GPR56 C-terminal fragment mediates signal received by N-terminal fragment of another adhesion GPCR Latrophilin1 in neurons.

Adhesion GPCRs (aGPCRs) are a subfamily of GPCRs that are involved in cell adhesion, cell proliferation, and cell migration in various tissues. G protein-coupled receptor proteolytic site (GPS) of aGPCR is required to cleave the extracellular domain autocatalytically, generating two fragments; a N-terminal fragment (NTF) and a C-terminal fragment (CTF) containing seven transmembrane structure. NTF can interact with CTF non-covalently after cleavage, however the physiological significance of the cleavage of aGPCR at GPS, and also the interaction between NTF and CTF have not been fully clarified yet. In this study, we first investigated the expression profiles of two aGPCRs, GPR56/ADGRG1, and LPHN1/ADGRL1 in mouse brain, and found that the NTF and CTF of GPR56 independently expressed in different brain region at different developmental stages. Immunoprecipitation of GPR56CTF co-immunoprecipitated LPHN1NTF from mouse brain and HEK293T cells expressing both fragments. Stimulation with LPHN1 ligand, α-Latrotoxin N4C (αLTXN4C), to cells expressing LPHN1NTF and GPR56CTF increased intracellular Ca2+ concentration ([Ca2+ ]i). We also demonstrated that GPR56KO mouse neurons attenuated their Ca2+ response to αLTXN4C. These results suggest the possibility of functional and chimeric complex containing LPHN1NTF and GPR56CTF in neuronal signal transduction.

Agonistic antibodies reveal the function of GPR56 in human glioma U87-MG cells.

GPR56 is a member of the adhesion G protein-coupled receptor (GPCR) and is highly expressed in parts of tumor cells. The involvement of GPR56 in tumorigenesis has been reported. We generated agonistic monoclonal antibodies against human GPR56 and analyzed the action and signaling pathway of GPR56. The antibodies inhibited cell migration through the Gq and Rho pathway in human glioma U87-MG cells. Co-immunoprecipitation analysis indicated that the interaction between the GPR56 extracellular domain and transmembrane domain was potentiated by agonistic antibodies. These results demonstrated that functional antibodies are invaluable tools for GPCR research and should open a new avenue for therapeutic treatment of tumors.

Increased ubiquitination and the crosstalk of G protein signaling in cardiac myocytes: involvement of Ric-8B in Gs suppression by Gq signal.

Hyperactivation of Gq signaling causes cardiac hypertrophy, and ß-adrenergic receptor-mediated Gs signaling is attenuated in hypertrophic cardiomyocytes. Here, we found the increase in a global ubiquitination in hypertrophic mouse heart. The activation of Gq signaling resulted in the ubiquitination of Gαs in neonatal rat cardiomyocytes, reduced Gαs expression, and suppressed cAMP response to ß-adrenergic receptor stimulation. Ectopic expression of Gαq induced a similar suppression, which is due to the degradation of Gαs by a ubiquitin-proteasome pathway. Co-expression of Ric-8B, a positive regulator of Gαs, effectively canceled the Gαq-induced ubiquitination of Gαs and recovered the cAMP accumulation. In vitro, Gαq competes for the binding of Gαs to Ric-8B. These data show a new role of Ric-8B in the crosstalk of two distinct G protein signaling pathways, which are possibly involved in a part of mechanisms of chronic heart failure.

Phosphorylation of doublecortin by protein kinase A orchestrates microtubule and actin dynamics to promote neuronal progenitor cell migration.

Doublecortin (DCX) is a microtubule-associated protein that is specifically expressed in neuronal cells. Genetic mutation of DCX causes lissencephaly disease. Although the abnormal cortical lamination in lissencephaly is thought to be attributable to neuronal cell migration defects, the regulatory mechanisms governing interactions between DCX and cytoskeleton in the migration of neuronal progenitor cells remain obscure. In this study we found that the G(s) and protein kinase A (PKA) signal elicited by pituitary adenylate cyclase-activating polypeptide promotes neuronal progenitor cells migration. Stimulation of G(s)-PKA signaling prevented microtubule bundling and induced the dissociation of DCX from microtubules in cells. PKA phosphorylated DCX at Ser-47, and the phospho-mimicking mutant DCX-S47E promoted cell migration. Activation of PKA and DCX-S47E induced lamellipodium formation. Pituitary adenylate cyclase-activating polypeptide and DCX-S47E stimulated the activation of Rac1, and DCX-S47E interacted with Asef2, a guanine nucleotide exchange factor for Rac1. Our data reveal a dual reciprocal role for DCX phosphorylation in the regulation of microtubule and actin dynamics that is indispensable for proper brain lamination.

Structural basis for the specific inhibition of heterotrimeric Gq protein by a small molecule.

Heterotrimeric GTP-binding proteins (G proteins) transmit extracellular stimuli perceived by G protein-coupled receptors (GPCRs) to intracellular signaling cascades. Hundreds of GPCRs exist in humans and are the targets of a large percentage of the pharmaceutical drugs used today. Because G proteins are regulated by GPCRs, small molecules that directly modulate G proteins have the potential to become therapeutic agents. However, strategies to develop modulators have been hampered by a lack of structural knowledge of targeting sites for specific modulator binding. Here we present the mechanism of action of the cyclic depsipeptide YM-254890, which is a recently discovered Gq-selective inhibitor. YM-254890 specifically inhibits the GDP/GTP exchange reaction of alpha subunit of Gq protein (Galphaq) by inhibiting the GDP release from Galphaq. X-ray crystal structure analysis of the Galphaqbetagamma-YM-254890 complex shows that YM-254890 binds the hydrophobic cleft between two interdomain linkers connecting the GTPase and helical domains of the Galphaq. The binding stabilizes an inactive GDP-bound form through direct interactions with switch I and impairs the linker flexibility. Our studies provide a novel targeting site for the development of small molecules that selectively inhibit each Galpha subunit and an insight into the molecular mechanism of G protein activation.

KappaB-Ras is a nuclear-cytoplasmic small GTPase that inhibits NF-kappaB activation through the suppression of transcriptional activation of p65/RelA.

NF-κB is an important transcription factor involved in various biological responses, including inflammation, cell differentiation, and tumorigenesis. κB-Ras was identified as an IκB-interacting small GTPase and is reported to disturb cytokine-induced NF-κB activation. In this study, we established that κB-Ras is a novel type of nuclear-cytoplasmic small GTPase that mainly binds to GTP, and its localization seemed to be regulated by its GTP/GDP-binding state. Unexpectedly, the GDP-binding form of the κB-Ras mutant exhibited a more potent inhibitory effect on NF-κB activation, and this inhibitory effect seemed to be due to suppression of the transactivation of a p65/RelA NF-κB subunit. κB-Ras suppressed phosphorylation at serine 276 on the p65/RelA subunit, resulting in decreased interaction between p65/RelA and the transcriptional coactivator p300. Interestingly, the GDP-bound κB-Ras mutant exhibited higher interactive affinity with p65/RelA and inhibited the phosphorylation of p65/RelA more potently than wild-type κB-Ras. Taken together, these findings suggest that the GDP-bound form of κB-Ras in cytoplasm suppresses NF-κB activation by inhibiting its transcriptional activation.

Ric-8B stabilizes the alpha subunit of stimulatory G protein by inhibiting its ubiquitination.

The alpha subunit of stimulatory G protein (G alpha(s)) activates adenylyl cyclase, which catalyzes cAMP production, and regulates many physiological aspects, such as cardiac regulation and endocrine systems. Ric-8B (resistance to inhibitors of cholinesterase 8B) has been identified as the G alpha(s)-binding protein; however, its role in G(s) signaling remains obscure. In this study, we present evidence that Ric-8B specifically and positively regulates G(s) signaling by stabilizing the G alpha(s) protein. An in vitro biochemical study suggested that Ric-8B does not possess guanine nucleotide exchange factor activity. However, knockdown of Ric-8B attenuated beta-adrenergic agonist-induced cAMP accumulation, indicating that Ric-8B positively regulates G(s) signaling. Interestingly, overexpression and knockdown of Ric-8B resulted in an increase and a decrease in the G alpha(s) protein, respectively, without affecting the G alpha(s) mRNA level. We found that the G alpha(s) protein is ubiquitinated and that this ubiquitination is inhibited by Ric-8B. This Ric-8B-mediated inhibition of G alpha(s) ubiquitination requires interaction between Ric-8B and G alpha(s) because Ric-8B splicing variants, which are defective for G alpha(s) binding, failed to inhibit the ubiquitination. Taken together, these results suggest that Ric-8B plays a critical and specific role in the control of G alpha(s) protein levels by modulating G alpha(s) ubiquitination and positively regulates G(s) signaling.

Signal transduction mediated through adhesion-GPCRs.

The signaling cascade of most adhesion-GPCRs remains uncharacterized, as the majority are still orphan receptors and further complicated by their unique structure containing a cleaved long extracellular domain (ECD) and a seven-transmembrane domain (7TM). In this chapter, we review previous reports which suggest G protein-dependent and -independent signaling pathways of adhesion-GPCRs and present our approach to investigate the signal transduction of the adhesion-GPCR, GPR56.

G-protein signalling negatively regulates the stability of aryl hydrocarbon receptor.

Aryl hydrocarbon receptor (AhR) is a transcription factor that works as a dioxin receptor and is also involved in various physiological phenomena, including development and cell proliferation. Here, we show that the Galpha13 signal destabilizes AhR by promoting the ubiquitination of AhR. Galpha13 interacts directly with AhR-interacting protein (AIP) and inhibits the interaction between AhR and AIP, a crucial interacting protein of AhR. Strikingly, a reporter gene assay and a quantitative reverse transcription-PCR analysis indicate that the Galpha13 signal shows a potent inhibitory effect on the ligand-induced transcriptional activation of AhR. Galpha13 results in the nuclear translocation of AhR in a ligand-independent manner. However, in the presence of active Galpha13, AhR fails to form the active transcriptional complex. Taken together, we propose a new negative regulation of dioxin signalling by the G protein.

Functions and regulatory mechanisms of Gq-signaling pathways.

Gq family members of heterotrimeric G protein activate beta isoforms of phospholipase C that hydrolyzes phosphatidylinositol phosphate to diacylglycerol and inositol trisphosphate, leading to the protein kinase C activation and intracellular Ca(2+) mobilization, respectively. To understand the functions and regulatory mechanisms of Gq-signaling pathways, we first introduce the Galphaq-interacting proteins, which function as the effectors and the modulators of Gq. Next, we describe the Pasteurella multocida toxin and YM-254890, which are useful tools to investigate Gq signaling as activator and inhibitor, respectively. Finally, we discuss the physiological function of Gq in developmental brain, especially in neural progenitor cells.

Domain-domain interaction of P-Rex1 is essential for the activation and inhibition by G protein betagamma subunits and PKA.

PtdIns(3, 4, 5)P(3)-dependent Rac exchanger (P-Rex) 1 is a guanine nucleotide exchange factor (GEF) for the small GTPase Rac. P-Rex1 is activated by G protein betagamma subunits (Gbetagamma), and the Gbetagamma-induced activation is inhibited by cAMP-dependent protein kinase A (PKA). However, the details of regulatory mechanism of P-Rex1 remain to be clarified. In the present study, we investigated the mechanism of activation and inhibition of P-Rex1 using various truncated and alanine-substituted mutants and found that the domain-domain interaction of P-Rex1 is important for Gbetagamma-induced activation and PKA-induced inhibition. Immunoprecipitation analysis showed that the second Disheveled/EGL-10/Pleckstrin (DEP) and first PSD-95/Dlg/ZO-1 (PDZ) domains of P-Rex1 associate with the inositol polyphosphate-4-phosphatase (IP4P) domain. Carboxyl-terminal truncation on the IP4P domain or mutations in the protein-binding pocket of the first PDZ domain abolished the association. Analysis of in vitro guanine nucleotide exchange assay, PAK1/2 phosphorylation, and Rac-specific actin reorganization revealed that Gbetagamma could activate a complex of the P-Rex1 mutant lacking the IP4P domain and the isolated IP4P domain as well as full-length P-Rex1. Moreover, PKA phosphorylation prevented the domain-domain interaction and Gbetagamma-binding. These results provide a new insight into the regulation of other Rho-family GEFs and cell responses induced by the heterotrimeric G protein.

Orphan G protein-coupled receptor GPR56 regulates neural progenitor cell migration via a G alpha 12/13 and Rho pathway.

In the developing forebrain, the migration and positioning of neural progenitor cells (NPCs) are regulated coordinately by various molecules. Mutation of these molecules, therefore, causes cortical malformation. GPR56 has been reported as a cortical malformation-related gene that is mutated in patients with bilateral frontoparietal polymicrogyria. GPR56 encodes an orphan G protein-coupled receptor, and its mutations reduce the cell surface expression. It has also been reported that the expression level of GPR56 is involved in cancer cell adhesion and metastasis. However, it remains to be clarified how GPR56 functions in brain development and which signaling pathways are activated by GPR56. In this study, we showed that GPR56 is highly expressed in NPCs and has the ability to inhibit NPC migration. We found that GPR56 coupled with Galpha(12/13) and induced Rho-dependent activation of the transcription mediated through a serum-responsive element and NF-kappaB-responsive element and actin fiber reorganization. The transcriptional activation and actin reorganization were inhibited by an RGS domain of the p115 Rho-specific guanine nucleotide exchange factor (p115 RhoGEF RGS) and dominant negative form of Rho. Moreover, we have demonstrated that a functional anti-GPR56 antibody, which has an agonistic activity, inhibited NPC migration. This inhibition was attenuated by p115 RhoGEF RGS, C3 exoenzyme, and GPR56 knockdown. These results indicate that GPR56 participates in the regulation of NPC movement through the Galpha(12/13) and Rho signaling pathway, suggesting its important role in the development of the central nervous system.

The lipid raft proteins flotillins/reggies interact with Galphaq and are involved in Gq-mediated p38 mitogen-activated protein kinase activation through tyrosine kinase.

The heterotrimeric G protein alpha q subunit (Galphaq) mediates a variety of cell functions by activating the effector molecule phospholipase Cbeta. Galphaq activity is regulated by G protein betagamma subunits, G protein-coupled receptors, RGS proteins, and Ric-8. In this study, we identified the lipid raft resident proteins, flotillin-1/reggie-2 and flotillin-2/reggie-1, as Galphaq-binding proteins. The interactions of Galphaq and flotillins were independent of the nucleotide-binding state of Galphaq, and the N-terminal portion of flotillins was critical for the interaction. A short interfering RNA-mediated knockdown of flotillins, particularly flotillin-2, attenuated the UTP-induced activation of p38 mitogen-activated protein kinase (MAPK) but not that of ERK1/2. The activation of p38 MAPK was inhibited by the Src family tyrosine kinase inhibitor PP2 and the cholesterol-depleting agent methyl-beta-cyclodextrin, which is generally used for the disruption of lipid rafts. In contrast, the activation of ERK1/2 was not inhibited by these compounds. These lines of evidence suggested that a Gq-coupled receptor activates specifically p38 MAPK through lipid rafts and Src kinase activation, in which flotillins positively modulate the Gq signaling.

Molecular loops in the galactic center: evidence for magnetic flotation.

The central few hundred parsecs of the Milky Way host a massive black hole and exhibit very violent gas motion and high temperatures in molecular gas. The origin of these properties has been a mystery for the past four decades. Wide-field imaging of the (12)CO (rotational quantum number J = 1 to 0) 2.6-millimeter spectrum has revealed huge loops of dense molecular gas with strong velocity dispersions in the galactic center. We present a magnetic flotation model to explain that the formation of the loops is due to magnetic buoyancy caused by the Parker instability. The model has the potential to offer a coherent explanation for the origin of the violent motion and extensive heating of the molecular gas in the galactic center.

Ric-8A potentiates Gq-mediated signal transduction by acting downstream of G protein-coupled receptor in intact cells.

RIC-8 was originally found by genetic studies on C. elegans mutants that were resistant to inhibitors of acetylcholinesterase and reported to act in vitro as a guanine nucleotide exchange factor for G protein alpha subunits. However, the physiological role of a mammalian homolog Ric-8A on G protein-coupled receptor signaling in intact cells is largely unknown. We isolated Ric-8A using a yeast two-hybrid system with Galphaq and examined the role of Ric-8A on Gq-mediated signaling. The small interfering RNA of Ric-8A diminished the Gq-coupled receptor-mediated ERK activation and intracellular calcium mobilization in 293T cells. Ric-8A was translocated to the cell membrane in response to the Gq-coupled receptor stimulation. The expression of the myristoylation sequence-conjugated Ric-8A mutant was located in the membranes and shown to enhance the Gq-coupled receptor-mediated ERK activation. Moreover, this enhancement on ERK activation and the guanine nucleotide exchange activity of Ric-8A for Galphaq were inhibited by Gq selective inhibitor YM-254890. These results suggested that Ric-8A potentiates Gq-mediated signal transduction by acting as a novel-type regulator in intact cells.

G protein-coupled receptor signaling through Gq and JNK negatively regulates neural progenitor cell migration.

In the early development of the central nervous system, neural progenitor cells divide in an asymmetric manner and migrate along the radial glia cells. The radial migration is an important process for the proper lamination of the cerebral cortex. Recently, a new mode of the radial migration was found at the intermediate zone where the neural progenitor cells become multipolar and reduce the migration rate. However, the regulatory signals for the radial migration are unknown. Using the migration assay in vitro, we examined how neural progenitor cell migration is regulated. Neural progenitor cells derived from embryonic mouse telencephalon migrated on laminin-coated dishes. Endothelin (ET)-1 inhibited the neural progenitor cell migration. This ET-1 effect was blocked by BQ788, a specific inhibitor of the ETB receptor, and by the expression of a carboxyl-terminal peptide of Galpha q but not Galpha i. The expression of constitutively active mutant of Galpha q, Galpha qR183C, inhibited the migration of neural progenitor cells. Moreover, the inhibitory effect of ET-1 was suppressed by the c-Jun N-terminal kinase (JNK) inhibitor SP600125 and the expression of the JNK-binding domain of JNK-interacting protein-1, a specific inhibitor of the JNK pathway. Using the slice culture system of embryonic brain, we demonstrated that ET-1 and the constitutively active mutant of Galpha q caused the retention of the neural progenitor cells in the intermediate zone and JNK-binding domain of JNK-interacting protein-1 abrogated the effect of ET-1. These results indicated that G protein-coupled receptor signaling negatively regulates neural progenitor cell migration through Gq and JNK.

The adaptor protein Nck1 mediates endothelin A receptor-regulated cell migration through the Cdc42-dependent c-Jun N-terminal kinase pathway.

Cell migration plays key roles in physiological and pathological phenomena, such as development and oncogenesis. The adaptor proteins Grb2, CrkII, and Nck1 are composed of only a single Src homology 2 domain and some Src homology 3 domains, giving specificity to each signal transduction pathway. However, little is known about the relationships between their adaptor proteins and cell migration, which are regulated by the G protein-coupled receptor. Here we showed that Nck1, but not Grb2 or CrkII, mediated the inhibition of cell migration induced by the endothelin-1 and endothelin A receptor. The small interference RNA and dominant negative mutants of Nck1 diminished the endothelin-1-induced inhibition of cell migration. Although overexpression of wild-type Nck1 was detected in the cytosol and did not affect cell migration, expression of the myristoylation signal sequence-conjugated Nck1 was detected in the membrane and induced activation of Cdc42 and c-Jun N-terminal kinase, inhibiting cell migration. Taken together, these results suggest that the endothelin A receptor transduces the signal of inhibition of cell migration through Cdc42-dependent c-Jun N-terminal kinase activation by using Nck1.