Regulated expression of active biotinylated G-protein coupled receptors in mammalian cells

We have developed a mammalian expression system suitable for the production of enzymatically biotinylated integral membrane proteins. The key feature of this system is the doxycycline (dox)-regulated co-expression of a secreted variant of Escherichia coli biotin ligase (BirA) and a target protein with a 13-residue biotin acceptor peptide (BioTag) appended to its extracellular domain. Here we describe the expression and functional analysis of three G-protein coupled receptors (GPCRs): protease-activated receptors (PARs) 1 and 2, and the platelet ADP receptor, P2Y(12). Clonal Chinese hamster ovary (CHO) Tet-On cell lines that express biotinylated GPCRs were rapidly isolated by fluorescence-activated cell sorting following streptavidin-FITC staining, thereby circumventing the need for manual colony picking. Analysis by Western blotting with streptavidin-HRP following endoglycosidase treatment revealed that all three GPCRs undergo N-linked glycosylation. The expression of biotinylated GPCRs on the cell surface was regulated by the concentration of dox in the medium, reaching a maximum at approximately 1 microg/mL dox. Similarly, the extent of GPCR biotinylation was dependent on biotin concentration, with maximum and complete biotinylation achieved upon supplementation with 50 microM biotin. Biotinylated PAR1 and PAR2 were readily and specifically cleaved on the surface of intact cells by their cognate proteases, and were capable of transducing extracellular stimuli, resulting in the downstream phosphorylation of extracellular signal-regulated kinase (ERK) 1/2. Notably, P2Y(12) mediated agonist-induced ERK phosphorylation only when it was expressed at low levels on the cell surface, highlighting the utility of regulated expression for the production of functionally active GPCRs in mammalian cells.     

Characterization of a G-protein alpha-subunit gene from the nematode Caenorhabditis elegans

A gene encoding the alpha-subunit of a guanine nucleotide binding regulatory protein (G-protein) was isolated from a library of genomic Caenorhabditis elegans DNA. The predicted coding region is colinear to related genes from mammals and the 356 amino acid residues show 63% sequence identity to e.g. rat Gi alpha 2. Three of the eight introns within the coding sequence are at exactly the same positions as those in a Drosophila G-protein alpha-subunit gene, and two of these are also conserved in the mammalian homologues. The nematode gene does not encode the cysteine residue that forms the substrate site for pertussis toxin-catalyzed ADP-ribosylation in several G-proteins. In spite of the similarity to mammalian G-protein alpha-subunit genes the gene can not unambiguously be categorized in one of the classes of G-proteins recognized in mammals (G alpha i, o, z, etc.). The position of the gene on the physical map of the animal was determined (chromosome V). The cloning and sequencing of this gene can be the starting point of reverse genetics experiments aimed at the isolation of animals mutated in a G-protein alpha-subunit gene.     

Contact inhibition, polyribosomes, and cell surface membranes in cultured mammalian cells

An “overlay” method for rapidly and synchronously inducing contact inhibition in normal cultured cells has been developed. Using this method, disaggregation of cytoplasmic polyribosomes has been observed to occur within a matter of hours after overlay, followed by a decrease in cellular ribosomal RNA. Polysome disaggregation was influenced by the extent of cell-cell interaction and was inhibited by pretreatment of overlay cells with cycloheximide. Treatment of underlay cells with cytosine arabinoside also induced polysome disaggregation, but only after an appreciable lag as compared to that observed in overlaid cultures. Disaggregation could be induced by this method in cultured cells derived from normal tissue but not in cells derived from cancerous tissue. Polysome synthesis in growing “normal” cells (as measured by incorporation of tracer uridine into RNA) was markedly decreased when a cell surface membrane preparation was added to cultures.     

Functional expression of cloned cDNA encoding the alpha-subunit of adenylate cyclase-stimulating G-protein

Cloned cDNA encoding the alpha-subunit of the adenylate cyclase-stimulating G-protein (Gs), carried by a simian virus 40 vector, has been introduced into the cyc- variant of S49 lymphoma cells by electroporation. In contrast to untransfected cys- cells, clones transformed with the cDNA exhibit an increase in intracellular cyclic AMP concentration in response to a beta-adrenergic agonist.     

Differential expression and interaction with the visual G-protein transducin of centrin isoforms in mammalian photoreceptor cells

Photoisomerization of rhodopsin activates a heterotrimeric G-protein cascade leading to closure of cGMP-gated channels and hyperpolarization of photoreceptor cells. Massive translocation of the visual G-protein transducin, Gt, between subcellular compartments contributes to long term adaptation of photoreceptor cells. Ca(2+)-triggered assembly of a centrin-transducin complex in the connecting cilium of photoreceptor cells may regulate these transducin translocations. Here we demonstrate expression of all four known, closely related centrin isoforms in the mammalian retina. Interaction assays revealed binding potential of the four centrin isoforms to Gtbetagamma heterodimers. High affinity binding to Gtbetagamma and subcellular localization of the centrin isoforms Cen1 and Cen2 in the connecting cilium indicated that these isoforms contribute to the centrin-transducin complex and potentially participate in the regulation of transducin translocation through the photoreceptor cilium. Binding of Cen2 and Cen4 to Gbetagamma of non-visual G-proteins may additionally regulate G-proteins involved in centrosome and basal body functions.     

Immunochemical analysis of the domain structure of CAD, the multifunctional protein that initiates pyrimidine biosynthesis in mammalian cells

CAD, is a multidomain polypeptide, with a molecular weight of over 200,000, that has glutamine-dependent carbamyl-phosphate synthetase, aspartate transcarbamylase, and dihydroorotase activity as well as regulatory sites that bind UTP and 5-phosphoribosyl 1-pyrophosphate. The protein thus catalyzes the first three steps of de novo pyrimidine biosynthesis and controls the activity of the pathway in higher eukaryotes. Controlled proteolysis of CAD isolated from Syrian hamster cells, cleaves the molecule into seven major proteolytic fragments that contain one or more of the functional domains. The two smallest fragments, which had molecular weights of 44,000 and 40,000, corresponded to the fully active dihydroorotase (DHO) and aspartate transcarbamylase (ATC) domains, respectively, but the larger fragments have not been previously characterized. In this study, enzymatic assays of partially fractionated digests and immunoblotting with antibodies specifically directed against the purified ATC domain, the purified dihydroorotase domain and an 80-kDa fragment of the putative carbamyl-phosphate synthetase domain established the precursor-product relationships among all of the major proteolytic fragments of CAD. These results indicate that 1) only the intact molecule had all of the functional domains, 2) a species with a molecular weight of 200,000 was produced in the first step of proteolysis which had glutamine-dependent carbamyl-phosphate synthetase and dihydroorotase activity, but neither aspartate transcarbamylase activity nor the antigenic determinants present on the isolated ATC domain, and 3) cleavage of the 200-kDa species produced a species, with a molecular mass of 150,000 which lacked both aspartate transcarbamylase and dihydroorotase domains. This 150-kDa species, containing the postulated carbamyl-phosphate synthetase, glutamine, and regulatory (UTP, 5-phosphoribosyl 1-pyrophosphate) domains, had two elastase-sensitive sites that divided this region of the polypeptide chain into 10-, 65-, and 80-kDa segments. The location of the functional sites on these segments has not yet been established. The immunochemical analysis also revealed the existence of possible precursors of the stable aspartate transcarbamylase and dihydroorotase domains, suggesting that the chain segments connecting the functional domains of CAD are extensive and that the overall size of the intact polypeptide chain has been underestimated. On the basis of these studies we have proposed a model of the domain structure of CAD.     

Immunochemical measurement of histone H3 in non-nucleosomal compartments of cultured mammalian cells

We measured histone H3 in the non-nucleosomal compartment of cultured mammalian cells by enzyme-linked immunoelectrotransfer blot assay of cytosolic proteins using affinity-purified rabbit anti-H3 IgG, and peroxidase-linked second antibodies. The cytosolic H3 level was estimated to be 0.5-1.0% of the nucleosomal H3 content in MH-134SC cells (mean generation time 11 h) and 3-4% in HeLa cells (mean generation time 22 h). It showed characteristic changes under the inhibitions of DNA and/or protein synthesis and during the cell cycle of HeLa cells. These indicate an inverse relationship between the cytosolic H3 level and the replicating activity of nuclear DNA. The possible implication of the non-nucleosomal histones in the regulation of histone gene expression is discussed.     

Distribution of 5-doxylstearic acid in the membranes of mammalian cells

Concentration-dependent spin broadening of ESR spectra of the nitroxide 5-doxylstearic acid has been used to evaluate the distribution of 5-doxylstearic acid in the membranes of intact mouse thymus-bone marrow (TB) and Chinese hamster ovary (CHO) cells. TB cells, CHO cells, erythrocytes, and isolated plasma membranes from CHO cells were labelled with 5-doxylstearic acid and the peak to peak linewidths of the central line of the resulting ESR spectra were measured. The measured line widths were linearly dependent on the amount of 5-doxylstearic acid incorporated into the sample over the range of 0-0.18 mol nitroxide per mol lipid. In erythrocytes, the relationship between linewidths approximated a linear function at lower concentrations of 5-doxylstearic acid, up to 0.07 mol nitroxide per mol lipid. The amount of broadening of the central line for a given amount of 5-doxylstearic acid was far less for intact cells than for either erythrocytes or plasma membrane, indicating that the 5-doxylstearic acid samples a much larger lipid pool in the intact cells. With the broad assumption that the mobility of the 5-doxylstearic acid is similar in different membranes, the size of the lipid pool sampled by 5-doxylstearic acid is approximately equal to the total cellular lipid in intact cells. If a given concentration of 5-doxylstearic acid sampled only the plasma membrane of TB or CHO cells, we would expect to see a linewidth corresponding to a 12-20-fold greater local concentration of 5-doxylstearic acid than was observed, since the plasma membranes of CHO and TB cells represent only 5-8 percent of the total cellular lipid. Therefore, the 5-doxylstearic acid must distribute into most or all cellular membranes of intact cells and is not localized in the plasma membrane alone.     

Mutations in the cone photoreceptor G-protein alpha-subunit gene GNAT2 in patients with achromatopsia

Achromatopsia is an autosomal recessively inherited visual disorder that is present from birth and that features the absence of color discrimination. We here report the identification of five independent families with achromatopsia that segregate protein-truncation mutations in the GNAT2 gene, located on chromosome 1p13. GNAT2 encodes the cone photoreceptor-specific alpha-subunit of transducin, a G-protein of the phototransduction cascade, which couples to the visual pigment(s). Our results demonstrate that GNAT2 is the third gene implicated in achromatopsia.     

Trichoderma atroviride G-protein alpha-subunit gene tga1 is involved in mycoparasitic coiling and conidiation

The soil fungus Trichoderma atroviride, a mycoparasite, responds to a number of external stimuli. In the presence of a fungal host, T. atroviride produces hydrolytic enzymes and coils around the host hyphae. In response to light or nutrient depletion, asexual sporulation is induced. In a biomimetic assay, different lectins induce coiling around nylon fibers; coiling in the absence of lectins can be induced by applying cyclic AMP (cAMP) or the heterotrimeric G-protein activator mastoparan. We isolated a T. atroviride G-protein alpha-subunit (Galpha) gene (tgal) belonging to the fungal subfamily with the highest similarity to the Galpha1 class. Generated transgenic lines that overexpress Galpha show very delayed sporulation and coil at a higher frequency. Furthermore, transgenic lines that express an activated mutant protein with no GTPase activity do not sporulate and coil at a higher frequency. Lines that express an antisense version of the gene are hypersporulating and coil at a much lower frequency in the biomimetic assay. The loss of Tgal in these mutants correlates with the loss of GTPase activity stimulated by the peptide toxin Mas-7. The application of Mas-7 to growing mycelial colonies raises intracellular cAMP levels, suggesting that Tgal can activate adenylyl cyclase. In contrast, cAMP levels and cAMP-dependent protein kinase activity drop when diffusible host signals are encountered and the mycoparasitism-related genes ech42 and prb1 are highly expressed. Mycoparasitic signaling is unlikely to be a linear pathway from host signals to increased cAMP levels. Our results demonstrate that the product of the tga1 gene is involved in both coiling and conidiation.     

Analysis by mRNA levels of the expression of six G protein alpha-subunit genes in mammalian cells and tissues.

The distribution and levels of expression of Gs alpha, Gi1 alpha, Gi2 alpha, Gi3 alpha, Go alpha, and Gx alpha mRNAs were compared by Northern blot analysis using several rat tissues and selected human and rat cell lines. Gi1 alpha, Go alpha, and Gx alpha, were detected in a limited number of tissue and cells whereas Gi2 alpha, Gi3 alpha, and Gs alpha, were expressed in all the tissues and cells tested albeit in varying amounts. The expression of these six genes appears to be differentially regulated during postnatal development of the rat brain. High expression levels particularly of Go alpha, in young rat brain may be related to the formation of neurites during differentiation of nerve cells.     

Primary structure of the alpha-subunit of bovine adenylate cyclase-stimulating G-protein deduced from the cDNA sequence

The primary structure of the alpha-subunit of the adenylate cyclase-stimulating G-protein (Gs) has been deduced from the nucleotide sequence of cloned DNA complementary to the bovine cerebral mRNA encoding the polypeptide. Comparison of the amino acid sequences of the alpha-subunits of Gs and transducin reveals that some of the highly conserved regions show sequence homology with elongation factor-Tu and ras p21 proteins and correspond to functional regions of guanine nucleotide-binding proteins.     

Genetic variability of proteins from plasma membranes and cytosols of mouse organs

The genetic variability of membrane proteins (structure-bound proteins) and cytosol proteins (water-soluble proteins) was investigated in two inbred strains of the mouse, C57BL/6J and DBA/2J. Membrane proteins and cytosol were isolated from the brain and liver of the mouse. The proteins were separated by two-dimensional electrophoresis. A high number of genetic variant proteins (brain, 30; liver, 72) was found in the cytosol. Most of these variants represented changes in the amount of proteins. Electrophoretic mobility changes occurred only in about 1% (brain, 6; liver, 9) of all protein spots of a two-dimensional pattern. In contrast to the cytosol proteins, no genetic variation was detected among the membrane proteins, not even for the quantitative characteristics of the protein spots. The results obtained for the two classes of proteins suggest that the degree of variability in the amount of proteins is related to the degree of variability in the structure of proteins.     

Primary structure of the alpha-subunit of bovine adenylate cyclase-inhibiting G-protein deduced from the cDNA sequence

The primary structure of the alpha-subunit of the adenylate cyclase-inhibiting G-protein (Gi) has been deduced from the nucleotide sequence of cloned DNA complementary to the bovine cerebral mRNA encoding the polypeptide. A much higher degree of amino acid sequence homology is observed between the alpha-subunits of Gi and transducin (68%) than between those of Gi and the adenylate cyclase-stimulating G-protein (Gs) (43%) or between those of transducin and Gs (42%).     

Immunochemical identification of the chick HPRT gene transferred from chick erythrocytes to mammalian somatic cells

Immunochemical methods were used to identify the genetic origin of hypoxanthine phosphoribosyltransferase (HPRT) expressed in heteroploid, HPRT-deficient mouse (A9) cells and Chinese hamster ovary (K627) cells, after these cells were fused with chick embryo erythrocytes and selected for resistance to hypoxanthine-aminopterin-thymidine (HAT) medium. All of the HAT-selected clones produced HPRT activity which was immunoprecipitable by an antiserum specific for chick HPRT, but not by an antiserum specific for mouse and hamster HPRT. Furthermore, the HPRT activity in these clones was electrophoretically indistinguishable from chick liver HPRT and clearly different from mouse liver HPRT. These data provide evidence that the HPRT activity expressed in cell hybrids produced by the fusion of HPRT-negative mammalian cells and chick erythrocytes containing genetically inactive nuclei is indeed coded by the chick HPRT gene and that an avian gene can be stably incorporated and correctly expressed in a mammalian cells.     

The cytosolic G-protein alpha-subunit in human neutrophils responds to treatment with guanine nucleotides and magnesium

The hydrodynamic properties of the Gi2 alpha-subunit in human neutrophil cytosol were analyzed. The S20,w value was 3.3 S at 30 degrees C and 4.1 S at 4 degrees C. Reconstitution of the cytosolic alpha-subunit with beta gamma-complex purified from porcine brain resulted in an S20,w value of 4.0 S at 30 degrees C and 4.2 S at 4 degrees C. Treatment of cytosol with G-protein-activating agents, GTP gamma S and MgCl2, decreased the S20,w value to 2.4 S at 30 degrees C and 2.9 S at 4 degrees C. Our results indicate that the cytosolic alpha-subunit of neutrophils is an inactive monomeric species at 30 degrees C capable of interacting with G-protein beta gamma-complex and responsive to treatment with activating agents leading to the two activated forms, alpha at 4 degrees C and alpha at 30 degrees C.