropin-releasing factor (CRF) receptor 1 (CRFR1) is a target for the treatment of psychiatric diseases such as depression, schizophrenia, anxiety disorder, and bipolar disorder. The carboxyl-terminal tail of the CRFR1 terminates in a PDZ-binding motif that provides a potential site for the interaction of PSD-95/Discs Large/Zona Occludens 1 (PDZ) domain-containing proteins. In this study, we found that CRFR1 interacts with synapse-associated protein 97 (SAP97; also known as DLG1) by co-immunoprecipitation in human embryonic 293 (HEK 293) cells and cortical brain lysates and that this interaction is dependent upon an intact PDZ-binding motif at the end of the CRFR1 carboxyl-terminal tail. Similarly, we demonstrated that SAP97 is recruited to the plasma membrane in HEK 293 cells expressing CRFR1 and that mutation of the CRFR1 PDZ-binding motif results in the redistribution of SAP97 into the cytoplasm. Overexpression of SAP97 antagonized agonist-stimulated CRFR1 internalization, whereas single hairpin (shRNA) knockdown of endogenous SAP97 in HEK 293 cells resulted in increased agonist-stimulated CRFR1 endocytosis. CRFR1 was internalized as a complex with SAP97 resulting in the redistribution of SAP97 to endocytic vesicles. Overexpression or shRNA knockdown of SAP97 did not significantly affect CRFR1-mediated cAMP formation, but SAP97 knockdown did significantly attenuate CRFR1-stimulated ERK1/2 phosphorylation in a PDZ interaction-independent manner. Taken together, our studies show that SAP97 interactions with CRFR1 attenuate CRFR1 endocytosis and that SAP97 is involved in coupling G protein-coupled receptors to the activation of the ERK1/2 signaling pathway.     

Sphingosine 1-Phosphate Receptor 4 Uses HER2 (ERBB2) to Regulate Extracellular Signal Regulated Kinase-1/2 in MDA-MB-453 Breast Cancer Cells

We demonstrate here that the bioactive lipid sphingosine 1-phosphate (S1P) uses sphingosine 1-phosphate receptor 4 (S1P₄) and human epidermal growth factor receptor 2 (HER2) to stimulate the extracellular signal regulated protein kinase 1/2 (ERK-1/2) pathway in MDA-MB-453 cells. This was based on several lines of evidence. First, the S1P stimulation of ERK-1/2 was abolished by JTE013, which we show here is an S1P_2/4 antagonist and reduced by siRNA knockdown of S1P₄. Second, the S1P-stimulated activation of ERK-1/2 was almost completely abolished by a HER2 inhibitor (ErbB2 inhibitor II) and reduced by siRNA knockdown of HER2 expression. Third, phyto-S1P, which is an S1P₄ agonist, stimulated ERK-1/2 activation in an S1P₄- and HER2-dependent manner. Fourth, FTY720 phosphate, which is an agonist at S1P_1,3,4,5 but not S1P₂ stimulated activation of ERK-1/2. Fifth, S1P stimulated the tyrosine phosphorylation of HER2, which was reduced by JTE013. HER2 which is an orphan receptor tyrosine kinase is the preferred dimerization partner of the EGF receptor. However, EGF-stimulated activation of ERK-1/2 was not affected by siRNA knockdown of HER2 or by ErbB2 (epidermal growth factor receptor 2 (or HER2)) inhibitor II in MDA-MB-453 cells. Moreover, S1P-stimulated activation of ERK-1/2 does not require an EGF receptor. Thus, S1P and EGF function in a mutually exclusive manner. In conclusion, the magnitude of the signaling gain on the ERK-1/2 pathway produced in response to S1P can be increased by HER2 in MDA-MB-453 cells. The linkage of S1P with an oncogene suggests that S1P and specifically S1P₄ may have an important role in breast cancer progression.     

GPR126 Protein Regulates Developmental and Pathological Angiogenesis through Modulation of VEGFR2 Receptor Signaling

Angiogenesis, the formation of new blood vessels from pre-existing ones, is essential for development, wound healing, and tumor progression. The VEGF pathway plays irreplaceable roles during angiogenesis, but how other signals cross-talk with and modulate VEGF cascades is not clearly elucidated. Here, we identified that Gpr126, an endothelial cell-enriched gene, plays an important role in angiogenesis by regulating endothelial cell proliferation, migration, and tube formation. Knockdown of Gpr126 in the mouse retina resulted in the inhibition of hypoxia-induced angiogenesis. Interference of Gpr126 expression in zebrafish embryos led to defects in intersegmental vessel formation. Finally, we identified that GPR126 regulated the expression of VEGFR2 by targeting STAT5 and GATA2 through the cAMP-PKA-cAMP-response element-binding protein signaling pathway during angiogenesis. Our findings illustrate that GPR126 modulates both physiological and pathological angiogenesis through VEGF signaling, providing a potential target for the treatment of angiogenesis-related diseases.     

Mitogen-activated Protein Kinase (MAPK) Hyperactivation and Enhanced NRAS Expression Drive Acquired Vemurafenib Resistance in V600E BRAF Melanoma Cells

Although targeting the V600E activating mutation in the BRAF gene, the most common genetic abnormality in melanoma, has shown clinical efficacy in melanoma patients, response is, invariably, short lived. To better understand mechanisms underlying this acquisition of resistance to BRAF-targeted therapy in previously responsive melanomas, we induced vemurafenib resistance in two V600E BRAF+ve melanoma cell lines, A375 and DM443, by serial in vitro vemurafenib exposure. The resulting approximately 10-fold more vemurafenib-resistant cell lines, A375rVem and D443rVem, had higher growth rates and showed differential collateral resistance to cisplatin, melphalan, and temozolomide. The acquisition of vemurafenib resistance was associated with significantly increased NRAS levels in A375rVem and D443rVem, increased activation of the prosurvival protein, AKT, and the MAPKs, ERK, JNK, and P38, which correlated with decreased levels of the MAPK inhibitor protein, GSTP1. Despite the increased NRAS, whole exome sequencing showed no NRAS gene mutations. Inhibition of all three MAPKs and siRNA-mediated NRAS suppression both reversed vemurafenib resistance significantly in A375rVem and DM443rVem. Together, the results indicate a mechanism of acquired vemurafenib resistance in V600E BRAF+ve melanoma cells that involves increased activation of all three human MAPKs and the PI3K pathway, as well as increased NRAS expression, which, contrary to previous reports, was not associated with mutations in the NRAS gene. The data highlight the complexity of the acquired vemurafenib resistance phenotype and the challenge of optimizing BRAF-targeted therapy in this disease. They also suggest that targeting the MAPKs and/or NRAS may provide a strategy to mitigate such resistance in V600E BRAF+ve melanoma.     

A Kinase Inhibitor Screen Reveals Protein Kinase C-dependent Endocytic Recycling of ErbB2 in Breast Cancer Cells

ErbB2 overexpression drives oncogenesis in 20–30% cases of breast cancer. Oncogenic potential of ErbB2 is linked to inefficient endocytic traffic into lysosomes and preferential recycling. However, regulation of ErbB2 recycling is incompletely understood. We used a high-content immunofluorescence imaging-based kinase inhibitor screen on SKBR-3 breast cancer cells to identify kinases whose inhibition alters the clearance of cell surface ErbB2 induced by Hsp90 inhibitor 17-AAG. Less ErbB2 clearance was observed with broad-spectrum PKC inhibitor Ro 31-8220. A similar effect was observed with Go 6976, a selective inhibitor of classical Ca²⁺-dependent PKCs (α, β1, βII, and γ). PKC activation by PMA promoted surface ErbB2 clearance but without degradation, and ErbB2 was observed to move into a juxtanuclear compartment where it colocalized with PKC-α and PKC-δ together with the endocytic recycling regulator Arf6. PKC-α knockdown impaired the juxtanuclear localization of ErbB2. ErbB2 transit to the recycling compartment was also impaired upon PKC-δ knockdown. PMA-induced Erk phosphorylation was reduced by ErbB2 inhibitor lapatinib, as well as by knockdown of PKC-δ but not that of PKC-α. Our results suggest that activation of PKC-α and -δ mediates a novel positive feedback loop by promoting ErbB2 entry into the endocytic recycling compartment, consistent with reported positive roles for these PKCs in ErbB2-mediated tumorigenesis. As the endocytic recycling compartment/pericentrion has emerged as a PKC-dependent signaling hub for G-protein-coupled receptors, our findings raise the possibility that oncogenesis by ErbB2 involves previously unexplored PKC-dependent endosomal signaling.     

Phosphorylation of G protein-coupled receptor kinase 1 (GRK1) is regulated by light but independent of phototransduction in rod photoreceptors

Phosphorylation of rhodopsin by G protein-coupled receptor kinase 1 (GRK1, or rhodopsin kinase) is critical for the deactivation of the phototransduction cascade in vertebrate photoreceptors. Based on our previous studies in vitro, we predicted that Ser(21) in GRK1 would be phosphorylated by cAMP-dependent protein kinase (PKA) in vivo. Here, we report that dark-adapted, wild-type mice demonstrate significantly elevated levels of phosphorylated GRK1 compared with light-adapted animals. Based on comparatively slow half-times for phosphorylation and dephosphorylation, phosphorylation of GRK1 by PKA is likely to be involved in light and dark adaptation. In mice missing the gene for adenylyl cyclase type 1, levels of phosphorylated GRK1 were low in retinas from both dark- and light-adapted animals. These data are consistent with reports that cAMP levels are high in the dark and low in the light and also indicate that cAMP generated by adenylyl cyclase type 1 is required for phosphorylation of GRK1 on Ser(21). Surprisingly, dephosphorylation was induced by light in mice missing the rod transducin α-subunit. This result indicates that phototransduction does not play a direct role in the light-dependent dephosphorylation of GRK1.     

Cyclic AMP Signaling Reduces Sirtuin 6 Expression in Non-small Cell Lung Cancer Cells by Promoting Ubiquitin-Proteasomal Degradation via Inhibition of the Raf-MEK-ERK (Raf/Mitogen-activated Extracellular Signal-regulated Kinase/Extracellular Signal-regulated Kinase) Pathway

The cAMP signaling system regulates various cellular functions, including metabolism, gene expression, and death. Sirtuin 6 (SIRT6) removes acetyl groups from histones and regulates genomic stability and cell viability. We hypothesized that cAMP modulates SIRT6 activity to regulate apoptosis. Therefore, we examined the effects of cAMP signaling on SIRT6 expression and radiation-induced apoptosis in lung cancer cells. cAMP signaling in H1299 and A549 human non-small cell lung cancer cells was activated via the expression of constitutively active Gα_s plus treatment with prostaglandin E2 (PGE2), isoproterenol, or forskolin. The expression of sirtuins and signaling molecules were analyzed by Western blotting. Activation of cAMP signaling reduced SIRT6 protein expression in lung cancer cells. cAMP signaling increased the ubiquitination of SIRT6 protein and promoted its degradation. Treatment with MG132 and inhibiting PKA with H89 or with a dominant-negative PKA abolished the cAMP-mediated reduction in SIRT6 levels. Treatment with PGE2 inhibited c-Raf activation by increasing inhibitory phosphorylation at Ser-259 in a PKA-dependent manner, thereby inhibiting downstream MEK-ERK signaling. Inhibiting ERK with inhibitors or with dominant-negative ERKs reduced SIRT6 expression, whereas activation of ERK by constitutively active MEK abolished the SIRT6-depleting effects of PGE2. cAMP signaling also augmented radiation-induced apoptosis in lung cancer cells. This effect was abolished by exogenous expression of SIRT6. It is concluded that cAMP signaling reduces SIRT6 expression by promoting its ubiquitin-proteasome-dependent degradation, a process mediated by the PKA-dependent inhibition of the Raf-MEK-ERK pathway. Reduced SIRT6 expression mediates the augmentation of radiation-induced apoptosis by cAMP signaling in lung cancer cells.     

Structural Basis for the Binding Specificity of Human Recepteur d'Origine Nantais (RON) Receptor Tyrosine Kinase to Macrophage-stimulating Protein

Recepteur d''origine nantais (RON) receptor tyrosine kinase and its ligand, serum macrophage-stimulating protein (MSP), play important roles in inflammation, cell growth, migration, and epithelial to mesenchymal transition during tumor development. The binding of mature MSPαβ (disulfide-linked α- and β-chains) to RON ectodomain modulates receptor dimerization, followed by autophosphorylation of tyrosines in the cytoplasmic receptor kinase domains. Receptor recognition is mediated by binding of MSP β-chain (MSPβ) to the RON Sema. Here we report the structure of RON Sema-PSI-IPT₁ (SPI₁) domains in complex with MSPβ at 3.0 Å resolution. The MSPβ serine protease-like β-barrel uses the degenerate serine protease active site to recognize blades 2, 3, and 4 of the β-propeller fold of RON Sema. Despite the sequence homology between RON and MET receptor tyrosine kinase and between MSP and hepatocyte growth factor, it is well established that there is no cross-reactivity between the two receptor-ligand systems. Comparison of the structure of RON SPI₁ in complex with MSPβ and that of MET receptor tyrosine kinase Sema-PSI in complex with hepatocyte growth factor β-chain reveals the receptor-ligand selectivity determinants. Analytical ultracentrifugation studies of the SPI₁-MSPβ interaction confirm the formation of a 1:1 complex. SPI₁ and MSPαβ also associate primarily as a 1:1 complex with a binding affinity similar to that of SPI₁-MSPβ. In addition, the SPI₁-MSPαβ ultracentrifuge studies reveal a low abundance 2:2 complex with ∼10-fold lower binding affinity compared with the 1:1 species. These results support the hypothesis that the α-chain of MSPαβ mediates RON dimerization.     

Macromolecular Composition Dictates Receptor and G Protein Selectivity of Regulator of G Protein Signaling (RGS) 7 and 9-2 Protein Complexes in Living Cells

Regulator of G protein signaling (RGS) proteins play essential roles in the regulation of signaling via G protein-coupled receptors (GPCRs). With hundreds of GPCRs and dozens of G proteins, it is important to understand how RGS regulates selective GPCR-G protein signaling. In neurons of the striatum, two RGS proteins, RGS7 and RGS9-2, regulate signaling by μ-opioid receptor (MOR) and dopamine D2 receptor (D2R) and are implicated in drug addiction, movement disorders, and nociception. Both proteins form trimeric complexes with the atypical G protein β subunit Gβ5 and a membrane anchor, R7BP. In this study, we examined GTPase-accelerating protein (GAP) activity as well as Gα and GPCR selectivity of RGS7 and RGS9-2 complexes in live cells using a bioluminescence resonance energy transfer-based assay that monitors dissociation of G protein subunits. We showed that RGS9-2/Gβ5 regulated both Gi and Go with a bias toward Go, but RGS7/Gβ5 could serve as a GAP only for Go. Interestingly, R7BP enhanced GAP activity of RGS7 and RGS9-2 toward Go and Gi and enabled RGS7 to regulate Gi signaling. Neither RGS7 nor RGS9-2 had any activity toward Gz, Gs, or Gq in the absence or presence of R7BP. We also observed no effect of GPCRs (MOR and D2R) on the G protein bias of R7 RGS proteins. However, the GAP activity of RGS9-2 showed a strong receptor preference for D2R over MOR. Finally, RGS7 displayed an four times greater GAP activity relative to RGS9-2. These findings illustrate the principles involved in establishing G protein and GPCR selectivity of striatal RGS proteins.     

Adaptor Protein LRAP25 Mediates Myotonic Dystrophy Kinase-related Cdc42-binding Kinase (MRCK) Regulation of LIMK1 Protein in Lamellipodial F-actin Dynamics

Myotonic dystrophy kinase-related Cdc42-binding kinase (MRCK) has been shown to localize to the lamella of mammalian cells through its interaction with an adaptor protein, leucine repeat adaptor protein 35a (LRAP35a), which links it with myosin 18A (MYO18A) for activation of the lamellar actomyosin network essential for cell migration. Here, we report the identification of another adaptor protein LRAP25 that mediates MRCK association with LIM kinase 1 (LIMK1). The lamellipodium-localized LRAP25-MRCK complex is essential for the regulation of local LIMK1 and its downstream F-actin regulatory factor cofilin. Functionally, inhibition of either MRCK or LRAP25 resulted in a marked suppression of LIMK1 activity and down-regulation of cofilin phosphorylation in response to aluminum fluoride induction in B16-F1 cells, which eventually resulted in deregulation of lamellipodial F-actin and reorganization of cytoskeletal structures causing defects in cell polarization and motility. These biochemical and functional characterizations thus underline the functional relevance of the LRAP25-MRCK complex in LIMK1-cofilin signaling and the importance of LRAP adaptors as key determinants of MRCK cellular localization and downstream specificities.     

Systematic Quantification of Negative Feedback Mechanisms in the Extracellular Signal-regulated Kinase (ERK) Signaling Network

Cell responses are actuated by tightly controlled signal transduction pathways. Although the concept of an integrated signaling network replete with interpathway cross-talk and feedback regulation is broadly appreciated, kinetic data of the type needed to characterize such interactions in conjunction with mathematical models are lacking. In mammalian cells, the Ras/ERK pathway controls cell proliferation and other responses stimulated by growth factors, and several cross-talk and feedback mechanisms affecting its activation have been identified. In this work, we take a systematic approach to parse the magnitudes of multiple regulatory mechanisms that attenuate ERK activation through canonical (Ras-dependent) and non-canonical (PI3K-dependent) pathways. In addition to regulation of receptor and ligand levels, we consider three layers of ERK-dependent feedback: desensitization of Ras activation, negative regulation of MEK kinase (e.g. Raf) activities, and up-regulation of dual-specificity ERK phosphatases. Our results establish the second of these as the dominant mode of ERK self-regulation in mouse fibroblasts. We further demonstrate that kinetic models of signaling networks, trained on a sufficient diversity of quantitative data, can be reasonably comprehensive, accurate, and predictive in the dynamical sense.