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1.
Nat Commun ; 13(1): 731, 2022 02 08.
Article in English | MEDLINE | ID: mdl-35136060

ABSTRACT

Lysophospholipids are bioactive lipids and can signal through G-protein-coupled receptors (GPCRs). The best studied lysophospholipids are lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P). The mechanisms of lysophospholipid recognition by an active GPCR, and the activations of lysophospholipid GPCR-G-protein complexes remain unclear. Here we report single-particle cryo-EM structures of human S1P receptor 1 (S1P1) and heterotrimeric Gi complexes formed with bound S1P or the multiple sclerosis (MS) treatment drug Siponimod, as well as human LPA receptor 1 (LPA1) and Gi complexes in the presence of LPA. Our structural and functional data provide insights into how LPA and S1P adopt different conformations to interact with their cognate GPCRs, the selectivity of the homologous lipid GPCRs for S1P versus LPA, and the different activation mechanisms of these GPCRs by LPA and S1P. Our studies also reveal specific optimization strategies to improve the MS-treating S1P1-targeting drugs.


Subject(s)
GTP-Binding Protein alpha Subunits, Gi-Go/metabolism , Receptors, Lysophosphatidic Acid/metabolism , Sphingosine-1-Phosphate Receptors/metabolism , Animals , Azetidines/pharmacology , Azetidines/therapeutic use , Benzyl Compounds/pharmacology , Benzyl Compounds/therapeutic use , Cryoelectron Microscopy , GTP-Binding Protein alpha Subunits, Gi-Go/genetics , GTP-Binding Protein alpha Subunits, Gi-Go/isolation & purification , GTP-Binding Protein alpha Subunits, Gi-Go/ultrastructure , Humans , Lysophospholipids/metabolism , Molecular Conformation/drug effects , Molecular Docking Simulation , Multiple Sclerosis/drug therapy , Multiple Sclerosis/pathology , Receptors, Lysophosphatidic Acid/genetics , Receptors, Lysophosphatidic Acid/isolation & purification , Receptors, Lysophosphatidic Acid/ultrastructure , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Recombinant Proteins/ultrastructure , Sf9 Cells , Single Molecule Imaging , Sphingosine/analogs & derivatives , Sphingosine/metabolism , Sphingosine-1-Phosphate Receptors/genetics , Sphingosine-1-Phosphate Receptors/isolation & purification , Sphingosine-1-Phosphate Receptors/ultrastructure , Spodoptera
2.
Nat Commun ; 10(1): 2234, 2019 05 20.
Article in English | MEDLINE | ID: mdl-31110175

ABSTRACT

The ß2 adrenergic receptor (ß2AR) signals through both Gs and Gi in cardiac myocytes, and the Gi pathway counteracts the Gs pathway. However, Gi coupling is much less efficient than Gs coupling in most cell-based and biochemical assays, making it difficult to study ß2AR-Gi interactions. Here we investigate the role of phospholipid composition on Gs and Gi coupling. While negatively charged phospholipids are known to enhance agonist affinity and stabilize an active state of the ß2AR, we find that they impair coupling to Gi3 and facilitate coupling to Gs. Positively charged Ca2+ and Mg2+, known to interact with the negative charge on phospholipids, facilitates Gi3 coupling. Mutational analysis suggests that Ca2+ coordinates an interaction between phospholipid and the negatively charged EDGE motif on the amino terminal helix of Gi3. Taken together, our observations suggest that local membrane charge modulates the interaction between ß2AR and competing G protein subtypes.


Subject(s)
Cell Membrane/metabolism , GTP-Binding Protein alpha Subunits, Gi-Go/metabolism , Membrane Lipids/metabolism , Receptors, Adrenergic, beta-2/metabolism , Amino Acid Motifs , Animals , Cations/metabolism , GTP-Binding Protein alpha Subunits, Gi-Go/chemistry , GTP-Binding Protein alpha Subunits, Gi-Go/isolation & purification , Membrane Lipids/chemistry , Myocytes, Cardiac/cytology , Myocytes, Cardiac/metabolism , Phospholipids/chemistry , Phospholipids/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Sf9 Cells , Spodoptera , Static Electricity
3.
Nat Commun ; 10(1): 2008, 2019 05 01.
Article in English | MEDLINE | ID: mdl-31043612

ABSTRACT

G protein-gated inwardly rectifying potassium channel (GIRK) plays a key role in regulating neurotransmission. GIRK is opened by the direct binding of the G protein ßγ subunit (Gßγ), which is released from the heterotrimeric G protein (Gαßγ) upon the activation of G protein-coupled receptors (GPCRs). GIRK contributes to precise cellular responses by specifically and efficiently responding to the Gi/o-coupled GPCRs. However, the detailed mechanisms underlying this family-specific and efficient activation are largely unknown. Here, we investigate the structural mechanism underlying the Gi/o family-specific activation of GIRK, by combining cell-based BRET experiments and NMR analyses in a reconstituted membrane environment. We show that the interaction formed by the αA helix of Gαi/o mediates the formation of the Gαi/oßγ-GIRK complex, which is responsible for the family-specific activation of GIRK. We also present a model structure of the Gαi/oßγ-GIRK complex, which provides the molecular basis underlying the specific and efficient regulation of GIRK.


Subject(s)
G Protein-Coupled Inwardly-Rectifying Potassium Channels/ultrastructure , GTP-Binding Protein alpha Subunits, Gi-Go/ultrastructure , GTP-Binding Protein beta Subunits/ultrastructure , GTP-Binding Protein gamma Subunits/ultrastructure , Ion Channel Gating/physiology , Bioluminescence Resonance Energy Transfer Techniques , G Protein-Coupled Inwardly-Rectifying Potassium Channels/metabolism , GTP-Binding Protein alpha Subunits, Gi-Go/isolation & purification , GTP-Binding Protein alpha Subunits, Gi-Go/metabolism , GTP-Binding Protein beta Subunits/isolation & purification , GTP-Binding Protein beta Subunits/metabolism , GTP-Binding Protein gamma Subunits/isolation & purification , GTP-Binding Protein gamma Subunits/metabolism , HEK293 Cells , Humans , Models, Molecular , Nuclear Magnetic Resonance, Biomolecular , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Recombinant Proteins/ultrastructure
4.
Prikl Biokhim Mikrobiol ; 50(4): 442-8, 2014.
Article in Russian | MEDLINE | ID: mdl-25707122

ABSTRACT

From the brain tissue of Wistar rats,we purified a bioregulator, which is active at ultralow doses. Using reversed-phase HPLC, we prepared a homogenous polypeptide with a molecular weight of 4749 +/- 2 Da, which is responsible for the biological activity of the bioregulator. Using the CD spectroscopy method, we calculated the percentage of canonical elements of the secondary polypeptide structure in a solution. Using the methods of proteomics, we revealed that the structure of the investigated polypeptide was similar to the N-terminal sequence of a fragment of guanine-nucleotide binding G0-protein subunit alpha-1.


Subject(s)
Brain/metabolism , GTP-Binding Protein alpha Subunits, Gi-Go/chemistry , GTP-Binding Protein alpha Subunits, Gi-Go/pharmacology , Liver/drug effects , Amino Acid Sequence , Animals , Brain Chemistry , Chromatography, High Pressure Liquid , Circular Dichroism , Female , GTP-Binding Protein alpha Subunits, Gi-Go/isolation & purification , Liver/metabolism , Male , Mice , Mice, Inbred C57BL , Molecular Sequence Data , Molecular Weight , Protein Structure, Secondary , Rats , Rats, Wistar , Tissue Culture Techniques
5.
Article in English | MEDLINE | ID: mdl-23295489

ABSTRACT

Regulator of G-protein signalling (RGS) proteins negatively regulate heterotrimeric G-protein signalling through their conserved RGS domains. RGS domains act as GTPase-activating proteins, accelerating the GTP hydrolysis rate of the activated form of Gα-subunits. Although omnipresent in eukaryotes, RGS proteins have not been adequately analysed in non-mammalian organisms. The Drosophila melanogaster Gαo-subunit and the RGS domain of its interacting partner CG5036 have been overproduced and purified; the crystallization of the complex of the two proteins using PEG 4000 as a crystallizing agent and preliminary X-ray crystallographic analysis are reported. Diffraction data were collected to 2.0 Šresolution using a synchrotron-radiation source.


Subject(s)
Drosophila Proteins/chemistry , Drosophila Proteins/metabolism , GTP-Binding Protein alpha Subunits, Gi-Go/chemistry , RGS Proteins/chemistry , Animals , Base Sequence , Cloning, Molecular , Crystallization/methods , Crystallography, X-Ray , GTP-Binding Protein alpha Subunits, Gi-Go/genetics , GTP-Binding Protein alpha Subunits, Gi-Go/isolation & purification , Molecular Sequence Data , Polyethylene Glycols/chemistry , Protein Structure, Tertiary , RGS Proteins/metabolism
6.
Arch Biochem Biophys ; 524(2): 93-8, 2012 Aug 15.
Article in English | MEDLINE | ID: mdl-22659491

ABSTRACT

Recombinant heterotrimeric G-protein α(i1), α(i2) and α(i3) subunits were purified in GDP-depleting conditions by affinity chromatography using StrepII-tagged ß1γ2 subunits. Real-time monitoring of fluorescence anisotropy of Bodipy-FL-GTPγS was used for characterization of nucleotide binding properties and inactivation of the purified proteins. All GDP-depleted α(i) were unstable at room temperature and therefore nucleotide binding could be characterized only in a nonequilibrium state. In comparison to Mg²âº, Mn²âº inhibited nucleotide binding to all α(i)-heterotrimers studied and accelerated nucleotide release. Mn²âº had stabilizing effect on the nucleotide free state of the α(i1) subunit, whereas both Mn²âº as well as G-protein activation by mastoparan destabilized the α(i2) subunit.


Subject(s)
GTP-Binding Protein alpha Subunits, Gi-Go/chemistry , GTP-Binding Protein alpha Subunits, Gi-Go/metabolism , Guanosine Diphosphate/metabolism , Guanosine Triphosphate/analogs & derivatives , Animals , Cell Line , Fluorescence Polarization , GTP-Binding Protein alpha Subunits, Gi-Go/genetics , GTP-Binding Protein alpha Subunits, Gi-Go/isolation & purification , Guanosine 5'-O-(3-Thiotriphosphate)/pharmacology , Guanosine Triphosphate/chemistry , Guanosine Triphosphate/metabolism , Humans , Intercellular Signaling Peptides and Proteins , Magnesium/pharmacology , Manganese/pharmacology , Peptides/pharmacology , Protein Multimerization/drug effects , Protein Stability/drug effects , Protein Structure, Quaternary , Protein Subunits/chemistry , Protein Subunits/metabolism , Wasp Venoms/pharmacology
7.
J Biol Chem ; 285(9): 6337-47, 2010 Feb 26.
Article in English | MEDLINE | ID: mdl-20026606

ABSTRACT

Accumulating evidence indicates that G protein-coupled receptors can assemble as dimers/oligomers but the role of this phenomenon in G protein coupling and signaling is not yet clear. We have used the purified leukotriene B(4) receptor BLT2 as a model to investigate the capacity of receptor monomers and dimers to activate the adenylyl cyclase inhibitory G(i2) protein. For this, we overexpressed the recombinant receptor as inclusion bodies in the Escherichia coli prokaryotic system, using a human alpha(5) integrin as a fusion partner. This strategy allowed the BLT2 as well as several other G protein-coupled receptors from different families to be produced and purified in large amounts. The BLT2 receptor was then successfully refolded to its native state, as measured by high-affinity LTB(4) binding in the presence of the purified G protein G alpha(i2). The receptor dimer, in which the two protomers displayed a well defined parallel orientation as assessed by fluorescence resonance energy transfer, was then separated from the monomer. Using two methods of receptor-catalyzed guanosine 5'-3-O-(thio)triphosphate binding assay, we clearly demonstrated that monomeric BLT2 stimulates the purified G alpha(i2) beta(1) gamma(2) protein more efficiently than the dimer. These data suggest that assembly of two BLT2 protomers into a dimer results in the reduced ability to signal.


Subject(s)
GTP-Binding Protein alpha Subunits, Gi-Go/metabolism , Receptors, Leukotriene B4/physiology , Fluorescence Resonance Energy Transfer , GTP-Binding Protein alpha Subunits, Gi-Go/chemistry , GTP-Binding Protein alpha Subunits, Gi-Go/isolation & purification , GTP-Binding Protein beta Subunits/metabolism , GTP-Binding Protein gamma Subunits/metabolism , Humans , Integrin alphaV , Protein Binding , Protein Multimerization , Signal Transduction
8.
Int J Biochem Cell Biol ; 41(7): 1495-501, 2009 Jul.
Article in English | MEDLINE | ID: mdl-19146979

ABSTRACT

Interaction between GAP-43 (growth associated protein-43) and Galpha(o) (alpha subunit of Go protein) influences the signal transduction pathways leading to differentiation of neural cells. GAP-43 is known to increase guanine nucleotide exchange by Galpha(o), which is a major component of neuronal growth cone membranes. However, it is not clear whether GAP-43 stimulation is related to the Galpha(o) palmitoylation or the conversion of Galpha(o) from oligmers to monomers, which was shown to be a necessary regulatory factor in GDP/GTP exchange of Galpha(o). Here we expressed and purified GAP-43, GST-GAP-43 and Galpha(o) proteins, detected their stimulatory effect on [(35)S]-GTPgammaS binding of Galpha(o). It was found that the EC(50) of both GAP-43 and GST-GAP-43 activation were tenfold lower in case of depalmitoylated Galpha(o) than palmitoylated Galpha(o). Non-denaturing gel electrophoresis and p-PDM cross-linking analysis revealed that addition of GST-GAP-43 induced disassociation of depalmitoylated Galpha(o) from oligomers to monomers, but did not influence the oligomeric state of palmitoylated Galpha(o), which suggests that palmitoylation is a key regulatory factor in GAP-43 stimulation on Galpha(o). These results indicated the interaction of GAP-43 and Galpha(o) could accelerate conversion of depalmitoylated Galpha(o) but not palmitoylated Galpha(o) from oligomers to monomers, so as to increase the GTPgammaS binding activity of Galpha(o). Results here provide new evidence about how signaling protein palmitoylation is involved in the G-protein-coupled signal transduction cascade, and give a useful clue on the participation of GAP-43 in G-protein cycle by its preferential activation of depalmitoylated Galpha(o).


Subject(s)
GAP-43 Protein/metabolism , GTP-Binding Protein alpha Subunits, Gi-Go/metabolism , Lipoylation , Animals , Blotting, Western , Cattle , Cross-Linking Reagents/pharmacology , Electrophoresis, Polyacrylamide Gel , GAP-43 Protein/isolation & purification , GTP-Binding Protein alpha Subunits, Gi-Go/chemistry , GTP-Binding Protein alpha Subunits, Gi-Go/isolation & purification , Guanosine 5'-O-(3-Thiotriphosphate)/metabolism , Lipoylation/drug effects , Maleimides/pharmacology , Protein Structure, Quaternary , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolism
9.
Biochem Biophys Res Commun ; 374(1): 128-33, 2008 Sep 12.
Article in English | MEDLINE | ID: mdl-18601900

ABSTRACT

Muscarinic receptor extracted from porcine atria in digitonin-cholate copurified with Galpha(o), Galpha(i1-3), and caveolins. The presence of complexes was confirmed by coimmunoprecipitation of the receptor, alpha-subunits, and caveolins in various combinations. Homooligomers of alpha(i2) were detected on Western blots, and heterooligomers of alpha(i2) and alpha(o) were identified by coimmunoprecipitation; thus, a complex may contain at least two alpha-subunits. Other combinations of alpha-subunit were not detected. The ratio of total alpha-subunit to receptor was near 1, as measured by [(35)S]GTPgammaS and the antagonist [(3)H]quinuclidinylbenzilate, and the binding of [(35)S]GTPgammaS was manifestly biphasic. The ratio of alpha(o) to alpha(i1,2) also was near 1, as determined from the intensity of Western blots. Cardiac muscarinic receptors therefore can be purified as a mixture of complexes that contain caveolins and oligomers of alpha-subunit, some of which are heteromeric. Each complex would appear to contain equal numbers of alpha-subunit and the receptor.


Subject(s)
GTP-Binding Protein alpha Subunits, Gi-Go/metabolism , Heart Atria/metabolism , Receptor, Muscarinic M2/metabolism , Swine/metabolism , Animals , Caveolins/metabolism , GTP-Binding Protein alpha Subunits, Gi-Go/isolation & purification , Muscarinic Antagonists/pharmacology , Protein Subunits/isolation & purification , Protein Subunits/metabolism , Quinuclidinyl Benzilate/pharmacology , Receptor, Muscarinic M2/antagonists & inhibitors , Receptor, Muscarinic M2/isolation & purification
10.
Proteins ; 71(4): 1732-43, 2008 Jun.
Article in English | MEDLINE | ID: mdl-18175322

ABSTRACT

G proteins are the molecular switches of G-protein-coupled signal transmembrane transduction, which plays a pivotal role in diverse cellular processes. The guanine nucleotide binding states of Galpha-subunits are considered key factors for their functions. We report here that phosphatidic acid (PA) inhibits the [(35)S]-GTPgammaS binding activity of Goalpha. To elucidate this inhibitory effect, biochemical analyses are carried out and a structure-based model is proposed. The experimental results show that PA particularly inhibits the activity of the Goalpha in a dose-dependent manner, whereas other lipids tested do not. Further analysis on the effects of PA analogs demonstrate that a phosphate head group together with at least one fatty acid chain is necessary for the inhibition. Using a lipid-protein binding assay, it is shown that Goalpha specifically and directly interacts with PA. In addition to these experimental studies, a 3D structure of Goalpha is constructed, based on sequence homology greater than 70% to E. coli Gialpha(1). Molecular docking is performed with PA and PA analogs, and the results are compared and analyzed. Collectively, the results of this investigation provide direct experimental evidence for an inhibitory effect of PA on GTP binding activity of Goalpha, and also suggest a structural model for the inhibitory mechanism. The lipid-protein model suggests that PA may occupy the channel for exchanging guanine nucleotides, thus leading to the inhibition. These findings reveal a potential new drug target for the diseases caused by genetic G-protein abnormalities.


Subject(s)
GTP-Binding Protein alpha Subunits, Gi-Go/metabolism , Guanosine 5'-O-(3-Thiotriphosphate)/antagonists & inhibitors , Models, Molecular , Phosphatidic Acids/pharmacology , Binding Sites , Computer Simulation , Databases, Factual , Dose-Response Relationship, Drug , Fluorescence Resonance Energy Transfer , GTP-Binding Protein alpha Subunits, Gi-Go/analysis , GTP-Binding Protein alpha Subunits, Gi-Go/chemistry , GTP-Binding Protein alpha Subunits, Gi-Go/isolation & purification , Guanosine 5'-O-(3-Thiotriphosphate)/metabolism , Hydrogen Bonding , Hydrophobic and Hydrophilic Interactions , Inhibitory Concentration 50 , Liposomes/metabolism , Lysophospholipids/chemistry , Lysophospholipids/metabolism , Phosphatidylserines/chemistry , Protein Binding , Protein Structure, Secondary , Protein Structure, Tertiary , Static Electricity , Sulfur Radioisotopes/metabolism
11.
FEBS J ; 274(23): 6106-15, 2007 Dec.
Article in English | MEDLINE | ID: mdl-17986258

ABSTRACT

We have investigated the existence of a precoupled form of the distal C-terminal truncated cannabinoid receptor 1 (CB1-417) and heterotrimeric G proteins in a heterologous insect cell expression system. CB1-417 showed higher production levels than the full-length receptor. The production levels obtained in our expression system were double the values reported in the literature. We also observed that at least the distal C-terminus of the receptor was not involved in receptor dimerization, as was predicted in the literature. Using fluorescence resonance energy transfer, we found that CB1-417 and Galpha(i1)beta(1)gamma(2) proteins were colocalized in the cells. GTPgammaS binding assays with the Sf9 cell membranes containing CB1-417 and the G protein trimer showed that the receptor could constitutively activate the Galpha(i1) protein in the absence of agonists. A CB1-specific antagonist (SR 141716A) inhibited this constitutive activity of the truncated receptor. We found that the CB1-417/Galpha(i1)beta(1)gamma(2) complex could be solubilized from Sf9 cell membranes and coimmunoprecipitated. In this study, we have proven that the receptor and G proteins can be coexpressed in higher yields using Sf9 cells, and that the protein complex is stable in detergent solution. Thus, our system can be used to produce sufficient quantities of the protein complex to start structural studies.


Subject(s)
Heterotrimeric GTP-Binding Proteins/metabolism , Receptor, Cannabinoid, CB1/chemistry , Receptor, Cannabinoid, CB1/metabolism , Spodoptera/cytology , Spodoptera/metabolism , Animals , Baculoviridae/genetics , Cell Membrane/chemistry , Cells, Cultured , Dimerization , Fluorescence Resonance Energy Transfer , GTP-Binding Protein alpha Subunits, Gi-Go/isolation & purification , GTP-Binding Protein alpha Subunits, Gi-Go/metabolism , Genetic Vectors , Guanosine 5'-O-(3-Thiotriphosphate)/metabolism , Microscopy, Confocal , Molecular Weight , Piperidines/pharmacology , Precipitin Tests , Protein Binding , Pyrazoles/pharmacology , Radioligand Assay , Receptor, Cannabinoid, CB1/antagonists & inhibitors , Receptors, G-Protein-Coupled/metabolism , Rimonabant , Viral Fusion Proteins/metabolism
12.
Protein Expr Purif ; 47(1): 303-10, 2006 May.
Article in English | MEDLINE | ID: mdl-16364655

ABSTRACT

G protein-mediated pathways are fundamental mechanisms of cell signaling. In this paper, the expression and the characterization of the alphai1, alphai3, alphao1, beta1, and gamma2 subunits of the human G protein are described. This approach was developed to evaluate the G protein activation profile of new compounds. pCR-TOPO T7 vectors, engineered to contain the target sequences, were used to transform Escherichia coli competent cells. Subunits were over-expressed in a preparative scale as fusion proteins with a six-histidine tag, and subsequently purified by metal chelate chromatography. Afterward, the His-tag was removed by enterokinase digestion, and the secondary structures of the recombinant subunits were analyzed by circular dichroism. To assess the functionality of the subunits, the rate of GTP hydrolysis and GTPgammaS binding were evaluated both in the absence and in the presence of two modulators: the peptidic activator Mastoparan and the non-peptidic activator N-dodecyl-lysinamide (ML250). Tests were conducted on isolated alpha-subunit and on heterotrimeric alphabetagamma complex, alone or reconstituted in phospholipidic vesicles. Our results show that recombinant subunits are stable, properly folded and, fully active, which makes them suitable candidates for functional studies.


Subject(s)
Cloning, Molecular , GTP-Binding Protein alpha Subunits, Gi-Go/biosynthesis , GTP-Binding Protein alpha Subunits, Gi-Go/genetics , Circular Dichroism , Escherichia coli , GTP-Binding Protein alpha Subunits, Gi-Go/isolation & purification , GTP-Binding Protein alpha Subunits, Gi-Go/metabolism , Humans , Intercellular Signaling Peptides and Proteins , Peptides/physiology , Plasmids , Wasp Venoms
13.
J Biomol NMR ; 32(1): 31-40, 2005 May.
Article in English | MEDLINE | ID: mdl-16041481

ABSTRACT

Heterologous expression systems are often employed to generate sufficient quantities of isotope-labeled proteins for high-resolution NMR studies. Recently, the interaction between the prodomain region of subtilisin and an active, mutant form of the mature enzyme has been exploited to develop a cleavable affinity tag fusion system for one-step generation and purification of full-length soluble proteins obtained by inducible prokaryotic expression. As a first step towards applying high-resolution NMR methods to study heterotrimeric G-protein alpha-subunit (G(alpha)) conformation and dynamics, the utility of this subtilisin prodomain fusion system for expressing and purifying an isotope-labeled G(alpha) chimera (approximately 40 kDa polypeptide) has been tested. The results show that a prodomain fused G(alpha) chimera can be expressed to levels approaching 6-8 mg/l in minimal media and that the processed, mature protein exhibits properties similar to those of G(alpha) isolated from natural sources. To assay for the functional integrity of the purified G(alpha) chimera at NMR concentrations and probe for changes in the structure and dynamics of G(alpha) that result from activation, 15N-HSQC spectra of the GDP/Mg2+ bound form of G(alpha) obtained in the absence and presence of aluminum fluoride, a well known activator of the GDP bound state, have been acquired. Comparisons of the 15N-HSQC spectra reveals a number of changes in chemical shifts of the 1HN, 15N crosspeaks that are discussed with respect to expected changes in the protein conformation associated with G(alpha) activation.


Subject(s)
GTP-Binding Protein alpha Subunits, Gi-Go/chemistry , GTP-Binding Protein alpha Subunits, Gi-Go/isolation & purification , Nitrogen Isotopes/chemistry , Protein Structure, Tertiary , GTP-Binding Protein alpha Subunits, Gi-Go/genetics , GTP-Binding Protein alpha Subunits, Gi-Go/metabolism , Guanosine Diphosphate/metabolism , Isotope Labeling , Magnesium/metabolism , Models, Molecular , Nuclear Magnetic Resonance, Biomolecular , Receptors, G-Protein-Coupled/metabolism , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Subtilisins/chemistry
14.
Brain Res Mol Brain Res ; 137(1-2): 89-97, 2005 Jun 13.
Article in English | MEDLINE | ID: mdl-15950765

ABSTRACT

GTP binding proteins play an important role in mediating signals transduced across the cell membrane by membrane-bound receptors. We previously described a partial sequence, termed Galphai2vest, obtained from rat vestibular tissue that was nearly identical to rat Galphai2. Using an experimental strategy to further characterize Galphai2vest (GenBank accession number AF189020) and identify other possible Galphai2-related transcripts expressed in the rat vestibular periphery, we employed a RecA-based gene enrichment protocol in place of conventional library screening techniques. We identified two novel Galphai2 splice variants, Galphai2(a) (GenBank accession number AY899210) and Galphai2(b) (GenBank accession number AY899211), that have most of exons 8 and 9 deleted, and exons 5 through 9 deleted, respectively. In situ hybridization studies were completed to determine the differential expression of Galphai2 between the vestibular primary afferent neurons and the vestibular end organs. Computer modeling and predicted 3D conformation of the wild type Galphai2 and the two splice variants were completed to evaluate the changes associated with the Gbetagamma and GTP binding sites. These two novel alternatively spliced isoforms of Galphai2 putatively encode truncated proteins that could serve unique roles in the physiology of the vestibular neuroepithelium. Galphai2vest was found to be a processed pseudogene.


Subject(s)
Alternative Splicing/genetics , GTP-Binding Protein alpha Subunits, Gi-Go/genetics , GTP-Binding Protein alpha Subunits, Gi-Go/metabolism , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , Vestibular Nerve/metabolism , Vestibule, Labyrinth/metabolism , Animals , Binding Sites/physiology , Exons/genetics , Female , GTP-Binding Protein alpha Subunit, Gi2 , GTP-Binding Protein alpha Subunits, Gi-Go/isolation & purification , Hair Cells, Vestibular/metabolism , Male , Models, Molecular , Molecular Sequence Data , Neurons, Afferent/metabolism , Protein Isoforms/genetics , Protein Isoforms/isolation & purification , Protein Isoforms/metabolism , Proto-Oncogene Proteins/isolation & purification , RNA, Messenger/genetics , RNA, Messenger/metabolism , Rats , Sequence Homology, Amino Acid , Sequence Homology, Nucleic Acid
15.
Biochemistry (Mosc) ; 68(1): 121-8, 2003 Jan.
Article in English | MEDLINE | ID: mdl-12693986

ABSTRACT

Myristoylated G(o)alpha was expressed in and highly purified from Escherichia coli strain JM109 cotransformed with pQE60 (G(o)alpha) and pBB131 (N-myristoyltransferase, NMT). Non-denaturing gel electrophoresis and gel filtration analysis revealed that the G(o)alpha, in its GDP-bound form, could form oligomers involving dimer, trimer, tetramer, pentamer, or hexamer and guanosine 5;-3-O-(thio)triphosphate (GTPgammaS) activation induced disaggregation of the G(o)alpha oligomers to monomers. The G(o)alpha was crosslinked by a cross-linker, N,N-1,4-phenylenedimaleimide (p-PDM), yielding multiple crosslinked products. In contrast, no obvious cross-linking occurred when G(o)alpha was pretreated with GTPgammaS. Immunoblot analysis also demonstrated oligomerization of the purified G(o)alpha proteins and its disaggregation triggered by GTPgammaS. These results provided direct evidence for the "disaggregation-coupling" theory and the disaggregation action of GTPgammaS may further elucidate the regulatory role of GDP/GTP exchange in G protein-coupled signal transduction pathways.


Subject(s)
GTP-Binding Protein alpha Subunits, Gi-Go/metabolism , Guanosine 5'-O-(3-Thiotriphosphate)/metabolism , Blotting, Western , Chromatography, Gel , Cross-Linking Reagents , Electrophoresis, Polyacrylamide Gel , GTP-Binding Protein alpha Subunits, Gi-Go/isolation & purification , Guanosine Diphosphate/metabolism , Maleimides , Myristic Acid/metabolism , Protein Binding , Signal Transduction
16.
Proc Natl Acad Sci U S A ; 98(13): 7617-22, 2001 Jun 19.
Article in English | MEDLINE | ID: mdl-11390975

ABSTRACT

Adenosine and its endogenous precursor ATP are main components of the purinergic system that modulates cellular and tissue functions via specific adenosine and ATP receptors (P1 and P2 receptors), respectively. Although adenosine inhibits excitability and ATP functions as an excitatory transmitter in the central nervous system, little is known about the ability of P1 and P2 receptors to form new functional structures such as a heteromer to control the complex purinergic cascade. Here we have shown that G(i/o) protein-coupled A1 adenosine receptor (A1R) and Gq protein-coupled P2Y1 receptor (P2Y1R) coimmunoprecipitate in cotransfected HEK293T cells, suggesting the oligomeric association between distinct G protein-coupled P1 and P2 receptors. A1R and P2Y2 receptor, but not A1R and dopamine D2 receptor, also were found to coimmunoprecipitate in cotransfected cells. A1R agonist and antagonist binding to cell membranes were reduced by coexpression of A1R and P2Y1R, whereas a potent P2Y1R agonist adenosine 5'-O-(2-thiotriphosphate) (ADPbetaS) revealed a significant potency to A1R binding only in the cotransfected cell membranes. Moreover, the A1R/P2Y1R coexpressed cells showed an ADPbetaS-dependent reduction of forskolin-evoked cAMP accumulation that was sensitive to pertussis toxin and A1R antagonist, indicating that ADPbetaS binds A1R and inhibits adenylyl cyclase activity via G(i/o) proteins. Also, a high degree of A1R and P2Y1R colocalization was demonstrated in cotransfected cells by double immunofluorescence experiments with confocal laser microscopy. These results suggest that oligomeric association of A1R with P2Y1R generates A1R with P2Y1R-like agonistic pharmacology and provides a molecular mechanism for an increased diversity of purine signaling.


Subject(s)
Receptors, Purinergic P1/physiology , Receptors, Purinergic P2/physiology , Adenosine/analogs & derivatives , Adenosine/pharmacology , Adenylyl Cyclases/metabolism , Animals , Cell Line , Cell Membrane/physiology , Cyclic AMP/metabolism , GTP-Binding Protein alpha Subunits, Gi-Go/chemistry , GTP-Binding Protein alpha Subunits, Gi-Go/isolation & purification , GTP-Binding Protein alpha Subunits, Gi-Go/physiology , Heterotrimeric GTP-Binding Proteins/chemistry , Heterotrimeric GTP-Binding Proteins/isolation & purification , Heterotrimeric GTP-Binding Proteins/physiology , Humans , Inositol 1,4,5-Trisphosphate/metabolism , Kinetics , Macromolecular Substances , Purinergic P1 Receptor Agonists , Radioligand Assay , Rats , Receptors, Dopamine D2/chemistry , Receptors, Dopamine D2/isolation & purification , Receptors, Dopamine D2/physiology , Receptors, Purinergic P1/isolation & purification , Receptors, Purinergic P2/chemistry , Receptors, Purinergic P2/isolation & purification , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Transfection , Tritium , Xanthines/pharmacokinetics
17.
Biochem Pharmacol ; 61(4): 485-91, 2001 Feb 15.
Article in English | MEDLINE | ID: mdl-11226383

ABSTRACT

Several studies have shown that stimulation of pertussis toxin (PTX)-sensitive G-proteins amplified alpha-adrenoceptor (alpha-AR) agonist-induced vasoconstriction in small muscular and resistance arteries. The aim of this study was to assess the potential involvement of PTX-sensitive G-proteins in norepinephrine (NE)-induced constriction in a large diameter artery, the rat aorta. PTX (1 microg/mL, 2 hr; 3 microg/mL, 4 hr) did not modify concentration-response curves to NE in endothelium-denuded aortic rings. However, several lines of evidence suggested that aortic smooth muscle cells (SMC) had a PTX-sensitive G-protein pathway. [alpha-(32)P]ADP-ribosylation of G(i/o)-proteins by PTX (3 microg/mL, 4 hr) was demonstrated in situ in the intact aorta without endothelium. alpha(i/o) subunits were identified in vitro by both immunoblotting and ADP-ribosylation experiments in rat aorta SMC membranes. The measurement of G(i/o)-specific GTPase activity evidenced an effective coupling between NE receptors and G(i/o)-proteins, as NE induced an increase in basal G(i/o)-specific GTPase activity (20.7 +/- 2.8 vs 7.2 +/- 2.2 pmol P(i)/mg protein at 5 min; P < 0.05 vs basal). Co-immunoprecipitation revealed the in vitro coupling between alpha(1D)-ARs and G(i)-protein in rat aorta SMC membranes. In conclusion, we identified a PTX-sensitive G(i/o)-protein pathway in rat endothelium-denuded aorta. We showed an effective coupling between NE receptors and G(i)-proteins via alpha(1D)-ARs. Since PTX has no effect on NE-induced vasoconstriction, the PTX-sensitive G(i)-protein pathway does not play a predominant role in NE-induced responses in rat aorta SMC in contrast to small diameter muscular and resistance arteries.


Subject(s)
GTP-Binding Protein alpha Subunits, Gi-Go/physiology , Heterotrimeric GTP-Binding Proteins/physiology , Muscle, Smooth, Vascular/physiology , Norepinephrine/pharmacology , Pertussis Toxin , Vasoconstriction/physiology , Virulence Factors, Bordetella/pharmacology , Adrenergic alpha-Agonists/pharmacology , Animals , Aorta/drug effects , Aorta/physiology , Dose-Response Relationship, Drug , Enzyme Activation , GTP Phosphohydrolases/metabolism , GTP-Binding Protein alpha Subunits, Gi-Go/isolation & purification , Heterotrimeric GTP-Binding Proteins/isolation & purification , In Vitro Techniques , Male , Muscle, Smooth, Vascular/drug effects , Prazosin/pharmacology , Radioligand Assay , Rats , Rats, Wistar , Receptors, Adrenergic/physiology , Receptors, Adrenergic, alpha-1/analysis , Tritium
18.
Biochem J ; 337 ( Pt 2): 289-95, 1999 Jan 15.
Article in English | MEDLINE | ID: mdl-9882627

ABSTRACT

Autophagic sequestration is controlled by the Galphai3 protein in human colon cancer HT-29 cells. Immunofluorescence and subcellular fractionation studies showed that the Galphai3 protein is preferentially associated with Golgi membranes but co-localization was also observed with the endoplasmic reticulum (ER) membrane. The Galphai2 protein, which is not involved in the control of autophagic sequestration, is associated with the plasma membrane. Transfection of chimaeric Galphai proteins (Galphai3/2, Galphai2/3) containing the N- and C-terminal parts of the relevant Galphai demonstrated that the C-terminal part of the Galphai3 protein, by governing its membrane localization [de Almeida, Holtzman, Peters, Ercolani, Ausiello and Stow (1994) J. Cell Sci. 107, 507-515], is important in the control of macroautophagic sequestration. G alpha interacting protein (GAIP),which stimulates the GTPase activity of the Galphai3 protein and favours macroautophagic sequestration in HT-29 cells,was shown, by immunofluorescence studies using confocal microscopy, to be confined to the cytoplasm. The cytoplasmic distribution of GAIP only partially overlaps with that of the Galphai3 protein. However, the presence of the two proteins on Golgi and ER membranes was confirmed by subcellular fractionation. These results point to the importance of the cytoplasmic localization of the Galphai3 protein and GAIP in controlling autophagic sequestration in HT-29 cells.


Subject(s)
Autophagy , Colonic Neoplasms/metabolism , GTP-Binding Protein alpha Subunits, Gi-Go/isolation & purification , Phosphoproteins/isolation & purification , Cell Compartmentation , Cell Fractionation , Endoplasmic Reticulum/chemistry , Endoplasmic Reticulum/ultrastructure , Fluorescent Antibody Technique , GTP-Binding Protein alpha Subunits, Gi-Go/genetics , Golgi Apparatus/chemistry , Golgi Apparatus/ultrastructure , HT29 Cells , Humans , Phosphoproteins/genetics , Phosphoproteins/immunology , RGS Proteins , Recombinant Fusion Proteins/isolation & purification
19.
J Biol Chem ; 273(37): 24181-9, 1998 Sep 11.
Article in English | MEDLINE | ID: mdl-9727041

ABSTRACT

The formyl peptide receptor (FPR) couples to pertussis toxin (PTX)-sensitive Gi-proteins to activate chemotaxis and exocytosis in neutrophils. PTX reduces not only formyl peptide-stimulated but also agonist-independent ("basal") Gi-protein activity, suggesting that the FPR is constitutively active. We aimed at identifying an inverse FPR agonist, i.e. a compound that suppresses constitutive FPR activity. In Sf9 insect cell membranes, the G-protein heterotrimer Gialpha2beta1gamma2 reconstituted N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP)-stimulated guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) binding and GTPgammaS-sensitive high affinity [3H]FMLP binding. The FPR "antagonist" cyclosporin H (CsH) potently and efficiently reduced basal GTPgammaS binding in Sf9 membranes. Another FPR antagonist, N-t-butoxycarbonyl-L-phenylalanyl-L-leucyl-L-phenylalanyl-L-leucyl-L- phenylalanine did not inhibit basal GTPgammaS binding but blocked the inhibitory effect of CsH on GTPgammaS binding. Na+ reduced basal GTPgammaS binding and eliminated the inhibitory effect of CsH. Similar effects of FMLP, CsH, and Na+ as in Sf9 membranes were observed with FPR expressed in the mammalian cell line HEK293. Our data show that the human FPR possesses high constitutive activity. CsH is an inverse FPR agonist and stabilizes the FPR in an inactive state. Na+ also stabilizes the FPR in an inactive state and, thereby, diminishes inverse agonist efficacy.


Subject(s)
GTP-Binding Proteins/metabolism , Guanosine 5'-O-(3-Thiotriphosphate)/pharmacology , N-Formylmethionine Leucyl-Phenylalanine/pharmacology , Receptors, Immunologic/metabolism , Receptors, Peptide/metabolism , Animals , Cell Line , Cell Membrane/physiology , Cyclosporine/pharmacology , GTP-Binding Protein alpha Subunits, Gi-Go/isolation & purification , GTP-Binding Protein alpha Subunits, Gi-Go/metabolism , Guanosine 5'-O-(3-Thiotriphosphate)/metabolism , Humans , Kinetics , Models, Chemical , N-Formylmethionine Leucyl-Phenylalanine/metabolism , Receptors, Formyl Peptide , Receptors, Immunologic/agonists , Receptors, Immunologic/antagonists & inhibitors , Receptors, Peptide/agonists , Receptors, Peptide/antagonists & inhibitors , Recombinant Proteins/metabolism , Sodium/pharmacology , Spodoptera , Transfection
20.
Neurochem Int ; 31(1): 21-5, 1997 Jul.
Article in English | MEDLINE | ID: mdl-9185160

ABSTRACT

A single molecular species of GTP-binding protein (G protein) has been purified from the bovine cerebral cortex. The immunoblot analysis indicated that the isolated G protein might be Gi1 or Gi2 but not Go, since it was reacted by specific antibodies, anti-Gi alpha 1-2 and anti-Gi alpha 1-1, but not anti-Go alpha. When the Gi protein was reconstituted into phospholipid vesicles with partially purified GABAB receptor and adenylyl cyclase, the stimulation of GABAB receptor by its agonists induced the inhibition of forskolin-stimulated cAMP accumulation. This GABA-induced inhibition was abolished by CGP 55845A, an antagonist of GABAB receptor. These results suggest that a Gi subtype, which was suggested to correspond to Gi1 or Gi2 may be functionally coupled with GABAB receptor/adenylyl cyclase system.


Subject(s)
Adenylyl Cyclases/metabolism , Cerebral Cortex/metabolism , GTP-Binding Protein alpha Subunits, Gi-Go/metabolism , GTP-Binding Proteins/metabolism , Receptors, GABA-A/metabolism , Animals , Cattle , GTP-Binding Protein alpha Subunits, Gi-Go/isolation & purification , GTP-Binding Proteins/isolation & purification , Liposomes , Phospholipids/metabolism , Receptors, GABA-B/metabolism
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