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• W2170720976 abstract "Article1 September 1998free access Temperature-sensitive Gβ mutants discriminate between G protein-dependent and -independent signaling mediated by serpentine receptors Tian Jin Tian Jin Department of Biological Chemistry, Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD, 21205 USA Search for more papers by this author Ron D.M. Soede Ron D.M. Soede Cell Biology Section, Institute for Molecular Plant Sciences, University of Leiden, Wassenaarseweg 64, 2333 AL, Leiden, The Netherlands Search for more papers by this author Jingchun Liu Jingchun Liu Laboratory of Cell and Developmental Biology, National Institutes of Health, Bethesda, MD, 20892 USA Search for more papers by this author Alan R. Kimmel Alan R. Kimmel Laboratory of Cell and Developmental Biology, National Institutes of Health, Bethesda, MD, 20892 USA Search for more papers by this author Peter N. Devreotes Peter N. Devreotes Department of Biological Chemistry, Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD, 21205 USA Search for more papers by this author Pauline Schaap Corresponding Author Pauline Schaap Cell Biology Section, Institute for Molecular Plant Sciences, University of Leiden, Wassenaarseweg 64, 2333 AL, Leiden, The Netherlands Search for more papers by this author Tian Jin Tian Jin Department of Biological Chemistry, Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD, 21205 USA Search for more papers by this author Ron D.M. Soede Ron D.M. Soede Cell Biology Section, Institute for Molecular Plant Sciences, University of Leiden, Wassenaarseweg 64, 2333 AL, Leiden, The Netherlands Search for more papers by this author Jingchun Liu Jingchun Liu Laboratory of Cell and Developmental Biology, National Institutes of Health, Bethesda, MD, 20892 USA Search for more papers by this author Alan R. Kimmel Alan R. Kimmel Laboratory of Cell and Developmental Biology, National Institutes of Health, Bethesda, MD, 20892 USA Search for more papers by this author Peter N. Devreotes Peter N. Devreotes Department of Biological Chemistry, Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD, 21205 USA Search for more papers by this author Pauline Schaap Corresponding Author Pauline Schaap Cell Biology Section, Institute for Molecular Plant Sciences, University of Leiden, Wassenaarseweg 64, 2333 AL, Leiden, The Netherlands Search for more papers by this author Author Information Tian Jin1, Ron D.M. Soede2, Jingchun Liu3, Alan R. Kimmel3, Peter N. Devreotes1 and Pauline Schaap 2 1Department of Biological Chemistry, Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD, 21205 USA 2Cell Biology Section, Institute for Molecular Plant Sciences, University of Leiden, Wassenaarseweg 64, 2333 AL, Leiden, The Netherlands 3Laboratory of Cell and Developmental Biology, National Institutes of Health, Bethesda, MD, 20892 USA *Corresponding author. [email protected] The EMBO Journal (1998)17:5076-5084https://doi.org/10.1093/emboj/17.17.5076 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Deletion of the single gene for the Dictyostelium G protein β-subunit blocks development at an early stage. We have now isolated temperature-sensitive alleles of Gβ to investigate its role in later development. We show that Gβ is directly required for adenylyl cyclase A activation and for morphogenetic signaling during the entire developmental program. Gβ was also essential for induction of aggregative gene expression by cAMP pulses, a process that is mediated by serpentine cAMP receptors (cARs). However, Gβ was not required for cAR-mediated induction of prespore genes and repression of stalk genes, and neither was Gβ needed for induction of prestalk genes by the differentiation inducing factor (DIF). cAMP induction of prespore genes and repression of stalk genes is mediated by the protein kinase GSK-3. GSK-3 also determines cell-type specification in insects and vertebrates and is regulated by the wingless/wnt morphogens that are detected by serpentine fz receptors. The G protein-dependent and -independent modes of cAR-mediated signaling reported here may also exist for the wingless/wnt signaling pathways in higher organisms. Introduction Serpentine receptors transduce extracellular signals to intracellular effectors by interacting with heterotrimeric G proteins. Receptor activation of G proteins requires both the α and βγ subunits and both Gα and Gβγ can directly regulate effectors (Birnbaumer, 1992). The roles of G proteins in vivo have been assessed in a variety of genetic systems. In Saccharomyces cerevisiae, deletion of the single gene for either the Gβ or Gγ subunits blocks the capacity of mating pheromone to activate gene expression (Whiteway et al., 1988), while in Caenorhabditis elegans, deletion of the single Gβ gene arrests development at gastrulation (Zwaal et al., 1996). In Drosophila melanogaster there are two identified Gβ subunits. Mutants defective in an eye-specific Gβ subunit display severe defects in light responsiveness (Dolph et al., 1994). In Dictyostelium discoideum, deletion of the single Gβ gene blocks entry into the developmental program and eliminates multiple chemoattractant-mediated responses (Wu et al., 1995a). These studies have been useful for elucidating the earliest requirement for Gβ function in different organisms. However, the functions of Gβ in later development remained elusive, because progression through the program is often too drastically impaired by earlier loss of function of Gβ. The D.discoideum life cycle consists of a vegetative stage where cells feed on bacteria, and a multicellular stage where cells aggregate and differentiate. Following starvation, cells start to secrete cAMP pulses and chemotax towards the cAMP source to form multicellular aggregates of up to 100 000 amoebae. Cells differentiate into prestalk and prespore cells, and the multicellular mounds undergo a number of morphological changes that lead to formation of freely migrating slugs and fruiting bodies. Extracellular cAMP also controls gene expression during the entire course of development. Before aggregation, cAMP pulses strongly enhance expression of aggregative genes, encoding components of the cAMP signaling system. After aggregation, cAMP induces entry into the spore differentiation pathway as well as synthesis of a secreted factor, DIF, which induces entry into the stalk differentiation pathway. All effects of extracellular cAMP on gene expression are mediated by serpentine cAMP receptors (see Firtel, 1995). Heterotrimeric G proteins have important regulatory functions during Dictyostelium development. Nine Gα subunits (Gα1–Gα9), one Gβ and one Gγ subunit have been identified (Devreotes, 1994; N.Zhang and P.N. Devreotes, unpublished results; J.Brzostowski and A.R. Kimmel, unpublished results). The single Gβ and Gγ subunits are expressed throughout growth and development, while Gα subunits are transiently expressed at specific stages. The functions of G protein subunits have been examined in deletion mutants. Gα1 null mutants are defective in adaptation of phospholipase C (Bominaar and Van Haastert, 1994). Gα2 mediates cAMP-induced activation of guanylyl cyclase and phospholipase C (Okaichi et al., 1992; Bominaar and Van Haastert, 1994) and by release of Gβγ of adenylyl cyclase (Wu et al., 1995a). Gα3 null mutants show an as yet uncharacterized defect in cAMP signal production (Brandon and Podgorski, 1997). Gα4 mediates activation of guanylyl cyclase and, via Gβγ, of adenylyl cyclase by folate, a chemoattractant secreted by bacteria (Hadwiger et al., 1994). Null mutants for the other Gαs either do not exhibit significant morphogenetic defects, or have not yet been investigated. It seems apparent that the Gβ subunit must have important functions throughout development. Gβ null cells (gβ−) fail to enter development and remain completely deficient in chemoattractant-induced responses, since Gβ is required for all Gαs. To investigate the functions of Gβ, we isolated temperature-sensitive Gβ mutants, which allow G protein functions to be turned off at any stage of development. Two strains expressing the temperature-sensitive Gβ subunits were used to examine the role of G proteins in adenylyl cyclase activation, postaggregative morphogenesis and cell-type specification. Results Screen for temperature-sensitive mutations in the Gβ subunit Cells of a gβ− parental strain were transformed with a library of randomly mutagenized Gβ cDNAs and ∼104 neoR transformants were collected. As shown in Figure 1, each transformant will have a Gβ protein, that is either fully functional, temperature-sensitive, conditional or non-functional. First, we incubated the transformants collectively on DB agar at the permissive temperature (22°C). The cells expressing functional or conditional Gβ subunits will develop into chimeric fruiting bodies, while the cells expressing non-functional Gβ subunits will not participate. Secondly, the spores from the chimeric fruiting bodies were collected, plated clonally on bacterial lawns and incubated at the restrictive temperature (27°C). Cells expressing functional Gβ subunits will form plaques with fruiting bodies (agg+), while those cells expressing a temperature-sensitive (ts) or otherwise conditional Gβ subunit will form smooth aggregation minus (agg−) plaques. We selected ∼100 independent transformants from this second plating, which made agg− plaques at 27°C. Thirdly, to distinguish the transformants expressing ts versus other conditional mutations, we replicated them individually on bacterial lawns and incubated them at both 22°C and 27°C. Those mutants which for various reasons can respond to, but not propagate, cAMP signals will be agg− at both temperatures. We selected ∼20 independent transformants which formed agg+ plaques at 22°C and agg− plaques at 27°C. To prove that these phenotypes are plasmid-dependent, we rescued plasmids from four candidates and then retransformed them into parental gβ− cells. All of the new transformants formed agg+ plaques at 22°C and agg− plaques at 27°C. These results confirmed that the developmental defects were dependent on the plasmids. Figure 1.Screening protocol for temperature-sensitive Gβ mutants. Approximately 104 transformants were amplified in shaking culture at 22°C. The mixture of ∼108 cells expressing temperature-sensitive (empty circle), fully functional (black circle), conditional (light-shaded circle) or non-functional (striped circle) Gβ subunits was plated on non-nutrient agar for development at 22°C. Fruiting bodies had formed in 24 h, the spores were collected, and heated twice at 45°C for 30 min to kill non-spore cells. 2×104 spores were plated clonally on bacterial lawns at 27°C. Cells that formed plaques with an aggregation minus (agg−) phenotype at 27°C were picked and plated on replica bacterial plates incubated at 22°C and 27°C. The cells which formed fruiting bodies within plaques at 22°C and agg− plaques at 27°C were isolated and grown in HL5 with G418. Download figure Download PowerPoint For further studies, we chose two of the new transformants, designated as gpbA1 and gpbA2, which formed fruiting bodies at 22°C. Figure 2A shows the development of wild- type, gβ−, gpbA1 and gpbA2 cells at the permissive (22°C) and restrictive (27°C) temperatures. Wild-type cells formed fruiting bodies at both temperatures, while the gβ− cells always failed to aggregate and remained as a monolayer. At 22°C, gpbA1 and gpbA2 cells formed fruiting bodies indistinguishable from those of wild-type, while at 27°C, gpbA1 and gpbA2 completely failed to enter the developmental program. In wild-type cells, the Gβ protein level is constant at all stages of growth and development (Lilly et al., 1993). Figure 2B shows that in both wild-type, gpbA1 and gpbA2 cells, the levels of Gβ protein were constant during development, and remained unchanged for 6 h after a shift to 27°C. We therefore assume that the Gβ subunits in the gpbA1 and gpbA2 cells show conformational instability at the restrictive temperature. Figure 2.Phenotype and Gβ protein levels of cells expressing ts Gβ mutations. (A) Developmental phenotype. Wild-type (a and e), gβ− (b and f), gpbA1 (c and g) and gpbA2 (d and h) cells were harvested from shaking cultures, plated on DB agar, incubated at either 22°C (a–d) or 27°C (e–h) for 72 h and photographed. (B) Immunoblot analysis of Gβ proteins. Wild-type (WT), gpbA1 and gpbA2 cells were incubated in DB at either 22°C or 27°C and stimulated with pulses of 50 nM cAMP at 6-min intervals. Samples were taken after the indicated periods of incubation. Total protein of 106 cells was size-fractionated by SDS–PAGE and Western blots were probed with Gβ antibodies. Download figure Download PowerPoint The gpbA1 and gpbA2 mutants allowed us to determine whether Gβ is required for postaggregative development. We incubated gpbA1 or gpbA2 cells on non-nutrient agar at 22°C until they formed aggregates (Figure 3a), tipped mounds (Figure 3d) or slugs (Figure 3g), and then shifted them to 27°C. gpbA2 aggregates formed abnormal mounds after 2.5 h at 27°C (Figure 3b), which showed no further progress for 26 h (Figure 3c). gpbA2 cells shifted at the tipped mound stage, formed a first finger after 2.5 h at 27°C (Figure 3e), but did not develop further (Figure 3f). gpbA1 cells shifted at the slug stage showed abnormal slug morphology after 2.5 h (Figure 3h), which became progressively more aberrant after 26 h (Figure 3i). These results suggested a continuous requirement for G protein-mediated signaling for proper tip-, slug- and fruiting body formation. Figure 3.Multicellular morphogenesis of gpbA1 and gpbA2 mutants. gpbA1 and gpbA2 cells were incubated on DB agar at 22°C until they had reached the late aggregation (a), tipped mound (d) and slug stage (g) and plates were then placed at 27°C and photographed after 2.5 h (b, e and h) and 26 h (c, f and i). gpbA1 cells were developed to tipped mounds 22°C (j), shifted to 27°C for 26 h (k), and back to 22°C for 22 h (l). gpbA2 cells were photographed in panels (a–f) and gpbA1 cells in panels (g–l). Download figure Download PowerPoint To determine whether the developmental block was reversible, we developed gpbA1 cells to tipped mounds at 22°C (Figure 3j), and then shifted them to 27°C for 26 h until development completely arrested (Figure 3k). We shifted cells back to 22°C and after another 22 h, fruiting bodies were observed (Figure 3l), demonstrating that the developmental block could be reversed. However, it should be noted that not all cells participated in fruiting body formation, so the reversal was not complete. Adenylyl cyclase A activation in Gβ ts mutants To demonstrate that G protein function was lost when gpbA1 and gpbA2 cells were shifted to the restrictive temperature, we assayed GTPγS activation of adenylyl cyclase A (ACA). In gβ− cells, cAR- and G protein-mediated activation of ACA is completely absent (Wu et al., 1995a). However, since the gβ− cells are blocked at an early stage of development, it might be argued that this defect is not directly due to the absence of Gβ, but to reduced expression of components other than Gβ, that are essential for ACA activation. Analysis of gpbA1 and gpbA2 ruled out this possibility and allowed us to assess how quickly G protein function was lost. We first stimulated gpbA1 and gpbA2 cells during 5 h at 22°C with cAMP pulses to allow them to express all aggregative genes, and then shifted them to 27°C to ‘turn-off’ the functions of the Gβ subunit. We measured activity in lysates stimulated with GTPγS to bypass the receptor and directly assess the G protein. When gpbA2 cells were incubated at 27°C for 1 h, GTPγS-stimulated ACA activity was reduced 3-fold when compared with cells incubated at 22°C (data not shown). We then extended incubation at 27°C to 2 h. Table I shows that in cells incubated at 22°C, GTPγS induced a 5.1- or 5.3-fold stimulation of ACA activity in gpbA1 and gpbA2 lysates, respectively. However, in cells incubated at 27°C, GTPγS stimulation was reduced to ∼1.3- and ∼1.7-fold in the gpbA1 and gpbA2 mutants, respectively. Under the same conditions, GTPγS significantly stimulated ACA activity in wild-type cells incubated at both 22°C and 27°C. These results indicate that a functional Gβ subunit is directly required for GTPγS activation of ACA. Table 1. Activation of adenylyl cyclase A (ACA) in wild-type (WT) cells and ts mutants Cell line Unstimulated ACA activitya Stimulated ACA activitya 22°C 27°C 22°C 27°C WT 11 23 (26) 89 86 (91) gpbA1 9 13 (20) 46 17 (22) gpbA2 12 13 (24) 63 22 (34) a Activity expressed in pmol/min/mg. Wild-type, gpbA1 and gpbA2 cells were stimulated for 5 h at 22°C with cAMP pulses to induce optimal expression of cAMP signaling components. Cells were subsequently pre-incubated at 22°C or 27°C for 2 h and lysed in the absence (unstimulated activity) or presence (stimulated activity) of GTPγS. Adenylyl cyclase activity in the lysates of wild-type, gpbA1 and gpbA2 cells was measured at 22°C as described in Materials and methods. Data in parentheses represent an adenylyl cyclase assay performed at 27°C instead of 22°C. All data represent the means of an assay performed in duplicate. An independent experiment was done and yielded similar results. Induction of aggregative gene expression in Gβ ts mutants The expression levels of several genes that encode components required for aggregation such as cAR1, Gα2, ACA, phosphodiesterase (PDE) and the cell adhesion glycoprotein csA/gp80, increase dramatically upon starvation. Cell density sensing factors, that are secreted upon starvation, induce sufficient levels of PDE, cAR1, Gα2 and ACA to initiate oscillatory cAMP secretion. cAMP pulses then enhance further transcription of the cAR1, Gα2 and PDE genes and induce transcription of the gp80 gene (Firtel, 1995). We first examined gp80 and cAR1 protein levels in wild-type cells and gβ−, gpbA1 and gpbA2 mutants, that were stimulated with cAMP pulses at 22°C and 27°C. In wild-type cells, gp80 (Figure 4A) and cAR1 (Figure 4B) proteins were induced at both 22°C and 27°C, while in gβ− cells, the level of gp80 and cAR1 protein was extremely low at both temperatures. In the gpbA1 and gpbA2 mutants, gp80 and cAR1 proteins were induced to the same levels as in wild-type cells at 22°C, but expression was strongly reduced at 27°C. We also measured basal and cAMP-stimulated levels of the gp80, cAR1 and PDE mRNAs in gpbA1 and gpbA2 cells (Figure 4C). There was a low level of cAR1 and a moderate level of PDE mRNA accumulation in the absence of cAMP stimuli at both 22°C and 27°C. Transcription of both cAR1 and gp80 was at least 10-fold stimulated by cAMP pulses at 22°C, but not at all at 27°C. Transcription of PDE was stimulated only ∼2-fold by cAMP pulses; this stimulation seemed to be reduced at 27°C. These data indicated that the Gβ subunit is required for stimulation of aggregative gene expression by cAMP pulses, but most likely not for basal induction by cell density sensing factors. Figure 4.Induction of aggregative gene expression by cAMP pulses. (A and B) gp80 and cAR1 protein levels in wild-type and mutant cells. Wild-type, gβ−, gpbA1 and gpbA2 cells were stimulated with 50 nM cAMP pulses at 6-min intervals for 5 h at either 22°C or 27°C. Samples were taken after the indicated incubation periods and subjected to SDS–PAGE and immunoblotting with gp80- and cAR1-specific antibodies. (C) Induction of gp80, cAR1 and PDE mRNA in ts mutants. gpbA1 and gpbA2 cells were incubated at 22°C or 27°C with or without 30 nM cAMP pulses at 6-min intervals. mRNA was isolated after 0, 2, 4 and 6 h of incubation and Northern blots were probed with 32P-labeled cAR1, gp80 and PDE cDNA probes. Download figure Download PowerPoint Prestalk and prespore gene expression in the Gβ ts mutants After aggregates have formed, cAMP continues to be essential for regulation of prespore and prestalk gene expression. The prespore genes psA and CotB are induced by cAMP and repressed by DIF. The prestalk gene ecmA is induced by DIF and cAMP in synergy. The stalk gene ecmB is also induced by DIF, but this induction is inhibited by cAMP (see Firtel, 1995). We determined whether either cAMP- or DIF-induced gene regulation requires the Gβ subunit. The prespore genes psA and cotB and the prestalk gene ecmA are optimally inducible in cells that have developed to the loose aggregate stage. Wild-type cells and the gpbA1 and gpbA2 mutants were developed on agar at 22°C until loose aggregates had formed, and were then incubated for 8 h in suspension at 22°C or 27°C with cAMP and/or DIF. As shown in Figure 5A and B, the expression of the prespore genes psA and cotB in wild-type and ts mutant cells was induced by cAMP and inhibited by DIF. The prestalk gene ecmA showed moderate levels of expression in the presence of DIF alone and optimal expression in the presence of both cAMP and DIF. These patterns were essentially the same at 22°C and 27°C. Prespore gene expression requires at least 4 h of incubation with cAMP, which leaves sufficient time for loss of Gβ function at 27°C, but ecmA induction by DIF occurs within 1 h (Williams et al., 1987). Figure 5C shows that some ecmA induction by DIF and cAMP was indeed evident in gpbA2 cells after 1 h, but mRNA levels increased progressively up to 5 h of incubation at both 22°C and 27°C, well after the inactivation of Gβ. Absolute induction levels were somewhat lower at 27°C, but this was also the case in wild-type cells (data not shown) and could be due to enhanced degradation of DIF at the higher temperature. In general, the results indicate that Gβ does not mediate regulation of prespore or prestalk genes by cAMP or DIF. Figure 5.Induction and maintenance of prestalk and prespore gene expression. (A) Prespore and ecmA mRNA accumulation. Wild-type, gpbA1 and gpbA2 cells were developed to loose aggregates, dissociated and resuspended in DB. Cells were shaken at 150 r.p.m. at either 22°C or 27°C and challenged with 100 nM DIF added once at the onset of the incubation, 300 μM cAMP added at 60-min intervals, or a combination of the two stimuli. After 0, 4, 6 and 8 h of incubation, RNA was isolated and probed to 32P-labeled psA, cotB or ecmA cDNA. Only the results at t = 0 and t = 6 h are shown in this panel. (B) Quantitation of the time-course of psA induction. psA RNA bands from three independent experiments were quantitated by densitometry. All optical density values were calculated as percentage of induction at t = 8 h in gpbA2 cells incubated in the presence of 300 μM cAMP at 27°C. The data are presented as mean ± SEM. (C) Time-course of ecmA induction. gpbA2 cells, developed to loose aggregates, were incubated with cAMP and DIF in shaken suspension at 22°C and 27°C. RNA was isolated at the indicated time points and probed to ecmA cDNA. (D) Maintenance of prespore gene expression. Wild-type and gpbA2 mutants were developed at 22°C until tight mounds had formed. Mounds were dissociated and cells were incubated in suspension at 22°C or 27°C with 300 μM cAMP for the indicated time periods. RNA was isolated and probed to 32P-labeled psA cDNA. Download figure Download PowerPoint Prespore differentiation requires the continued presence of cAMP, since transcription ceases and prespore mRNAs become destabilized in its absence (Mangiarotti et al., 1983). We investigated whether maintenance of prespore gene expression required the Gβ subunit. Wild-type and gpbA2 cells were developed to the tight mound stage, when the prespore gene psA is already expressed. Cells were then incubated at either 22°C or 27°C in the presence and absence of cAMP. Figure 5D shows that in wild-type and gpbA2 cells, cAMP stabilized psA mRNA at both 22°C and 27°C, suggesting that cAR-mediated mRNA stabilization is also independent of the Gβ subunit. To study regulation of the prestalk gene ecmB, cells were prestimulated in monolayers with 5 mM cAMP during 16 h at 22°C (Berks and Kay, 1988). cAMP was removed and cells were incubated at 22°C and 27°C with 100 nM DIF and 1 μM Sp-cAMPS. Sp-cAMPS mimics the nanomolar cAMP levels, which are most effective to repress ecmB induction (Soede et al., 1996). Figure 6 shows that DIF induced ecmB expression in both wild-type cells and the ts mutants, while Sp-cAMPS almost completely inhibited this induction. These effects were essentially the same at 22°C and 27°C, and indicated that neither DIF induction nor Sp-cAMPS repression of ecmB required the Gβ subunit. Figure 6.Regulation of ecmB gene expression. Wild-type, gpbA1 and gpbA2 cells were first incubated for 16 h with 5 mM cAMP in stalk salts. Aggregates were dissociated into single cells, which were thoroughly washed and resuspended in stalk salts. Cells were incubated in Petri dishes at either 22°C or 27°C and challenged with 100 nM DIF and 1 μM Sp-cAMPS. RNA was isolated after 8 h and probed to 32P-labeled ecmB cDNA. The experiment was repeated twice with similar results for gpbA1 and gpbA2 and equal levels of ecmB mRNA at 22°C and 27°C for wild-type cells. Download figure Download PowerPoint Determination of mutations in the ts Gβ subunits We sequenced the Gβ genes on the plasmids rescued from the gpbA1 and gpbA2 cells, and found that each contained multiple mutations. Figure 7A shows the amino acid alignment of bovine Gtβ, human Gβ2, mouse Gβ4 and D.discoideum Gβ, and the amino acid substitutions in gpbA1 and gpbA2 mutants. The gpbA1 mutant contained three amino acid substitutions: M48T, I52T and E310G, and the gpbA2 contained seven, namely R13G, N74S, S79P, H149R, V178A, V194R and F293L. Figure 7B illustrates the positions of these mutations superimposed on a ribbon structure of the bovine Gtβγ dimer. Figure 7.Sequence identification of ts alleles in Gβ mutants. (A) Amino acid sequence alignment of four Gβ subunits and the substitutions in ts Gβ subunits. (B) Ribbon diagram showing the positions of the substitutions in ts Gβ subunits. Download figure Download PowerPoint Although it is not our purpose here to investigate structure, we separated some of the mutations. Using a convenient restriction site in the Gβ gene, we replaced a portion of the coding regions of gpbA1 and gpbA2 (from aa 187–346, or from aa 1–186) with the corresponding coding regions of the wild-type gene. These swaps yielded the mutant Gβ genes gpbA1a (M48T and I52T), gpbA1b (E310G), gpbA2a (R13G, N74S, S79P, H149R and V179A) and gpbA2b (V194R and F293L). We transformed the gβ− cells with plasmids of gpbA1a, gpbA1b, gpbA2a and gpbA2b, and examined the development of these transformants at 22°C and 27°C. At 22°C, all of these transformants made agg+ plaques on bacterial lawns, suggesting that they expressed a functional Gβ protein at the permissive temperature. At 27°C, gpbA1a and gpbA2a formed agg− plaques, while gpbA1b and gpbA2b formed plaques with small aggregates. When plated on DB agar, cells of gpbA1a and gpbA2a remained as a monolayer at 27°C and formed fruiting bodies at 22°C, while cells of gpbA1b and gpbA2b formed aggregates at 27°C. These observations suggested that gpbA1a with M48T and I52T mutations, and gpbA2a with the R13G, N74S, S79P, H149R, V178A mutations resulted in temperature-sensitive Gβ subunits, while gpbA1b with E310G, and gpbA2b with V194R and F293L substitutions showed only weak temperature sensitivity. The crystal structure of a Gtβγ heterodimer showed that Gβ forms extensive, mainly hydrophobic, interactions with Gγ (Sondek et al., 1996). For gpbA1a, the substitutions M48T and I52T are located at a loop that links the N-terminal helix to the first β sheet. Interestingly, the residue corresponding to I52 has been implicated in the formation of a specific hydrophobic interaction between Gβ and Gγ (Sondek et al., 1996), the substitutions in gpbA1a may weaken or disrupt the hydrophobic interaction, and therefore possibly destabilize the Gβγ complex. For gpbA2a, substitution of N74S changed the diverged N in the Gβ of D.discoideum back to the conserved S of other Gβ subunits, and mutation of V179A occurred at the residue which is V, I or L on the other Gβ subunits, while mutations of R13G, S79P and H149R represent more significant substitutions occurring at highly conserved residues in the identified Gβ subunits of all organisms. Thus, it is likely that the temperature-sensitive character of gpbA2 is due to the R13G, S79P or H149R mutations. None of the changes in gpbA2 is in positions that have been implicated in interaction with γ or α subunits and functions of βγ. Discussion We designed a screening procedure based on the essential role of the Gβ subunit in early Dictyostelium development and isolated the temperature-sensitive Gβ mutants gpbA1 and gpbA2. Both mutant Gβ subunits function at the permissive temperature, but could be ‘turned off’ by shifting the cells to restrictive temperature. The mutants completed development and formed normal fruiting bodies at 22°C, but failed to aggregate at 27°C. We used these conditional mutants to investigate the roles of the Gβ in cell–cell signaling and gene regulation during the entire course of development. The Gβ subunit directly mediates ACA activation At the aggregation stage, cAR1-mediated activation of ACA reaches its highest level" @default.
• W2170720976 created "2016-06-24" @default.
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• W2170720976 date "1998-09-01" @default.
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• W2170720976 title "Temperature-sensitive Gbeta mutants discriminate between G protein-dependent and -independent signaling mediated by serpentine receptors" @default.
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