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The phosphotyrosine (Tyr(P)) form of insulin receptor substrate 1 (IRS-1) is a key component in insulin signaling. Our previous study revealed that Tyr(P) IRS-1 binds to the widely distributed tyrosine phosphatase PTP2C through the src homology 2 (SH2) domains of the latter. In the present study, we examined the activity of this enzyme and of a truncated form lacking the SH2 domains (delta PTP2C) toward IRS-1 and also toward the cytoplasmic domain of the insulin receptor. Tyr(P) IRS-1 was prepared by phosphorylation of recombinant IRS-1 with recombinant cytoplasmic insulin receptor kinase (CIRK). PTP2C rapidly dephosphorylated Tyr(P) IRS-1; dephosphorylation by delta PTP2C was approximately one-third as fast. Other substrates, including Tyr(P) CIRK, were not dephosphorylated as rapidly by PTP2C; moreover, delta PTP2C was at least 10 times more active than PTP2C toward CIRK and other substrates. These results indicate that the binding of Tyr(P) residues on IRS-1 to the SH2 domain(s) of PTP2C enhances its activity toward IRS-1 and suggest that PTP2C is the phosphatase responsible for the dephosphorylation of IRS-1 in vivo. In addition, with the expectation that a PTP2C-resistant form of IRS-1 will be useful in investigations of IRS-1 function, we determined that IRS-1 can be thiophosphorylated with adenosine 5'-O-(3-thiotriphosphate) and CIRK and that this form of IRS-1 is resistant to PTP2C.

The insulin and insulin-like growth factor-I (IGF-I) receptors are tyrosine kinases. Consequently, an approach to investigating signaling pathways from these receptors is to characterize proteins rapidly phosphorylated on tyrosine in response to insulin and IGF-I. In many cell types the most prominent phosphotyrosine (Ptyr) protein, in addition to the receptors themselves, is a protein of approximately 160 kD, now known as the insulin receptor substrate 1 (IRS-1). We have purified IRS-1 from mouse 3T3-L1 adipocytes, obtained the sequences of tryptic peptides, and cloned its cDNA based on this information. Mouse IRS-1 is a protein of 1,231 amino acids. It contains 12 tyrosine residues in sequence contexts typical for tyrosine phosphorylation sites. Six of these begin the sequence motif YMXM and two begin the motif YXXM. Recent studies have shown that the enzyme phosphatidylinositol 3-kinase (PI 3-kinase) binds tightly to the activated platelet-derived growth factor (PDGF) and colony-stimulating factor-1 (CSF-1) receptors, through interaction of the src homology 2 (SH2) domains on the 85 kD subunit of PI 3-kinase with Ptyr in one of these motifs on the receptors. We have found that, upon insulin treatment of 3T3-L1 adipocytes, a portion of the Ptyr form of IRS-1 becomes tightly complexed with PI 3-kinase. Since IRS-1 binds to fusion proteins containing the SH2 domains of PI 3-kinase, association most likely occurs through this domain. The association of IRS-1 with PI 3-kinase activates the enzyme about fivefold.(ABSTRACT TRUNCATED AT 250 WORDS)

Insulin stimulates the tyrosine phosphorylation of a 60-kDa protein (pp60) in rat adipocytes. After insulin treatment of these cells, pp60, as well as the 160-kDa insulin receptor substrate-1 (IRS-1), were found to be associated with the enzyme phosphatidylinositol 3-kinase (PtdIns-3-kinase) in separate complexes. By contrast, pp60 was not detected in insulin-treated mouse 3T3-L1 adipocytes, which contain abundant IRS-1. PtdIns-3-kinase complex. The pp60.PtdIns 3-kinase complex was located in both the soluble and membrane fractions of the rat adipocytes. Fusion proteins containing the isolated src homology 2 domains from the 85-kDa subunit of PtdIns-3-kinase bound to pp60 in lysates of insulin-treated rat adipocytes. This finding indicates that the most likely mode of association of pp60 with PtdIns-3-kinase is through binding of phosphotyrosine residues in pp60 to these domains. By immunoaffinity chromatography on a monoclonal antibody against phosphotyrosine, pp60 was purified in high percentage yield from insulin-stimulated rat adipocytes, but the low amount of the protein obtained (about 3 ng from the adipocytes of one rat) precluded sequence analysis.

The interactions of the phosphotyrosine (Tyr(P))-containing proteins in basal and insulin-stimulated 3T3-L1 adipocytes with src homology 2 (SH2) domains from phosphatidylinositol 3-kinase (PI3K), ras GTPase-activating protein (GAP), and phospholipase C gamma have been examined. The Tyr(P) forms of the insulin receptor and its 160-kDa substrate protein (pp160) associated with fusion proteins containing either or both the SH2 domains of PI3K, but not with fusion proteins containing the two SH2 domains of GAP or phospholipase C gamma. These results demonstrate a specificity for the association of the Tyr(P) form of the insulin receptor and pp160 with SH2 domains that parallels the reported effects of insulin on PI3K, GAP, and phospholipase C gamma in vivo. Immunoprecipitates of pp160 from the cytosol of insulin-treated, but not basal, 3T3-L1 adipocytes contained PI3K activity. Moreover, the Tyr(P) form of pp160 with associated PI3K activity migrated at 10 S on a sucrose velocity gradient, whereas the Tyr(P) form without associated activity migrated at 6 S. These findings indicate that the Tyr(P) form of pp160 associates directly with PI3K in vivo.

Recently, the alpha-subunit of the inhibitory guanine-nucleotide-binding protein Gi2 (alpha-Gi2) has been shown to be a substrate for phosphorylation both by protein kinase C and also by other unidentified kinase(s) which are activated as a result of elevated cyclic AMP levels in intact rat hepatocytes [Bushfield, Murphy, Lavan, Parker, Hruby, Milligan & Houslay (1990) Biochem. J. 268, 449-457]. Here we show that the incorporation of [32P]Pi into alpha-Gi2 was enhanced 3-fold by incubation of intact hepatocytes with the tumour promoter and protein phosphatase (1 and 2A) inhibitor, okadaic acid. This action was both time- and concentration-dependent and was accompanied by a loss of guanine-nucleotide-induced inhibition of adenylate cyclase. The increased labelling of alpha-Gi2 induced by okadaic acid was partially additive with that elicited by 8-bromo cyclic AMP, but not with that elicited by the protein kinase C activator phorbol 12-myristate 13-acetate. We suggest that, in the absence of hormones, the activity of alpha-Gi2 is under the control of a dynamic phosphorylation/dephosphorylation system involving protein kinase C and protein phosphatases 1 and/or 2A. This highlights the regulation of kinases and phosphatases as both providing potentially important mechanisms for causing ‘cross-talk’ between different signalling systems, in this instance controlling cellular responsiveness through regulation of alpha-Gi2 phosphorylation.

Hepatocytes contain the Gi2 and Gi3 forms of the ‘Gi-family’ of guanine-nucleotide-binding proteins (G-proteins), but not Gi1. The anti-peptide antisera AS7 and I3B were shown to immunoprecipitate Gi2 and Gi3 selectively, and the antiserum CS1 immunoprecipitated the stimulatory G-protein Gs. Treatment of intact, 32P-labelled hepatocytes with one of glucagon, TH-glucagon ([1-N-alpha-trinitrophenylhistidine, 12-homoarginine]glucagon), Arg-vasopressin, angiotensin-II, the phorbol ester TPA (12-O-tetradecanoylphorbol 13-acetate) and 8-bromo-cyclic AMP elicited a time- and dose-dependent increase in the labelling of the alpha-subunit of immunoprecipitated Gi2 which paralleled the loss of ability of low concentrations of the non-hydrolysable GTP analogue guanosine 5′-[beta gamma-imido]triphosphate (p[NH]ppG) to inhibit forskolin-stimulated adenylate cyclase activity (‘Gi’-function). The immunoprecipitation of phosphorylated Gi-2 alpha-subunit by the antiserum AS7 was blocked in a dose-dependent fashion by the inclusion of the C-terminal decapeptide of transducin, but not that of Gz (a ‘Gi-like’ G-protein which lacks the C-terminal cysteine group which is ADP-ribosylated by pertussis toxin in other members of the Gi family), in the immunoprecipitation assay. No labelling of the alpha-subunits of either Gi3 or Gs was observed. alpha-Gi2 was labelled in the basal state and this did not change over 15 min in the absence of ligand addition. In contrast to the monophasic dose-effect curves seen with vasopressin, angiotensin and TPA, the dose-effect curve for the glucagon-mediated increase in the labelling of alpha-Gi2 was markedly biphasic where the loss of Gi function paralleled the high-affinity component of the labelling of alpha-Gi2 caused by glucagon. TPA, TH-glucagon, angiotensin-II and vasopressin achieved similar maximal increases in the labelling of alpha-Gi2, which was approximately half that found after treatment of hepatocytes with either high glucagon concentrations (1 microM) or 8-bromocyclic AMP. Analysis of the phosphoamino acid content of immunoprecipitated alpha-Gi2 showed the presence of phosphoserine only. Incubation of hepatocyte membranes with [gamma-32P]ATP and purified protein kinase C, but not protein kinase A, led to the incorporation of label into immunoprecipitated alpha-Gi2. This labelling was abolished if membranes were obtained from cells which had received prior treatment with ligands shown to cause the phosphorylation of alpha-Gi2 in intact cells. We suggest that there are two possible sites for the phosphorylation of alpha-Gi2; one for C-kinase and the other for an unidentified kinase whose action is triggered by A-kinase activation.

A transient increase in the intracellular concentrations of cyclic AMP occurs as a result of the challenge of hepatocytes with glucagon. This event is determined by the initial rapid activation of adenylate cyclase, which is responsible for the production of cyclic AMP within the cell. Following on from this we observe the desensitization of adenylate cyclase; the A-kinase-mediated activation of the ‘dense-vesicle’, high affinity cyclic AMP phosphodiesterase; the phosphorylation and functional inactivation of the inhibitory G-protein Gi-2 and the establishment of a ‘selective’ insulin-resistant state. These events identify ‘interplay’ or ‘cross-talk’ occurring between distinct cellular signalling systems.

DEAE chromatography of a high speed supernatant fraction from a homogenate of rat liver, prepared under isotonic conditions in the presence of protease inhibitors, yielded three peaks of cyclic nucleotide phosphodiesterase activity (PDE activity). The first peak could be resolved on Affi-gel Blue chromatography to yield a Ca2+/calmodulin stimulated cyclic GMP specific PDE and a cyclic AMP and cyclic GMP hydrolysing PDE whose activity was insensitive to Ca2+/calmodulin. These two activities could also be clearly resolved by Mono-Q chromatography of soluble extracts from both liver and hepatocytes. These had different molecular weights, kinetics of substrate utilization, thermostabilities, dependence on Mg2+ and inhibitor sensitivities. The cyclic AMP and cyclic GMP utilizing PDE resolved in these procedures appears to be a novel enzyme form (PDE-MQ-I) which is insensitive to inhibition by the so-called non-selective PDE inhibitor IBMX and displays catalytic activity in the absence of Mg2+. None of the inhibitors tested were capable of inhibiting this form showing that the catalytic activity of this species could be distinguished from all the other soluble activities. This novel enzyme hydrolysed both cyclic AMP and cyclic GMP with Km values of 25 μM and 237 μM, respectively. The Vmax ratio of hydrolysis of cyclic GMP/cyclic AMP was above unity (1.4). It accounted for 30% of the soluble cyclic AMP PDE activity and 10% of the cyclic GMP PDE activity assessed at 1 μM substrate. Gel filtration of PDE-MQ-I indicated a size of 33,150 Da, in contrast to the size of 237,500 Da observed for the Ca2+/calmodulin PDE-MQ-II. Thermal inactivation of PDE-MQ-I and PDE-MQ-II yielded single exponential decays with t 1 2 values of 6.33 min and 0.7 min at 60° respectively. In the presence of saturating Ca2+, PDE-MQ-II was activated by calmodulin with an EC 50 of ca. 30 ng/ml. In the presence of calmodulin, PDE-MQ-II was activated by Ca2+ with an EC 50 of ca. 20 μM. Chromatography of homogenates on Mono-Q also identified a cyclic GMP-activated cyclic nucleotide PDE (PDE-MQ-III) and two cyclic AMP specific activities (PDE-MQ-IV and PDE-MQ-V). These exhibited very different inhibitor sensitivities and could be readily distinguished using the compound Ro-20-1724 which yielded IC 50 values for inhibition of >500 μM, 13 μM and 1.5 μM, respectively, for the hepatocyte enzymes. PDE-MQ-III to -V exhibited Km values for the hydrolysis of cyclic AMP of 37.7 μM, 0.62 μM and 0.62 μM and Hill coefficients of 1.62, 0.69 and 1.01, respectively. Rolipram was found to potently inhibit both the cyclic AMP specific forms. The compound ICI 118233 failed to inhibit any of the isolated soluble phosphodiesterases. A similar multiplicity of phosphodiesterase species was observed in the soluble fraction from both rat liver homogenates and isolated hepatocytes. We suggest that care be taken in interpreting experiments performed to assess adenylate cyclase activity in intact cells by monitoring intracellular cyclic AMP accumulation in the presence of IBMX due to the possible presence of an IBMX-insensitive phosphodiesterase.

T h e detailed pathways which link occupancy of the insulin receptor with actions on target cells remain t o be defined. The insulin receptor expresses a tyrosyl kinase activity for which considerable evidence has accrued indicating that it plays a fundamental role in transducing at least certain o f insulin's actions. Nevertheless, the mechanism whcrcby tyrosyl phosphorylation o f proteins might lead to cellular responses is still as much of an enigma a s the physiological role o f the few substrates that have been identified 11.21. T h e functioning o f ii wide variety of cell surface receptors for hormones and neurotransmitters is transduced by their interaction with specific guanine nucleotide regulatory proteins (G-proteins) I3,4]. Not only that, but considerable evidence suggests that there is substantial 'cross-talk' bctwecn the machinery o f cellular signalling systems [ 51. This can lead to heterologous desensitization or t o supersensitization depending upon the signalling system and cell type. Here we discuss evidence which supports the contention we made earlier 16, 71, that insulin can interact with the G-protein system. This shows that ( i ) insulin, acting through its receptor can interact with both individual G-proteins and specific G-protein-controlled systems, ( i i ) that a specific G-protein may be involved in transducing certain of insulin's actions and ( i i i ) that in diabetes and in insulin-resistant states one sees the functional inactivation o f the inhibitory Gprotein G, in both liver 18.91 and white adipose tissue. MARY-ANNE McGREGOR. ELAINE KILGOUR.

Polyclonal-antibody preparations DV1 and PM1, raised against purified preparations of rat liver insulin-stimulated ‘dense-vesicle’ and peripheral-plasma-membrane cyclic AMP phosphodiesterases, were used to analyse rat liver homogenates by Western-blotting techniques. The antibody DV1 identified only the 63 kDa native subunit of the ‘dense-vesicle’ enzyme, and the antibody PM1 only the 52 kDa subunit of the plasma-membrane enzyme. These antibodies also detected the subunits of these two enzymes in homogenates of kidney, heart and white adipose tissue from rat. Quantitative immunoblotting demonstrated that the amount of these enzymes (by wt.) varied in these different tissues, as did the expression of these two enzymes, relative to each other, by a factor of as much as 7-fold. The ratio of the dense-vesicle enzyme to the peripheral-plasma-membrane enzyme was lowest in liver and kidney and highest in heart and white adipose tissue. ICI 118233 was shown to inhibit selectively the ‘dense-vesicle’ cyclic AMP phosphodiesterase in liver. It did this in a competitive fashion, with a Ki value of 3.5 microM. Inhibition of tissue-homogenate cyclic AMP phosphodiesterase activity by ICI 118233 was used as an index of the contribution to activity by the ‘dense-vesicle’ enzyme. By this method, a tissue distribution of the ‘dense-vesicle’ enzyme was obtained which was similar to that found by using the immunoblotting technique. The differential expression of isoenzymes of cyclic AMP phosphodiesterase activity in various tissues might reflect a functional adaptation, and may provide the basis for the different physiological actions of compounds which act as selective inhibitors.

A phosphorylated form of alpha-Gi-2 (the alpha-subunit of Gi-2), immunoprecipitated from hepatocytes under basal conditions, migrated as a single species of pI approximately 5.7, the labelling of which increased approximately 2-fold in cells challenged with either vasopressin or phorbol 12-myristate 13-acetate (PMA); agents which activate protein kinase C. In contrast, treatment of hepatocytes with 8-bromo-cyclic AMP produced a more acidic species of phosphorylated alpha-Gi-2 having a pI of approximately 5.4 and whose labelling was increased approximately 3-fold. Trypsin digestion of labelled alpha-Gi-2 isolated from hepatocytes under basal conditions identified, on two-dimensional peptide analyses, three positively charged phosphoserine-containing peptides (C1, C2 and C3), with only peptides C1 and C2 being evident upon less extensive digestion with trypsin. These are suggested to reflect a single site of phosphorylation, with proteolysis by trypsin being incomplete, and where C2 is larger than C1, which is larger than C3. An identical pattern of tryptic phosphopeptides was seen in hepatocytes treated with either vasopressin or PMA, although labelling of this group of peptides was increased by approximately 2-fold compared with the basal state. In contrast, treatment of hepatocytes with glucagon, 8-bromo-cyclic AMP or forskolin not only resulted in increased labelling of the ‘basal’ sites approximately 3-fold, but identified a novel positively charged tryptic phosphoserine-containing peptide (AN). All four tryptic peptides were susceptible to proteolysis by V8 protease. Treatment of labelled alpha-Gi-2 from basal and PMA-treated cells produced a pattern of peptides which was identical with those found when the tryptic phosphopeptide was treated with V8 protease. We tentatively suggest that, on alpha-Gi-2, Ser144 is phosphorylated through the action of protein kinase C and Ser207 is phosphorylated upon elevation of the intracellular concentrations of cyclic AMP.

A DNA encoding the human alpha 2-C10 adrenergic receptor was transfected into Rat 1 fibroblasts and clones selected on the basis of resistance to G418 sulfate. Two clones, one of which (1C) expressed some 3.5 pmol/mg membrane protein of the receptor as assessed by the specific binding of [3H]yohimbine and one (4D) which did not express detectable amounts of the receptor were selected for further study. When cholera toxin-catalyzed ADP-ribosylation was performed with [32P]NAD on membranes of these cells in the absence of added guanine nucleotides, radioactivity was incorporated into a polypeptide(s) of 40 kDa in addition to the 45- and 42-kDa forms of Gs alpha. Addition of the selective alpha 2 receptor agonist U.K.14304 enhanced markedly, in a dose-dependent manner, the cholera toxin-catalyzed [32P]ADP-ribosylation of the 40-kDa polypeptide(s), but not the 45- or 42-kDa polypeptides, in membranes of the 1C cells. Dose response curves for U.K.14304 enhancement of cholera toxin-labeling of the 40-kDa polypeptide(s) and stimulation of high affinity GTPase activity were identical. By contrast, U.K.14304 was ineffective in either assay in membranes from the 4D cells, demonstrating this effect to be dependent upon receptor activation. Furthermore, the alpha 2 receptor antagonist yohimbine blocked all effects of U.K.14304. The agonist promotion of cholera toxin-catalyzed ADP-ribosylation of Gi was completely blocked by guanine nucleotides. Whether GDP or GDP + fluoroaluminate (as a mimic of GTP) was used, blockade of the agonist effect was complete and indeed both conditions prevented agonist-independent labeling by cholera toxin of the 40-kDa polypeptide(s). Mg2+ produced an agonist-independent cholera toxin-catalyzed [32P]ADP-ribosylation of the 40-kDa polypeptide(s) but even in the presence of [Mg2+], agonist-stimulation of cholera toxin-labeling of the 40-kDa polypeptide(s) was observed and was additive with the effect of [Mg2+]. Agonist stimulation of cholera toxin-catalyzed ADP-ribosylation of Gi was completely attenuated by pretreatment of the cells with pertussis toxin, which prevents contact between receptors and G-proteins which are substrates for this toxin. By contrast, pretreatment of the cells with concentrations of cholera toxin able to "down-regulate" essentially all of the membrane-associated Gs alpha did not prevent agonist stimulation of cholera toxin-catalyzed ADP-ribosylation of Gi.(ABSTRACT TRUNCATED AT 400 WORDS)