Andy V. Babwah

β-Arrestins regulate a Ral-GDS–Ral effector pathway that mediates cytoskeletal reorganization

β-Arrestins are important in chemoattractant receptor-induced granule release, a process that may involve Ral-dependent regulation of the actin cytoskeleton. We have identified the Ral GDP dissociation stimulator (Ral-GDS) as a β-arrestin-binding protein by yeast two-hybrid screening and co-immunoprecipitation from human polymorphonuclear neutrophilic leukocytes (PMNs). Under basal conditions, Ral-GDS is localized to the cytosol and remains inactive in a complex formed with β-arrestins. In response to formyl-Met-Leu-Phe (fMLP) receptor stimulation, β-arrestin–Ral-GDS protein complexes dissociate and Ral-GDS translocates with β-arrestin from the cytosol to the plasma membrane, resulting in the Ras-independent activation of the Ral effector pathway required for cytoskeletal rearrangement. The subsequent re-association of β-arrestin–Ral-GDS complexes is associated with the inactivation of Ral signalling. Thus, β-arrestins regulate multiple steps in the Ral-dependent processes that result in chemoattractant-induced cytoskeletal reorganization.

Regulation of angiotensin II type 1A receptor intracellular retention, degradation and recycling by Rab5, Rab7 and Rab11 GTPases

Previous studies have demonstrated that the interaction of the angiotensin II type 1A receptor (AT1AR) carboxyl-terminal tail with Rab5a may modulate Rab5a activity, leading to the homotypic fusion of endocytic vesicles. Therefore, we have investigated whether AT1AR/Rab5a interactions mediate the retention of AT1AR·β-arrestin complexes in early endosomes and whether the overexpression of Rab7 and Rab11 GTPases influences AT1AR lysosomal degradation and plasma membrane recycling. We found that internalized AT1AR was retained in Rab5a-positive early endosomes and was neither targeted to lysosomes nor recycled back to the cell surface, whereas a mutant defective in Rab5a binding, AT1AR-(1-349), was targeted to lysosomes for degradation. However, the loss of Rab5a binding to the AT1AR carboxyl-terminal tail did not promote AT1AR recycling. Rather, it was the stable binding of β-arrestin to the AT1AR that prevented, at least in part, AT1AR recycling. The overexpression of wild-type Rab7 and Rab7-Q67L resulted in both increased AT1AR degradation and AT1AR targeting to lysosomes. The Rab7 expression-dependent transition of “putative” AT1AR·β-arrestin complexes to late endosomes was blocked by the expression of dominant-negative Rab5a-S34N. Rab11 overexpression established AT1AR recycling and promoted the redistribution of AT1AR·β-arrestin complexes from early to recycling endosomes. Taken together, our data suggest that Rab5, Rab7, and Rab11 work in concert with one another to regulate the intracellular trafficking patterns of the AT1AR.

β-Arrestin/Ral Signaling Regulates Lysophosphatidic Acid-Mediated Migration and Invasion of Human Breast Tumor Cells

The lipid mediator lysophosphatidic acid (LPA) plays a role in cancer progression and signals via specific G protein–coupled receptors, LPA1-3. LPA has been shown to enhance the metastasis of breast carcinoma cells to bone. However, the mechanisms by which LPA receptors regulate breast cancer cell migration and invasion remain unclear. Breast cancer cell proliferation has been shown to be stimulated by Ral GTPases, a member of the Ras superfamily. Ral activity can be regulated by the multifunctional protein β-arrestin. We now show that HS578T and MDA-MB-231 breast cancer cells and MDA-MB-435 melanoma cells have higher expression of β-arrestin 1 mRNA compared with the nontumorigenic mammary MCF-10A cells. Moreover, we found that the mRNA levels of LPA1, LPA2, β-arrestin 2, and Ral GTPases are elevated in the advanced stages of breast cancer. LPA stimulates the migration and invasion of MDA-MB-231 cells, but not of MCF-10A cells, and this is mediated by pertussis toxin–sensitive G proteins and LPA1. However, ectopic expression of LPA1 in MCF-10A cells caused these cells to acquire an invasive phenotype. Gene knockdown of either β-arrestin or Ral proteins significantly impaired LPA-stimulated migration and invasion. Thus, our data show a novel role for β-arrestin/Ral signaling in mediating LPA-induced breast cancer cell migration and invasion, two important processes in metastasis. (Mol Cancer Res 2009;7(7):1064–77)

Small GTP-binding protein-coupled receptors

Heterotrimeric GPCRs (G-protein-coupled receptors) form the largest group of integral membrane receptor proteins and mediate diverse physiological processes. In addition to signalling via heterotrimeric G-proteins, GPCRs can also signal by interacting with various small G-proteins to regulate downstream effector pathways. The small G-protein superfamily is structurally classified into at least five families: the Ras, Rho/Rac/cdc42, Rab, Sar1/Arf and Ran families. They are monomeric G-proteins with molecular masses over the range 20-30 kDa, which function as molecular switches to control many eukaryotic cell functions. Several studies have provided evidence of crosstalk between GPCRs and small G-proteins. It is well documented that GPCR signalling through heterotrimeric G-proteins can lead to the activation of Ras and Rho GTPases. In addition, RhoA, Rabs, ARFs and ARF GEFs (guanine nucleotide-exchange factors) can associate directly with GPCRs, and GPCRs may also function as GEFs for small GTPases. In this review, we summarize the recent progress made in understanding the interaction between GPCRs and small GTPases, focusing on understanding how the association of small G-proteins with GPCRs and GPCR-regulatory proteins may influence GPCR signalling and intracellular trafficking.

Spatial-Temporal Patterning of Metabotropic Glutamate Receptor-mediated Inositol 1,4,5-Triphosphate, Calcium, and Protein Kinase C Oscillations PROTEIN KINASE C-DEPENDENT RECEPTOR PHOSPHORYLATION IS NOT REQUIRED

The metabotropic glutamate receptors (mGluR), mGluR1a and mGluR5a, are G protein-coupled receptors that couple via Gq to the hydrolysis of phosphoinositides, the release of Ca2+ from intracellular stores, and the activation of protein kinase C (PKC). We show here that mGluR1/5 activation results in oscillatory G protein coupling to phospholipase C thereby stimulating oscillations in both inositol 1,4,5-triphosphate formation and intracellular Ca2+ concentrations. The mGluR1/5-stimulated Ca2+ oscillations are translated into the synchronized repetitive redistribution of PKCβII between the cytosol and plasma membrane. The frequency at which mGluR1a and mGluR5a subtypes stimulate inositol 1,4,5-triphosphate, Ca2+, and PKCβII oscillations is regulated by the charge of a single amino acid residue localized within their G protein-coupling domains. However, oscillatory mGluR signaling does not involve the repetitive feedback phosphorylation and desensitization of mGluR activity, since mutation of the putative PKC consensus sites within the first and second intracellular loops as well as the carboxyl-terminal tail does not prevent mGluR1a-stimulated PKCβII oscillations. Furthermore, oscillations in Ca2+ continued in the presence of PKC inhibitors, which blocked PKCβII redistribution from the plasma membrane back into the cytosol. We conclude that oscillatory mGluR signaling represents an intrinsic receptor/G protein coupling property that does not involve PKC feedback phosphorylation.

Ral and Phospholipase D2-Dependent Pathway for Constitutive Metabotropic Glutamate Receptor Endocytosis

G-protein-coupled receptors play a central role in the regulation of neuronal cell communication. Class 1 metabotropic glutamate receptors (mGluRs) mGluR1a and mGluR5a, which are coupled with the hydrolysis of phosphoinositides, are essential for modulating excitatory neurotransmission at glutamatergic synapses. These receptors are constitutively internalized in heterologous cell cultures, neuronal cultures, and intact neuronal tissues. We show here that the small GTP-binding protein Ral, its guanine nucleotide exchange factor RalGDS (Ral GDP dissociation stimulator), and phospholipase D2 (PLD2) are constitutively associated with class 1 mGluRs and regulate constitutive mGluR endocytosis. Moreover, both Ral and PLD2 are colocalized with mGluRs in endocytic vesicles in both human embryonic kidney 293 (HEK 293) cells and neurons. Ral and PLD2 activity is required for the internalization of class 1 mGluRs but is not required for the internalization of the β2-adrenergic receptor. Constitutive class 1 mGluR internalization is not dependent on the downstream Ral effector proteins Ral-binding protein 1 and PLD1 or either ADP-ribosylation factors ARF1 or ARF6. The treatment of HEK 293 cells and neurons with small interfering RNA both downregulates PLD2 expression and blocks mGluR1a and mGluR5a endocytosis. The constitutive internalization of mGluR1a and mGluR5a is also attenuated by the treatment of cells with 1-butanol to prevent PLD2-mediated phosphatidic acid formation. We propose that the formation of a mGluR-scaffolded RalGDS/Ral/PLD2 protein complex provides a novel alternative mechanism to β-arrestins for the constitutive endocytosis of class 1 mGluRs.

Regulation of GPR54 Signaling by GRK2 and β-Arrestin

Kisspeptin and its receptor, GPR54, are major regulators of the hypothalamic-pituitary-gonadal axis as well as regulators of human placentation and tumor metastases. GPR54 is a G(q/11)-coupled G protein-coupled receptor (GPCR), and activation by kisspeptin stimulates phosphatidy linositol 4, 5-biphosphate hydrolysis, Ca(2+) mobilization, arachidonic acid release, and ERK1/2 MAPK phosphorylation. Physiological evidence suggests that GPR54 undergoes agonist-dependent desensitization, but underlying molecular mechanisms are unknown. Furthermore, very little has been reported on the early events that regulate GPR54 signaling. The lack of information in these important areas led to this study. Here we report for the first time on the role of GPCR serine/threonine kinase (GRK)2 and beta-arrestin in regulating GPR54 signaling in human embryonic kidney (HEK) 293 cells, a model cell system for studying the molecular regulation of GPCRs, and genetically modified MDA MB-231 cells, an invasive breast cancer cell line expressing about 75% less beta-arrestin-2 than the control cell line. Our study reveals that in HEK 293 cells, GPR54 is expressed both at the plasma membrane and intracellularly and also that plasma membrane expression is regulated by cytoplasmic tail sequences. We also demonstrate that GPR54 exhibits constitutive activity, internalization, and association with GRK2 and beta- arrestins-1 and 2 through sequences in the second intracellular loop and cytoplasmic tail of the receptor. We also show that GRK2 stimulates the desensitization of GPR54 in HEK 293 cells and that beta-arrestin-2 mediates GPR54 activation of ERK1/2 in MDA-MB-231 cells. The significance of these findings in developing molecular-based therapies for treating certain endocrine-related disorders is discussed.

Taurine prevents cardiomyocyte death by inhibiting NADPH oxidase-mediated calpain activation

Taurine has been shown to prevent cardiomyocyte apoptosis. This study investigated the effects of taurine on NADPH oxidase and calpain activation in mediating apoptosis in cardiomyocytes. Apoptosis was induced by norepinephrine (NE) in cultured adult rat ventricular cardiomyocytes. NE (5 microM) increased NADPH oxidase activation and reactive oxygen species (ROS) production and induced apoptosis. These effects of NE on cardiomyocytes were diminished by taurine (0.5 mg/kg) but not beta-alanine. Inhibition of gp91(phox)-NADPH oxidase or ROS production protected cardiomyocytes from apoptosis. NE also induced calpain-1 activation in cardiomyocytes. This effect of NE on calpain was abrogated by gp91(phox)-NADPH oxidase inhibition or ROS scavengers and was mimicked by H(2)O(2) (25 microM) in cardiomyocytes. Pharmacological inhibitors of calpain or overexpression of calpastatin, a specific calpain inhibitor, blocked calpain activation and prevented cardiomyocyte apoptosis during NE stimulation. Furthermore, taurine treatment inhibited NE- or H(2)O(2)-induced calpain activation in cardiomyocytes. In conclusion, NADPH oxidase induces calpain activation, leading to apoptosis in NE-induced cardiomyocytes. Taurine inhibits NADPH oxidase and calpain activation. Thus, inhibition of NADPH oxidase-mediated calpain activation may be an important mechanism for taurines antiapoptotic action in cardiomyocytes.

Decorin-Mediated Inhibition of Proliferation and Migration of the Human Trophoblast via Different Tyrosine Kinase Receptors

Decorin (DCN), a decidua-derived TGFbeta-binding proteoglycan, negatively regulates proliferation, migration, and invasiveness of human extravillous trophoblast (EVT) cells in a TGFbeta-independent manner. The present study examined underlying mechanisms, in particular possible roles of epidermal growth factor receptor (EGFR), IGF receptor (IGFR)-I, and vascular endothelial growth factor receptor (VEGFR)-2. EVT cell sprouting from first-trimester chorionic villus explants in the presence or absence of TGFbeta-neutralizing antibody was inhibited with DCN, suggesting its negative regulatory role in situ. Inhibition of migration of the human EVT cell line HTR-8/SVneo in transwells undercoated with fibronectin was stronger when cells were briefly preincubated with DCN at 4 C (known to retard dissociation of receptor-ligand complex) than at 37 C, suggesting possible DCN action by cell membrane binding. Pretreatment of cells with an IGFR-I blocking agent, but not two EGFR blocking agents or a VEGFR blocking agent, significantly abrogated migration inhibitory effects of DCN, suggesting the involvement of IGFR-I but not EGFR or VEGFR in migration inhibition by DCN. On the other hand, pretreatment with either of the EGFR blocking agents, or the VEGFR blocking agent but not the IGFR-I blocking agent, blocked proliferation inhibitory effects of DCN, indicating the roles of EGFR and VEGFR, but not IGFR-I in antiproliferative action of DCN. EVT cells expressed EGFR, IGFR-I, and VEGFR-2. IGFR-I and VEGF-R2 were phosphorylated in the presence of their natural ligands as well as DCN, and these events were blocked by pretreatment with respective receptor blocking agents indicating DCN-mediated activation of these receptors. In conclusion, DCN effects on EVT cells are mediated selectively by multiple tyrosine kinase receptors.

GPR54 (KISS1R) transactivates EGFR to promote breast cancer cell invasiveness.

Kisspeptins (Kp), peptide products of the Kisspeptin-1 (KISS1) gene are endogenous ligands for a G protein-coupled receptor 54 (GPR54). Previous findings have shown that KISS1 acts as a metastasis suppressor in numerous cancers in humans. However, recent studies have demonstrated that an increase in KISS1 and GPR54 expression in human breast tumors correlates with higher tumor grade and metastatic potential. At present, whether or not Kp signaling promotes breast cancer cell invasiveness, required for metastasis and the underlying mechanisms, is unknown. We have found that kisspeptin-10 (Kp-10), the most potent Kp, stimulates the invasion of human breast cancer MDA-MB-231 and Hs578T cells using Matrigel-coated Transwell chamber assays and induces the formation of invasive stellate structures in three-dimensional invasion assays. Furthermore, Kp-10 stimulated an increase in matrix metalloprotease (MMP)-9 activity. We also found that Kp-10 induced the transactivation of epidermal growth factor receptor (EGFR). Knockdown of the GPCR scaffolding protein, β-arrestin 2, inhibited Kp-10-induced EGFR transactivation as well as Kp-10 induced invasion of breast cancer cells via modulation of MMP-9 secretion and activity. Finally, we found that the two receptors associate with each other under basal conditions, and FRET analysis revealed that GPR54 interacts directly with EGFR. The stability of the receptor complex formation was increased upon treatment of cells by Kp-10. Taken together, our findings suggest a novel mechanism by which Kp signaling via GPR54 stimulates breast cancer cell invasiveness.