Inhibition of insulin receptor phosphorylation by PC-1 is not mediated by the hydrolysis of adenosine triphosphate or the generation of adenosine.

Individuals with insulin resistance show increased levels of PC-1 expression in skeletal muscle and fibroblasts, and in transfected cell lines that overexpress PC-1 there is a reduction in the insulin-stimulated insulin receptor tyrosine phosphorylation. As PC-1 is a type II transmembrane protein with extracellular phosphodiesterase and pyrophosphatase activity, increased expression of PC-1 at the cell surface will decrease extracellular adenosine triphosphate levels and increase extracellular adenosine levels. Consequently it is possible that PC-1-mediated insulin resistance could be caused either by a decrease in adenosine triphosphate or an indirect increase in adenosine levels. We have tested this hypothesis and find that the PC-1-mediated inhibition of insulin-stimulated insulin receptor autophosphorylation is not altered by agents that alter the level or action of adenosine. Further, a mutated PC-1 with a single amino acid change that abolishes the phosphodiesterase and pyrophosphatase activities is still able to inhibit insulin-stimulated insulin receptor phosphorylation. The results of these experiments indicate that the phosphodiesterase activity of PC-1 is not involved in the inhibition of insulin receptor autophosphorylation.


Individuals with insulin resistance show increased levels of PC-1 expression in skeletal muscle and fibroblasts, and in transfected cell lines that overexpress PC-1 there is a reduction in the insulin-stimulated insulin receptor tyrosine phosphorylation. As PC-1 is a type II transmembrane protein with extracellular phosphodiesterase and pyrophosphatase activity, increased expression of PC-1 at the cell surface will decrease extracellular adenosine triphosphate levels and increase extracellular adenosine levels. Consequently it is possible that PC-1-mediated insulin resistance could be caused either by a decrease in adenosine triphosphate
or an indirect increase in adenosine levels. We have tested this hypothesis and find that the PC-1-mediated inhibition of insulin-stimulated insulin receptor autophosphorylation is not altered by agents that alter the level or action of adenosine. Further, a mutated PC-1 with a single amino acid change that abolishes the phosphodiesterase and pyrophosphatase activities is still able to inhibit insulin-stimulated insulin receptor phosphorylation. The results of these experiments indicate that the phosphodiesterase activity of PC-1 is not involved in the inhibition of insulin receptor autophosphorylation.
Insulin controls glucose homeostasis by regulating the production of glucose by the liver and the uptake of glucose into muscle and fat. If these actions of insulin are inadequate, glucose levels rise and diabetes mellitus develops. The most common form of diabetes mellitus is non-insulin-dependent diabetes mellitus (type II diabetes). While the cause(s) of noninsulin-dependent diabetes mellitus is unknown, this form of diabetes is accompanied by insulin resistance, an inability of insulin to appropriately trigger cellular responses. Except in rare individuals, this insulin resistance is not the result of alterations of the number or structure of the insulin receptors; rather the defect lies distal to the insulin receptor in the insulin signaling pathway (for reviews see Refs. [1][2][3][4]. We have demonstrated that in muscle and fibroblasts of many patients with insulin resistance there is increased expression of PC-1 (5). When the mammary epithelial cell line, MCF-7, is transfected with a PC-1 cDNA expression vector, expression of PC-1 is increased and the cells become insulinresistant (5). PC-1 is a type II (extracellular C terminus) transmembrane glycoprotein (6). Although initially identified in plasma cells, it is now known to be expressed in a wide variety of cell types (7). PC-1 is also known as nucleotide pyrophosphatase/alkaline phosphodiesterase I, which has pyrophosphatase as well as phosphodiesterase activity (8). These activities of PC-1 suggested two hypotheses as to how PC-1 could decrease insulin receptor phosphorylation. One possibility is that the pyrophosphatase activity leads to a loss of ATP either at the cell membrane or in an intracellular trafficking pool, and this reduction in ATP could impact negatively on insulin signaling. Another possibility is that extracellular ATP hydrolysis could lead indirectly to an increase in extracellular adenosine, which could act through one of the adenosine receptors to reduce the response of PC-1 expressing cells to insulin. Both of these hypotheses rely on the pyrophosphatase activity of PC-1.
We have tested these hypotheses in two ways. First we have used pharmacological agents to either alter the level of extracellular adenosine or the signaling through the adenosine receptor. None of these manipulations altered the insulin resistance in either control MCF7 cells or in MCF7 cell lines that express PC-1. In addition we have introduced an amino acid change into PC-1 that abolishes the pyrophosphatase and phosphodiesterase activity. When an expression plasmid encoding this variant is introduced into MCF7 cells, the protein is expressed without these two enzymatic activities. This variant is, however, as active as the wild type PC-1 protein in its ability to reduce the insulin-stimulated autophosphorylation of the insulin receptor kinase. Taken together these data suggest that the phosphodiesterase activity of PC-1 is not involved in the inhibition of insulin receptor phosphorylation.

MATERIALS AND METHODS
Cell Lines-The human mammary epithelial cell line MCF7 was cultured in Dulbecco's modified Eagle's medium/F-12 medium supplemented with 10% fetal calf serum, nonessential amino acids, and antibiotics. To increase the level of PC-1, cultures of MCF7 cells were transfected by electroporation using plasmids with either the antibiotic resistance gene neo under the control of the cytomegalovirus promoter or this plasmid and one that contained the human PC-1 cDNA under the control of the cytomegalovirus promoter. The PC-1 cDNA used was one in which the 5Ј-most ATG was included (9). A plasmid that contained the more 3Ј-ATG was provided by Dr. Jim Goding (Monash University, Melbourne). The missing sequence for the 5Ј-ATG was added using a synthetic oligonucleotide. This part of the plasmid was sequenced. The numbering used this N-terminal-most methionine as 1 and indicates a protein of 925 amino acids in length. Clones of cells resistant to the antibiotic G418 were identified and grown under selective conditions and assayed for the expression of the PC-1 protein by Western analysis and by enzymatic assay.
ATP Pyrophosphatase Assay-The enzymatic assay for the PC-1 protein used a modification of the phosphoadenosine phosphosulfate hydrolysis assay (5). In this assay the radioactive substrate phosphoadenosine phosphosulfate was replaced by [␥-33 P]ATP. The final concentration of ATP in the assay was 0.25 mM. The unlabeled ATP was supplemented with 0.01 mCi of [ 33 P]ATP that had a specific activity of 2000 Ci/mmol. The reaction and determination of activity were carried out as described (5).
Insulin-stimulated Insulin Receptor Phosphorylation-MCF7 cells were grown to approximately 80% confluence in six-well plates. Eight-* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. een hours prior to the assay the cells were washed with PBS 1 and cultured overnight in 2.0 ml of Dulbecco's modified Eagle's medium/ F-12 medium (low glucose) that was supplemented with antibiotics and 0.1% bovine serum albumin. Insulin was added to these cultures (final concentrations from 0 to 100 nM insulin). Fifteen minutes after the addition of the insulin, the medium was rapidly removed and the cells washed with PBS at 4°C. The cells were lysed in 300 l of PBS with 1% Triton X-100 supplemented with 1 mM sodium vanadate and the protease inhibitors leupeptin, aprotinin, and 4-(2 aminoethyl)-benzenesulfonyl fluoride hydrochloride (ICN). The lysate was centrifuged at 12,000 ϫ g and the supernatant assayed for insulin receptor phosphorylation.
Assay for Insulin-stimulated Insulin Receptor Phosphorylation-Insulin-stimulated tyrosine phosphorylation of the insulin receptor was assayed using a plate-based system and a combination of an antiinsulin receptor monoclonal antibody and an anti-phosphotyrosine monoclonal antibody. Capture plates were coated overnight with the anti-insulin receptor monoclonal antibody AB3 (Oncogene Science, 2 g/ml) diluted 1/500 in a carbonate buffer. The plates were then incubated for 1 h in a blocking buffer (0.5% bovine serum albumin in PBS) and washed 6 times in 0.05% Tween 20 in PBS. 85 l of the MCF7 cell extract (above) was added to the capture plates and incubated for 2 h with gentle agitation. The wells were then washed 10 times with wash buffer, and 100 l of the second antibody (anti-phosphotyrosine (4G10, UBI, diluted 1:2000) were added. After 2 h of incubation the wells were washed 6 times with the wash buffer, and color was developed with streptavidin-horseradish peroxidase. Color development was monitored at 650 nm. This assay is specific for insulin-stimulated phosphorylation of the insulin receptor: 1) a base-line OD 650 is taken in the absence of added insulin, and the increase in OD 650 as a function of the physiologically relevant insulin concentrations is taken as the insulin-dependent response; 2) insulin receptor specificity is determined by the use of a monoclonal antibody that is specific for the insulin receptor (10); 3) detection of tyrosine phosphorylation is assured by the use of a monoclonal antibody to phosphotyrosine. The two monoclonal antibodies used are the same two that are routinely used for assaying insulinstimulated insulin receptor phosphorylation by immunoprecipitation and Western blotting. The insulin dose response seen using the platebased assay is comparable with that seen by immunoprecipitation/ Western analysis ( Fig. 1 and data not shown).
Mutagenesis-Site-directed mutagenesis of the PC-1-cDNA was performed using dutϪ ungϪ Escherichia coli as described (11). Uracil containing single-stranded pRK7-PC-1 DNA was isolated from the E. coli strain CJ236. The oligonucleotide 5Ј-GATTGGGGAAAGATTTTGT-TGGA-3Ј was used to produce the mutagenized PC-1 version PC-1-T256S. The region of the oligonucleotide was sequenced to verify that no other changes occurred in the reading frame.
Western Blot Analysis-The PC-1-derived peptide PVSDILKLKTH-LPTFSQED corresponding to amino acids 907-925 (see above for the numbering used) was synthesized using solid phase methodology and coupled to keyhole limpet hemocyanin. The conjugate was injected into rabbits and the antiserum assayed using an enzyme-linked immunosorbent assay format with the peptide as the coat. The antiserum recognizes both the unfolded PC-1 by Western analysis (see Fig. 3) and native PC-1 (Western analysis following electrophoresis in a non-denaturing gel (data not shown)). This antiserum was used at a dilution of 1:2,500 and detected using a horseradish peroxidase-coupled anti-rabbit secondary antibody.
Pharmacological Agents-The MCF7 cells were incubated with the indicated agents under one of two conditions. Either (a) the cells were incubated with the compounds for 1 h prior to the 15-min incubation with insulin or (b) the cells were exposed to the compound for 18 h after which the medium was replaced and the compound replaced at the same concentration for a further hour prior to the 15-min incubation with insulin. In both cases the compounds were present during the insulin treatment period. Adenosine was used at either 10 or 100 M (12,14), adenosine deaminase at 2.5 or 5.0 units/ml (14,23), and 8-phenyltheophylline (8-PT) at either 1, 2, or 3 M (12, 13). The cells were stimulated with insulin at the indicated concentrations and assayed for insulin receptor phosphorylation as described above.

RESULTS AND DISCUSSION
An increase in the expression of PC-1 in MCF7 cells causes these cells to have a diminished response to insulin as measured by the ability of insulin to stimulate phosphorylation of the insulin receptor (5). To facilitate an analysis of the mechanism by which the PC-1-mediated inhibition occurs we have developed a 96-well plate-based assay that specifically measures insulin-mediated insulin receptor phosphorylation (Fig.  1). As described under "Materials and Methods" the assay is dependent on an insulin-mediated signal and is determined by the use of two well characterized monoclonal antibodies. The insulin-dependent response seen in this assay parallels the response determined by Western blotting analysis (data not shown).
It has been reported that adenosine can alter insulin sensitivity (see below), and it is known that ATP is required for phosphorylation of the insulin receptor. Thus we hypothesized that the pyrophosphatase activity associated with PC-1 could cause insulin resistance by increasing adenosine or by decreasing ATP. This hypothesis was tested initially using adenosine receptor agonists and antagonists. Insulin at 100 nM induced a comparable phosphorylation of the insulin receptor whether or not adenosine was present. This lack of effect of adenosine was observed when the adenosine was present for 19 h at either 100 or 10 mM ( Fig. 2 and data not shown) or for 1 h at 100 or 10 mM ( Fig. 2 and data not shown). Extracellular adenosine is converted to inosine by exogenous adenosine deaminase. If PC-1 were leading to an increased level of extracellular adenosine and this was causing the insulin resistance, then PC-1-transfected cells would have an increase in their response to insulin in the presence of adenosine deaminase. As can be seen in Fig.  2 the presence of adenosine deaminase in the culture medium for the 19 h prior to the insulin response assay did not significantly alter the sensitivity of either wild type or the PC-1transfected cells. We also tested whether the adenosine receptor antagonist 8-PT was able to alter the response of the cells to insulin. As shown in Fig. 2, the PC-1-induced insulin resistance was not altered by the presence of 8-PT when present for 19 h at 3 M (Fig. 2) or 1 M (data not shown).
These experiments were all carried out at 0, 1, and 100 nM insulin (data not shown for 0 and 1 nM insulin). To determine whether an adenosine-related effect could be detected at other insulin concentrations, the experiments were repeated using adenosine, adenosine deaminase, or 8-PT at doses that are known to be effective and a range of insulin concentrations. As can be seen in Fig. 1 there was no significant alteration in the insulin-stimulated insulin receptor phosphorylation at insulin doses ranging from 0.5 to 100 nM. This lack of effect was consistently noted (n ϭ 3) in both the wild type MCF7 cells and in the PC-1-transfected cells.
These pharmacological interventions suggested that the in- 1 The abbreviations used are: PBS, phosphate-buffered saline; IR, insulin receptor; PD, phosphodiesterase; 8-PT, 8-phenyltheophylline. sulin resistance was not mediated through an increase in the production of adenosine. To test this possibility in a more direct fashion and also to test the possibility that the PC-1 effect was due to a reduction in ATP levels, we introduced a single amino acid change into the PC-1 protein that allowed protein expression but abolished the pyrophosphatase and phosphodiesterase activity. The relevant amino acids of PC-1 were identified based on homology with bovine intestinal 5Ј-nucleotide phosphodiesterase. It has been demonstrated that a threonine at the active site is required for catalysis mediated by this bovine phosphodiesterase (15). The threonine is thought to contribute to catalysis by forming an intermediate bond with the phosphate. A conserved series of amino acids including a threonine (at amino acid 256) is present in PC-1. Accordingly this threonine was changed to a serine. The cDNAs encoding either the wild type PC-1 (Thr-256) or the variant (Ser-256) were cloned into an expression vector under the control of the cytomegalovirus promoter, and the plasmids co-transfected with a plasmid that confers G418 resistance into MCF7 cells. Clones were selected in the presence of G418, expanded, and tested for PC-1 expression, first by Western analysis and then for phosphodiesterase activity. Three clones that expressed the wild type PC-1 protein and three clones that expressed the Ser-256 variant were identified by Western blot analysis using anti-peptide antiserum (see "Materials and Methods"). As seen in Fig. 3 there was a small amount of PC-1 in the control cell lines (neo5 and neo8) as detected by Western blot analysis. The three cell lines expressing wild type PC-1 (PC6, PC8, and PC22) and the three cell lines expressing the mutant PC-1 (PD-F1, PD-A2, PD-A7) all had comparable immunoreactive PC-1 of the appropriate size. All of these cell lines were then assayed for pyrophosphatase activity using radioactive ATP as a substrate (see "Materials and Methods"). The two control cell lines expressing the neomycin-selective marker had activities of 7.5 and 7. These three groups of cell lines (expressing either neo, wild type, PC-1, or the phosphodiesterase-deficient version of PC-1) were then assayed for the ability of insulin to stimulate phosphorylation of the insulin receptor. As demonstrated (Figs. 1 and 2), the expression of wild type PC-1 led to a clear reduction in the ability of insulin to stimulate the phosphorylation of the insulin receptor (Fig. 3). When the cell lines expressing the pyrophosphatase-deficient version of PC-1 were assayed for insulin-stimulated IR phosphorylation, we found that these cell lines were as resistant to insulin-stimulated insulin receptor phosphorylation as were the cell lines that expressed the wild type PC-1.
To evaluate whether the results obtained with the cell lines expressing the phosphodiesterase-deficient PC-1 were dependent on the insulin concentration the experiment was repeated using a range of insulin concentrations. The PC-1-mediated inhibition of insulin-stimulated insulin receptor phosphorylation could be detected at insulin doses ranging from 0.5 to 100 nM insulin. A comparable dose-dependent inhibition was also observed for all of the cell lines expressing the pyrophosphatase-deficient PC-1 (Fig. 4).
We had hypothesized that PC-1 could reduce the response to insulin by either reducing ATP or by increasing adenosine. Intracellular ATP is required for the autophosphorylation of the insulin receptor, and it might be expected that PC-1 could locally reduce ATP levels sufficiently to reduce IR phosphoryl- ation. This possibility is unlikely since PC-1 is a type II transmembrane protein with the phosphodiesterase and pyrophosphatase activity outside the cell (6). It has been shown, however, that increased expression of PC-1 can lead to an increase in intracellular pyrophosphate levels possibly within vesicles associated with the movement of PC-1 to the plasma membrane (16). A related hypothesis was that a PC-1-mediated increase in extracellular adenosine could mediate the effects on insulin-stimulated IR phosphorylation. Adenosine interacts with and signals through the P 1 -purinergic receptors, which are further categorized as either A 1 , A 2 , A 3 , or A 4 adenosine receptors on the basis of selective binding of pharmacological agents. A further distinction between the various adenosine receptors is made on the basis of the intracellular signaling pathways used. The A 2 receptor appears to couple exclusively to and activates adenylate cyclase. In contrast A 1 and A 3 activation decreases adenylate cyclase activity, and in addition the A 1 receptor affects potassium and calcium channels and can couple to a variety of intracellular messenger systems including G proteins (17)(18)(19). That adenosine can influence insulinmediated signals is well established although whether adenosine acts to increase or decrease insulin sensitivity appears to be very much dependent on the cells of the organ system used and which insulin-mediated event is measured. Adenosine increases insulin sensitivity in isolated fat cells as measured by insulin-mediated glucose phosphorylation and insulin-mediated anti-lipolysis (20 -22). In contrast, the effects of adenosine on insulin-stimulated glucose uptake in muscle are less clear. Challiss and colleagues (23) have shown that adenosine receptor antagonists (adenosine deaminase, 8-PT) will increase the sensitivity of isolated muscle strips to insulin. In contrast, Vergauwen et al. (24) reported that adenosine receptor antagonists (caffeine, 8-cyclopentyl-1,3-dipropylxanthine) will decrease the effectiveness of insulin in vivo. In this latter study the adenosine had no effect on insulin action when the muscle was at rest but did decrease insulin-stimulated glucose uptake when the muscles were electrically stimulated.
At least for MCF7 cells, there does not appear to be any effect of adenosine on the response to insulin, as measured by the autophosphorylation of the insulin receptor. In addition, data from both our pharmacological intervention studies and the analysis of the cell lines expressing the pyrophosphatase-negative PC-1 are concordant in demonstrating that the PC-1mediated inhibition of insulin-stimulated insulin receptor autophosphorylation is not mediated either by the hydrolysis of ATP or the generation of adenosine.
The mechanism by which PC-1 inhibits insulin receptor autophosphorylation is not known. It has been suggested that PC-1 has both intrinsic kinase and phosphatase activities (25,26) and that PC-1 interacts directly with the fibroblast growth factor receptor (25). As it is likely that any kinase or phosphatase activity associated with PC-1 would be extracellular, it is unclear how these kinase/phosphatase activities would impact on insulin signaling. Furthermore, it has been demonstrated that PC-1 may not be a true kinase; the radiolabeling by [␥-32 P]ATP may be due to the presence of a covalent ATP-PC-1 intermediate formed during the cleavage of the pyrophosphate bond in the ATP (27). The PC-1 protein also includes amino acid sequence motifs that are characteristic of tyrosine kinase phosphorylation sites, somatomedin B domain signature motifs, and divalent cation binding domains (28). 2 We are currently determining whether these regions may be involved in the inhibition of insulin-stimulated phosphorylation of the insulin receptor.