G-protein Α-subunit Research Articles

The Active Conformation Of Opsin (ops∗): Role In Signal Transduction And Regeneration

Functional modules are specific subsets of the proteome, which fulfil an autonomous function in living cells. Functional modules in retinal photoreceptors include those devoted to phototransduction and regeneration [1]. In signal transduction, rhodopsin is kept inactive in the dark by a set of structural elements and residues conserved in the E(D)RY and NPxxY(x)5,6F regions of the receptor protein. On light activation, the same residues are crucial determinants of the Meta II state [2], with the hallmark of a motion of the sixth of the seven transmembrane helices [3]. The same elements stabilize the ligand free active form of the opsin apoprotein, Ops∗ [4], and build up new interactions of Ops∗ with the G-protein α-subunit C-terminal interaction site [5]. The Ops∗ state can thus act as the key element in both functional modules of the rod cell. In signal transduction, the Ops∗ conformation is present in the highly active Meta II photoproduct. In rhodopsin regeneration, Ops∗ facilitates the uptake of 11-cis-retinal into the binding site [6] and causes the visual system to behave as if it is experiencing an “equivalent background” [7]. 1. Hofmann, K.P., et al., Trends Biochem Sci, 2006. 31, 497. 2. Knierim, B., et al., PNAS 2007 104, 4967. 3. Altenbach, C., et al., PNAS 2008. 105, 7439. 4. Park, J.H., et al., Nature, 2008. 454, 183. 5. Scheerer, P., et al., Nature, 2008. 455, 497. 6. Kefalov, V.J., et al., Neuron, 2001, 29, 749. 7. Lamb, T.D. & E.N. Pugh, Jr.,Prog Retin Eye Res, 2004. 23, 307.

Heterotrimeric G protein α and β subunits antagonistically modulate stomatal density in Arabidopsis thaliana

Stomata are essential for efficient gas and water-vapor exchange between the atmosphere and plants. Stomatal density and movement are controlled by a series of signal molecules including phytohormones and peptides as well as by environmental stimuli. It is known that heterotrimeric G-proteins play an important role in the ABA-inhibited stomatal opening. In this study, the G-protein signaling pathway was also found to regulate stomatal density on the lower epidermis of Arabidopsis cotyledons. The loss-of-function mutation of the G-protein α-subunit ( GPA1) showed a reduction in stomatal density, while overexpression of the constitutively active form of GPA1 QL increased stomatal density, indicating a positive role of the active form of GPA1 in stomatal development. In contrast, stomatal density increased in the null mutant of the G-protein β-subunit ( AGB1) but decreased in transgenic lines that overexpressed AGB1. Stomatal analysis of the gpa1 agb1 double mutants displayed an average value of stomatal density compared to the single mutants. Taken together, these results suggest that the stomatal density in Arabidopsis is modulated by GPA1 and AGB1 in an antagonistic manner.

Prevalence of mutations in TSHR, GNAS, PRKAR1A and RAS genes in a large series of toxic thyroid adenomas from Galicia, an iodine-deficient area in NW Spain

Toxic thyroid adenoma (TA) is a common cause of hyperthyroidism. Mutations in the TSH receptor (TSHR) gene, and less frequently in the adenylate cyclase-stimulating G alpha protein (GNAS) gene, are well established causes of TA in Europe. However, genetic causes of TA remain unknown in a small percentage of cases. We report the first study to investigate mutations in TSHR, GNAS, protein kinase, cAMP-dependent, regulatory, type I alpha (PRKAR1A) and RAS genes, in a large series of TA from Galicia, an iodine-deficient region in NW Spain. Eighty-five TA samples were obtained surgically from 77 hyperthyroid patients, operated on for treatment of non-autoimmune toxic nodular goitre. After DNA extraction, all coding exons of TSHR, GNAS and PRKAR1A genes, and exons 2 and 3 of HRAS, KRAS and NRAS were amplified by PCR and sequenced. Previously unreported mutants were cloned in expression vectors and their basal constitutive activities were determined by quantification of cAMP response element (CRE)-luciferase activity in CO7 cells transfected with wild-type and mutant plasmids. TSHR gene mutations were found in 52 (61.2%) samples, GNAS gene mutations in 4 (4.71%) samples and no PRKAR1A or RAS mutations were found. Only three previously unreported mutations were found, two affecting the TSHR, A623F and I635V, and one affecting the G-protein alpha-subunit (Gsalpha), L203P. All mutant proteins showed higher CRE-luciferase activity than their wild-type counterparts. TA in a hyperthyroid population living in Galicia, a Spanish iodine-deficient region, harbours elevated frequencies of TSHR and GNAS mutations activating the cAMP pathway. However, the genetic cause of TA was undetermined in 34% of the TA samples.

Functional reconstitution of G-protein-coupled receptor-mediated adenylyl cyclase activation by a baculoviral co-display system

Recently, evidence has accumulated in support of the heterologous expression of functional membrane proteins and their complexes on extracellular baculovirus particles (budded virus, BV). In this study, we attempted to apply this BV display system to detect G-protein-coupled receptor (GPCR) signaling. We infected Sf9 cells with a combination of four recombinant baculoviruses individually encoding the dopamine D1 receptor (DR-D1), G-protein α-subunit (Gα s), G-protein β 1γ 2 subunit dimer (Gβ 1γ 2), and adenylyl cyclase type VI (ACVI). The recovered BV fraction produced cAMP in response to the stimulation with dopamine. Co-expression of all three G-protein subunits in addition to receptor and ACVI led to a maximal response. BV co-expressing DR-D1, Gα s, Gβ 1γ 2, and ACVI also responded to dopamine agonists and an antagonist. Furthermore, BV expressing two other Gα s-coupled receptors together with Gα s, Gβ 1γ 2, and ACVI also produced cAMP in response to their specific ligands. These results indicate the functional coupling of receptor, Gα s and ACVI is reconstituted on BV. Since BV is essentially free of endogenous GPCRs, this BV co-display system should prove highly useful for the development of functional assay systems for GPCRs.

Cloning and Characterization of Sdga Gene Encoding .ALPHA.-Subunit of Heterotrimeric Guanosine 5'-Triphosphate-Binding Protein Complex in Scoparia dulcis

A homology-based cloning strategy yielded Sdga, a cDNA clone presumably encoding alpha-subunit of heterotrimeric guanosine 5'-triphosphate-binding protein complex, from leaf tissues of Scoparia dulcis. Phylogenetic tree analysis of G-protein alpha-subunits from various biological sources suggested that, unlike in animal cells, classification of Galpha-proteins into specific subfamilies could not be applicable to the proteins from higher plants. Restriction digests of genomic DNA of S. dulcis showed a single hybridized signal in Southern blot analysis, suggesting that Sdga is a sole gene encoding Galpha-subunit in this plant. The expression level of Sdga appeared to be maintained at almost constant level after exposure of the leaves to methyl jasmonate as analyzed by reverse-transcription polymerase chain reaction. These results suggest that Sdga plays roles in methyl jasmonate-induced responses of S. dulcis without a notable change in the transcriptional level.

N-Terminal Fatty Acylation of Transducin Profoundly Influences Its Localization and the Kinetics of Photoresponse in Rods

N-terminal acylation of the alpha-subunits of heterotrimeric G-proteins is believed to play a major role in regulating the cellular localization and signaling of G-proteins, but physiological evidence has been lacking. To examine the functional significance of N-acylation of a well understood G-protein alpha-subunit, transducin (G alpha(t)), we generated transgenic mice that expressed a mutant G alpha(t) lacking N-terminal acylation sequence (G alpha(t)G2A). Rods expressing G alpha(t)G2A showed a severe defect in transducin cellular localization. In contrast to native G alpha(t), which resides in the outer segments of dark-adapted rods, G alpha(t)G2A was found predominantly in the inner compartments of the photoreceptor cells. Remarkably, transgenic rods with the outer segments containing G alpha(t)G2A at 5-6% of the G alpha(t) levels in wild-type rods showed only a sixfold reduction in sensitivity and a threefold decrease in the amplification constant. The much smaller than predicted reduction may reflect an increase in the lateral diffusion of transducin and an increased activation rate by photoexcited rhodopsin or more efficient activation of cGMP phosphodiesterase 6 by G alpha(t)G2A; alternatively, nonlinear relationships between concentration and the activation rate of transducin also potentially contribute to the mismatch between the amplification constant and quantitative expression analysis of G alpha(t)G2A rods. Furthermore, the G2A mutation reduced the GTPase activity of transducin and resulted in two to three times slower than normal recovery of flash responses of transgenic rods, indicating the role of G alpha(t) membrane tethering for its efficient inactivation by the regulator of G-protein signaling 9 GTPase-activating protein complex. Thus, N-acylation is critical for correct compartmentalization of transducin and controls the rate of its deactivation.

Polycystic kidney disease: genes, proteins, animal models, disease mechanisms and therapeutic opportunities

An increased understanding of the genetic, molecular and cellular mechanisms responsible for the development of polycystic kidney disease has laid out the foundation for the development of rational therapies. Many animal models where these therapies can be tested are currently available. This review summarizes the rationale for these treatments, the results of preclinical trials and the prospects for clinical trials, some already in early phases of implementation.

Syndromes of hormone resistance in the hypothalamic–pituitary–thyroid axis

Forty years have elapsed since the first description of a syndrome of resistance in the hypothalamic-pituitary-thyroid axis, i.e., resistance to thyroid hormone action. In the last two decades many other types of resistance have been discovered, including resistance to the action of thyrotropin-releasing hormone (TRH), of thyroid-stimulating hormone (TSH), and of thyroid hormones (THs); the latter can be due not only to thyroid hormone receptor defects but also to alteration in genes encoding TH-specific transporters or components involved in metabolic pathways of THs. Moreover, alteration in genes encoding for second messengers may cause forms of resistance other than those due to receptor mutations, the most important one being that of an inactivating mutation in the G-protein alpha-subunit leading to TSH resistance in the setting of pseudohypoparathyroidism type 1a. Recognition of these rare thyroid disorders is of great importance not only for informed genetic counselling but also for avoiding diagnostic mistakes that may lead to incorrect and potentially dangerous treatments.

The First Luminal Domain of Vesicular Monoamine Transporters Mediates G-protein-dependent Regulation of Transmitter Uptake

The activity of vesicular monoamine transporters (VMATs) is down-regulated by the G-protein alpha-subunits of G(o2) and G(q), but the signaling pathways are not known. We show here that no such regulation is observed when VMAT1 or VMAT2 are expressed in Chinese hamster ovary (CHO) cells. However, when the intracellular compartments of VMAT-expressing CHO cells are preloaded with different monoamines, transport becomes susceptible to G-protein-dependent regulation, with differences between the two transporter isoforms. Epinephrine induces G-protein-mediated inhibition of transmitter uptake in CHOVMAT1 cells but prevents inhibition induced by dopamine in CHOVMAT2 cells. Epinephrine also antagonizes G-protein-mediated inhibition of monoamine uptake by VMAT2 expressing platelets or synaptic vesicles. In CHOVMAT2 cells G-protein-mediated inhibition of monoamine uptake can be induced by 5-hydroxytryptamine (serotonin) 1B receptor agonists, whereas alpha1 receptor agonists modulate uptake into CHOVMAT1 cells. Accordingly, 5-hydroxytryptamine 1B receptor antagonists prevent G-protein-mediated inhibition of uptake in partially filled platelets and synaptic vesicles expressing VMAT2. CHO cells expressing VMAT mutants with a shortened first vesicular loop transport monoamines. However, no or a reduced G-protein regulation of uptake can be initiated. In conclusion, vesicular content is involved in the activation of vesicle associated G-proteins via a structure sensing the luminal monoamine content. The first luminal loop of VMATs may represent a G-protein-coupled receptor that adapts vesicular filling.

The Receptor-Bound “Empty Pocket” State of the Heterotrimeric G-Protein α-Subunit Is Conformationally Dynamic

Heterotrimeric G-protein activation by a G-protein-coupled receptor (GPCR) requires the propagation of structural signals from the receptor-interacting surfaces to the guanine nucleotide-binding pocket. To probe conformational changes in the G-protein alpha-subunit (G(alpha)) associated with activated GPCR (R*) interactions and guanine nucleotide exchange, high-resolution solution NMR methods are being applied in studying signaling of the G-protein, transducin, by light-activated rhodopsin. Using these methods, we recently demonstrated that an isotope-labeled G(alpha) reconstituted heterotrimer forms functional complexes under NMR experimental conditions with light-activated, detergent-solubilized rhodopsin and a soluble mimic of R*, both of which trigger guanine nucleotide exchange [Ridge, K. D., et al. (2006) J. Biol. Chem. 281, 7635-7648]. Here, it is shown that both light-activated rhodopsin and the soluble mimic of R form trapped intermediate complexes with a GDP-released "empty pocket" state of the heterotrimer in the absence of GTP (or GTPgammaS). In contrast to guanine nucleotide-bound forms of G(alpha), the NMR spectra of the GDP-released, R-bound empty pocket state of G(alpha) display severe line broadening suggestive of a dynamic intermediate state. Interestingly, the conformation of a GDP-depleted, Mg(2+)-bound state of G(alpha) generated in a manner independent of R* does not exhibit a similar degree of line broadening but rather appears structurally similar to the GDP/Mg(2+)-bound form of the protein. Taken together, these results suggest that R*-mediated changes in the receptor-interacting regions of G(alpha), and not the absence of bound guanine nucleotide, are the predominant factors which dictate G(alpha) conformation and dynamics in this R*-bound state of the heterotrimer.

Expression of the G-protein α-subunit gustducin in mammalian spermatozoa

Although chemotaxis has been proposed to guide sperm to egg throughout the animal kingdom, sperm attractants released from mammalian eggs have not been identified. Since the G protein subunit alpha-gustducin is accepted as a marker of chemosensitive cells, attempts were made to explore whether alpha-gustducin is also expressed in spermatozoa of mammals. Immunohistochemical approaches using an anti-alpha-gustducin-specific antibody revealed the most intense immunoreactivity in differentiating spermatids. Further evidence for the alpha-gustducin expression was obtained analyzing testicular and sperm-derived tissue preparations in western blot analyses. To elucidate whether alpha-gustducin is retained in mature spermatozoa, epididymal mouse and rat sperm were subjected to immunocytochemistry as well as immunogold electron microscopy. A specific staining was obtained within the circumference of the midpiece-localized mitochondria, on the axoneme and the outer dense fibers surrounding the microtubules of this region, whereas no labeling was detectable in the end piece regions. The analysis of ejaculated bovine and human sperm revealed a comparable segmental distribution pattern for alpha-gustducin. Although a possible function for alpha-gustducin has yet to be determined, the axonemal-associated localization within the midpiece and principal piece of different mammalian spermatozoa raises the possibility that this G protein alpha-subunit may process intracellular signals controlling sperm motility.

NMR analysis of rhodopsin–transducin interactions

Heterotrimeric G-protein activation by an agonist-stimulated G-protein coupled receptor (R ∗) requires the propagation of structural signals from the receptor interacting surfaces to the guanine nucleotide-binding pocket. Employing high-resolution NMR methods, we are probing heterotrimer-associated and rhodopsin-stimulated changes in an isotope-labeled G-protein α-subunit (G α). A key aspect of the work involves the trapping and interrogation of discrete R ∗-bound conformations of G α. Our results demonstrate that functionally important changes in G α structure and dynamics can be detected and characterized by NMR, enabling the generation of robust models for the global and local structural changes accompanying signal transfer from R ∗ to the G-protein.

Differential Roles of Arabidopsis Heterotrimeric G-Protein Subunits in Modulating Cell Division in Roots

Signaling through heterotrimeric G proteins is conserved in diverse eukaryotes. Compared to vertebrates, the simpler repertoire of G-protein complex and accessory components in Arabidopsis (Arabidopsis thaliana) offers a unique advantage over all other multicellular, genetic-model systems for dissecting the mechanism of G-protein signal transduction. One of several biological processes that the G-protein complex regulates in Arabidopsis is cell division. We determined cell production rate in the primary root and the formation of lateral roots in Arabidopsis to define individually the types of modulatory roles of the respective G-protein alpha- and beta-subunits, as well as the heterotrimer in cell division. The growth rate of the root is in part a consequence of cell cycle maintenance in the root apical meristem (RAM), while lateral root production requires meristem formation by founder pericycle cells. Thus, a comparison of these two parameters in various genetic backgrounds enabled dissection of the role of the G-protein subunits in modulation of cell division, both in maintenance and initiation. Cell production rates were determined for the RAM and lateral root formation in gpa1 (Arabidopsis G-protein alpha-subunit) and agb1 (Arabidopsis G-protein beta-subunit) single and double mutants, and in transgenic lines overexpressing GPA1 or AGB1 in agb1 or gpa1 mutant backgrounds, respectively. We found in the RAM that the heterotrimeric complex acts as an attenuator of cell proliferation, whereas the GTP-bound form of the Galpha-subunit's role is a positive modulator. In contrast, for the formation of lateral roots, the Gbetagamma-dimer acts largely independently of the Galpha-subunit to attenuate cell division. These results suggest that Arabidopsis heterotrimeric G-protein subunits have differential and opposing roles in the modulation of cell division in roots.

Conformational Changes Associated with Receptor-stimulated Guanine Nucleotide Exchange in a Heterotrimeric G-protein α-Subunit: NMR ANALYSIS OF GTPγS-BOUND STATES

Solution NMR studies of a (15)N-labeled G-protein alpha-subunit (G(alpha)) chimera ((15)N-ChiT)-reconstituted heterotrimer have shown previously that G-protein betagamma-subunit (G(betagamma)) association induces a "pre-activated" conformation that likely facilitates interaction with the agonist-activated form of a G-protein-coupled receptor (R*) and guanine nucleotide exchange (Abdulaev, N. G., Ngo, T., Zhang, C., Dinh, A., Brabazon, D. M., Ridge, K. D., and Marino, J. P. (2005) J. Biol. Chem. 280, 38071-38080). Here we demonstrated that the (15)N-ChiT-reconstituted heterotrimer can form functional complexes under NMR experimental conditions with light-activated, detergent-solubilized rhodopsin (R*), as well as a soluble mimic of R*. NMR methods were used to track R*-triggered guanine nucleotide exchange and release of guanosine 5'-O-3-thiotriphosphate (GTPgammaS)/Mg(2+)-bound ChiT. A heteronuclear single quantum correlation (HSQC) spectrum of R*-generated GTPgammaS/Mg(2+)-bound ChiT revealed (1)HN, (15)N chemical shift changes relative to GDP/Mg(2+)-bound ChiT that were similar, but not identical, to those observed for the GDP.AlF(4)(-)/Mg(2+)-bound state. Line widths observed for R*-generated GTPgammaS/Mg(2+)-bound (15)N-ChiT, however, indicated that it is more conformationally dynamic relative to the GDP/Mg(2+)- and GDP.AlF(4)(-)/Mg(2+)-bound states. The increased dynamics appeared to be correlated with G(betagamma) and R* interactions because they are not observed for GTPgammaS/Mg(2+)-bound ChiT generated independently of R*. In contrast to R*, a soluble mimic that does not catalytically interact with G-protein (Abdulaev, N. G., Ngo, T., Chen, R., Lu, Z., and Ridge, K. D. (2000) J. Biol. Chem. 275, 39354-39363) is found to form a stable complex with the GTPgammaS/Mg(2+)-exchanged heterotrimer. The HSQC spectrum of (15)N-ChiT in this complex displays a unique chemical shift pattern that nonetheless shares similarities with the heterotrimer and GTPgammaS/Mg(2+)-bound ChiT. Overall, these results demonstrated that R*-induced changes in G(alpha) can be followed by NMR and that guanine nucleotide exchange can be uncoupled from heterotrimer dissociation.

Rho GEF Lsc is required for normal polarization, migration, and adhesion of formyl-peptide–stimulated neutrophils

Neutrophil migration requires continuous reorganization of the cytoskeleton and cellular adhesion apparatus. Chemoattractants initiate intracellular signals that direct this reorganization. The signaling pathways that link chemoattractant receptors to the cytoskeleton and cellular adhesion apparatus are now being defined. Formyl-peptide chemoattractants released from bacteria stimulate G-protein-linked receptors on the surface of neutrophils and regulate the neutrophil cytoskeleton and adhesion apparatus through RhoA-dependent pathways. Lsc is a RhoA guanine nucleotide exchange factor that binds the heterotrimeric G-protein alpha-subunits, Galpha12 and Galpha13. We have disrupted the Lsc gene and demonstrated that formyl-peptide-stimulated Lsc knock-out (KO) neutrophils are unable to generate and sustain a single-dominant pseudopod and migrate with increased speed and reduced directionality. Unexpectedly, we also found that Lsc is required for normal beta2- and beta1-integrin-dependent neutrophil adhesion. Lsc-deficient mice have a peripheral leukocytosis and extramedullary hematopoiesis, demonstrating that Lsc is required for leukocyte homeostasis. Lsc-deficient neutrophils are recruited normally to sites of bacterial peritonitis and chemical dermatitis, indicating that other signaling pathways compensate for the Lsc deficiency in some forms of inflammation. These results demonstrate that Lsc links formyl-peptide receptors to RhoA signaling pathways that regulate polarization, migration, and adhesion in neutrophils and that Lsc is required for leukocyte homeostasis.

Global Patterns of Gene Expression in the Aleurone of Wild-Type and dwarf1 Mutant Rice

The cereal aleurone layer is a model system for studying the regulation of transcription by gibberellin (GA) and abscisic acid (ABA). GA stimulates and ABA prevents the transcription of genes for alpha-amylases and other secreted hydrolytic enzymes, but how GA and ABA affect the transcription of other genes is largely unknown. We characterized gene expression in rice (Oryza sativa) aleurone using a half-genome rice microarray. Of the 23,000 probe sets on the chip, approximately 11,000 hybridized with RNA from rice aleurone treated with ABA, GA, or no hormone. As expected, GA regulated the expression of many genes, and 3 times as many genes were up-regulated by GA at 8 h than were down-regulated. Changes in gene expression resulting from ABA treatment were not consistent with the hypothesis that the role of ABA in this tissue is primarily to repress gene expression, and 10 times more genes were up-regulated by ABA at 8 h than were down-regulated by ABA. We also measured transcript abundance in aleurone of dwarf1 (d1) mutant rice. The d1 protein is the sole alpha-subunit of heterotrimeric G-proteins in rice. Genes up-regulated by GA or ABA had higher expression in wild type than in d1 aleurone, and genes down-regulated by GA had lower expression in wild type relative to d1 aleurone. The d1 mutation did not result in a decrease in sensitivity to GA at the level of transcription. Rather, changes in transcript abundance were smaller in the d1 mutant than in wild type.

Heterotrimeric G-protein α-Subunit Adopts a “Preactivated” Conformation When Associated with βγ-Subunits

Heterotrimeric G-protein α-Subunit Adopts a “Preactivated” Conformation When Associated with βγ-Subunits

Deficiency of the G-protein α-Subunit Gsα in Osteoblasts Leads to Differential Effects on Trabecular and Cortical Bone

The G-protein alpha-subunit G(s)alpha is required for the intracellular cAMP responses to hormones and other agonists. G(s)alpha is known to mediate the cAMP response to parathyroid hormone and other hormones and cytokines in bone and cartilage. To analyze the in vivo role of G(s)alpha signaling in osteoblasts, we developed mice with osteoblast/osteocyte-specific G(s)alpha deficiency (BGsKO) by mating G(s)alpha-floxed mice with collagen Ialpha1 promoter-Cre recombinase transgenic mice. Early skeletal development was normal in BGsKO mice, because formation of the initial cartilage template and bone collar was unaffected. The chondrocytic zones of the growth plates also appeared normal in BGsKO mice. BGsKO mice had a defect in the formation of the primary spongiosa with reduced immature osteoid (new bone formation) and overall length, which led to reduced trabecular bone volume. In contrast, cortical bone was thickened with narrowing of the bone marrow cavity. This was probably due to decreased cortical bone resorption, because osteoclasts were markedly reduced on the endosteal surface of cortical bone. In addition, the expression of alkaline phosphatase, an early osteoblastic differentiation marker, was normal, whereas the expression of the late osteoblast differentiation markers osteopontin and osteocalcin was reduced, suggesting that the number of mature osteoblasts in bone is reduced. Expression of the osteoclast-stimulating factor receptor activator of NF-kappaB ligand was also reduced. Overall, our findings have similarities to parathyroid hormone null mice and confirm that the differential effects of parathyroid hormone on trabecular and cortical bone are primarily mediated via G(s)alpha in osteoblasts. Osteoblast-specific G(s)alpha deficiency leads to reduced bone turnover.

Phosducin-like Protein Regulates G-Protein βγ Folding by Interaction with Tailless Complex Polypeptide-1α: DEPHOSPHORYLATION OR SPLICING OF PhLP TURNS THE SWITCH TOWARD REGULATION OF Gβγ FOLDING

Phosducin-like Protein Regulates G-Protein βγ Folding by Interaction with Tailless Complex Polypeptide-1α: DEPHOSPHORYLATION OR SPLICING OF PhLP TURNS THE SWITCH TOWARD REGULATION OF Gβγ FOLDING

Bacterial expression and one-step purification of an isotope-labeled heterotrimeric G-protein α-subunit

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.