PKC regulates turnover rate of rabbit intestinal Na+-glucose transporter expressed in COS-7 cells

1999 ◽  
Vol 276 (5) ◽  
pp. C1053-C1060 ◽  
Author(s):  
Steven Vayro ◽  
Mel Silverman
Keyword(s):  
Cell Surface ◽  
Binding Affinity ◽  
Turnover Rate ◽  
Glucose Transporter ◽  
Sugar Uptake ◽  
Transport Activity ◽  
Pkc Inhibitor ◽  
High Affinity ◽  
Kinase C ◽  
Menten Constant

We have used the recombinant NH2-terminal myc-tagged rabbit Na+-glucose transporter (SGLT1) to study the regulation of this carrier expressed in COS-7 cells. Treatment of cells with a protein kinase C (PKC) agonist, phorbol 12-myristate 13-acetate (PMA), caused a significant decrease (38.03 ± 0.05%) in methyl α-d-glucopyranoside transport activity that could not be emulated by 4α-phorbol 12,13-didecanoate. The decrease in sugar uptake stimulated by PMA was reversed by the PKC inhibitor bisindolylmaleimide I. The maximal rate of Na+-glucose cotransport activity ( V max) was decreased from 1.29 ± 0.09 to 0.85 ± 0.04 nmol ⋅ min−1 ⋅ mg protein−1 after PMA exposure. However, measurement of high-affinity Na+-dependent phloridzin binding revealed that there was no difference in the number of cell surface transporters after PMA treatment; maximal binding capacities were 1.54 ± 0.34 and 1.64 ± 0.21 pmol/mg protein for untreated and treated cells, respectively. The apparent sugar binding affinity (Michaelis-Menten constant) and phloridzin binding affinity (dissociation constant) were not affected by PMA. Because PKC reduced V max without affecting the number of cell surface SGLT1 transporters, we conclude that PKC has a direct effect on the carrier, resulting in a lowering of the transporter turnover rate by a factor of two.

scholarly journals Nedd4-2 but not Nedd4-1 is critical for protein kinase C-regulated ubiquitination, expression, and transport activity of human organic anion transporter 1

2016 ◽  
Vol 310 (9) ◽  
pp. F821-F831 ◽  
Author(s):  
Da Xu ◽  
Haoxun Wang ◽  
Qiang Zhang ◽  
Guofeng You
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Cell Surface ◽  
Organic Anion ◽  
The Body ◽  
Organic Anion Transporter ◽  
Transport Activity ◽  
Kinase C ◽  
Anion Transporter ◽  
In Cells

Human organic anion transporter 1 (hOAT1) expressed at the membrane of the kidney proximal tubule cells mediates the body disposition of a diverse array of clinically important drugs, including anti-HIV therapeutics, antitumor drugs, antibiotics, antihypertensives, and antiinflammatories. Therefore, understanding the regulation of hOAT1 will provide significant insights into kidney function and dysfunction. We previously established that hOAT1 transport activity is inhibited by activation of protein kinase C (PKC) through accelerating hOAT1 internalization from cell surface into intracellular endosomes and subsequent degradation. We further established that PKC-induced hOAT1 ubiquitination is an important step preceding hOAT1 internalization. In the current study, we identified two closely related E3 ubiquitin ligases, neural precursor cell expressed, developmentally downregulated 4-1 and 4-2 (Nedd4-1 and Nedd4-2), as important regulators for hOAT1: overexpression of Nedd4-1 or Nedd4-2 enhanced hOAT1 ubiquitination, reduced the hOAT1 amount at the cell surface, and suppressed hOAT1 transport activity. In further exploring the relationship among PKC, Nedd4-1, and Nedd4-2, we discovered that PKC-dependent changes in hOAT1 ubiquitination, expression, and transport activity were significantly blocked in cells transfected with the ligase-dead mutant of Nedd4-2 (Nedd4-2/C821A) or with Nedd4-2-specific siRNA to knockdown endogenous Nedd4-2 but not in cells transfected with the ligase-dead mutant of Nedd4-1 (Nedd4-1/C867S) or with Nedd4-1-specific siRNA to knockdown endogenous Nedd4-1. In conclusion, this is the first demonstration that both Nedd4-1 and Nedd4-2 are important regulators for hOAT1 ubiquitination, expression, and function. Yet they play distinct roles, as Nedd4-2 but not Nedd4-1 is a critical mediator for PKC-regulated hOAT1 ubiquitination, expression, and transport activity.

scholarly journals Trafficking of glucose transporters in 3T3-L1 cells. Inhibition of trafficking by phenylarsine oxide implicates a slow dissociation of transporters from trafficking proteins

1992 ◽  
Vol 281 (3) ◽  
pp. 809-817 ◽  
Author(s):  
J Yang ◽  
A E Clark ◽  
R Harrison ◽  
I J Kozka ◽  
G D Holman
Keyword(s):  
Cell Surface ◽  
Glucose Transport ◽  
Glucose Transporter ◽  
Fluid Phase ◽  
Transport Activity ◽  
Phenylarsine Oxide ◽  
Fluid Phase Endocytosis ◽  
Surface Labelling ◽  
Slow Dissociation ◽  
Stimulation Of

We have compared the rates of insulin stimulation of cell-surface availability of glucose-transporter isoforms (GLUT1 and GLUT4) and the stimulation of 2-deoxy-D-glucose transport in 3T3-L1 cells. The levels of cell-surface transporters have been assessed by using the bismannose compound 2-N-[4-(1-azi-2,2,2-trifluoroethyl)benzoyl]-1,3-bis(D-mannos -4-yloxy) propyl-2-amine (ATB-BMPA). At 27 degrees C the half-times for the appearance of GLUT1 and GLUT4 at the cell surface were 5.7 and 5.4 min respectively and were slightly shorter than that for the observed stimulation of transport activity (t 1/2 8.6 min). This lag may be due to a slow dissociation of surface transporters from trafficking proteins responsible for translocation. When fully-insulin-stimulated cells were subjected to a low-pH washing procedure to remove insulin at 37 degrees C, the cell-surface levels of GLUT1 and GLUT4 decreased, with half-times of 9.2 and 6.8 min respectively. These times correlated well with decrease in 2-deoxy-D-glucose transport activity that occurred during this washing procedure (t1/2 6.5 min). When fully-insulin-stimulated cells were treated with phenylarsine oxide (PAO), a similar decrease in transport activity occurred (t1/2 9.8 min). However, surface labelling showed that this corresponded with a decrease in GLUT4 only (t1/2 7.8 min). The cell-surface level of GLUT1 remained high throughout the PAO treatment. Light-microsome membranes were isolated from cells which had been cell-surface-labelled with ATB-BMPA. Internalization of both transporter isoforms to this pool occurred when cells were maintained in the presence of insulin for 60 min. In contrast with the surface-labelling results, we have shown that the transfer to the light-microsome pool of both transporters occurred in cells treated with insulin and PAO. These results suggest that both transporters are recycled by fluid-phase endocytosis and exocytosis. PAO may inhibit this recycling at a stage which involves the re-emergence of internalized transporters at the plasma membrane. The GLUT1 transporters that are recycled to the surface in insulin- and PAO-treated cells appear to have low transport activity. This may be because of a failure to dissociate fully from trafficking proteins at the cell surface. GLUT4 transporters appear to have a greater tendency to remain internalized if the normal mechanisms that commit transporters to the cell surface, such as dissociation from trafficking proteins, are uncoupled.

scholarly journals Insulin-sensitive regulation of glucose transport and GLUT4 translocation in skeletal muscle of GLUT1 transgenic mice

1998 ◽  
Vol 337 (1) ◽  
pp. 51-57 ◽  
Author(s):  
Garret J. ETGEN ◽  
William J. ZAVADOSKI ◽  
Geoffrey D. HOLMAN ◽  
E. Michael GIBBS
Keyword(s):  
Skeletal Muscle ◽  
Cell Surface ◽  
Transgenic Mice ◽  
Glucose Transport ◽  
Hind Limb ◽  
Glucose Transporter ◽  
Glut4 Translocation ◽  
Transport Activity ◽  
Fast Twitch Muscle ◽  
Fast Twitch

Skeletal muscle glucose transport was examined in transgenic mice overexpressing the glucose transporter GLUT1 using both the isolated incubated-muscle preparation and the hind-limb perfusion technique. In the absence of insulin, 2-deoxy-d-glucose uptake was increased ∼ 3–8-fold in isolated fast-twitch muscles of GLUT1 transgenic mice compared with non-transgenic siblings. Similarly, basal glucose transport activity was increased ∼ 4–14-fold in perfused fast-twitch muscles of transgenic mice. In non-transgenic mice insulin accelerated glucose transport activity ∼ 2–3-fold in isolated muscles and to a much greater extent (∼ 7–20-fold) in perfused hind-limb preparations. The observed effect of insulin on glucose transport in transgenic muscle was similarly dependent upon the technique used for measurement, as insulin had no effect on isolated fast-twitch muscle from transgenic mice, but significantly enhanced glucose transport in perfused fast-twitch muscle from transgenic mice to ∼ 50–75% of the magnitude of the increase observed in non-transgenic mice. Cell-surface glucose transporter content was assessed via 2-N-4-(l-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis-(d -mannos-4-yloxy)-2-propylamine photolabelling methodology in both isolated and perfused extensor digitorum longus (EDL). Cell-surface GLUT1 was enhanced by as much as 70-fold in both isolated and perfused EDL of transgenic mice. Insulin did not alter cell-surface GLUT1 in either transgenic or non-transgenic mice. Basal levels of cell-surface GLUT4, measured in either isolated or perfused EDL, were similar in transgenic and non-transgenic mice. Interestingly, insulin enhanced cell-surface GLUT4 ∼ 2-fold in isolated EDL and ∼ 6-fold in perfused EDL of both transgenic and non-transgenic mice. In summary, these results reveal differences between isolated muscle and perfused hind-limb techniques, with the latter method showing a more robust responsiveness to insulin. Furthermore, the results demonstrate that muscle overexpressing GLUT1 has normal insulin-induced GLUT4 translocation and the ability to augment glucose-transport activity above the elevated basal rates.

scholarly journals Substrate specificity and kinetic parameters of GLUT3 in rat cerebellar granule neurons

1996 ◽  
Vol 315 (3) ◽  
pp. 827-831 ◽  
Author(s):  
Fran MAHER ◽  
Theresa M. DAVIES-HILL ◽  
Ian A. SIMPSON
Keyword(s):  
Glucose Transporter ◽  
Cerebellar Granule Neurons ◽  
Granule Neurons ◽  
Transport Activity ◽  
Turnover Number ◽  
Cerebellar Granule ◽  
Apparent Affinity ◽  
High Affinity ◽  
Transport Assay ◽  
Transport Method

This study examines the apparent affinity, catalytic-centre activity (‘turnover number’) and stereospecificity of the neuronal glucose transporter GLUT3 in primary cultured cerebellar granule neurons. Using a novel variation of the 3-O-[14C]methylglucose transport assay, by measuring zero-trans kinetics at 25 °C, GLUT3 was determined to be a high-apparent-affinity, high-activity, glucose transporter with a Km of 2.87±0.23 mM (mean±S.E.M.) for 3-O-methylglucose, a Vmax of 18.7± 0.48 nmol/min per 106 cells, and a corresponding catalytic-centre activity of 853 s-1. Transport of 3-O-methylglucose was competed by glucose, mannose, 2-deoxyglucose and galactose, but not by fructose. This methodology is compared with the more common 2-[3H]deoxyglucose methodology and the [U-14C]glucose transport method. The high affinity and transport activity of the neuronal glucose transporter GLUT3 appears to be an appropriate adaptation to meet the demands of neuronal metabolism at prevailing interstitial brain glucose concentrations (1–2 mM).

scholarly journals Phosphorylation of the adipose/muscle-type glucose transporter (GLUT4) and its relationship to glucose transport activity

1992 ◽  
Vol 285 (1) ◽  
pp. 223-228 ◽  
Author(s):  
A Schürmann ◽  
G Mieskes ◽  
H G Joost
Keyword(s):  
Protein Kinase ◽  
Glucose Transport ◽  
Plasma Membranes ◽  
Glucose Transporter ◽  
Low Density ◽  
Transport Activity ◽  
Muscle Type ◽  
Kinase C ◽  
Glucose Transporter Glut4

The effects of protein phosphorylation and dephosphorylation on glucose transport activity reconstituted from adipocyte membrane fractions and its relationship to the phosphorylation state of the adipose/muscle-type glucose transporter (GLUT4) were studied. In vitro phosphorylation of membranes in the presence of ATP and protein kinase A produced a stimulation of the reconstituted glucose transport activity in plasma membranes and low-density microsomes (51% and 65% stimulation respectively), provided that the cells had been treated with insulin prior to isolation of the membranes. Conversely, treatment of membrane fractions with alkaline phosphatase produced an inhibition of reconstituted transport activity. However, in vitro phosphorylation catalysed by protein kinase C failed to alter reconstituted glucose transport activity in membrane fractions from both basal and insulin-treated cells. In experiments run under identical conditions, the phosphorylation state of GLUT4 was investigated by immunoprecipitation of glucose transporters from membrane fractions incubated with [32P]ATP and protein kinases A and C. Protein kinase C stimulated a marked phosphate incorporation into GLUT4 in both plasma membranes and low-density microsomes. Protein kinase A, in contrast to its effect on reconstituted glucose transport activity, produced a much smaller phosphorylation of the GLUT4 in plasma membranes than in low-density microsomes. The present data suggest that glucose transport activity can be modified by protein phosphorylation via an insulin-dependent mechanism. However, the phosphorylation of the GLUT4 itself was not correlated with changes in its reconstituted transport activity.

scholarly journals Thrombin-induced translocation of GLUT3 glucose transporters in human platelets

1997 ◽  
Vol 328 (2) ◽  
pp. 511-516 ◽  
Author(s):  
R. Lynn SORBARA ◽  
Theresa M. DAVIES-HILL ◽  
Ellen M. KOEHLER-STEC ◽  
J. Susan VANNUCCI ◽  
K. McDonald HORNE ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Glucose Transport ◽  
Glucose Transporter ◽  
Glucose Transporters ◽  
Human Platelets ◽  
Half Time ◽  
Transport Activity ◽  
Kinase C ◽  
Adipose Cells

Platelets derive most of their energy from anaerobic glycolysis; during activation this requirement rises approx. 3-fold. To accommodate the high glucose flux, platelets express extremely high concentrations (155±18 pmol/mg of membrane protein) of the most active glucose transporter isoform, GLUT3. Thrombin, a potent platelet activator, was found to stimulate 2-deoxyglucose transport activity 3-5-fold within 10 min at 25 °C, with a half-time of 1-2 min. To determine the mechanism underlying the increase in glucose transport activity, an impermeant photolabel, [2-3H]2N-4-(1-azi-2,2,2-trifluoethyl)benzoyl-1,3,-bis-(d-mannose-4-ylozy)-2-propylamine, was used to covalently bind glucose transporters accessible to the extracellular milieu. In response to thrombin, the level of transporter labelling increased 2.7-fold with a half-time of 1-2 min. This suggests a translocation of GLUT3 transporters from an intracellular site to the plasma membrane in a manner analogous to that seen for the translocation of GLUT4 in insulin-stimulated rat adipose cells. To investigate whether a similar signalling pathway was involved in both systems, platelets and adipose cells were exposed to staurosporin and wortmannin, two inhibitors of GLUT4 translocation in adipose cells. Thrombin stimulation of glucose transport activity in platelets was more sensitive to staurosporin inhibition than was insulin-stimulated transport activity in adipose cells, but it was totally insensitive to wortmannin. This indicates that the GLUT3 translocation in platelets is mediated by a protein kinase C not by a phosphatidylinositol 3-kinase mechanism. In support of this contention, the phorbol ester PMA, which specifically activates protein kinase C, fully stimulated glucose transport activity in platelets and was equally sensitive to inhibition by staurosporin. This study provides a cellular mechanism by which platelets enhance their capacity to import glucose to fulfil the increased energy demands associated with activation.

scholarly journals Protein kinase C regulates organic anion transporter 1 through phosphorylating ubiquitin ligase Nedd4–2

2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Zhou Yu ◽  
Chenchang Liu ◽  
Jinghui Zhang ◽  
Zhengxuan Liang ◽  
Guofeng You
Keyword(s):  
Protein Kinase C ◽  
Cell Surface ◽  
Ubiquitin Ligase ◽  
Organic Anion ◽  
Surface Expression ◽  
Organic Anion Transporter ◽  
Cell Surface Expression ◽  
Transport Activity ◽  
Kinase C ◽  
Anion Transporter

Abstract Background Organic anion transporter 1 (OAT1) is a drug transporter expressed on the basolateral membrane of the proximal tubule cells in kidneys. It plays an essential role in the disposition of numerous clinical therapeutics, impacting their pharmacological and toxicological properties. The activation of protein kinase C (PKC) is shown to facilitate OAT1 internalization from cell surface to intracellular compartments and thereby reducing cell surface expression and transport activity of the transporter. The PKC-regulated OAT1 internalization occurs through ubiquitination, a process catalyzed by a E3 ubiquitin ligase, neural precursor cell expressed developmentally down-regulated 4–2 (Nedd4–2). Nedd4–2 directly interacts with OAT1 and affects ubiquitination, expression and stability of the transporter. However, whether Nedd4–2 is a direct substrate for PKC-induced phosphorylation is unknown. Results In this study, we investigated the role of Nedd4–2 phosphorylation in the PKC regulation of OAT1. The results showed that PKC activation enhanced the phosphorylation of Nedd4–2 and increased the OAT1 ubiquitination, which was accompanied by a decreased OAT1 cell surface expression and transport function. And the effects of PKC could be reversed by PKC-specific inhibitor staurosporine. We further discovered that the quadruple mutant (T197A/S221A/S354A/S420A) of Nedd4–2 partially blocked the effects of PKC on Nedd4–2 phosphorylation and on OAT1 transport activity. Conclusions Our investigation demonstrates that PKC regulates OAT1 likely through direct phosphorylation of Nedd4–2. And four phosphorylation sites (T197, S221, S354, and S420) of Nedd4–2 in combination play an important role in this regulatory process.

scholarly journals Transmembrane segments 1, 5, 7 and 8 are required for high-affinity glucose transport by Saccharomyces cerevisiae Hxt2 transporter

2003 ◽  
Vol 372 (1) ◽  
pp. 247-252 ◽  
Author(s):  
Toshiko KASAHARA ◽  
Michihiro KASAHARA
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Glucose Transport ◽  
Glucose Transporter ◽  
Amino Acid Identity ◽  
Major Facilitator Superfamily ◽  
Transport Activity ◽  
Transmembrane Segments ◽  
High Affinity ◽  
Yeast Transformants ◽  
Major Facilitator

Hxt2 is a high-affinity facilitative glucose transporter of Saccharomyces cerevisiae and belongs to the major facilitator superfamily. Hxt1 shares ≈ 70% amino acid identity with Hxt2 in its transmembrane segments (TMs) and inter-TM loops, but transports d-glucose with an affinity about one-tenth of that of Hxt2. To determine which TMs of Hxt2 are important for high-affinity glucose transport, we constructed chimaeras of Hxt2 and Hxt1 by randomly replacing each of the 12 TMs of Hxt2 with the corresponding segment of Hxt1, for a total of 4096 different transporters. Among > 20000 yeast transformants screened, 39 different clones were selected by plate assays of high-affinity glucose-transport activity and sequenced. With only two exceptions, the selected chimaeras contained Hxt2 TMs 1, 5, 7 and 8. We then constructed chimaeras corresponding to all 16 possible combinations of Hxt2 TMs 1, 5, 7 and 8. Only one chimaera, namely that containing all four Hxt2 TMs, exhibited transport activity comparable with that of Hxt2. The Km and Vmax values for d-glucose transport, and the substrate specificity of this chimaera were almost identical with those of Hxt2. These results indicate that TMs 1, 5, 7 and 8 are necessary for exhibiting high-affinity glucose-transport activity of Hxt2.

scholarly journals Identification and characterization of a soluble c-kit receptor produced by human hematopoietic cell lines

Blood ◽  
1995 ◽  
Vol 85 (8) ◽  
pp. 2052-2058 ◽  
Author(s):  
AM Turner ◽  
LG Bennett ◽  
NL Lin ◽  
J Wypych ◽  
TD Bartley ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Cell Surface ◽  
Human Serum ◽  
Binding Affinity ◽  
Cell Lines ◽  
Hematopoietic Cells ◽  
High Affinity ◽  
Kit Receptor ◽  
Soluble C

Stem cell factor (SCF) triggers cell growth by binding to cell surface c-kit receptors. Soluble forms of several cytokine receptors have been described and may play a role in the modulation of cytokine activity in vivo. For these reasons, we investigated whether human hematopoietic cells produce soluble c-kit receptors. The human leukemia cell lines OCIM1 and MO7e display approximately 80,000 and approximately 35,000 high-affinity cell surface c-kit receptors, respectively. Soluble c-kit receptors were detected by enzyme immunoassay in OCIM1 and MO7e culture supernatants. We determined the molecular weight and binding affinity of soluble c-kit receptor produced by OCIM1 cells, soluble c-kit receptor purified from human serum, and recombinant soluble c-kit receptor expressed in CHO cells. The three soluble c-kit receptors each have a molecular weight of 98 kD. Quantitative binding experiments with 125I-SCF indicate that the soluble c-kit receptors obtained from human serum or OCIM1 cells have binding affinities for SCF of approximately 200 to 300 pmol/L, in contrast to the recombinant form, which has a binding affinity of approximately 1.5 nmol/L. All three forms of the soluble c-kit receptor were able to compete with c-kit receptors on OCIM1 cells for 125I-SCF binding. Thus human hematopoietic cells can produce a soluble form of the c-kit receptor that retains high-affinity SCF binding activity. We speculate that the soluble c-kit receptor may bind SCF and function as a receptor antagonist in vivo.

scholarly journals Regulation of cell surface GLUT4 in skeletal muscle of transgenic mice

1997 ◽  
Vol 321 (1) ◽  
pp. 75-81 ◽  
Author(s):  
Joseph T. BROZINICK ◽  
Scott C. McCOID ◽  
Thomas H. REYNOLDS ◽  
Cindy M. WILSON ◽  
Ralph W. STEVENSON ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Cell Surface ◽  
Glucose Transport ◽  
Glucose Transporter ◽  
Subcellular Fractionation ◽  
Muscle Membrane ◽  
Transport Activity ◽  
Hindlimb Muscles ◽  
Subcellular Membrane ◽  
Glucose Transporter Glut4

Marked overexpression of the glucose transporter GLUT4 in skeletal muscle membrane fractions of GLUT4 transgenic (TG) mice is accompanied by disproportionately small increases in basal and insulin-stimulated glucose transport activity. Thus we have assessed cell surface GLUT4 by photolabelling with the membrane-impermeant reagent 2-N-[4-(1-azi-2,2,2-trifluoroethyl)benzoyl]-1,3-bis(d-mannos-4-yloxy)-2-propylamine (ATB-BMPA) and measured the corresponding glucose transport activity using 2-deoxyglucose in isolated extensor digitorum longus (EDL) muscles from non-transgenic (NTG) and GLUT4 TG mice in the absence and presence of 13.3 nM (2000 µ-units/ml) insulin, without or with hypoxia as a model of muscle contraction. TG mice displayed elevated rates of glucose transport activity under basal and insulin-stimulated conditions, and in the presence of insulin plus hypoxia, compared with NTG mice. Photoaffinity labelling of cell surface GLUT4 indicated corresponding elevations in plasma membrane GLUT4 in the basal and insulin-stimulated states, and with insulin plus hypoxia, but no difference in cell surface GLUT4 during hypoxia stimulation. Subcellular fractionation of hindlimb muscles confirmed the previously observed 3-fold overexpression of GLUT4 in the TG compared with the NTG mice. These results suggest that: (1) alterations in glucose transport activity which occur with GLUT4 overexpression in EDL muscles are directly related to cell surface GLUT4 content, regardless of the levels observed in the corresponding subcellular membrane fractions, (2) while overexpression of GLUT4 influences both basal and insulin-stimulated glucose transport activity, the response to hypoxia/contraction-stimulated glucose transport is unchanged, and (3) subcellular fractionation provides little insight into the subcellular trafficking of GLUT4, and whatever relationship is demonstrated in EDL muscles from NTG mice is disrupted on GLUT4 overexpression.

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