The N-terminal coiled-coil domain of the cytohesin/ARNO family of guanine nucleotide exchange factors interacts with Gαq

Cytohesins are guanine-nucleotide exchange factors (GEF) for the Arf family of GTPases. One member of the Arf family, ARF6, plays an active role in the intracellular trafficking of G protein-coupled receptors. We have previously reported that Gαq signaling leads to the activation of ARF6, possibly through a direct interaction with cytohesin-2/ARNO. Here, we report that Gαq can directly interact with cytohesin-1, another Arf-GEF of the ARNO/cytohesin family. Cytohesin-1 preferentially associated with a constitutively active mutant of Gαq (Gαq-Q209L) compared to wild-type Gαq in HEK293 cells. Stimulation of TPβ, a Gαq-coupled receptor, to activate Gαq resulted in the promotion of a protein complex between Gαq and cytohesin-1. Confocal immunofluorescence microscopy revealed that wild-type Gαq and cytohesin-1 co-localized in intracellular compartments and at or near the plasma membrane. In contrast, expression of Gαq-Q209L induced a drastic increase in the localization of cytohesin-1 at the plasma membrane. Expression of a dominant-negative mutant of cytohesin-1 reduced by 40% the agonist-induced internalization of TPβ, a process that we previously demonstrated to be dependent on Gαq-mediated signaling and Arf6 activation. Using deletion mutants, we show that cytohesin-1 interacts with Gαq through its N-terminal coiled-coil domain. Cytohesin-1 and cytohesin-2/ARNO mutants lacking the coiled-coil domain were unable to relay Gαq-mediated activation of Arf6. This is the first report of an interaction between the coiled-coil domain of the cytohesin/ARNO family of Arf-GEFs and a member of the heterotrimeric G proteins.     

G protein-coupled receptors and resistance to inhibitors of cholinesterase-8A (Ric-8A) both regulate the regulator of g protein signaling 14 RGS14·Gαi1 complex in live cells

Regulator of G protein Signaling 14 (RGS14) is a multifunctional scaffolding protein that integrates both conventional and unconventional G protein signaling pathways. Like other RGS (regulator of G protein signaling) proteins, RGS14 acts as a GTPase accelerating protein to terminate conventional Gα(i/o) signaling. However, unlike other RGS proteins, RGS14 also contains a G protein regulatory/GoLoco motif that specifically binds Gα(i1/3)-GDP in cells and in vitro. The non-receptor guanine nucleotide exchange factor Ric-8A can bind and act on the RGS14·Gα(i1)-GDP complex to play a role in unconventional G protein signaling independent of G protein-coupled receptors (GPCRs). Here we demonstrate that RGS14 forms a Gα(i/o)-dependent complex with a G(i)-linked GPCR and that this complex is regulated by receptor agonist and Ric-8A (resistance to inhibitors of cholinesterase-8A). Using live cell bioluminescence resonance energy transfer, we show that RGS14 functionally associates with the α(2A)-adrenergic receptor (α(2A)-AR) in a Gα(i/o)-dependent manner. This interaction is markedly disrupted after receptor stimulation by the specific agonist UK14304, suggesting complex dissociation or rearrangement. Agonist-mediated dissociation of the RGS14·α(2A)-AR complex occurs in the presence of Gα(i/o) but not Gα(s) or Gα(q). Unexpectedly, RGS14 does not dissociate from Gα(i1) in the presence of stimulated α(2A)-AR, suggesting preservation of RGS14·Gα(i1) complexes after receptor activation. However, Ric-8A facilitates dissociation of both the RGS14·Gα(i1) complex and the Gα(i1)-dependent RGS14·α(2A)-AR complex after receptor activation. Together, these findings indicate that RGS14 can form complexes with GPCRs in cells that are dependent on Gα(i/o) and that these RGS14·Gα(i1)·GPCR complexes may be substrates for other signaling partners such as Ric-8A.     

Structure,Function, and Dynamics of the Gα Binding Domain of Ric-8A

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Activation of the regulator of G protein signaling 14-Gαi1-GDP signaling complex is regulated by resistance to inhibitors of cholinesterase-8A

RGS14 is a brain scaffolding protein that integrates G protein and MAP kinase signaling pathways. Like other RGS proteins, RGS14 is a GTPase activating protein (GAP) that terminates Gαi/o signaling. Unlike other RGS proteins, RGS14 also contains a G protein regulatory (also known as GoLoco) domain that binds Gαi1/3-GDP in cells and in vitro. Here we report that Ric-8A, a nonreceptor guanine nucleotide exchange factor (GEF), functionally interacts with the RGS14-Gαi1-GDP signaling complex to regulate its activation state. RGS14 and Ric-8A are recruited from the cytosol to the plasma membrane in the presence of coexpressed Gαi1 in cells, suggesting formation of a functional protein complex with Gαi1. Consistent with this idea, Ric-8A stimulates dissociation of the RGS14-Gαi1-GDP complex in cells and in vitro using purified proteins. Purified Ric-8A stimulates dissociation of the RGS14-Gαi1-GDP complex to form a stable Ric-8A-Gαi complex in the absence of GTP. In the presence of an activating nucleotide, Ric-8A interacts with the RGS14-Gαi1-GDP complex to stimulate both the steady-state GTPase activity of Gαi1 and binding of GTP to Gαi1. However, sufficiently high concentrations of RGS14 competitively reverse these stimulatory effects of Ric-8A on Gαi1 nucleotide binding and GTPase activity. This observation correlates with findings that show RGS14 and Ric-8A share an overlapping binding region within the last 11 amino acids of Gαi1. As further evidence that these proteins are functionally linked, native RGS14 and Ric-8A coexist within the same hippocampal neurons. These findings demonstrate that RGS14 is a newly appreciated integrator of unconventional Ric-8A and Gαi1 signaling.     

A conserved α helix of Bcs1, a mitochondrial AAA chaperone,is required for the Respiratory Complex III maturation

Bcs1 is a transmembrane chaperone in the mitochondrial inner membrane, and is required for the mitochondrial Respiratory Chain Complex III assembly. It has been shown that the highly-conserved C-terminal region of Bcs1 including the AAA ATPase domain in the matrix side is essential for the chaperone function. Here we describe the importance of the N-terminal short segment located in the intermembrane space in the Bcs1 function. Among the N-terminal 44 amino acid residues of yeast Bcs1, the first 37 residues are dispensable whereas a hydrophobic amino acid in the residue 38 is essential for integration of Rieske Iron-sulfur Protein into the premature Complex III from the mitochondrial matrix. Substitution of the residue 38 by a hydrophilic amino acid residue affects conformation of Bcs1 and interactions with other proteins. The evolutionarily-conserved short α helix of Bcs1 in the intermembrane space is an essential element for the chaperone function.     

CCL2 −2518 A/G single nucleotide polymorphism as a risk factor for breast cancer

The contribution of the CCL2 −2518 A>G (rs 1024611) polymorphism in the occurrence and progression of various cancers has been found to be discordant. We studied the prevalence of the CCL2 −2518 A>G polymorphism in patients with breast cancer (n = 160) and controls (n = 323) in a sample of the Polish population. There were no significant differences in CCL2 −2518 A>G genotypes between patients with breast tumors and controls. Odds ratio (OR) for patients bearing the GG genotype was 1.481 (95% CI = 0.7711–2.845, P = 0.2358), and OR of the GG and AG genotypes was 0.7269 (95% CI = 0.4967–1.064, P = 0.1002). There was also no significant distinction in the prevalence of alleles between patients and healthy individuals. OR for the CCL2 −2518 G allele frequency was 0.8903 (95% CI = 0.6611–1.199, P = 0.4441). Analysis of the association between tumor size, lymph node metastases, histological grade, and distribution of genotypes and alleles for the CCL2 −2518 A>G polymorphism also did not show significant differences. Our results did not show association of the CCL2 −2518 A>G polymorphism with breast cancer occurrence and clinical characteristics in a sample of the Polish cohort.     

A novel splice variant of Gαq-coupled Bombyx CAPA-PVK receptor 1 functions as a specific Gαi/o-linked receptor for CAPA-PK

Alternative splicing enables G protein-coupled receptor (GPCR) genes to greatly increase the number of structurally and functionally distinct receptor isoforms. However, the functional role and relevance of the individual GPCR splice variants in regulating physiological processes are still to be assessed. A naturally occurring alternative splice variant of Bombyx CAPA-PVK receptor, BomCAPA-PVK-R1-Δ341, has been shown to act as a dominant-negative protein to regulate cell surface expression and function of the canonical CAPA-PVK receptor. Herein, using functional assays, we identify the splice variant Δ341 as a specific receptor for neuropeptide CAPA-PK, and upon activation, Δ341 signals to ERK1/2 pathway. Further characterization demonstrates that Δ341 couples to Gαi/o, distinct from the Gαq-coupled canonical CAPA-PVK receptor, triggering ERK1/2 phosphorylation through Gβγ-PI3K-PKCζ signaling cascade. Moreover, our ELISA data show that the ligand-dependent internalization of the splice variant Δ341 is significantly impaired due to lack of GRKs-mediated phosphorylation sites. Our findings highlight the potential of this knowledge for molecular, pharmacological and physiological studies on GPCR splice variants in the future.     

Cool-1/βPIX functions as a guanine nucleotide exchange factor in the cycling of Cdc42 to regulate insulin secretion

Second-phase insulin release requires the sustained mobilization of insulin granules from internal storage pools to the cell surface for fusion with the plasma membrane. However, the detailed mechanisms underlying this process remain largely unknown. GTP-loading of the small GTPase Cdc42 is the first glucose-specific activation step in the process, although how glucose triggers Cdc42 activation is entirely unknown. In a directed candidate screen for guanine nucleotide exchange factors (GEFs), which directly activate small GTPases, Cool-1/βPix was identified in pancreatic islet beta cells. In support of its role as the beta cell Cdc42 GEF, βPix coimmunoprecipitated with Cdc42 in human islets and MIN6 beta cells in a glucose-dependent manner, peaking just prior to Cdc42 activation. Furthermore, RNAi-mediated βPix reduction by 50% corresponded to full ablation of glucose-induced Cdc42 activation and significant attenuation of basal and glucose-stimulated insulin secretion. Of the two Cdc42 guanine nucleotide dissociation inhibitor (GDI) proteins identified in beta cells, βPix competed selectively with caveolin-1 (Cav-1) but not RhoGDI in coimmunoprecipitation and GST-Cdc42-GDP interaction assays. However, a phospho-deficient Cav-1-Y14F mutant failed to compete with βPix; Cav-1(Tyr14) is an established phosphorylation site for Src kinase. Taken together, these data support a new model, wherein glucose stimulates Cav-1 and induces its dissociation from Cdc42, possibly via Src kinase activation to phosphorylate Cav-1(Tyr14), to promote Cdc42-βPix binding and Cdc42 activation, and to trigger downstream signaling and ultimately sustain insulin release.     

Nucleobindin 1 Is a Calcium-regulated Guanine Nucleotide Dissociation Inhibitor of Gαi1

Nucleobindin 1 (NUCB1) is a widely expressed multidomain calcium-binding protein whose precise physiological and biochemical functions are not well understood. We engineered and heterologously expressed a soluble form of NUCB1 (sNUCB1) and characterized its biophysical and biochemical properties. We show that sNUCB1 exists as a dimer in solution and that each monomer binds two divalent calcium cations. Calcium binding causes conformational changes in sNUCB1 as judged by circular dichroism and fluorescence spectroscopy experiments. Earlier reports suggested that NUCB1 might interact with heterotrimeric G protein α subunits. We show that dimeric calcium-free sNUCB1 binds to expressed Gα_i1 and that calcium binding inhibits the interaction. The binding of sNUCB1 to Gα_i1 inhibits its basal rate of GDP release and slows its rate and extent of GTPγS uptake. Additionally, our tissue culture experiments show that sNUCB1 prevents receptor-mediated Gα_i-dependent inhibition of adenylyl cyclase. Thus, we conclude that sNUCB1 is a calcium-dependent guanine nucleotide dissociation inhibitor (GDI) for Gα_i1. To our knowledge, sNUCB1 is the first example of a calcium-dependent GDI for heterotrimeric G proteins. We also show that the mechanism of GDI activity of sNUCB1 is unique and does not arise from the consensus GoLoco motif found in RGS proteins. We propose that cytoplasmic NUCB1 might function to regulate heterotrimeric G protein trafficking and G protein-coupled receptor-mediated signal transduction pathways.     

The molecular chaperone Hsp90α is required for meiotic progression of spermatocytes beyond pachytene in the mouse

The molecular chaperone Hsp90 has been found to be essential for viability in all tested eukaryotes, from the budding yeast to Drosophila. In mammals, two genes encode the two highly similar and functionally largely redundant isoforms Hsp90α and Hsp90β. Although they are co-expressed in most if not all cells, their relative levels vary between tissues and during development. Since mouse embryos lacking Hsp90β die at implantation, and despite the fact that Hsp90 inhibitors being tested as anti-cancer agents are relatively well tolerated, the organismic functions of Hsp90 in mammals remain largely unknown. We have generated mouse lines carrying gene trap insertions in the Hsp90α gene to investigate the global functions of this isoform. Surprisingly, mice without Hsp90α are apparently normal, with one major exception. Mutant male mice, whose Hsp90β levels are unchanged, are sterile because of a complete failure to produce sperm. While the development of the male reproductive system appears to be normal, spermatogenesis arrests specifically at the pachytene stage of meiosis I. Over time, the number of spermatocytes and the levels of the meiotic regulators and Hsp90 interactors Hsp70-2, NASP and Cdc2 are reduced. We speculate that Hsp90α may be required to maintain and to activate these regulators and/or to disassemble the synaptonemal complex that holds homologous chromosomes together. The link between fertility and Hsp90 is further supported by our finding that an Hsp90 inhibitor that can cross the blood-testis barrier can partially phenocopy the genetic defects.     

The interaction of Gα13 with integrin β1 mediates cell migration by dynamic regulation of RhoA

Heterotrimeric G protein Gα₁₃ is known to transmit G protein–coupled receptor (GPCR) signals leading to activation of RhoA and plays a role in cell migration. The mechanism underlying the role of Gα₁₃ in cell migration, however, remains unclear. Recently we found that Gα₁₃ interacts with the cytoplasmic domain of integrin β₃ subunits in platelets via a conserved ExE motif. Here we show that a similar direct interaction between Gα₁₃ and the cytoplasmic domain of the integrin β₁ subunit plays a critical role in β₁-dependent cell migration. Point mutation of either glutamic acid in the Gα₁₃-binding ⁷⁶⁷EKE motif in β₁ or treatment with a peptide derived from the Gα₁₃-binding sequence of β₁ abolished Gα₁₃–β₁ interaction and inhibited β₁ integrin–dependent cell spreading and migration. We further show that the Gα₁₃-β₁ interaction mediates β₁ integrin–dependent Src activation and transient RhoA inhibition during initial cell adhesion, which is in contrast to the role of Gα₁₃ in mediating GPCR-dependent RhoA activation. These data indicate that Gα₁₃ plays dynamic roles in both stimulating RhoA via a GPCR pathway and inhibiting RhoA via an integrin signaling pathway. This dynamic regulation of RhoA activity is critical for cell migration on β₁ integrin ligands.     

The solid-state catalytic isotope exchange of hydrogen in α-conotoxin G1 by the tritium spillover

Tritium-labeled α-conotoxin G1 with a molar radioactivity of 35 Ci/mmol and full biological activity (according to the binding to nicotinic acetylcholine receptor) was obtained by the high-temperature solid-state catalytic isotope exchange (HSCIE). The tritium distribution in the molecule of α-conotoxin G1 was revealed by³H NMR spectroscopy. Tritium was found in all amino acid residues except for the Asn4-Pro5-Ala6 fragment. The data on the comparative reactivity of C-H bonds, theab initio quantum-chemical calculation of the hydrogen exchange reaction, and the information on the spatial structures of α-conotoxin G1 in solution and in crystal state allowed us to establish that the reactivity of H atoms may be increased by their interaction with the electron donor O and N atoms at the transition state of the HSCIE reaction. A decrease in the rate of the HSCIE reaction could be caused by both a poor spatial accessibility of C-H bonds and a limited mobility of the peptide fragment containing these bonds.     

A Highlights from MBoC Selection: Drosophila Ric-8 interacts with the Gα12/13 subunit,Concertina, during activation of the Folded gastrulation pathway

Heterotrimeric G proteins, composed of α, β, and γ subunits, are activated by exchange of GDP for GTP on the Gα subunit. Canonically, Gα is stimulated by the guanine-nucleotide exchange factor (GEF) activity of ligand-bound G protein–coupled receptors. However, Gα subunits may also be activated in a noncanonical manner by members of the Ric-8 family, cytoplasmic proteins that also act as GEFs for Gα subunits. We used a signaling pathway active during Drosophila gastrulation as a model system to study Ric-8/Gα interactions. A component of this pathway, the Drosophila Gα_12/13 subunit, Concertina (Cta), is necessary to trigger actomyosin contractility during gastrulation events. Ric-8 mutants exhibit similar gastrulation defects to Cta mutants. Here we use a novel tissue culture system to study a signaling pathway that controls cytoskeletal rearrangements necessary for cellular morphogenesis. We show that Ric-8 regulates this pathway through physical interaction with Cta and preferentially interacts with inactive Cta and directs its localization within the cell. We also use this system to conduct a structure–function analysis of Ric-8 and identify key residues required for both Cta interaction and cellular contractility.