The beta-adrenergic receptor of turkey erythrocyte membranes: conformational modification by beta-adrenergic agonists.

The β-adrenergic receptors of turkey erythrocyte membranes have been identified by the specific binding of the radiolabeled antagonist (?)-|³H|-dihydroalprenolol. Pretreatment of these membranes with either the alkylating agent N-ethylmaleimide or with β-adrenergic agonists does not affect (?)-|³H|-dihydroalprenolol binding to the receptor sites. However, the simultaneous presence of both types of products causes a 50% decline in the number of binding sites. A less pronounced decline occurs when the membranes are pretreated with N-ethylmaleimide in presence of the partial agonist (?)-phenylephrine, and no decline in the presence of the antagonist (?)-|³H|-dihydroalprenolol. β-adrenergic agonists thus appear to induce a conformational change of their receptor, with results in an increased susceptibility to inactivation by N-ethylmaleimide.     

Leishmanial protein kinases phosphorylate components of the complement system.

Externally oriented protein kinases are present on the plasma membrane of the human parasite, Leishmania. Since activation of complement plays an important role in the survival of these parasites, we examined the ability of protein kinases from Leishmania major to phosphorylate components of the human complement system. The leishmanial protein kinase-1 (LPK-1) isolated from promastigotes of L. major was able to phosphorylate purified human C3, C5 and C9. Only the alpha-chain of C3 and C5 was phosphorylated. The beta-chain appeared not to be a substrate for this enzyme. C3b which is formed by proteolytic cleavage of C3 was not phosphorylated by LPK-1. Trypsin treatment of phosphorylated C3 (P-C3) resulted in the disappearance of 32P from the alpha-chain. This was correlated with the conversion of the C3 alpha-chain to the alpha'-chain of C3b, and the appearance of a 9 kDa 32P fragment comigrating with the C3a fragment of C3. P-C3 was more resistant to cleavage by trypsin than nonphosphorylated C3. LPK-1 phosphorylated purified C3a and two synthetic peptides, C3a21R and YA-C3a10R, derived from its COOH-terminal end, which contain the C3a binding site to leukocytes and platelets. LPK-1 did not phosphorylate C3a8R. Phosphoamino acid analysis of the synthetic peptides indicated that serine 71 of C3a was phosphorylated by LPK-1. Treatment of C3 with either methylamine or freeze-thaw C3 (H2O) prevented phosphorylation by the LPK-1 suggesting that substrate conformation may be involved in recognition by the leishmanial enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Desensitization of beta-adrenergic stimulated adenylate cyclase in turkey erythrocytes.

Desensitization of catecholamine stimulated adenylate cyclase (AC) activity is demonstrated in membranes derived from turkey erythrocytes pre-treated with isoproterenol. Membranes from desensitized cells had a loss in maximal catecholamine stimulated adenylate cyclase activity of 104 +/- 13 (pmols/mg protein/10', p less than .001) compared with controls. When adenylate cyclase was maximally stimulated with NaF or Gpp(NH)p, the decrements were 84 +/- 19 (p less than .005) and 92 +/- 32 (p less than .05) pmol/mg protein/10' respectively. There was no change in beta-adrenergic receptor number in membranes derived from treated cells. While the molecular mechanism accounting for the desensitization is uncertain, the data is consistent with the hypothesis that there is a lesion distal to the beta-adrenergic receptor, possibly involving the nucleotide site or the catalytic subunit of adenylate cyclase, causing the desensitization in the isoproterenol treated cells.     

Isolation and characterization of two growth factor-stimulated protein kinases that phosphorylate the epidermal growth factor receptor at threonine 669.

A growth factor-stimulated protein kinase activity that phosphorylates the epidermal growth factor (EGF) receptor at Thr669 has been described (Countaway, J. L., Northwood, I. C., and Davis, R. J. (1989) J. Biol. Chem. 264, 10828-10835). Anion-exchange chromatography demonstrated that this protein kinase activity was accounted for by two enzymes. The first peak of activity eluted from the column corresponded to the microtubule-associated protein 2 (MAP2) kinase. However, the second peak of activity was found to be a distinct enzyme. We present here the purification of this enzyme from human tumor KB cells by sequential ion-exchange chromatography. The isolated protein kinase was identified as a 46-kDa protein by polyacrylamide gel electrophoresis and silver staining. Gel filtration chromatography demonstrated that the enzyme was functional in a monomeric state. A kinetic analysis of the purified enzyme was performed at 22 degrees C using a synthetic peptide substrate based on the primary sequence of the EGF receptor (KREL VEPLT669PSGEAPNQALLR). The Km(app) for ATP was 40 +/- 5 microM (mean +/- S.D., n = 3). GTP was not found to be a substrate for the purified enzyme. The Km(app) for the synthetic peptide substrate was 260 +/- 40 microM (mean +/- S.D., n = 3). The Vmax(app) for the isolated protein kinase was determined to be 400-900 nmol/mg/min. The purified enzyme was designated EGF receptor Thr669 (ERT) kinase. It is likely that the MAP2 and ERT kinases account for the phosphorylation of the EGF receptor at Thr669 observed in cultured cells. The marked stimulation of protein kinase activity caused by growth factors indicates that these enzymes may have an important function during signal transduction.     

Catecholamine-induced desensitization of turkey erythrocyte adenylate cyclase. Structural alterations in the beta-adrenergic receptor revealed by photoaffinity labeling

Preincubation of turkey erythrocytes with isoproterenol results in an impaired ability of beta-adrenergic agonists to stimulate adenylate cyclase in membranes prepared from these cells. The biochemical basis for this agonist-induced desensitization was investigated using the new beta-adrenergic antagonist photoaffinity label [125I]p-azidobenzylcarazolol ([125I]PABC). Exposure of [125I]PABC-labeled turkey erythrocyte membranes to high intensity light leads to specific covalent incorporation of the labeled compound into two polypeptides, Mr approximately equal to 38,000 and 50,000, as determined by sodium dodecyl sulfate-polyacrylamide electrophoresis. Incorporation of [125I]PABC into these two polypeptides is completely blocked by a beta-adrenergic agonist and antagonist consistent with covalent labeling of the beta-adrenergic receptor. After desensitization of the turkey erythrocyte by preincubation with 10(-5) M isoproterenol, the beta-adrenergic receptor polypeptides specifically labeled by [125I]PABC in membranes prepared from desensitized erythrocytes were of larger apparent molecular weight (Mr approximately equal to 42,000 versus 38,000, and 53,000 versus 50,000) compared to controls. When included during the preincubation of the erythrocytes with isoproterenol, the antagonist propranolol (10(-5) M) inhibited both agonist-promoted desensitization of the adenylate cyclase and the altered mobility of the [125I]PABC-labeled receptor polypeptides. These data indicate that structural alterations in the beta-adrenergic receptor accompany the desensitization process in turkey erythrocytes.     

Mechanisms altered beta-adrenergic responsiveness in the hyperthyroid and hypothyroid turkey erythrocyte

Studies on the relationship between thyroid hormone and the beta-adrenergic catecholamines have been carried out in the turkey erythrocyte. Conditions of thyroid hormone excess and deficiency were examined with respect to their effects on the beta receptor itself, as well as to their effects on associated biochemical and physiological indices of beta receptor function, including agonist stimulated adenylate cyclase activity, cellular cyclic AMP generation, and catecholamine-induced stimulation of potassium ion influx. Erythrocytes obtained from hypothyroid turkeys showed a marked (approximately 50%) reduction in beta receptor number without any change in receptor affinity for agonists or antagonists. Catecholamine-sensitive adenylate cyclase activity and cellular cyclic AMP levels were similarly reduced. The sensitivity of these cells to agonist-stimulated potassium influx was significantly decreased, but maximal agonist-stimulated transport rate was unchanged. Analysis of the quantitative relationship between beta receptor number, agonist concentration, and level of catecholamine-stimulated potassium influx indicates that, at any given absolute level of receptor occupancy, the level of agonist-stimulated potassium influx is identical in hypothyroid and normal erythrocytes, and that the diminished physiological sensitivity of the hypothyroid cell is attributable in its entirety to a reduction in beta receptor number per se. The results obtained in the hyperthyroid turkey erythrocyte were strikingly different. Here, beta receptor number, binding affinity for agonists and antagonists, catecholamine-sensitive adenylate cyclase activity, and maximal cyclic AMP levels were all unchanged. In contrast, maximal agonist-stimulated potassium ion transport was markedly reduced, while the concentration of isoproterenol required for half-maximal stimulation was only slightly increased. Analysis of the relationship between beta receptor number, agonist concentration, and catecholamine-stimulated potassium influx rate indicates that, at all absolute levels of beta receptor occupancy, the stimulation of monovalent cation influx is markedly blunted in the hyperthyroid cell. In contrast to the findings in the hypothyroid cell, where decreased physiologic sensitivity to catecholamines is directly attributable to a reduction in beta receptor number, the primary abnormality responsible for diminished catecholamine responsiveness in the hyperthyroid cell would appear to be located at a point "distal" to the beta receptor itself.     

Desensitization of turkey erythrocyte adenylate cyclase. Beta-adrenergic receptor phosphorylation is correlated with attenuation of adenylate cyclase activity

Preincubation of turkey erythrocytes with beta-adrenergic agonists leads to an attenuation of the responsiveness of adenylate cyclase to subsequent hormonal stimulation. Recently, our laboratory has shown (Stadel, J. M., Nambi, P., Shorr, R. G. L., Sawyer, D. D., Caron, M. G., and Lefkowitz, R. J. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 3173-3177) using 32Pi incorporation that phosphorylation of the beta-adrenergic receptor accompanies this desensitization process. We now report that, as determined from intracellular [gamma-32P] ATP specific activity measurements, this phosphorylation reaction occurs in a stoichiometric fashion. Under basal conditions there exists 0.75 +/- 0.1 mol of phosphate per mol of receptor whereas under maximally desensitized conditions this ratio increases to 2.34 +/- 0.13 mol/mol. This phosphorylation of the receptor is dose-dependent with respect to isoproterenol and exhibits a dose-response curve coincidental with that for isoproterenol-induced desensitization of adenylate cyclase. The time courses for receptor phosphorylation and adenylate cyclase desensitization are identical. In addition, the rate of resensitization of adenylate cyclase activity is comparable to the rate of return of the phosphate/receptor stoichiometries to control levels. Both the phosphorylation and desensitization reactions are pharmacologically specific as indicated by the high degree of stereoselectivity, rank order of catecholamines, and blockade by the specific beta-adrenergic antagonist, propranolol. Incubation of turkey erythrocytes with cAMP and cAMP analogs maximally activates cAMP-dependent protein kinase but only partially mimics isoproterenol in promoting phosphorylation of the receptor in concordance with their partial effects in inducing desensitization. Conversely, activators or inhibitors of Ca2+/calmodulin kinase or protein kinase C do not affect the isoproterenol-induced desensitization. These results indicate that desensitization of turkey erythrocyte adenylate cyclase is highly correlated with phosphorylation of the beta-adrenergic receptor and that these events are mediated, at least partially, by cAMP.     

Affinity chromatography of the beta-adrenergic receptor from turkey erythrocytes.

The beta 1-adrenergic receptors of turkey erythrocyte membranes have been identified by binding of the radioactively labeled antagonist (--)-[3H]dihydroalprenolol, solubilized by treatment of the membranes with the detergent digitonin, and purified by affinity chromatography. Binding of (--)-[3H]dihydroalprenolol to the membranes occurred to a single class of non-cooperative binding sites (0.2--0.3 pmol/mg protein) with a equilibrium dissociation constant (Kd) of 8 (+/- 2) nM. These sites were identified as the functional, adenylate-cyclase-linked beta 1-adrenergic receptors on the basis of: firstly, the fast association and dissociation binding kinetics at 30 degrees C; secondly, the stereospecific displacement of bound (--)-[3H]dihydroalprenolol by beta-adrenergic agonists and antagonists; and thirdly, the order of potencies for agonists to displace bound tracer (isoproterenol congruent to protokylol greater than norepinephrine congruent to epinephrine) similar to the one found for adenylate cyclase activation, and typical for beta 1-adrenergic receptors. Treatment of the membranes with the detergent digitonin solubilized 30% of the receptors in an active form. Digitonin solubilized also adenylate cyclase activity with a yield of 20 to 30%, provided the membranes were first treated with an effector known to produce a persistent active state of the enzyme: e.g. sodium fluoride. Binding sites for guanine nucleotides ([3H]p[NH]ppG) were solubilized as well. Their concentration (24 pmol/mg protein) was in large excess over the concentration of solubilized receptors (0.30--0.45 pmol/mg protein). Solubilized receptors were purified 500--2000-fold by affinity chromatography with a 25 to 35% yield, using an alprenolol-agarose affinity matrix. Affinity purified receptors were devoid of measurable adenylate cyclase activity and guanine nucleotide binding sites, thus showing that receptors and adenylate cyclase are distinct membrane constituents, and that guanine nucleotides apparently do not bind directly to the receptor molecules. Membrane-bound, solubilized and purified receptors were sensitive to inactivation by dithiothreitol, but not by N-ethylmaleimide, suggesting that receptors are at least partly constituted of protein molecules, with essential disulfide bonds.     

Immunologic mapping of the amino- and carboxy-termini of the turkey erythrocyte beta-adrenergic receptor: selective proteolysis of both domains.

Peptide-directed antibodies were used to map the N- and C-termini of the turkey erythrocyte beta-adrenergic receptor, the full length recombinant receptor expressed in Sf9 cells, and a mutant that terminates after residue 424 (T424). Both forms of the natural receptor (P40 and P50) were proteolytically clipped between residues 419 and 424. P40, but not P50, is also proteolyzed between residues 14 and 28. Truncation mutants, but not full length receptors, also display both large and small forms. The short form of T424 is formed by proteolysis after residue 14, but neither form is proteolyzed in the C-terminal region. The wild type recombinant receptor is not proteolyzed.     

Autophosphorylation profiling of Arabidopsis protein kinases using the cell-free system

Protein phosphorylation is one of the main process in the signal transduction pathway. In recent years, there has been increasing attention to plant phosphorylation signaling and many laboratories are trying to elucidate pathways using various approaches. Although more than 1000 protein kinase (PK) genes have been annotated in the Arabidopsis genome, biochemical characterization of those PKs is limited. In this work, we demonstrate high-throughput profiling of serine/threonine autophosphorylation activity by a combination of the 759N-terminal biotinylated proteins library, produced using a wheat germ cell-free protein production system, and a commercially available luminescence system. Luminescent analysis revealed that 179 of the 759 PKs had autophosphorylation activity. From these 179 PKs, 67 of the most active PKs were analyzed to determine their function using the PlantP database. This analysis revealed that 35 (53%) of the proteins were classified as non-transmembrane protein kinases, and 15 (23%) were receptor-like protein kinases. Additionally, PKs from Group 4.4-MAP3K, Group 1.6, Group 4.5-MAPK/CDC/CK2/GSK kinases and Group 1.10-receptor like cytoplasmic kinases contained the highest percentage of autophosphorylated activity. Next, to get a better overview of the annotated 67 PKs, we used the gene ontology annotation search on the TAIR website to classify the 67 PKs into functional category. As a result, some of these PKs may be involved in phospho-signaling pathways such as signal transduction, stress response, and the regulation of cell division. Information from this study may shed light on many unknown plant PKs. This study will be a basis for understanding the function of PKs in phosphorylation network for future research.     

The insulin receptor of the turkey erythrocyte. Characterization of the membrane-bound receptor.

The insulin receptor of the turkey erythrocyte has previously been shown to be very similar to that of the mammalian insulin receptors. As a first step in the isolation of this receptor a highly purified plasma membrane fraction has been prepared. The binding characteristics of the purified membrane-bound receptor were identical to those found with intact erythrocytes, but the membrane preparation had very little insulin-degrading activity. Isolation of the membrane by the methods described gave a 100-fold purification of the insulin receptor with 67% yield.     

Enhancing multiple disulfide bonded protein folding in a cell-free system

A recombinant plasminogen activator (PA) protein with nine disulfide bonds was expressed in our cell-free protein synthesis system. Due to the unstable and reducing environment in the initial E. coli-based cell-free system, disulfide bonds could not be formed efficiently. By treating the cell extract with iodoacetamide and utilizing a mixture of oxidized and reduced glutathione, a stabilized redox potential was optimized. Addition of DsbC, replacing polyethylene glycol with spermidine and putrescine to create a more natural environment, adding Skp, an E. coli periplasmic chaperone, and expressing PA at 30 degrees C increased the solubility of the protein product as well as the yield of active PA. Taken together, the modifications enabled the production of more than 60 microg/mL of bioactive PA in a simple 3-h batch reaction.     

Desensitization of the epidermal adenylate cyclase system: agonists and phorbol esters desensitize by independent mechanisms.

Exposure of pig epidermis to adenylate cyclase stimulators results in receptor-specific desensitization. We investigated the nature of the agonist-induced desensitization, which was compared with the phorbol ester-induced, receptor-nonspecific desensitization. Both phorbol ester-induced desensitization and the agonist-induced desensitization were accompanied by an increase in forskolin- and cholera toxin-induced cyclic AMP accumulations. The magnitude of the increase in the agonist-induced desensitization was parallel to the degree of the initial cyclic AMP accumulation; histamine and adenosine, which increase more cyclic AMP than epinephrine, resulted in a more marked increase in forskolin- and cholera toxin-induced cyclic AMP accumulations. Similarly, epidermis desensitized to multiple receptors revealed more marked forskolin- and cholera toxin-induced cyclic AMP accumulations than epidermis desensitized to a single receptor. In contrast to the phorbol ester-induced desensitization, agonist-induced desensitization was not affected by the protein kinase C inhibitors H-7 and staurosporin. Further, agonist-induced desensitization was still inducible in phorbol ester-desensitized epidermis and vice versa. In contrast to the agonist-induced desensitization, which is accompanied by the preceding adenylate cyclase stimulation, no evidence for the stimulation of the adenylate cyclase during phorbol ester treatment was obtained. Neither agonist-induced desensitization nor phorbol ester-induced desensitization affected the content of inhibitory guanine nucleotide binding protein of the epidermis, which was monitored by the pertussis toxin (IAP)-catalyzed ADP ribosylation reaction. Our results indicate that agonist-induced desensitization and the phorbol ester-induced desensitization are independent of each other. Although both processes are characterized by increased forskolin- and toxin-induced cyclic AMP accumulations, the former is accompanied by initial cyclic AMP accumulation; the latter is not.     

Mitogen-activated protein kinases phosphorylate nuclear lamins and display sequence specificity overlapping that of mitotic protein kinase p34cdc2.

Members of the mitogen-activated protein (MAP) kinase family are implicated in mediating entry of cells into the cell cycle, as well as passage through meiotic M phase. These kinases have attracted much interest because their activation involves phosphorylation on both tyrosine and threonine residues, but little is known about their physiological targets. In this study, two distinct members of the MAP kinase family (p44mpk and p42mapk) are shown to phosphorylate chicken lamin B2 at a single site identified as Ser16. Moreover, these MAP kinases cause depolymerization of in-vitro-assembled longitudinal lamin head-to-tail polymers. Ser16 was previously shown to be phosphorylated during mitosis in vivo, and to be a target of the mitotic protein kinase p34cdc2 in vitro. Accordingly, lamins were proposed to be direct in vivo substrates of p34cdc2. This proposal is supported by quantitative analyses indicating that lamin B2, when assayed in vitro, is a substantially better substrate for p34cdc2 than for MAP kinases. Nevertheless, a physiological role of MAP kinases in lamin phosphorylation is not excluded. The observation that members of the MAP kinase family display sequence specificities overlapping that of p34cdc2 raises the possibility that some of the purported substrates of p34cdc2 may actually be physiological substrates of MAP kinases.     

Cell-free desensitization of catecholamine-sensitive adenylate cyclase. Agonist- and cAMP-promoted alterations in turkey erythrocyte beta-adrenergic receptors

Conditions have been developed for desensitizing the beta-adrenergic receptor-coupled adenylate cyclase of turkey erythrocytes in a cell-free system. Desensitization is observed when cell lysates are incubated with isoproterenol or cAMP analogs for 30 min at 37 degrees C. Maximally effective concentrations of isoproterenol produce a 41.0 +/- 1.55% loss of iosproterenol-stimulated and a 15.0 +/- 2.35% loss of fluoride-stimulated enzyme activity. cAMP causes a 26.5 +/- 1.5% fall in isoproterenol-stimulated and a 21.5 +/- 4.4% fall in fluoride-sensitive activity. Desensitization by isoproterenol is dose-dependent, stereospecific, and blocked by the beta-adrenergic antagonist propranolol. Cell-free desensitization required ATP, Mg2+, and factor(s) present in the soluble fraction of the cell. Nonphosphorylating analogs of ATP did not support desensitization. Desensitization by agonist or cAMP in the cell-free system caused structural alterations in the beta-adrenergic receptor peptides apparent as an altered mobility of the photoaffinity labeled receptor peptides on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. As with the desensitization reaction, supernatant factors and ATP were also required for the agonist or cAMP-promoted receptor alterations. These data indicate that beta-adrenergic agonists promote a cAMP-mediated process which leads to receptor alterations and desensitization. The reactions involved in this process require ATP and soluble cellular factors. Additional processes must also occur to account for decreases in fluoride-sensitive enzyme activity. The availability of this cell-free system should facilitate elucidation of the molecular mechanisms involved in these processes.