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PubMed1999

Involvement of the carboxyl terminus of the third intracellular loop of the cannabinoid CB1 receptor in constitutive activation of Gs.

The human cannabinoid receptor CB1 functionally couples primarily to Gi-, but also to Gs-mediated pathways to modulate intracellular cyclic AMP (cAMP) levels. To probe the features of the receptor that may be involved in promoting interactions with one G protein type over another, we generated the L341A/A342L mutant CB1 receptor. The double mutation involved the swap in position of two adjacent residues in the carboxyl-terminal segment of the third intracellular loop of CB1. This resulted in partial constitutive activation of the receptor and an agonist-independent enhancement in cAMP levels. Characterization following treatment with either pertussis or cholera toxin indicated that the constitutive activity is selective for a Gs- and not a Gi-mediated pathway. Treatment with the CB1-specific inverse agonist SR141716A inhibited the basal accumulation of cAMP in the presence of pertussis toxin, establishing that the effect is CB1 mediated. The binding of the agonist CP-55,940 to the L341A/A342L receptor was not markedly different from that for the wild-type receptor despite the constitutive Gs activity. This may reflect a preference of this ligand for an activated receptor state associated with the Gi coupling form and underscores the potential for developing therapeutics that selectively activate one pathway over another.

Animals↗

PubMed1998

Pathogenesis of toxic thyroid adenomas and nodules: relevance of activating mutations in the TSH-receptor and Gs-alpha gene, the possible role of iodine deficiency and secondary and TSH-independent molecular mechanisms.

In iodine deficiency areas, activating mutations in the TSH receptor and Gs-alpha gene are found in the majority of toxic thyroid adenomas and in some toxic goiter nodules. Since TSH receptor and Gs-alpha gene mutations are very rare in areas with high iodine supply, iodine deficiency has been suspected to favor the occurrence of these mutations by yet unknown pathways. However, TSH receptor and Gs-alpha gene mutations alone are not sufficient to cause toxic adenomas and nodules. There is compelling evidence that other secondary and cAMP-independent mechanisms, including enhanced expression of various growth factors, their corresponding receptors and of signaling proteins, may affect the mutated cell and thus promote cell proliferation and in turn generation of the tumor.

Adenoma↗

PubMed1999

A Gsalpha mutant designed to inhibit receptor signaling through Gs.

Hormonal signals activate trimeric G proteins by substituting GTP for GDP bound to the G protein alpha subunit (Galpha), thereby generating two potential signaling molecules, Galpha-GTP and free Gbetagamma. The usefulness of dominant negative mutations for investigating Ras and other monomeric G proteins inspired us to create a functionally analogous dominant negative Galpha mutation. Here we describe a mutant alpha subunit designed to inhibit receptor-mediated hormonal activation of Gs, the stimulatory regulator of adenylyl cyclase. To construct this mutant, we introduced into the alpha subunit (alphas) of Gs three separate mutations chosen because they impair alphas function in complementary ways: the A366S mutant reduces affinity of alphas for binding GDP, whereas the G226A and E268A mutations impair the protein's ability to bind GTP and to assume an active conformation. The triple mutant robustly inhibits (by up to 80%) Gs-dependent hormonal stimulation of adenylyl cyclase in cultured cells. Inhibition is selective in that it does not affect cellular responses to expression of a constitutively active alphas mutant (alphas-R201C) or to agonists for receptors that activate Gq or Gi. This alphas triple mutant and cognate Galpha mutants should provide specific tools for dissection of G protein-mediated signals in cultured cells and transgenic animals.

Adenylyl Cyclase Inhibitors↗

PubMed1995

Coupling of human D-1 dopamine receptors to different guanine nucleotide binding proteins. Evidence that D-1 dopamine receptors can couple to both Gs and G(o).

Coupling between D-1 dopamine receptors and G proteins in cell lines expressing human D-1 receptors and different G proteins was examined. Pertussis toxin (PTX) treatment of rat pituitary GH4C1 cells significantly reduced, but did not abolish, agonist high affinity binding sites of the D-1 dopamine receptor; in SK-N-MC neuroblastoma cells, PTX failed to have any effect on D-1 high affinity sites. Cholera toxin (CTX) treatment of GH4C1 cells reduced but did not abolish the high affinity sites of D-1 receptors, while in SK-N-MC cells, treatment with CTX abolished all the high affinity sites. Western blot analyses with specific antisera indicated that Gs alpha, Gi1 alpha, Gi3 alpha, and Gq alpha were expressed in both cell lines, while Gi2 alpha and G(o) alpha were expressed in GH4C1 but not SK-N-MC cells. Antisera NEI-805 (anti-Gs alpha) and 9072 (anti-G(o) alpha) immunoprecipitated 24 +/- 4.3 and 34.4 +/- 6.9%, respectively, of G protein-associated D-1 dopamine receptors. Antisera 3646 (anti-Gi1 alpha), 1521 (anti-Gi2 alpha), 1518 (anti-Gi3 alpha), and 0941 (anti-Gq alpha) failed to coimmunoprecipitate appreciable levels of soluble receptors. These data indicate that D-1 dopamine receptors are coupled to both Gs alpha and G(o) alpha but not to Gq alpha.

Animals↗

PubMed1998

Comparison of rabbit and human corneas stored in Optisol-GS: changes in stromal sodium.

PURPOSE: To evaluate the hydration, and the levels of free, total and bound sodium in fresh rabbit corneal stromas and also those preserved for up to 21 days in Optisol-GS. The effect of epithelial removal on stromal sodium and hydration parameters was also evaluated. Trends in stromal hydration and sodium environment were compared to results we previously obtained using human eyes stored under identical conditions. METHODS: Stromal hydration was evaluated thermogravimetrically. A sodium-specific electrode and an atomic absorption spectrophotometer were used to determine the amounts of free and total stromal sodium, respectively. In one cornea of each pair, the epithelium was removed prior to placement in the storage media. After 3, 7, 14 or 21 days at 4 degrees C, corneas were removed from the Optisol-GS, at which time sodium and hydration measurements were obtained. RESULTS: With an intact epithelium, the hydration of the rabbit stromas was elevated significantly at each day of storage compared to fresh corneas. Free and total sodium levels of rabbit stromas did not differ statistically from fresh values, however the bound sodium values did increase during storage. In the absence of the epithelium, the stromal hydration and sodium content (free, total and bound) were significantly elevated and the increase was much greater than in corneas stored with an intact epithelium. These findings differ from those we measured previously using human tissue. CONCLUSIONS: Rabbit corneas responded differently from human corneas to storage in Optisol-GS. The hydration levels increased to a greater level in rabbit than human corneas under both storage conditions. The trends in amounts of both free and total sodium were similar between the species, although the absolute amounts differed. The largest discrepancy was observed in the amount of bound sodium, with the rabbit corneas experiencing large increases not documented in the human tissue. These results suggest that direct comparisons of stromal hydration and ionic environment between the species should be approached with caution.

Animals↗

PubMed1992

Oxygen free radical injury and Gs mediated signal transduction in the stunned porcine myocardium.

OBJECTIVE: The aim was to investigate involvement of oxygen free radicals and any changes in the Gs mediated beta adrenergic signalling system of stunned porcine myocardium. METHODS: Myocardial stunning was induced in eight pentobarbitone anaesthetised pigs by brief occlusions of the distal left anterior descending coronary artery for periods of up to 10 min. Segment length function was measured in the ischaemic region and in a control region supplied by the circumflex artery. Left ventricular biopsies were obtained from the two regions 1 h after the last occlusion for ultrastructural and biochemical studies. Timolol has been used to prevent arrhythmia during ischaemia. RESULTS: At the time when biopsies were obtained, percent systolic shortening was reduced to 58% in the region subjected to ischaemia and was only minimally reduced in the control region. In the biopsies from the stunned region: (1) electron microscopy showed mild and reversible intracellular changes in the stunned myocardium; (2) the activities of superoxide dismutase and glutathione peroxidase were decreased by 66% and 52%, respectively; (3) the content of malondialdehyde was increased by 49%; (4) neither density nor affinity of beta adrenoceptors showed any changes; (5) there were no alterations in messenger RNA encoding for the alpha subunit of the stimulatory guanine nucleotide binding protein (Gs), demonstrated by northern and dot-blot hybridisations; (6) ELISA technique utilising a specific antipeptide antibody showed no quantitative change in Gs; (7) the activity of adenyl cyclase was unchanged. CONCLUSIONS: Even though the stunned porcine myocardium showed substantial evidence of free radical injury, the beta adrenergic signalling system was intact.

Adenylyl Cyclases↗

PubMed2003

GS-Aligner: a novel tool for aligning genomic sequences using bit-level operations.

A novel algorithm, GS-Aligner, that uses bit-level operations was developed for aligning genomic sequences. GS-Aligner is efficient in terms of both time and space for aligning two very long genomic sequences and for identifying genomic rearrangements such as translocations and inversions. It is suitable for aligning fairly divergent sequences such as human and mouse genomic sequences. It consists of several efficient components: bit-level coding, search for matching segments between the two sequences as alignment anchors, longest increasing subsequence (LIS), and optimal local alignment. Efforts have been made to reduce the execution time of the program to make it truly practical for aligning very long sequences. Empirical tests suggest that for relatively divergent sequences such as sequences from different mammalian orders or from a mammal and a nonmammalian vertebrate GS-Aligner performs better than existing methods. The program and data can be downloaded from http://pondside.uchicago.edu/~lilab/ and http://webcollab.iis.sinica.edu.tw/~biocom.

Algorithms↗

PubMed2001

Co-expression of P2X receptor subunits on rat nodose neurons that bind the isolectin GS-I-B4.

Triple fluorescent histochemistry was used to describe the types of overlap in visceral sensory neurons (nodose ganglion) for the labeling of the isolectin B4 from Griffonia simplicifolia type one (GS-I-B4) and their immunoreactivity (IR) for two of the ATP receptor subunits (P2X1/3 or P2X2/3). The vast majority of nodose neurons expressed GS-I-B4-binding and most of these displayed P2X receptor IR. Most of the P2X-IR was co-expressed on these individual nodose neurons (P2X1/P2X3 or P2X2/P2X3). A very small subpopulation of neurons that were GS-I-B4 negative but P2X positive displayed a very high relative intensity of P2X3-IR. The functional role that these expression patterns play in visceral sensory processing is currently unclear.

Animals↗

PubMed2000

Impact of transportation on short-term preserved corneas preserved in Optisol-GS, Likorol, Likorol-DX, and MK-medium.

PURPOSE: To investigate the impact of transportation, simulated under laboratory conditions, on the corneal endothelium preserved by Optisol-GS, Likorol-DX, Likorol, and MK-Medium. METHODS: Three hundred twenty corneas from freshly slaughtered pigs were stored in Optisol-GS. Likorol-DX, Likorol. and MK-Medium for 1, 3, 6, or 10 days. After short-term preservation, the transportation was simulated on a laboratory shaker at 4 degrees C with an acceleration of 0-100 km/h in 16 seconds for 5 hours. Mate corneas served as the control. Corneal endothelial cell density was determined at the time of dissection, directly before the experiment, and after subsequent organ culture. RESULTS: A significant cell loss induced by transportation simulation was not observed in any experimental group. Preservation in MK-Medium starting at 3 days of short-term preservation resulted in a significant cell loss. Storage for up to 6 days in Optisol-GS, Likorol-DX, and Likorol did not result in a significant decrease in cell density. CONCLUSION: Short-term preserved corneas can be routinely distributed without a reevaluation, if the tissue is preserved for a shorter time as recommended by the manufacturers.

Animals↗

PubMed1994

Chronic sympathectomy of canine cardiac ventricles affects Gs-adenylyl cyclase coupling and muscarinic receptor density.

The effect of chronic ventricular sympathectomy on sarcolemmal muscarinic receptor (MR) and beta-adrenoceptor densities and coupling of these receptors to adenylyl cyclase was examined. Microsomal membranes were isolated from right and left ventricles of control dogs (sham- and nonoperated) and dogs with ventricles sympathectomized 4 weeks earlier. Relative to control membranes, MR density was decreased in left but not right ventricular (LV, RV) membranes from sympathectomized hearts. Relative carbachol inhibition of adenylyl cyclase was similar in RV and LV membranes from both heart groups, however, Although beta-adrenoceptor densities and ratio of beta 1- and beta 2-adrenoceptor subtypes did not change, basal adenylyl cyclase activity was 40% less in sympathectomized membranes as compared with control membranes. Furthermore, relative stimulation of adenylyl cyclase by isoproterenol was twofold greater in sympathectomized heart membranes. Because maximally stimulated adenylyl cyclase activity by NaF or MnCl2 was identical in sympathectomized and control membranes, the reduction in basal activity may not be related to a decrease in Gs and adenylyl cyclase. In support of this hypothesis, Gs alpha content as estimated from optimal cholera toxin-catalyzed ADP-ribosylation was similar in control and sympathectomized membranes. Therefore, an alteration in Gs interaction with adenylyl cyclase may account for the reduction in basal adenylyl cyclase activity and the increased relative responsiveness of adenylyl cyclase to isoproterenol in chronically sympathectomized ventricular membranes.

Adenylyl Cyclases↗

PubMed2006

The effects of epithelial viability on stromal keratocyte apoptosis in porcine corneas stored in Optisol-GS.

PURPOSE: To investigate the effect of corneal epithelium on the viability of corneal stromal keratocytes in Optisol-GS. METHODS: After sterilization, corneoscleral buttons were excised and stored in Optisol-GS for various time periods. Group 1 corneas (n = 40) underwent mechanical corneal epithelial debridement before storage while group 2 corneas (n = 40) were stored with intact epithelium. Changes in corneal thickness, keratocyte density, and keratocyte apoptosis were investigated immediately, at 4 hours, and on days 1, 2, 3, 5, 7, and 14 in the preservation medium. The differences between group 1 and 2 corneas were analyzed. RESULTS: Corneal thickness increased significantly in the second week of preservation in both groups, though more substantially in group 1. Significant corneal epithelial apoptosis was noticed in the first week in group 2 corneas. Corneal stromal keratocyte density decreased with prolonged preservation time. DNA laddering was detected by ligation-mediated polymerase chain reaction throughout the experiment periods in both groups, but the increase of keratocyte apoptosis was more significant after 5 days of preservation, especially in group 1. CONCLUSIONS: Stromal keratocytes underwent apoptosis in Optisol-GS. The absence of corneal epithelium during preservation further increased the stromal keratocyte apoptosis.

Animals↗

PubMed1988

Gi affects the agonist-binding properties of beta-adrenoceptors in the presence of Gs.

Pertussis-toxin-catalyzed ADP-ribosylation of Gi in S49 membranes, but not in S49AC- membranes, which lack Gs, induces a threefold reduction of isoproterenol affinity to the beta-adrenoceptors. A similar treatment of turkey erythrocyte membranes, which are devoid of functional Gi, has no effect on beta-agonist affinity to their beta-adrenoceptors. Non-hydrolyzable analogs such as GTP[S] induce a larger decrease in beta-adrenoceptor affinity in S49 cells towards the agonist isoproterenol as compared to pertussis-toxin-catalyzed ADP-ribosylation of Gi. These results suggest that Gi affects beta-adrenoceptor affinity to its agonist and that this interaction requires the presence of Gs. It seems, therefore, that Gi physically interacts with Gs to exert its effects on the receptor and probably on adenylate cyclase as well. Our ability to detect (a) the effect of pertussis-toxin-catalyzed ADP-ribosylation in S49 cells on beta-agonist affinity and (b) the quantitative difference between the effect of pertussis toxin (approx. threefold) and GTP[S] (fivefold to sevenfold) depends on the use of a simple but rigorous method to study in detail the affinity of beta-agonists to their receptors. This method seems to be superior to the analysis of displacement curves as a means to examine receptor-ligand interactions.

Adenylate Cyclase Toxin↗

PubMed1990

Segregation of discrete GS alpha-mediated responses that accompany homologous or heterologous desensitization in two related somatic hybrids.

1. Prostacyclin and adenosine A2 receptors activate adenylate cyclase in the neuroblastoma hybrid cell lines NG108-15 and NCB-20. Prolonged exposure of NG108-15 cells to iloprost (a stable analogue of prostacyclin) results in a subsequent reduction in the capacity for adenylate cyclase activation by iloprost, the adenosine analogue 5'-(N-ethyl)-carboxamidoadenosine (NECA) or NaF. In contrast prolonged exposure of NCB-20 cells to iloprost results only in the loss of iloprost responsiveness. 2. Iloprost pretreatment of NG108-15 cells also magnified the morphine-dependent inhibition of iloprost-stimulated adenylate cyclase activity from 36 to 48%. This change was not due to lower iloprost stimulation following desensitization, since the % inhibition of adenylate cyclase activity by morphine in control cells was constant irrespective of enzyme activity. 3. These heterologous effects observed in NG108-15 cells following iloprost pretreatment may involve changes in the GS alpha protein, since there was a reduction of about 30% in the cholera toxin-induced [32P]-ADP-ribosylation of a 45 kDa protein from cell membranes (corresponding to the extent of loss of NECA or NaF responsiveness). A similar reduction was not observed in NCB-20 cells. 4. These results indicate that iloprost pretreatment induces different forms of desensitization in NG108-15 and NCB-20 cell lines. The heterologous desensitization in the former may, like the human platelet, involve a functional loss of GS alpha from the cell membrane. Changes in the activity of GS alpha may also account for the heterologous effects on receptors that mediate inhibition of adenylate cyclase.

Adenosine↗

PubMed1986

Isolation and characterization of a fructosyltransferase gene from Streptococcus mutans GS-5.

A fructosyltransferase (FTF) gene from Streptococcus mutans GS-5 has been isolated from a lambda L47.1 clone bank. The gene was contained on an 11.7-kilobase GS-5 DNA fragment and was initially subcloned into plasmid pACYC184 as a 5.4-kilobase HindIII fragment. However, further analysis revealed that transcription of the FTF gene was initiated at the P1 promoter contained on the vector. It was possible to subclone the FTF gene with its presumed promoter as a 3.4-kilobase EcoRI fragment to produce the chimeric plasmid pSS22 expressing FTF activity. The cloned enzyme was purified to apparent homogeneity after ammonium sulfate precipitation, gel filtration, and DEAE-Bio-Gel-A chromatography followed by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme displayed a lower molecular weight (63,000) compared with the multiple activities detected in the culture fluids of strain GS-5. In addition, storage of the purified enzyme resulted in the formation of even lower-molecular-weight enzymatically active species. These results suggested that proteolytic degradation of the FTF occurs both in S. mutans and in Escherichia coli. In addition, a comparison of the properties of the cloned enzyme with those previously characterized from another serotype c S. mutans strain suggests that multiple FTF genes may be present in these organisms.

Cell Compartmentation↗

PubMed1979

Comparative analysis of GS and BK virus genomes.

Analysis of heteroduplexing between the genomes of GS virus, a BK-group virus, and the prototype BK virus revealed one region of nonhomology. Further analysis by cleavage of viral DNA with the restriction endonucleases EcoRI, HindIII, and HaeIII revealed that base changes in the GS virus genome spanned 0.6 to 0.7 map unit from the EcoRI site. Large T and small t antigens of GS virus appear to be similar in size to the BK virus antigens.

Antigens, Neoplasm↗

PubMed1990

Mutation of the Gs protein alpha subunit NH2 terminus relieves an attenuator function, resulting in constitutive adenylyl cyclase stimulation.

G-proteins couple hormonal activation of receptors to the regulation of specific enzymes and ion channels. Gs and Gi are G-proteins which regulate the stimulation and inhibition, respectively, of adenylyl cyclase. We have constructed two chimeric cDNAs in which different lengths of the alpha subunit of Gs (alpha s) have been replaced with the corresponding sequence of the Gi alpha subunit (alpha i2). One chimera, referred to as alpha i(54)/s' replaces the NH2-terminal 61 amino acids of alpha s with the first 54 residues of alpha i. Within this sequence there are 7 residues unique to alpha s, and 16 of the remaining 54 amino acids are nonhomologous between alpha i and alpha s. The second chimera, referred to as alpha i/s(Bam), replaces the first 234 amino acids of alpha s with the corresponding 212 residues of alpha i. Transient expression of alpha i(54)/s in COS-1 cells resulted in an 18- to 20-fold increase in cyclic AMP (cAMP) levels, whereas expression of either alpha i/s(Bam) or the wild-type alpha s polypeptide resulted in only a 5- to 6-fold increase in cellular cAMP levels. COS-1 cells transfected with alpha i showed a small decrease in cAMP levels. Stable expression of the chimeric alpha i(54)/s polypeptide in Chinese hamster ovary (CHO) cells constitutively increased both cAMP synthesis and cAMP-dependent protein kinase activity. CHO clones expressing transfected alpha i/s(Bam) or the wild-type alpha s and alpha i cDNAs exhibited cAMP levels and cAMP-dependent protein kinase activities similar to those in control CHO cells. Therefore, the alpha i(54)/s chimera behaves as a constitutively active alpha s polypeptide, whereas the alpha i/s(Bam) polypeptide is regulated similarly to wild-type alpha s. Expression in cyc-S49 cells, which lack expression of wild-type alpha s, confirmed that the alpha i(54)/s polypeptide is a highly active alpha s molecule whose robust activity is independent of any change in intrinsic GTPase activity. The difference in phenotypes observed upon expression of alpha i(54)/s or alpha i/s(Bam) indicates that the NH2-terminal moieties of alpha s and alpha i function as attenuators of the effector enzyme activator domain which is within the COOH-terminal half of the alpha subunit. Mutation at the NH2 terminus of alpha s relieves the attenuator control of the Gs protein and results in a dominant active G-protein mutant.

1-Methyl-3-isobutylxanthine↗

PubMed1993

Modulation of coronary smooth muscle KCa channels by Gs alpha independent of phosphorylation by protein kinase A.

The occupancy of beta-receptors in the smooth muscle membrane of the coronary arteries produces vasodilation and a concomitant hyperpolarization. Large conductance calcium-activated K (KCa) channels are likely to be involved in such hyperpolarization, since they are densely distributed in coronary myocytes, and they are targets of beta-adrenergic stimulation in other smooth muscles. We sought to explore if coronary smooth muscle KCa channels are modulated by beta-agonists and we studied the mechanisms of their activation. We found that KCa channels reconstituted into lipid bilayers were activated in the presence of GTP by the beta-adrenergic receptor agonist isoproterenol. KCa channels were also stimulated on non-specific activation of an endogenous G protein(s) with guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), on addition of a purified activated stimulatory G protein (Gs alpha), and when the catalytic subunit of protein kinase A (PKA) was added. Inhibition of PKA activity prevented KCa channel stimulation by PKA, but not by endogenous G protein or by exogenous Gs alpha. These results indicate that beta-adrenoceptor activation of coronary smooth muscle KCa channels results from a dual control: 1) a membrane delimited, possibly direct action of Gs, independent of PKA-mediated phosphorylation; and 2) by PKA-dependent phosphorylation.

Animals↗

PubMed2003

Use of bioinformatics to predict a function for the GS element in Mycobacterium avium subspecies paratuberculosis.

Mycobacterium avium subsp. Paratuberculosis (MAP) is a member of the Mycobacterium avium complex (MAC) and causes the inflammatory bowel disease, Johne's disease, in livestock. MAP has also been implicated as the causative agent of a similar disease, Crohn's disease, in humans. One of three major genetic differences between MAP and non-pathogenic MAC is the 6496-bp GS element. Based on the output from freely available protein sequence and structural bioinformatics tools, and the close homology of GS genes with the SER2 region of the closely related Mycobacterium avium subsp. Avium (MAA), we predict that GS encoded enzymes are involved in the biosynthesis of GDP-fucose, and the addition to, and modification of fucose on, the oligosaccharide moiety of GPL. GPL is a major constituent of the cell wall of the MAC and has immunomodulatory properties. Therefore, the enzymes involved in its synthesis may provide novel drug targets against MAP and other pathogenic MAC members.

Amino Acid Sequence↗