Scanning mutagenesis of regions in the Galpha protein Gpa1 that are predicted to interact with yeast mating pheromone receptors.

The mechanism by which receptors activate heterotrimeric G proteins was examined by scanning mutagenesis of the Saccharomyces cerevisiae pheromone-responsive Galpha protein (Gpa1). The juxtaposition of high-resolution structures for rhodopsin and its cognate G protein transducin predicted that at least six regions of Galpha are in close proximity to the receptor. Mutagenesis was targeted to residues in these domains in Gpa1, which included four loop regions (beta2-beta3, alpha2-beta4, alpha3-beta5, and alpha4-beta6) as well as the N and C termini. The mutants displayed a range of phenotypes from nonsignaling to constitutive activation of the pheromone pathway. The constitutive activity of some mutants could be explained by decreased production of Gpa1, which permits unregulated signaling by Gbetagamma. However, the constitutive activity caused by the F344C and E335C mutations in the alpha2-beta4 loop and F378C in the alpha3-beta5 loop was not due to decreased protein levels, and was apparently due to defects in sequestering Gbetagamma. The strongest loss of the function mutant, which was not detectably induced by a pheromone, was caused by a K314C substitution in the beta2-beta3 loop. Several other mutations caused weak signaling phenotypes. Altogether, these results suggest that residues in different interface regions of Galpha contribute to activation of signaling.     

N‐terminal residues of the yeast pheromone receptor,Ste2p,mediate mating events independently of G1‐arrest signaling

In Saccharomyces cerevisiae, mechanisms modulating the mating steps following cell cycle arrest are not well characterized. However, the N‐terminal domain of Ste2p, a G protein‐coupled pheromone receptor, was recently proposed to mediate events at this level. Toward deciphering receptor mechanisms associated with this mating functionality, scanning mutagenesis of targeted regions of the N‐terminal domain has been completed. Characterization of ste2 yeast overexpressing Ste2p variants indicated that residues Ile 24 and Ile 29 as well as Pro 15 are critical in mediating mating efficiency. This activity was shown to be independent of Ste2p mediated G1 arrest signaling. Further analysis of Ile 24 and Ile 29 highlight the residues' solvent accessibility, as well as the importance of the hydrophobic nature of the sites, and in the case of Ile 24 the specific size and shape of the side chain. Mutation of these Ile's led to arrest of mating after cell contact, but before completion of cell wall degradation. We speculate that these extracellular residues mediate novel receptor interactions with ligand or proteins, leading to stimulation of alternate signaling effector pathways. J. Cell. Biochem. 107: 630–638, 2009. © 2009 Crown in the right of Canada.     

Dominant negative mutations in the alpha-factor receptor, a G protein-coupled receptor encoded by the STE2 gene of the yeast Saccharomyces cerevisiae.

The α-mating pheromone receptor encoded by the STE2 gene of the yeast Saccharomyces cerevisiae is a G protein-coupled receptor (GPCR) that is homologous to the large family of GPCRs that mediate multiple types of signal transduction in mammals. We have screened libraries of mutant receptors to identify dominant negative alleles that are capable of interfering with the function of a co-expressed normal receptor. Two dominant negative alleles have been recovered in this manner. In addition, we find that previously isolated loss-of-function mutations in the α-factor receptor exhibit dominant negative effects. Detection of the dominant effects requires high-level expression of the mutant receptors but does not require a high ratio of mutant to normal receptors. Cellular levels of the normal receptors are not affected by co-expression of the dominant negative alleles. Expression of the mutant receptors does not interfere with constitutive signaling in a strain that lacks the G protein α subunit encoded by GPA1, indicating that interference with signaling occurs at the level of the receptor or the interacting G protein. Expression of increased levels of G protein subunits partially reverses the dominant negative effects. The dominant negative behavior of the mutant receptors is diminished by deletion of the SST2 gene, which encodes an RGS (Regulator of G protein Signaling) protein involved in desensitization of pheromone signaling. The most likely explanation for the dominant negative effects of the mutations appears to be the existence of an interaction between unactivated receptors and the trimeric G protein that titrates the G protein away from the normal receptors or renders the G protein insensitive to receptor activation. This interaction appears to be mediated by the SST2 gene product. Received: 15 January 1999 / Accepted: 25 March 1999     

CHK1相互作用蛋白的筛选

将CHK1基因克隆入酵母双杂交载体中,转化入酵母菌AH109,将转化有CHK1基因的酵母菌AH109培养并再转化人前列腺cDNA库质粒,检测报告基因的表达情况,筛选与CHK1相互作用的蛋白。共转化子中有4个β-半乳糖苷酶活性分析和α-半乳糖苷酶活性分析结果为阳性的克隆,但测序结果其中有2个克隆为相同序列。本实验筛选到3个与CHK1相互作用的蛋白,此结果为进一步研究与CHK1相互作用的蛋白奠定了基础。     

Receptor‐Like Kinase Phosphorylation of Arabidopsis Heterotrimeric G‐Protein Gα ‐Subunit AtGPA1

As molecular on–off switches, heterotrimeric G protein complexes, comprised of a Gα subunit and an obligate Gβγ dimer, transmit extracellular signals received by G protein–coupled receptors (GPCRs) to cytoplasmic targets that respond to biotic and abiotic stimuli. Signal transduction is modulated by phosphorylation of GPCRs and G protein complexes. In Arabidopsis thaliana, the Gα subunit AtGPA1 is phosphorylated by the receptor‐like kinase (RLK) BRI1‐associated Kinase 1 (BAK1), but the extent that other RLKs phosphorylates AtGPA1 is unknown. Twenty‐two trans‐phosphorylation sites on AtGPA1 are mapped by 12 RLKs hypothesized to act in the Arabidopsis G protein signaling pathway. Cis‐phosphorylation sites are also identified on these RLKs, some newly shown to be dual specific kinases. Multiple sites are present in the core AtGPA1 functional units, including pSer52 and/or pThr53 of the conserved P‐loop that directly binds nucleotide/phosphate, pThr164, and pSer175 from αE helix in the intramolecular domain interface for nucleotide exchange and GTP hydrolysis, and pThr193 and/or pThr194 in Switch I (SwI) that coordinates nucleotide exchange and protein partner binding. Several AtGPA1 S/T phosphorylation sites are potentially nucleotide‐dependent phosphorylation patterns, such as Ser52/Thr53 in the P‐loop and Thr193 and/or Thr194 in SwI.     

Identification of proteins that interact with a protein of interest: Applications of the yeast two-hybrid system

The yeast two-hybrid system is a molecular genetic test for protein interaction. Here we describe a step by step procedure to screen for proteins that interact with a protein of interest using the two-hybrid system. This process includes, construction and testing of the bait plasmid, screening a plasmid library for interacting fusion proteins, elimination of false positives and deletion analysis of true positives. This procedure is designed to allow investigators to identify proteins and their encoding cDNAs that have a biologically significant interaction with your protein of interest.     

Clathrin interactions with C-terminal regions of the yeast AP-1 beta and gamma subunits are important for AP-1 association with clathrin coats

Heterotetrameric adaptor (AP) complexes are thought to coordinate cargo recruitment and clathrin assembly during clathrin-coated vesicle biogenesis. We have identified, and characterized the physiological significance of clathrin-binding activities in the two large subunits of the AP-1 complex in Saccharomyces cerevisiae . Using GST-fusion chromatography, two clathrin-binding sites were defined in the β1 subunit that match consensus clathrin-binding sequences in other mammalian and yeast clathrin-binding proteins. Clathrin interactions were also identified with the C-terminal region of the γ subunit. When introduced into chromosomal genes, point mutations in the β1 clathrin-binding motifs, or deletion of the γ C-terminal region, reduced association of AP-1 with clathrin in coimmunoprecipitation assays. The β1 mutations or the γ truncation individually produced minor effects on AP-1 distribution by subcellular fractionation. However, when β1 and γ mutations were combined, severe defects were observed in AP-1 association with membranes and incorporation into clathrin-coated vesicles. The combination of subunit mutations accentuated growth and α-factor pheromone maturation defects in chc1-ts cells, though not to the extent caused by complete loss of AP-1 activity. Our results suggest that both the β1 and γ subunits contribute interactions with clathrin that are important for stable assembly of AP-1 complexes into clathrin coats in vivo .     

Mutagenic mapping of helical structures in the transmembrane segments of the yeast alpha-factor receptor

The alpha-mating pheromone receptor encoded by the yeast STE2 gene is a G protein coupled receptor that initiates signaling via a MAP kinase pathway that prepares haploid cells for mating. To establish the range of allowed amino acid substitutions within transmembrane segments of this receptor, we conducted extensive random mutagenesis of receptors followed by screening for receptor function. A total of 157 amino acid positions in seven different mutagenic libraries corresponding to the seven predicted transmembrane segments were analyzed, yielding 390 alleles that retain at least 60 % of normal signaling function. These alleles contained a total of 576 unique amino acid substitutions, including 61 % of all the possible amino acid changes that can arise from single base substitutions. The receptor exhibits a surprising tolerance for amino acid substitutions. Every amino acid in the mutagenized regions of the transmembrane regions could be substituted by at least one other residue. Polar amino acids were tolerated in functional receptors at 115 different positions (73 % of the total). Hydrophobic amino acids were tolerated in functional receptors at all mutagenized positions. Substitutions introducing proline residues were recovered at 53 % of all positions where they could be brought about by single base changes. Residues with charged side-chains could also be tolerated at 53 % of all positions where they were accessible through single base changes. The spectrum of allowed amino acid substitutions was characterized in terms of the hydrophobicity, radius of gyration, and charge of the allowed substitutions and mapped onto alpha-helical structures. By comparing the patterns of allowed substitutions with the recently determined structure of rhodopsin, structural features indicative of helix-helix interactions can be discerned in spite of the extreme sequence divergence between these two proteins.     

Disease-causing V(2) vasopressin receptors are retained in different compartments of the early secretory pathway

The G protein-coupled V(2) vasopressin receptor is crucially involved in water reabsorption in the renal collecting duct. Mutations in the human V(2) vasopressin receptor gene cause nephrogenic diabetes insipidus. Many of the disease-causing mutants are retained intracellularly by the quality control system of the early secretory pathway. It was previously thought that quality control system is restricted to the endoplasmic reticulum (ER). Here, we have examined the retention mechanisms of eight V(2) vasopressin receptor mutants. We show that mutants L62P, DeltaL62-R64 and S167L are trapped exclusively in the ER. In contrast, mutants R143P, Y205C, InsQ292, V226E and R337X reach the ER/Golgi intermediate compartment (ERGIC) and are rerouted to the ER. The ability of the mutant receptors to reach the ERGIC is independent of their expression levels. Instead, it is determined by their folding state. Mutant receptors in the ERGIC may be sorted into retrograde transport vesicles by an interaction of an RXR motif in the third intracellular loop with the coatomer complex I. Our data show that disease-causing mutants of a particular membrane protein may be retained in different compartments of the early secretory pathway and that the folding states of the proteins determine their retention mechanism.     

Activation of the heterotrimeric G protein α-subunit GPA1 suppresses the ftsh-mediated inhibition of chloroplast development in Arabidopsis

Heterotrimeric G protein knock-out mutants have no phenotypic defect in chloroplast development, and the connection between the G protein signaling pathway and chloroplast development has only been inferred from pharmaceutical evidence. Thus, whether G protein signaling plays a role in chloroplast development remains an open question. Here, we present genetic evidence, using the leaf-variegated mutant thylakoid formation 1 ( thf1 ), indicating that inactivation or activation of the endogenous G protein α-subunit (GPA1) affects chloroplast development, as does the ectopic expression of the constitutively active Gα-subunit (cGPA1). Molecular biological and genetic analyses showed that FtsH complexes, which are composed of type-A (FtsH1/FtsH5) and type-B (FtsH2/FtsH8) subunits, are required for cGPA1-promoted chloroplast development in thf1 . Furthermore, the ectopic expression of cGPA1 rescues the leaf variegation of ftsh2 . Consistent with this finding, microarray analysis shows that ectopic expression of cGPA1 partially corrects mis-regulated gene expression in thf1 . This overlooked function of G proteins provides new insight into our understanding of the integrative signaling network, which dynamically regulates chloroplast development and function in response to both intracellular and extracellular signals.     

Netrin-1-independent adenosine A2b receptor activation regulates the response of axons to netrin-1 by controlling cell surface levels of UNC5A receptors

Growth cone response to the bifunctional guidance cue netrin-1 is regulated by the activity of intracellular signaling intermediates such as protein kinase C-alpha (PKCα) and adenylyl cyclase. Among the diverse cellular events these enzymes regulate is receptor trafficking. Netrin-1, itself, may govern the activity of these signaling intermediates, thereby regulating axonal responses to itself. Alternatively, other ligands, such as activators of G protein-coupled receptors, may regulate responses to netrin-1 by governing these signaling intermediates. Here, we investigate the mechanisms controlling activation of PKCα and the subsequent downstream regulation of cell surface UNC5A receptors. We report that activation of adenosine receptors by adenosine analogs, or activation of the putative netrin-1 receptor, the G protein-coupled receptor adenosine A2b receptor (A2bR) results in PKCα-dependent removal of UNC5A from the cell surface. This decrease in cell surface UNC5A reduces the number of growth cones that collapse in response to netrin-1 and converts repulsion to attraction. We show these A2bR-mediated alterations in axonal response are not because of netrin-1 because netrin-1 neither binds A2bR, as assayed by protein overlay, nor stimulates PKCα-dependent UNC5A surface loss. Our results demonstrate that netrin-1-independent A2bR signaling governs the responsiveness of a neuron to netrin-1 by regulating the levels of cell surface UNC5A receptor.     

Strained contacts with the cell membrane may influence ligand affinity to G protein coupled receptors: a case of free fatty acid receptor 1 agonists

A set of 1,3,4-thiadiazole-2-carboxamides bearing a substituted biphenyl in the amide portion was synthesised and tested for agonistic activity towards free fatty acid receptor 1 (FFA1). The observed activity trends were impossible to rationalised based solely on the docking energy scores of Glide SP. On the contrary, when the phospholipid cell membrane bilayer was reconstructed around FFA1, it became apparent that inactive compounds displayed significant strained contacts with the membrane while for active compounds the strain was noticeably lower. These findings justify using the improved docking protocol for modelling GPCR-ligand interactions which uses the crystal structure of the receptor and a reconstructed portion of a cell membrane.     

Glucose-induced posttranslational activation of protein phosphatases PP2A and PP1 in yeast

The protein phosphatases PP2A and PP1 are major regulators of a variety of cellular processes in yeast and other eukaryotes. Here, we reveal that both enzymes are direct targets of glucose sensing. Addition of glucose to glucose-deprived yeast cells triggered rapid posttranslational activation of both PP2A and PP1. Glucose activation of PP2A is controlled by regulatory subunits Rts1, Cdc55, Rrd1 and Rrd2. It is associated with rapid carboxymethylation of the catalytic subunits, which is necessary but not sufficient for activation. Glucose activation of PP1 was fully dependent on regulatory subunits Reg1 and Shp1. Absence of Gac1, Glc8, Reg2 or Red1 partially reduced activation while Pig1 and Pig2 inhibited activation. Full activation of PP2A and PP1 was also dependent on subunits classically considered to belong to the other phosphatase. PP2A activation was dependent on PP1 subunits Reg1 and Shp1 while PP1 activation was dependent on PP2A subunit Rts1. Rts1 interacted with both Pph21 and Glc7 under different conditions and these interactions were Reg1 dependent. Reg1-Glc7 interaction is responsible for PP1 involvement in the main glucose repression pathway and we show that deletion of Shp1 also causes strong derepression of the invertase gene SUC2. Deletion of the PP2A subunits Pph21 and Pph22, Rrd1 and Rrd2, specifically enhanced the derepression level of SUC2, indicating that PP2A counteracts SUC2 derepression. Interestingly, the effect of the regulatory subunit Rts1 was consistent with its role as a subunit of both PP2A and PP1, affecting derepression and repression of SUC2, respectively. We also show that abolished phosphatase activation, except by reg1Δ, does not completely block Snf1 dephosphorylation after addition of glucose. Finally, we show that glucose activation of the cAMP-PKA (protein kinase A) pathway is required for glucose activation of both PP2A and PP1. Our results provide novel insight into the complex regulatory role of these two major protein phosphatases in glucose regulation.     

GIT1 is a novel MEK1–ERK1/2 scaffold that localizes to focal adhesions

Cell polarity is critical for cell migration and requires localized signal transduction in subcellular domains. Recent evidence demonstrates that activation of ERK1/2 (extracellular‐signal‐regulated kinase 1/2) in focal adhesions is essential for cell migration. GIT1 (G‐protein‐coupled receptor kinase‐interacting protein 1) has been shown to bind paxillin and regulate focal‐adhesion disassembly. We have previously reported that GIT1 binds to MEK1 [MAPK (mitogen‐activated protein kinase)/ERK kinase 1] and acts as a scaffold to enhance ERK1/2 activation in response to EGF (epidermal growth factor). In the present study we show that GIT1 associates with ERK1/2 in focal adhesions and this association increases after EGF stimulation. The CC (coiled‐coil) domain of ERK1/2 is required for association with GIT1, translocation to focal adhesions, and cell spreading and migration. Immunofluorescent staining showed that, after EGF stimulation, GIT1 co‐localized with pERK1/2 (phosphorylated ERK1/2) in focal adhesions. The binding of GIT1 and ERK1/2 was functionally important, since transfecting an ERK2 mutant lacking the CC domain [ERK2(del CC)] significantly decreased pERK1/2 translocation to focal adhesions, cell spreading and migration induced by EGF. In summary, the CC domain of ERK1/2 is necessary for binding to GIT1, for ERK1/2 activation in focal adhesions, and for cell spreading and migration.     

Agonist-induced internalization of metabotropic glutamate receptor 1A: structural determinants for protein kinase C- and G protein-coupled receptor kinase-mediated internalization

To investigate the role of the intracellular C-terminal tail of the rat metabotropic glutamate receptor 1a (mGlu1a) in receptor regulation, we constructed three C-terminal tail deletion mutants (Arg847stop, DM-I; Arg868stop, DM-II; Val893stop, DM-III). Quantification of glutamate-induced internalization provided by ELISA indicated that DM-III, like the wild-type mGlu1a, underwent rapid internalization whilst internalization of DM-I and DM-II was impaired. The selective inhibitor of protein kinase C (PKC), GF109203X, which significantly reduced glutamate-induced mGlu1a internalization, had no effect on the internalization of DM-I, DM-II, or DM-III. In addition activation by carbachol of endogenously expressed M1 muscarinic acetylcholine receptors, which induces PKC- and Ca2+-calmodulin-dependent protein kinase II-dependent internalization of mGlu1a, produced negligible internalization of the deletion mutants. Co-expression of a dominant negative mutant form of G protein-coupled receptor kinase 2 (DNM-GRK2; Lys220Arg) significantly attenuated glutamate-induced internalization of mGlu1a and DM-III, whilst internalization of DM-I and DM-II was not significantly affected. The glutamate-induced internalization of mGlu1a and DM-III, but not of DM-I or DM-II, was inhibited by expression of DNM-arrestin [arrestin-2(319-418)]. In addition glutamate-induced rapid translocation of arrestin-2-Green Fluorescent Protein (arr-2-GFP) from cytosol to membrane was only observed in cells expressing mGlu1a or DM-III. Functionally, in cells expressing mGlu1a, glutamate-stimulated inositol phosphate accumulation was increased in the presence of PKC inhibition, but so too was that in cells expressing DM-II and DM-III. Together these results indicate that different PKC mechanisms regulate the desensitization and internalization of mGlu1a. Furthermore, PKC regulation of mGlu1a internalization requires the distal C terminus of the receptor (Ser894-Leu1199), whilst in contrast glutamate-stimulated GRK- and arrestin-dependent regulation of this receptor depends on a region of 25 amino acids (Ser869-Val893) in the proximal C-terminal tail.     

Illuminating the role of the Gα heterotrimeric G protein subunit,RGA1, in regulating photoprotection and photoavoidance in rice

We studied physiological mechanisms of photoavoidance and photoprotection of a dwarf rice mutant with erect leaves, d1, in which the RGA1 gene, which encodes the Gα subunit of the heterotrimeric G protein, is non‐functional. Leaves of d1 exhibit lower leaf temperature and higher photochemical reflectance index relative to wild type (WT), indicative of increased photoavoidance and more efficient light harvesting. RNA sequencing analysis of flag leaves revealed that messenger RNA levels of genes encoding heat shock proteins, enzymes associated with chlorophyll breakdown, and ROS scavengers were down‐regulated in d1. By contrast, genes encoding proteins associated with light harvesting, Photosystem II, cyclic electron transport, Photosystem I, and chlorophyll biosynthesis were up‐regulated in d1. Consistent with these observations, when WT and d1 plants were experimentally subjected to the same light intensity, d1 plants exhibited a greater capacity to dissipate excess irradiance (increased nonphotochemical quenching) relative to WT. The increased capacity in d1 for both photoavoidance and photoprotection reduced sustained photoinhibitory damage, as revealed by a higher F_v/F_m. We therefore propose RGA1 as a regulator of photoavoidance and photoprotection mechanisms in rice and highlight the prospect of exploiting modulation of heterotrimeric G protein signalling to increase these characteristics and improve the yield of cereals in the event of abiotic stress.     

The role of PLDα1 in providing specificity to signal‐response coupling by heterotrimeric G‐protein components in Arabidopsis

Heterotrimeric G‐proteins comprised of Gα, Gβ and Gγ subunits are important signal transducers in all eukaryotes. In plants, G‐proteins affect multiple biotic and abiotic stress responses, as well as many developmental processes, even though their repertoire is significantly limited compared with that in metazoan systems. One canonical and three extra‐large Gα, 1 Gβ and 3 Gγ proteins represent the heterotrimeric G‐protein complex in Arabidopsis, and a single regulatory protein, RGS1, is one of the few known biochemical regulators of this signaling complex. This quantitative disparity between the number of signaling components and the range of processes they influence is rather intriguing. We now present evidence that the phospholipase Dα1 protein is a key component and modulator of the G‐protein complex in affecting a subset of signaling pathways. We also show that the same G‐protein subunits and their modulators exhibit distinct physiological and genetic interactions depending on specific signaling and developmental pathways. Such developmental plasticity and interaction specificity likely compensates for the lack of multiplicity of individual subunits, and helps to fine tune the plants' responses to constantly changing environments.     

Rapid Communication: A cis-Acting Element, Tα-1, in the Upstream Region of Rod α-Transducin Gene that Binds a Developmentally Regulated Retina-Specific Nuclear Factor

Abstract: The G protein transducin (T) is an integral component of the signal transduction pathway in photoreceptors. We have identified a cis -acting element, Ta-1, in the upstream region of the mouse rod a-T (T_rα) gene that may be important for tissue-specific expression. Tα-1 binds a retina-specific nuclear factor of apparent molecular mass of 90 kDa. Binding to the Tα-1 site is developmentally regulated and peaks between postnatal days 6 and 9. This corresponds to the time of rod photoreceptor maturation and the rise in T_rα gene expression. The sequence of Tα-1 shows homology with RET-1, a cis -acting element in the proximal promoter of opsin gene that binds a distinct retina-specific factor. Tα-1 and RET-1 sequences may have been derived from a prototype Tα-1/RET-1 sequence, evolved to confer photoreceptor specificity on retina-specific genes.