Glucose-induced cAMP signalling in yeast requires both a G-protein coupled receptor system for extracellular glucose detection and a separable hexose kinase-dependent sensing process

In Saccharomyces cerevisiae, glucose activation of cAMP synthesis requires both the presence of the G-protein-coupled receptor (GPCR) system, Gpr1-Gpa2, and uptake and phosphorylation of the sugar. In a hxt-null strain that lacks all physiologically important glucose carriers, glucose transport as well as glucose-induced cAMP signalling can be restored by constitutive expression of the galactose permease. Hence, the glucose transporters do not seem to have a regulatory function but are only required for glucose uptake. We established a system in which the GPCR-dependent glucose-sensing process is separated from the glucose phosphorylation process. It is based on the specific transport and hydrolysis of maltose providing intracellular glucose in the absence of glucose transport. Preaddition of a low concentration (0.7 mM) of maltose to derepressed hxt-null cells and subsequent addition of glucose restored the glucose-induced cAMP signalling, although there was no glucose uptake. Addition of a low concentration of maltose itself does not increase the cAMP level but enhances Glu6P and apparently fulfils the intracellular glucose phosphorylation requirement for activation of the cAMP pathway by extracellular glucose. This system enabled us to analyse the affinity and specificity of the GPCR system for fermentable sugars. Gpr1 displayed a very low affinity for glucose (apparent Ka = 75 mM) and responded specifically to extracellular alpha and beta D-glucose and sucrose, but not to fructose, mannose or any glucose analogues tested. The presence of the constitutively active Gpa2val132 allele in a wild-type strain bypassed the requirement for Gpr1 and increased the low cAMP signal induced by fructose and by low glucose up to the same intensity as the high glucose signal. Therefore, the low cAMP increases observed with fructose and low glucose in wild-type cells result only from the low sensitivity of the Gpr1-Gpa2 system and not from the intracellular sugar kinase-dependent process. In conclusion, we have shown that the two essential requirements for glucose-induced activation of cAMP synthesis can be fulfilled separately: an extracellular glucose detection process dependent on Gpr1 and an intracellular sugar-sensing process requiring the hexose kinases.     

Internal acidification and cAMP increase are not correlated in Saccharomyces cerevisiae

Addition of glucose to a yeast suspension can produce both an increase in the level of cAMP and a decrease in the intracellular pH. This observation led to the idea that internal acidification triggers the cAMP increase. We have tested this hypothesis using different approaches. To study the effect of sugar metabolism on internal pH we added to the yeast either glucose or a sugar, like xylose, that cannot be phosphorylated. We also utilized yeast strains lacking hexose kinases or phosphoglucose isomerase. We found that phosphorylation of the sugar added is a requisite for internal acidification but not for the cAMP increase. Internal acidification is due to an imbalance between the rate of the metabolic reactions that generate protons and the rate at which protons can be pumped out of the cell. We have manipulated the excretion of protons by using yeast harvested at different phases of growth and resuspended in a medium with or without added K+. Addition of glucose produced a marked drop in internal pH only when the yeast was harvested in the stationary phase of growth and transferred to a medium without added K+. In contrast an increase in cAMP was observed in all situations. We conclude that in yeast there is no correlation between internal acidification and cAMP increase.     

Involvement of distinct G-proteins, Gpa2 and Ras, in glucose- and intracellular acidification-induced cAMP signalling in the yeast Saccharomyces cerevisiae.

Adenylate cyclase activity in Saccharomyces cerevisiae is dependent on Ras proteins. Both addition of glucose to glucose-deprived (derepressed) cells and intracellular acidification trigger an increase in the cAMP level in vivo. We show that intracellular acidification, but not glucose, causes an increase in the GTP/GDP ratio on the Ras proteins independent of Cdc25 and Sdc25. Deletion of the GTPase-activating proteins Ira1 and Ira2, or expression of the RAS2(val19) allele, causes an enhanced GTP/GDP basal ratio and abolishes the intracellular acidification-induced increase. In the ira1Delta ira2Delta strain, intracellular acidification still triggers a cAMP increase. Glucose also did not cause an increase in the GTP/GDP ratio in a strain with reduced feedback inhibition of cAMP synthesis. Further investigation indicated that feedback inhibition by cAPK on cAMP synthesis acts independently of changes in the GTP/GDP ratio on Ras. Stimulation by glucose was dependent on the Galpha-protein Gpa2, whose deletion confers the typical phenotype associated with a reduced cAMP level: higher heat resistance, a higher level of trehalose and glycogen and elevated expression of STRE-controlled genes. However, the typical fluctuation in these characteristics during diauxic growth on glucose was still present. Overexpression of Ras2(val19) inhibited both the acidification- and glucose-induced cAMP increase even in a protein kinase A-attenuated strain. Our results suggest that intracellular acidification stimulates cAMP synthesis in vivo at least through activation of the Ras proteins, while glucose acts through the Gpa2 protein. Interaction of Ras2(val19) with adenylate cyclase apparently prevents its activation by both agonists.     

Tyrosine phosphorylation in semaphorin signalling: shifting into overdrive

The semaphorins constitute a large family of molecular signals with regulatory functions in neuronal development, angiogenesis, cancer progression and immune responses. Accumulating data indicate that semaphorins might trigger multiple signalling pathways, and mediate different and sometimes opposing effects, depending on the cellular context and the particular plexin-associated subunits of the receptor complex, which can include receptor-type or cytoplasmic tyrosine kinases such as MET, ERBB2, VEGFR2, FYN, FES, PYK2 and SRC. It has also been shown that a specific plexin can alternatively associate with different tyrosine kinase receptors, eliciting divergent signalling pathways and functional outcomes. Tyrosine phosphorylation is a pivotal post-translational protein modification that regulates intracellular signalling. Therefore, phosphorylation of tyrosines in the intracellular domain of plexins could determine or modify their interactions with additional signal transducers. Here, we discuss the potential relevance of tyrosine phosphorylation in semaphorin-induced signalling, with an emphasis on its probable role in dictating the choice between multiple pathways and functional outcomes. The identification of implicated tyrosine kinases will pave the way to target individual semaphorin-mediated functions.     

The high-affinity glucose uptake system is not required for induction of the RAS-mediated cAMP signal by glucose in cells of the yeast Saccharomyces cerevisiae

Addition of glucose or related fermentable sugars to yeast cells grown on non-fermentable carbon sources, triggers a RAS-protein mediated cAMP signal which induces a protein phosphorylation cascade. The high-affinity glucose uptake system in yeast cells is known to be glucose-repressible and only functional in strains containing at least one active kinase. In strains containing point or disruption mutations in the SNF3 gene, which codes for the high-affinity glucose carrier, the glucose-induced cAMP signal is still present. This indicates that the previously demonstrated requirement of a functional kinase for the induction of the cAMP signal, does not reflect requirement of high-affinity sugar transport. It also indicates that the unknown glucose-repressible protein in the induction sequence of the RAS-mediated cAMP signal is not the high-affinity sugar carrier.     

Glucose as a regulator of insulin-sensitive hexose uptake in 3T3 adipocytes

In the present study we examined the role of glucose in the regulation of its own transport activity in the cultured 3T3 fat cell. A regulatory control of glucose became apparent after these cells were cultured in the absence of glucose. Glucose deprivation of the cells was accompanied by a specific time and protein synthesis-dependent increase in dGlc (2-deoxyglucose) uptake (up to 5-fold), which was due to an increase in the apparent Vmax of the transport system. Concomitantly, the stimulatory effect of insulin on hexose uptake almost completely disappeared. Addition of glucose to the glucose-deprived cells rapidly reversed the deprivation effects. Cycloheximide experiments revealed that the glucose deprivation-induced increase in hexose uptake required protein synthesis as well as a protein synthesis-independent response to glucose deprivation that retarded the turnover of hexose transport activity. Taken together, these data indicate that glucose deprivation is accompanied by retardation of the rate of degradation, internalization, or inactivation of hexose transporters while the increase in dGlc uptake requires at least the continuation of protein synthesis-dependent de novo synthesis, insertion, or activation of hexose transporters. Hexose competitively taken up with dGlc, including the nonmetabolizable glucose analogue 3-O-methylglucose, could replace glucose in the process of prevention and reversal of the deprivation effects, indicating that competitive transport but not the metabolism of hexose is a prerequisite for the regulatory effect of glucose on the activity of its own transport system. In conclusion, our results indicate that in cultured 3T3 fat cells glucose itself is involved in the regulation of the activity of its own transport system by influencing the rate of degradation, internalization, or inactivation of hexose transporters by a protein synthesis-independent mechanism.     

Involvement of the CDC25 gene product in the signal transmission pathway of the glucose-induced RAS-mediated cAMP signal in the yeast Saccharomyces cerevisiae.

Addition of glucose or related fermentable sugars to derepressed cells of the yeast Saccharomyces cerevisiae triggers a RAS-protein-mediated cAMP signal, which induces a protein phosphorylation cascade. Yeast strains without a functional CDC25 gene were deficient in basal cAMP synthesis and in the glucose-induced cAMP signal. Addition of dinitrophenol, which in wild-type strains strongly stimulates in vivo cAMP synthesis by lowering intracellular pH, did not enhance the cAMP level. cdc25 disruption mutants, in which the basal cAMP level was restored by the RAS2val19 oncogene or by disruption of the gene (PDE2) coding for the high-affinity phosphodiesterase, were still deficient in the glucose- and acidification-induced cAMP responses. These results indicate that the CDC25 gene product is required not only for basal cAMP synthesis in yeast but also for specific activation of cAMP synthesis by the signal transmission pathway leading from glucose to adenyl cyclase. They also show that intracellular acidification stimulates the pathway at or upstream of the CDC25 protein. When shifted to the restrictive temperature, cells with the temperature sensitive cdc25-5 mutation lost their cAMP content within a few minutes. After prolonged incubation at the restrictive temperature, cells with this mutation, and also those with the temperature sensitive cdc25-1 mutation, arrested at the 'start' point (in G1) of the cell cycle, and subsequently accumulated in the resting state G0. In contrast with cdc25-5 cells, however, the cAMP level did not decrease and normal glucose- and acidification-induced cAMP responses were observed when cdc25-1 cells were shifted to the restrictive temperature.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of the cAMP level in the yeast Saccharomyces cerevisiae: the glucose-induced cAMP signal is not mediated by a transient drop in the intracellular pH

Addition of glucose to derepressed cells of the yeast Saccharomyces cerevisiae is known to cause a rapid, transient increase in the cAMP level, which lasts for 1-2 min and induces a cAMP-dependent protein phosphorylation cascade. The glucose-induced cAMP signal cannot be explained solely on the basis of an increased ATP level. Transient membrane depolarization and transient intracellular acidification have been suggested as possible triggers for the cAMP peak. Addition of glucose to cells in which the plasma membrane had been depolarized still produced the increase in the cAMP level excluding membrane depolarization as the possible trigger. Using in vivo 31P NMR-spectroscopy we followed phosphate metabolism and the time course of the drop in the intracellular pH after addition of glucose with a time resolution of 15 s. Under aerobic conditions the initial pH and ATP level were high. On addition of glucose, they both showed a rapid, transient drop, which lasted for about 30 s. Under anaerobic conditions, the initial pH and ATP level were low and on addition of glucose they both increased relatively slowly compared to aerobic conditions. Several conditions were found in which the pH drop which occurs under aerobic conditions could be blocked completely without effect on the cAMP signal or without completely preventing it: addition of NH4Cl together with glucose at high extracellular pH and addition of a low concentration of glucose before a high concentration. Also, when glucose was added twice to the same cells no consistent relationship was observed between the pH drop and the cAMP peak. These results appear to exclude transient intracellular acidification as the trigger for the cAMP signal. Hence, we conclude that the effect of glucose cannot be explained on the basis of effects known to be caused by the membrane depolarizing compounds which cause increases in the cAMP level. A new, more specific kind of interaction appears to be involved.     

Endogenous regulation of 2-deoxyglucose uptake in C6 glioma cells correlates with cytoskeleton-mediated changes of surface morphology

The cellular basis of the membrane-limited state of glucose utilization and the mechanism of the endogenous regulation of hexose uptake in dense monolayers of C6 glioma cells were investigated. In an earlier study, it was shown that at high rates of glucose transport and phosphorylation combined with the inhibition of glycolytic adenosine triphosphate (ATP) production by iodoacetate, an endogenous regulatory response occurred that resulted in rapid, periodic variations of the glucose uptake rates (Lange et al., 1982). Similar time-dependent periodic changes of uptake rates also occurred during incubation of C6 glioma cells with 2 mM 2-deoxyglucose (2-DG) without pretreatment of the cells with iodoacetate. These changes were accompanied by variations of the intracellular ATP content, by distinct alterations of the shape and arrangement of microvilli and lamellae (lamellipodia) on the cell surface, and by changes of the cytoskeletal F-actin content. Because the changes of 2-DG uptake rates occurred independent of the intracellular 2-DG concentration, the bulk of this 2-DG pool was assumed to be localized apart from the membranal transport sites. Downregulation of 2-DG uptake appeared to be triggered by a rapid decrease of a small pool of the cellular ATP involved in the phosphorylation of transported hexose. Scanning and transmission electron microscopic observations of cells fixed in different states of the endogenous uptake regulation supported the assumption that the interior of lamellae and microvilli may represent a small entrance compartment for transported hexoses in which occurred the observed close coupling between hexose transport and phosphorylation as well as the rapid variations of ATP content. Hexose uptake is supposed to be regulated by cytoskeleton-mediated changes of volume and diffusional accessibility of this compartment, modulating the degree of its metabolic coupling with the cytoplasmic main compartment.     

The mechanism of intracellular acidification induced by glucose in Saccharomyces cerevisiae

Addition of glucose or fructose to cells of Saccharomyces cerevisiae adapted to grow in the absence of glucose induced an acidification of the intracellular medium. This acidification appeared to be due to the phosphorylation of the sugar since: (i) glucose analogues which are not efficiently phosphorylated did not induce internal acidification; (ii) glucose addition did not cause internal acidification in a mutant deficient in all the three sugar-phosphorylating enzymes; (iii) fructose did not affect the intracellular pH in a double mutant having only glucokinase activity; (iv) glucose was as effective as fructose in inducing the internal pH drop in a mutant deficient in phosphoglucose isomerase activity; and (v) in strains deficient in two of the three sugar-phosphorylating activities, there was a good correlation between the specific glucose- or fructose-phosphorylating activity of cell extracts and the sugar-induced internal acidification. In addition, in whole cells any of the three yeast sugar kinases were capable of mediating the internal acidification described. Glucose-induced internal acidification was observed even when yeast cells were suspended in growth medium and in cells suspended in buffer containing K+, which supports the possible signalling function of the glucose-induced internal acidification. Evaluation of internal pH by following fluorescence changes of fluorescein-loaded cells indicated that the change in intracellular pH occurred immediately after addition of sugar. The apparent Km for glucose in this process was 2 mM. Changes in both the internal and external pH were determined and it was found that the internal acidification induced by glucose was followed by a partial alkalinization coincident with the initiation of H+ efflux. This reversal of acidification could be due to the activity of the H+-ATPase, since it was inhibited by diethylstilboestrol. Coincidence between internal alkalinization and the H+ efflux was also observed after addition of ethanol.     

Role of calcium in serum-stimulation of hexose transport in muscle cells

Serum stimulates glucose uptake into several cells in culture. In intact muscle, an increase in cytosolic free Ca2+ has been proposed to mediate the activation of glucose uptake by hormones and other stimuli [Cell Calcium (1980) 1, 311-325]. We report that hexose (2-deoxy-D-glucose) uptake into L6 muscle cells in culture is enhanced several-fold by fetal calf serum. The increase in uptake is due to stimulation of transmembrane transport, since serum also stimulated uptake of the non-metabolizable hexose 3-O-methyl-D-glucose. The role of Ca2+ in this stimulation was assessed: (i) stimulation of transport by serum was independent of the presence of extracellular Ca2+ during the incubation with serum; (ii) the intracellular levels of free Ca2+, measured by the fluorescence of the novel Ca-indicator quin-2, were identical in serum-stimulated and control cells. It is concluded that hexose transport can increase in muscle cells without concomitant changes in cytoplasmic free Ca2+.     

Glycolysis and glucose uptake in intact outer segments isolated from bovine retinal rods

Glucose transport across the plasma membrane of isolated bovine rod outer segments (ROS) was measured by uptake of 14C-labeled 3-O-methylglucose and 2-deoxyglucose and was inferred from deenergization of ROS with 2-deoxyglucose. Glucose transport was mediated by a facilitated diffusion glucose transporter that equilibrated external and internal free hexose concentrations. Glucose transport in ROS displayed two components as judged from kinetic analysis of hexose equilibration and as judged from inhibition by cytochalasin B and phloretin. Transport under exchange conditions was considerably faster as compared with net hexose uptake, similar to that observed for the erythrocyte glucose transporter. Sensitivity to cytochalasin B and affinity to 3-O-methylglucose were similar to those observed for the hepatocyte glucose transporter. The cytochalasin-insensitive component appears unique to ROS and did not reflect leakage transport as judged from a comparison with L-glucose uptake. Glucose transport feeds glycolysis localized to ROS. We suggest that a major role for glycolysis in ROS is phosphorylation of GDP to GTP via pyruvate kinase and PEP, while phosphorylation of ADP to ATP can use the creatine kinase/phosphocreatine pathway as well.     

Activation of glucose transport during simulated ischemia in H9c2 cardiac myoblasts is mediated by protein kinase C isoforms

Glucose transport into cells may be regulated by a variety of conditions, including ischemia. We investigated whether some enzymes frequently involved in the metabolic adaptation to ischemia are also required for glucose transport activation. Ischemia was simulated by incubating during 3 h H9c2 cardiomyoblasts in a serum- and glucose-free medium in hypoxia. Under these conditions 2-deoxy-d-[2,6-³H]-glucose uptake was increased (57% above control levels, p < 0.0001) consistently with GLUT1 and GLUT4 translocation to sarcolemma. Tyrosine kinases inhibition via tyrphostin had no effect on glucose transport up-regulation induced by simulated ischemia. On the other hand, chelerythrine, a broad range inhibitor of protein kinase C isoforms, and rottlerin, an inhibitor of protein kinase C delta, completely prevented the stimulation of the transport rate. A lower activation of hexose uptake (19%, p < 0.001) followed also treatment with Gö6976, an inhibitor of conventional protein kinases C. Finally, PD98059-mediated inhibition of the phosphorylation of ERK 1/2, a downstream mitogen-activated protein kinase (MAPK), only partially reduced the activation of glucose transport induced by simulated ischemia (31%, p < 0.01), while SB203580, an inhibitor of p38 MAPK, did not exert any effect. These results indicate that stimulation of protein kinase C delta is strongly related to the up-regulation of glucose transport induced by simulated ischemia in cultured cardiomyoblasts and that conventional protein kinases C and ERK 1/2 are partially involved in the signalling pathways mediating this process.     

A mutation in Saccharomyces cerevisiae adenylate cyclase, Cyr1K1876M, specifically affects glucose- and acidification-induced cAMP signalling and not the basal cAMP level

In the yeast Saccharomyces cerevisiae, the addition of glucose to derepressed cells and intracellular acidification trigger a rapid increase in the cAMP level within 1 min. We have identified a mutation in the genetic background of several related 'wild-type' laboratory yeast strains (e.g. ENY.cat80-7A, CEN.PK2-1C) that largely prevents both cAMP responses, and we have called it lcr1 (for lack of cAMP responses). Subsequent analysis showed that lcr1 was allelic to CYR1/CDC35, encoding adenylate cyclase, and that it contained an A to T substitution at position 5627. This corresponds to a K1876M substitution near the end of the catalytic domain in adenylate cyclase. Introduction of the A5627T mutation into the CYR1 gene of a W303-1A wild-type strain largely eliminated glucose- and acidification-induced cAMP signalling and also the transient cAMP increase that occurs in the lag phase of growth. Hence, lysine1876 of adenylate cyclase is essential for cAMP responses in vivo. Lysine1876 is conserved in Schizosaccharomyces pombe adenylate cyclase. Mn2+-dependent adenylate cyclase activity in isolated plasma membranes of the cyr1met1876 (lcr1) strain was similar to that in the isogenic wild-type strain, but GTP/Mg2+-dependent activity was strongly reduced, consistent with the absence of signalling through adenylate cyclase in vivo. Glucose-induced activation of trehalase was reduced and mobilization of trehalose and glycogen and loss of stress resistance were delayed in the cyr1met1876 (lcr1) mutant. During exponential growth on glucose, there was little effect on these protein kinase A (PKA) targets, indicating that the importance of glucose-induced cAMP signalling is restricted to the transition from gluconeogenic/respiratory to fermentative growth. Inhibition of growth by weak acids was reduced, consistent with prevention of the intracellular acidification effect on cAMP by the cyr1met1876 (lcr1) mutation. The mutation partially suppressed the effect of RAS2val19 and GPA2val132 on several PKA targets. These results demonstrate the usefulness of the cyr1met1876 (lcr1) mutation for epistasis studies on the signalling function of the cAMP pathway.     

Hexose phosphorylation and the putative calcium channel component Mid1p are required for the hexose-induced transient elevation of cytosolic calcium response in Saccharomyces cerevisiae

Saccharomyces cerevisiae responds to environ-mental stimuli such as an exposure to pheromone or to hexoses after carbon source limitation with a transient elevation of cytosolic calcium (TECC) response. In this study, we examined whether hexose transport and phosphorylation are necessary for the TECC response. We found that a mutant strain lacking most of the known hexose transporters was unable to carry out the TECC response when exposed to glucose. A mutant strain that lacked the ability to phosphorylate glucose was unable to respond to glucose addition, but displayed a normal TECC response after the addition of galactose. These results indicate that hexose uptake and phosphorylation are required to trigger the hexose-induced TECC response. We also found that the TECC response was significantly smaller than normal when the level of environmental calcium was reduced, and was abolished in a mid1 mutant that lacked a subunit of the high-affinity calcium channel of the yeast plasma membrane. These results indicate that most or all of the TECC response is mediated by an influx of calcium from the extracellular space. Our results indicate that this transient increase in plasma membrane calcium permeability may be linked to the accumulation of Glc-1-P (or a related glucose metabolite) in yeast.     

Absence of glucose-induced cAMP signaling in the Saccharomyces cerevisiae mutants cat1 and cat3 which are deficient in derepression of glucose-repressible proteins

Addition of glucose to derepressed cells of the yeast Saccharomyces cerevisiae induces a transient, specific cAMP signal. Intracellular acidification in these cells, as caused by addition of protonophores like 2,4-dinitrophenol (DNP) causes a large, lasting increase in the cAMP level. The effect of glucose and DNP was investigated in glucose-repressed wild type cells and in cells of two mutants which are deficient in derepression of glucose-repressible proteins, cat1 and cat3. Addition of glucose to cells of the cat3 mutant caused a transient increase in the cAMP level whereas cells of the cat1 mutant and in most cases also repressed wild type cells did not respond to glucose addition with a cAMP increase. The glucose-induced cAMP increase in cat3 cells and the cAMP increase occasionally present in repressed wild type cells however could be prevented completely by addition of a very low level of glucose in advance. In derepressed wild type cells this does not prevent the specific glucose-induced cAMP signal at all. These results indicate that repressed cells do not show a true glucose-induced cAMP signal. When DNP was added to glucose-repressed wild type cells or to cells of the cat1 and cat3 mutants no cAMP increase was observed. Addition of a very low level of glucose before the DNP restored the cAMP increase which points to lack of ATP as the cause for the absence of the DNP effect. These data show that intracellular acidification is able to enhance the cAMP level in repressed cells. The glucose-induced artefactual increase occasionally observed in repressed cells is probably caused by the fact that their low intracellular pH is only restored after the ATP level has increased to such an extent that it is no longer limiting for cAMP synthesis. It is unclear why the artefactual increases are not always observed. Measurement of glucose- and DNP-induced activation of trehalase confirmed the physiological validity of the changes observed in the cAMP level. Our results are consistent with the idea that the glucose-induced signaling pathway contains a glucose-repressible protein and that the protein is located before the point where intracellular acidification triggers activation of the pathway.Abbreviations CCCP carbonyl cyanide m-chlorophenylhydrazone - DNP 2,4-dinitrophenol - Mes 4-morpholineethanesulfonic acid