Moshmi Bhattacharya

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.

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.

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.

The human gonadotropin releasing hormone type I receptor is a functional intracellular GPCR expressed on the nuclear membrane

The mammalian type I gonadotropin releasing hormone receptor (GnRH-R) is a structurally unique G protein-coupled receptor (GPCR) that lacks cytoplasmic tail sequences and displays inefficient plasma membrane expression (PME). Compared to its murine counterparts, the primate type I receptor is inefficiently folded and retained in the endoplasmic reticulum (ER) leading to a further reduction in PME. The decrease in PME and concomitant increase in intracellular localization of the mammalian GnRH-RI led us to characterize the spatial distribution of the human and mouse GnRH receptors in two human cell lines, HEK 293 and HTR-8/SVneo. In both human cell lines we found the receptors were expressed in the cytoplasm and were associated with the ER and nuclear membrane. A molecular analysis of the receptor protein sequence led us to identify a putative monopartite nuclear localization sequence (NLS) in the first intracellular loop of GnRH-RI. Surprisingly, however, neither the deletion of the NLS nor the addition of the Xenopus GnRH-R cytoplasmic tail sequences to the human receptor altered its spatial distribution. Finally, we demonstrate that GnRH treatment of nuclei isolated from HEK 293 cells expressing exogenous GnRH-RI triggers a significant increase in the acetylation and phosphorylation of histone H3, thereby revealing that the nuclear-localized receptor is functional. Based on our findings, we conclude that the mammalian GnRH-RI is an intracellular GPCR that is expressed on the nuclear membrane. This major and novel discovery causes us to reassess the signaling potential of this physiologically and clinically important receptor.

GPR54 Regulates ERK1/2 Activity and Hypothalamic Gene Expression in a Gαq/11 and β-Arrestin-Dependent Manner

G protein-coupled receptor 54 (GPR54) is a Gq/11-coupled 7 transmembrane-spanning receptor (7TMR). Activation of GPR54 by kisspeptin (Kp) stimulates PIP2 hydrolysis, Ca2+ mobilization and ERK1/2 MAPK phosphorylation. Kp and GPR54 are established regulators of the hypothalamic-pituitary-gonadal (HPG) axis and loss-of-function mutations in GPR54 are associated with an absence of puberty and hypogonadotropic hypogonadism, thus defining an important role of the Kp/GPR54 signaling system in reproductive function. Given the tremendous physiological and clinical importance of the Kp/GPR54 signaling system, we explored the contributions of the GPR54-coupled Gq/11 and β-arrestin pathways on the activation of a major downstream signaling molecule, ERK, using Gq/11 and β-arrestin knockout mouse embryonic fibroblasts. Our study revealed that GPR54 employs the Gq/11 and β-arrestin-2 pathways in a co-dependent and temporally overlapping manner to positively regulate ERK activity and pERK nuclear localization. We also show that while β-arrestin-2 potentiates GPR54 signaling to ERK, β-arrestin-1 inhibits it. Our data also revealed that diminished β-arrestin-1 and -2 expression in the GT1-7 GnRH hypothalamic neuronal cell line triggered distinct patterns of gene expression following Kp-10 treatment. Thus, β-arrestin-1 and -2 also regulate distinct downstream responses in gene expression. Finally, we showed that GPR54, when uncoupled from the Gq/11 pathway, as is the case for several naturally occurring GPR54 mutants associated with hypogonadotropic hypogonadism, continues to regulate gene expression in a G protein-independent manner. These new and exciting findings add significantly to our mechanistic understanding of how this important receptor signals intracellularly in response to kisspeptin stimulation.

Dual regulation of lysophosphatidic acid (LPA1) receptor signalling by Ral and GRK.

Lysophosphatidic acid (LPA) is a major constituent of blood and is involved in a variety of physiological and pathophysiological processes. LPA signals via the ubiquitously expressed G protein-coupled receptors (GPCRs), LPA(1) and LPA(2) that are specific for LPA. However, in large, the molecular mechanisms that regulate the signalling of these receptors are unknown. We show that the small GTPase RalA associates with both LPA(1) and LPA(2) in human embryonic kidney (HEK 293) cells and that stimulation of LPA(1) receptors with LPA triggers the activation of RalA. While RalA was not found to play a role in the endocytosis of LPA receptors, we reveal that LPA(1) receptor stimulation promoted Ral-dependent phospholipase C activity. Furthermore, we found that GRK2 is required for the desensitization of LPA(1) and LPA(2) and have identified a novel interaction between RalA and GRK2, which is promoted by LPA(1) receptor activity. Taken together, these results establish RalA and GRK2 as key regulators of LPA receptor signalling and demonstrate for the first time that LPA(1) activity facilitates the formation of a novel protein complex between these two proteins.