Modification of functional arginine residues in purified bovine testicular hyaluronidase with butane-2, 3-dione

The effect of mononucleotides on the cytosolic rat liver glucocorticoid receptor has been studied by the use of aqueous dextran-poly(ethylene glycol) two-phase partitioning. During incubations in 0.4 M KCl at 0 degrees C, millimolar concentrations of ADP and ATP, but not AMP, CTP, UTP and GTP, inhibit the increase in the receptor partition coefficient associated with receptor activation. This inhibition is counteracted by millimolar concentrations of theophylline and MgCl2. Two nonhydrolyzable analogues of ATP, alpha, beta-Methyleneadenosine 5'-triphosphate and beta, gamma-methyleneadenosine 5'-triphosphate, also inhibit the increase in the partition coefficient. alpha, beta-Methyleneadenosine 5'-triphosphate is much more potent than ATP in doing so, and this compound was also shown to reduce the amount of receptor to bind to DNA-Sepharose after the incubations. Thus, adenine nucleotides induce a change in the state of the receptor, apparently consisting in an inhibition of receptor activation.     

Aluminum fluoride inhibition of glucocorticoid receptor inactivation and transformation

Fluoride, in the presence of aluminum ions, reversibly inhibits the temperature-mediated inactivation of unoccupied glucocorticoid receptors in cytosol preparations from mouse L cells. The effect is concentration-dependent, with virtually complete stabilization of specific glucocorticoid-binding capacity at 2 mM fluoride and 100 microM aluminum. These concentrations of aluminum and fluoride are ineffective when used separately. Aluminum fluoride also stabilizes receptors toward inactivation by gel filtration and ammonium sulfate precipitation. Aluminum fluoride prevents temperature-dependent transformation of steroid-receptor complexes to the DNA-binding state. Aluminum fluoride does not inhibit calf intestine alkaline phosphatase, and unoccupied receptors inactivated by this enzyme in the presence of aluminum fluoride can be completely reactivated by dithiothreitol. The effects of aluminum fluoride are due to stabilization of the complex between the glucocorticoid receptor and the 90-kDa mammalian heat-shock protein hsp90, which suggests that aluminum fluoride interacts directly with the receptor. Endogenous thermal inactivation of receptors in cytosol is not accompanied by receptor dephosphorylation. However, inactivation is correlated with dissociation of hsp90 from the unoccupied receptor. These results support the proposal that hsp90 is required for the receptor to bind steroid and dissociation of hsp90 is sufficient to inactivate the unoccupied receptor.     

Interaction of rat liver glucocorticoid receptor with sodium tungstate.

Effects of sodium tungstate on various properties of rat liver glucocorticoid receptor were examined at pH7 and pH 8. At pH 7, [3H]triamcinolone acetonide binding in rat liver cytosol preparations was completely blocked in the presence of 10--20 mM-sodium tungstate at 4 degrees C, whereas at 37 degrees C a 30 min incubation of cytosol receptor preparation with 1 mM-sodium tungstate reduced the loss of unoccupied receptor by 50%. At pH 8.0, tungstate presence during the 37 degrees C incubation maintained the steroid-binding capacity of unoccupied glucocorticoid receptor at control (4 degrees C) levels. In addition, heat-activation of cytosolic glucocorticoid-receptor complex was blocked by 1 mM- and 10 mM-sodium tungstate at pH 7 and pH 8 respectively. The DNA-cellulose binding by activated receptor was also inhibited completely and irreversibly by 5 mM-tungstate at pH 7, whereas at pH 8 no significant effect was observed with up to 20 mM-tungstate. The entire DNA-cellulose-bound glucocorticoid-receptor complex from control samples could be extracted by incubation with 1 mM- and 20 mM-tungstate at pH 7 and pH 8 respectively, and appeared to sediment as a 4.3--4.6 S molecule, both in 0.01 M- and 0.3 M-KCl-containing sucrose gradients. Tungstate effects are, therefore, pH-dependent and appear to involve an interaction with both the non-activated and the activated forms of the glucocorticoid receptor.     

ATP-dependent activation of L cell glucocorticoid receptors to the steroid binding form.

The specific glucocorticoid binding capacity in cytosols prepared from L929 mouse fibroblasts (L cells) is inactivated with a half-life of approximately 2 h at 25 degrees C. As previously published, this inactivation can be prevented with 10 mM molybdate and markedly slowed by addition of other phosphatase inhibitors such as glucose 1-phosphate and fluoride. We have now found that ATP (5 to 10 mM) also slows the rate of this inactivation. After extensively inactivating the receptor by preincubating cytosol at 25 degrees C for 4 and preventing further inactivation by addition of molybdate, addition of ATP results in reactivation of the steroid binding capacity. Maximal reactivation of 40 to 70% is achieved with 5 to 10 mM ATP. The activation is temperature-dependent and specific for ATP. ADP, GTP, CTP, and UTP do not cause activation and preliminary results indicate no effect of cyclic nucleotides in this system. If activation is prevented by addition of 10 mM EDTA to the cytosol, addition of 3 to 10 mM magnesium permits ATP-dependent activation of the binding capacity. The level of reactivation can be enhanced by addition of a heat-stable factor prepared from the same L cell supernatant. These results support the proposal that L cell glucocorticoid receptors can be activated to the glucocorticoid binding state by an ATP-dependent phosphorylation mechanism.     

Phosphorylation of L-cell glucocorticoid receptors in immune complexes: evidence that the receptor is not a protein kinase

Two phosphoproteins are absorbed to protein A-Sepharose when cytosol from 32P-labeled L-cells is incubated with a monoclonal antibody against the glucocorticoid receptor: one is a 98K phosphoprotein that contains the steroid binding site, and the other is a 90K non-steroid-binding phosphoprotein that is associated with the molybdate-stabilized receptor [Housley, P. R., Sanchez, E. R., Westphal, H. M., Beato, M., & Pratt, W. B. (1985) J. Biol. Chem. 260, 13810-13817]. In this paper we have incubated L-cell cytosol with rabbit antiserum against the mouse glucocorticoid receptor and show that incubation of protein A-Sepharose-bound immune complexes with [gamma-32P]ATP and Mg2+ results in phosphorylation of the 98K steroid-binding protein but not of the 90K receptor-associated protein. Phosphorylation occurs regardless of whether the receptor is unoccupied or is present as the untransformed or transformed steroid-receptor complex. No phosphorylation occurs in the presence of Ca2+ instead of Mg2+. If protein A-Sepharose-bound immune complexes prepared with a monoclonal antibody against the receptor are incubated with [gamma-32P]ATP and Mg2+, neither protein is phosphorylated. If the protein A-Sepharose pellet is obtained from molybdate-stabilized cytosol that has been incubated both with monoclonal antibody to provide the 98K receptor and its 90K associated protein and with preimmune rabbit serum, which causes the nonspecific adsorption of an L-cell protein kinase, then incubation with [gamma-32P]ATP and Mg2+ causes receptor phosphorylation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modulators of the glucocorticoid receptor also regulate mineralocorticoid receptor function.

Modulators are proposed to be novel ether aminophosphoglycerides that stabilize unoccupied and occupied glucocorticoid receptor steroid binding and inhibit glucocorticoid receptor complex activation. Two isoforms, modulator 1 and modulator 2, have been purified from rat liver cytosol [Bodine, P.V., & Litwack, G. (1990) J. Biol. Chem. 265, 9544-9554]. Since the mineralocorticoid receptor is relatively resistant to activation, modulator's effect on rat distal colon mineralocorticoid receptor function was examined. Warming of unoccupied receptor decreased residual specific [3H]aldosterone binding by 86 +/- 2%. Both modulator isoforms completely prevented this destabilization with Km's of 2 +/- 1 microM modulator 1 and 24 +/- 5 microM modulator 2. Warming of occupied mineralocorticoid receptors decreased [3H]aldosterone binding by 56 +/- 3%. Modulator only partially stabilized occupied receptor binding with Km's of 10 +/- 2 microM modulator 1 and 68 +/- 8 microM modulator 2. Modulator inhibited receptor activation with Km's of 3 +/- 1 microM modulator 1 and 33 +/- 10 microM modulator 2. Double-reciprocal analysis showed linear kinetics, and mixing modulator isoforms together had additive effects on unoccupied and occupied receptor steroid binding stabilization and activation inhibition. Colon cytosol contained a low molecular weight, heat-stable factor(s) which inhibited receptor activation and stabilized occupied receptor steroid binding. Molybdate completely stabilized unoccupied mineralocorticoid receptor steroid binding and inhibited activation with half-maximal effects at 3-4 mM but only stabilized occupied receptor binding by approximately 40%. These data indicate that (i) apparent physiologic concentrations of modulator stabilize mineralocorticoid receptor steroid binding and inhibit receptor activation, (ii) an aldosterone-responsive tissue contains a modulator-like activity, and (iii) molybdate mimics the effects of modulator.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of a macromolecular inhibitor of glucocorticoid-receptor complex activation in rat liver cytosol

We have identified an endogenous regulator of the glucocorticoid receptor following fractionation of dialyzed rat liver cytosol on DEAE-cellulose. The macromolecular regulator, purified approximately 20-fold as judged by Lowry-reactive material, inhibits activation of glucocorticoid-receptor complexes when assayed by DNA-cellulose binding and by chromatography on DEAE-cellulose minicolumns. In addition the active DEAE-cellulose fraction stabilizes the unoccupied glucocorticoid receptor against heat inactivation. Evidence is presented that the observed inhibition of activation by the active DEAE-cellulose fraction is not due to concentration of cytosolic proteases or RNA. The inhibitory molecule in the active fraction is not stable to heating at 90 degrees C (15 min) and is partially inactivated at 45 degrees C (15-60 min).     

ATP-dependent activation of glucocorticoid receptor from rat liver cytosol.

The glucocorticoid--receptor complex from freshly prepared rat liver cytosol is in a non-activated form, with very little affinity to bind to isolated nuclei. When such preparations were incubated with 5--10 mM-ATP at 4 degrees C, the receptor complex acquired the properties of an 'activated' transformed form, which readily bound to nuclei, ATP--Sepharose, phosphocellulose and DNA--cellulose. This transformation was comparable with the activation achieved by warming the steroid--receptor complex at 23 degrees C. The effect of ATP was specific, as it was more effective than ADP, whereas AMP had no such effect on activation. The process of receptor activation was sensitive to the presence of 10 mM-sodium molybdate; the latter blocked activation by both ATP and heat. Bivalent cations had no observable effect on the receptor activation at low temperature, but they decreased the extent of activation by ATP. The steroid-binding properties of glucocorticoid receptor remained intact under the above conditions. However, a significant increase in steroid binding occurred when ATP was preincubated with cytosol receptor before the addition of [3H]triamcinolone acetonide. ATP also stabilized the glucocorticoid--receptor complexes at 23 degrees C. These results suggest a role for ATP in receptor function and offer a convenient method of studying the activation process of glucocorticoid receptor under mild assay conditions.     

Reversible Inhibition of Cytosolic Glucocorticoid Receptors by Mercurial Agents. Application in the Measurement of Total and Unoccupied Receptors

Abstract

Recently developed “exchange assays” have been used to measure total cytosolic glucocorticoid receptor (GR) binding activity as compared to standard GR assays which measure unoccupied receptor. In the current study we modified these methods and extended the applications of such assays. Experiments defined the conditions whereby two sulfhydryl-binding agents, p-hydroxymercuribenzoate (PHMB) and mersalyl, completely inhibited binding of the glucocorticoid receptor to ligand in mouse renal cytosol. Reactivation of steroid-binding activity was restored by addition of dithiothreitol. The present study demonstrates 12% higher GR binding activity when this exchange assay is performed using saturated glucocorticoid-receptor complexes, rather than standard cytosol. Combining the data from the standard and exchange mouse renal cytosolic GR assays, it was determined that, at physiologic tissue corticosterone levels, the respective mean concentrations of unoccupied, occupied, and total GR were 467, 89, and 556 fmol/mg cytosol protein. Measurement of receptor concentrations by the use of these methods permits precise experimental differentiation of factors which affect total, as well as unoccupied GR.     

Physicochemical characteristics of the interaction of the glucocorticoid antagonist RU38486 with glucocorticoid receptors in vitro, and the role of molybdate

The physicochemical properties of complexes formed between the glucocorticoid antagonist, RU38486, and the glucocorticoid receptor in rat thymus cytosol were investigated and compared with those of complexes formed with the potent agonist, triamcinolone acetonide. The equilibrium dissociation constant for the interaction of [3H]RU38486 with the molybdate-stabilized glucocorticoid receptor was lower than that for [1,2,4-3H]triamcinolone acetonide at 0 degree C but higher at 25 degrees C, suggesting that hydrophobic interactions play a major role in the binding of RU38486. Differences in equilibrium constants were reflected in corresponding differences in dissociation rate constants; association rate constants for the two steroids were similar. The rate of dissociation of [3H]RU38486 from the glucocorticoid receptor was higher in the absence of molybdate than in its presence both at 0 degree C and at 25 degrees C, suggesting that molybdate modifies the physical state of the antagonist-receptor complex, but other physical properties were similar both in the presence and in the absence of molybdate. The rate of inactivation of the unoccupied glucocorticoid receptor at 25 degrees C in the absence of molybdate was lower in phosphate buffer than in Tris-HCl buffer but the rate of dissociation of [3H]RU38486 was the same in both buffers. The binding of RU38486 afforded little, if any, protection against inactivation in either buffer; [3H]RU38486 dissociated irreversibly from the inactivated receptor at the same rate as from the non-inactivated complex but molybdate had no effect on the dissociation kinetics of the inactivated complex. It is concluded that RU38486 interacts with the ground state of the glucocorticoid receptor in a manner which neither promotes receptor transformation nor prevents receptor inactivation.     

Purification and structural analysis of the modulator of the glucocorticoid-receptor complex. Evidence that modulator is a novel phosphoglyceride

Modulator is the low molecular weight heat-stable inhibitor of glucocorticoid-receptor complex activation. We have purified modulator to apparent homogeneity from heated rat liver cytosol. This was accomplished using Sephadex G-15 gel filtration, Dowex 1 anion-exchange chromatography, and preparative silica high-performance liquid chromatography. The modulator preparation was judged to be homogeneous by analytical silica high-performance liquid chromatography, two-dimensional silica thin-layer chromatography, and proton nuclear magnetic resonance spectroscopy. The apparent concentration of modulator in rat liver cytosol is 6.5 microM. The purified modulator inhibits heat activation of the rat liver glucocorticoid-receptor complex and stabilizes the steroid binding ability of the unoccupied rat liver glucocorticoid receptor in a dose-dependent manner. At a concentration of 5-6.5 microM, modulator inhibits receptor activation and stabilizes the unoccupied receptor by 50%. At a concentration of 500-630 microM, sodium molybdate also inhibits receptor activation and stabilizes the unoccupied receptor by 50%. Thus, modulator appears to be the endogenous factor that exogenous sodium molybdate mimics in vitro. Chemical analysis of the purified modulator following two-dimensional silica thin-layer chromatography indicates that modulator is an aminophospholipid. Physical analysis of the purified modulator by infrared and nuclear magnetic resonance spectroscopy, as well as mass spectrometry, demonstrates that modulator is an ether aminophosphoglyceride.     

Activation of thymocyte glucocorticoid receptors to the steroid binding form. The roles of reduction agents, ATP, and heat-stable factors.

The specific glucocorticoid binding capacity in cytosol preparations of rat thymocytes decays with a half-life of 4 h at 0 degrees C or 20 min at 25 degrees C. Phosphatase inhibitors (molybdate, fluoride, glucose 1-phosphate) added alone do not prevent this inactivation. Dithiothreitol (2 mM) has a large stabilizing effect on the binding capacity at 0 degrees C but only a small effect at 25 degrees C. Addition of 10 mM molybdate plus 2 mM dithiothreitol totally prevents inactivation for at least 8 h at 25 degrees C as well as at 0 degrees C. Fluoride (100 mM) also retards the inactivation if added with dithiothreitol. Addition of dithiothreitol at 25 degrees C to inactivated cytosol receptors results in partial activation of the binding capacity. Addition of dithiothreitol to receptors inactivated at 25 degrees C in the presence of molybdate allows total reactivation of the binding capacity to the maximum zero time value. If binding capacity is inactivated by preincubation of the cytosol at 25 degrees C, addition of ATP with dithiothreitol enhances the activation observed with only dithiothreitol. This ATP stimulated activation is optimal at 1 to 3 mM. ATP (10 mM) is required when molybdate is added to prevent simultaneous inactivation. ADP, GTP, CTP, and UTP have some activating capacity but the effects of all nucleotides are inhibited by the ATP analog, adenyl-5'-yl (beta, gamma-methylene)diphosphonate. ATP-dependent activation can also be prevented with 50 mM EDTA, and addition of magnesium partially overcomes the EDTA inhibition. Dithiothreitol activation of thymocyte glucocorticoid binding capacity can also be enhanced by addition of a heat-stable preparation from thymocytes, L cells, or liver. Sephadex G-25 chromatography, assay of ATP, and inhibition of the activation with adenyl-5'-yl (beta, gamma-methylene)diphosphonate suggest that these preparations contain varying amounts of endogenous reducing equivalents and ATP as well as a larger heat stable factor. Maximum activation is obtained by adding dithiothreitol, ATP, molybdate, and the larger heat-stable factor. These results suggest that stabilization and activation of glucocorticoid binding capacity in thymocytes requires phosphorylation as well as reduction of the receptor itself or of some other component required for the steroid binding reaction.     

Effects of metal ions and chelating agents on in vitro stability of glucocorticoid receptors in brain cytosol

In vitro studies in a variety of tissues and cell types suggest that glucocorticoid receptor binding capacity is not static and that binding sites are subject to up- and down-regulatory mechanisms. The interpretation of such studies, however, is often complicated by factors affecting the stability of the receptor. This situation is particularly acute in the absence of ligand because of the increased lability of the unoccupied receptor. Studies reported here investigate effects of various metal ions and chelating agents on the stability of unoccupied [3H]dexamethasone binding sites in whole mouse brain cytosol. Variation in the ionic strength of cytosol, as created by the additions of various monovalent cations (Li+, Na+, K+, Rb+ and Cs+), was found to be an important factor affecting the increased stability of the receptor in vitro. Additions of divalent (Mg++, Ca++, Ba++, and Mn++) and trivalent (La , Cr and Al ) cations to cytosol, however, were generally found to produce a dose-dependent decrease in the stability of the unoccupied receptor. Additions of the chelating agents EDTA, EGTA and 1,10-phenanthroline to cytosol, resulted in differential, and sometimes complex, dose-dependent effects on receptor stability. The complex effects of various combinations of cations and the chelator EDTA were also investigated.     

ATP-induced activation of purified rat hepatic glucocorticoid receptors

We have utilized unactivated rat hepatic glucocorticoid receptor complexes purified to near homogeneity by a three-step scheme which includes affinity chromatography, gel filtration and anion exchange chromatography, to demonstrate for the first time that ATP can interact directly with the receptor protein in stimulating activation. This stimulation is reflected by an increase in DNA-cellulose binding as well as by a shift in the elution profile of the purified receptor complexes from DEAE-cellulose. A concentration of 10 mM Na2MoO4 is able to block both of these effects. ATP stimulates activation in a dose-dependent manner (maximally at 10 mM), and elicits maximal activation within 30 min at 15 degrees C. There appears to be no nucleotide specificity since GTP, CTP and UTP, as well as ADP and GDP also stimulate activation. All of these observations closely parallel data obtained from similar activation experiments performed with crude rat hepatic receptors. ATP does not appear to stimulate activation of receptors (crude or purified) by initiating a phosphorylation reaction since hydrolysis-resistant analogues of ATP are also effective. Pyrophosphate (PPi) is as effective as ATP in promoting receptor activation, since it elicits similar increases in DNA-cellulose binding, shifts in elution patterns from DEAE-cellulose, and dose-response relationships. None of the compounds tested stimulate activation indirectly by pH or ionic strength effects. Despite the fact that high ATP concentrations (3-4-fold higher than those present in vivo) are necessary to stimulate maximal activation, a physiological role of ATP in directly regulating in vivo activation of glucocorticoid receptors cannot be ruled out.     

Hydrogen peroxide stabilizes the steroid-binding state of rat liver glucocorticoid receptors by promoting disulfide bond formation

Hydrogen peroxide and diamide inactivate the steroid-binding capacity of unoccupied glucocorticoid receptors in rat liver cytosol at 0 degrees C, and steroid-binding capacity is reactivated with dithiothreitol. Treatment of cytosol with peroxide or sodium molybdate, but not diamide, inhibits the irreversible inactivation (i.e., inactivation not reversed by dithiothreitol) of steroid-binding capacity that occurs when cytosol is incubated at 25 degrees C. Pretreatment of cytosol with the thiol derivatizing agent methyl methanethiosulfonate at 0 degrees C prevents the ability of peroxide, but not molybdate, to stabilize binding capacity at 25 degrees C. As derivatization of thiol groups prevents peroxide stabilization of steroid-binding capacity and as treatment with dithiothreitol reverses the effect, we propose that peroxide acts by promoting the formation of new disulfide linkages. The receptor in our rat liver cytosol preparations is present as three major degradation products of Mr 40,000, 52,000, and 72,000 in addition to the Mr 94,000 intact receptor. Like the intact receptor, these three forms exist in the presence of molybdate as an 8-9S complex, they bind glucocorticoid in a specific manner, and they copurify with the intact Mr 94,000 receptor on sequential phosphocellulose and DNA-cellulose chromatography. Despite the existence of receptor cleavage products, it is clear that peroxide does not stabilize steroid-binding capacity by inhibiting receptor cleavage.     

Modulation of glucocorticoid receptor activity by cyclic nucleotides and its implications on the regulation of human skin fibroblast growth and protein synthesis

The modulation of glucocorticoid receptor activity by cyclic nucleotides was studied in cultured human skin fibroblasts. The receptors appeared to be activated in the presence of dibutyryl-cAMP and inactivated by dibutyryl-cGMP. Significantly, the cGMP content of the fibroblasts increased during cell growth, with a concomitant decrease in the glucocorticoid receptor activity, while when the cells reached early confluency the decrease in cGMP content was accompanied by an increase in cAMP and increased activity of the glucocorticoid receptors. In addition, cortisol induced (2'-5')oligoadenylate synthetase in these cells and raised the cellular (2'-5')oligoadenylate concentrations. This resulted in a decrease in both DNA and protein synthesis activity in the cells, a response which correlated with the (2'-5')oligoadenylate concentration. The combination of cortisol and dibutyryl-cAMP had a synergetic stimulatory effect on the (2'-5')oligoadenylate concentration and a synergetic inhibitory effect on protein synthesis. In conclusion, it is demonstrated here that cyclic nucleotides can modulate glucocorticoid receptor activity in cultured human skin fibroblasts, and thus these compounds may indirectly affect cellular metabolism by regulating the cellular responses to glucocorticoids.     

ATP and UTP at low concentrations strongly inhibit bone formation by osteoblasts: a novel role for the P2Y2 receptor in bone remodeling

There is increasing evidence that extracellular nucleotides act on bone cells via multiple P2 receptors. The naturally-occurring ligand ATP is a potent agonist at all receptor subtypes, whereas ADP and UTP only act at specific receptor subtypes. We have reported that the formation and resorptive activity of rodent osteoclasts are stimulated powerfully by both extracellular ATP and its first degradation product, ADP, the latter acting at nanomolar concentrations, probably via the P2Y1 receptor subtype. In the present study, we investigated the actions of ATP, ADP, adenosine, and UTP on osteoblastic function. In 16-21 day cultures of primary rat calvarial osteoblasts, ADP and the selective P2Y1 agonist 2-methylthioADP were without effect on bone nodule formation at concentrations between 1 and 125 microM, as was adenosine. However, UTP, a P2Y2 and P2Y4 receptor agonist, known to be without effect on osteoclast function, strongly inhibited bone nodule formation at concentrations >or= 1 microM. ATP was inhibitory at >or= 10 microM. Rat osteoblasts express P2Y2, but not P2Y4 receptor mRNA, as determined by in situ hybridization. Thus, the low-dose effects of extracellular nucleotides on bone formation and bone resorption appear to be mediated via different P2Y receptor subtypes: ADP, signalling through the P2Y1 receptor on both osteoclasts and osteoblasts, is a powerful stimulator of osteoclast formation and activity, whereas UTP, signalling via the P2Y2 receptor on osteoblasts, blocks bone formation by osteoblasts. ATP, the 'universal' agonist, can simultaneously stimulate resorption and inhibit bone formation. These findings suggest that extracellular nucleotides could function locally as important negative modulators of bone metabolism, perhaps contributing to bone loss in a number of pathological states.     

Inhibition of protein kinase activity of phorboid and ingenoid receptor by di(adenosine-5')oligophosphate

Di(adenosine-5')oligophosphate nucleotides of general structure ApnA (n = 3-6) inhibited the protein kinase activity of homogeneous phorboid receptor. These nucleotides did not affect the phorboid binding activity. Ap4A competed for an ATP binding site on the phorboid receptor. Km for ATP was increased from 0.5 to 2 microM in the presence of 0.2 mM of Ap4A. KI was calculated to be approximately 0.1 mM. Ap4A-elicited inhibition of phorboid receptor kinase activity was independent of receptor concentration as well as of phosphoacceptor substrate concentration.     

Allosteric modulation by ATP of the bovine adrenal natriuretic factor R1 receptor functions.

Atrial natriuretic factor (ANF-R1) receptor is a 130-kDa protein that contains a cytoplasmic guanylate cyclase domain. We report that ATP interacts in an allosteric manner with the ANF-R1 receptor, resulting in reduced ANF binding and enhanced ANF-stimulated guanylate cyclase activity. The modulatory properties of various nucleotides indicate a preference for the adenine family with a rank order of potency of ATP greater than App(NH)p greater than or equal to ADP greater than or equal to AMP while cyclic and guanine nucleotides except GTP are inactive. The negative modulation by ATP of ANF binding is specific for the ANF-R1 receptor subtype since the amount of ANF bound by the guanylate cyclase uncoupled ANF-R2 subtype is increased in the presence of ATP. Furthermore, the effects of ATP on ANF-R1 receptor binding function are still observed with the affinity-purified ANF-R1 receptor, suggesting an allosteric binding site for ATP on the ANF-R1 receptor. In intact membranes, limited proteolysis of the ANF-R1 receptor with trypsin dose-dependently prevents the ATP-induced decrease in ANF binding concomitantly with the formation of a membrane-associated ANF-binding fragment of 70 kDa. These results confirm the direct modulatory role of ATP on hormone binding activity of ANF-R1 receptor and suggest that the nucleotide regulatory binding site is located in the intracellular domain vicinal to the protease-sensitive region.