Turkey Erythrocyte Ghosts Research Articles

Characterization of a membrane-associated, receptor and G-protein responsive phosphoinositide-specific phospholipase C from avian erythrocytes

We describe the reconstitution and purification of a membrane-associated phosphoinositide-specific phospholipase C (PIC) from turkey erythrocyte ghosts. This PIC is responsive to a G-protein coupled to P 2y purinergic receptors which are expressed in turkey erythrocytes. Reconstitution is achieved by adding partially purified PIC to [ 3H]inositol-prelabelled turkey erythrocyte membranes depleted of their endogenous PIC (acceptor membranes). PIC activity is associated with a 52 kDa polypeptide on SDS-polyacrylamide gel electrophoresis. Addition of a 307-fold purified enzyme to the acceptor membranes has no effect on basal PIC activity, but markedly increases the response to GTPγS and P 2y-purinergic receptor activation.

Association of a receptor and G-protein-regulated phospholipase C with the cytoskeleton

Approximately 98% of turkey erythrocyte phospholipase C (PLC) is cytosolic and is released by hypotonic lysis of the cells and extensive washing of the resultant erythrocyte ghosts. Well washed turkey erythrocyte ghosts retain a fraction of tightly associated PLC, which is activated by the P2y-purinergic receptor and G-protein present in ghost membranes. The particulate PLC is sufficient to couple to all the available purinergic receptor-regulated G-protein. In contrast to ghosts, turkey erythrocyte plasma membrane preparations contain no detectable PLC. To investigate the subcellular location of the ghost-associated PLC, cytoskeletons were prepared by Triton X-100 extraction of turkey erythrocyte ghosts. The ghost-associated PLC was quantitatively recovered in cytoskeleton preparations. Cytoskeleton-associated PLC was solubilized by sodium cholate extraction, partially purified, and shown to reconstitute with PLC-free plasma membrane preparations in an agonist and guanine nucleotide-dependent fashion, indicating that the cytoskeleton-associated PLC is G-protein-regulated. Dissociation of erythrocyte ghost cytoskeletons with the actin-binding protein DNase 1 resulted in a dose-dependent inhibition of agonist and guanine nucleotide-stimulated PLC responses in ghosts and caused release of PLC from ghost or cytoskeleton preparations. These data demonstrate the specific association of a receptor and G-protein-regulated PLC with a component of the detergent-insoluble cytoskeleton and indicate that the integrity of the actin cytoskeleton is important for localization and effective coupling of PLC to the relevant G-protein.

G-protein-mediated activation of turkey erythrocyte phospholipase C by beta-adrenergic and P2y-purinergic receptors.

Isoprenaline, previously known only to stimulate adenylate cyclase via the stimulatory G-protein, Gs, activates turkey erythrocyte ghost phospholipase C (PLC) in a dose-dependent manner when GTP or guanosine 5'-[gamma-thio]triphosphate (GTP[S]) is present. The effect is specific in that it is abolished by beta-adrenergic-receptor antagonists. Stimulation of adenosine receptors, which also couple to adenylate cyclase via Gs in turkey erythrocytes, does not activate PLC, indicating that the stimulation observed in the presence of isoprenaline is not due to Gs activation. Furthermore, the stimulation seen is independent of cyclic AMP production. Purified turkey erythrocyte PLC is activated in an adenosine 5'-[beta-thio]diphosphate (ADP[S]; a P2y-purinergic-receptor agonist)- or isoprenaline-regulated manner when reconstituted with turkey erythrocyte ghosts, demonstrating that a single species of PLC effector enzyme can be regulated by both the purinergic and the beta-adrenergic receptor populations present in turkey erythrocyte membranes. Pretreatment of intact turkey erythrocytes with the P2y agonist ADP[S] causes decreased PLC responsiveness of subsequent ghost preparations to ADP[S] stimulation, although responses to isoprenaline are unaffected (homologous desensitization). In contrast, pretreatment of intact erythrocytes with isoprenaline results in heterologous desensitization of both the P2y and the beta-adrenergic receptors. These effects occur at the level of receptor-G-protein coupling, since PLC stimulation by GTP[S] (which directly activates G-proteins) in the absence of agonists is unaffected.

A receptor and G-protein-regulated polyphosphoinositide-specific phospholipase C from turkey erythrocytes. II. P2Y-purinergic receptor and G-protein-mediated regulation of the purified enzyme reconstituted with turkey erythrocyte ghosts.

The preceding paper describes purification and properties of a 150-kDa polyphosphoinositide-specific phospholipase C from a cytosolic fraction of turkey erythrocytes (Morris, A. J., Waldo, G. L., Downes, C. P., and Harden, T. K. (1990) J. Biol. Chem. 265, 13501-13507). Turkey erythrocytes express a P2Y-purinergic receptor that employs an unidentified G-protein to activate phospholipase C (Boyer, J. L., Downes, C. P., and Harden, T. K. (1989) J. Biol. Chem. 264, 884-890; Cooper, C. L., Morris, A. J., and Harden, T. K. (1989) J. Biol. Chem. 264, 6202-6206). This paper describes receptor and G-protein regulation of the purified turkey erythrocyte phospholipase C after reconstitution of the enzyme using [3H]inositol pre-labeled turkey erythrocyte ghosts as acceptor membranes. These membranes contain polyphosphoinositides labeled to high specific radioactivity and display reduced responsiveness of their endogenous phospholipase C to P2Y-purinergic receptor agonists and guanine nucleotides. Reconstitution of purified enzyme had no effect on basal inositol phosphate production, but markedly increased P2Y-purinergic receptor agonist and guanine nucleotide-dependent accumulation of inositol phosphates. Reconstitution of 5 ng of purified phospholipase C with 10 micrograms of acceptor membrane protein produced half-maximal effects, and maximal activity was observed with reconstitution of 100 ng of purified enzyme. Agonist and guanine nucleotide-regulated phospholipase C activity measured using a reconstitution assay co-purified with phospholipase C activity detected using exogenously provided phosphatidylinositol 4,5-bisphosphate during purification of the 150-kDa protein. Only the maximal rate of inositol phosphate formation attained upon activation was increased in the presence of the purified phospholipase C. K0.5 values for adenosine 5'-O-(2-thiodiphosphate), guanosine 5'-3-O-(thio)triphosphate, and A1F4- activation of the purified enzyme were the same as for the endogenous phospholipase C activity of the acceptor membranes.

A receptor and G-protein-regulated polyphosphoinositide-specific phospholipase C from turkey erythrocytes. II. P2Y-purinergic receptor and G-protein-mediated regulation of the purified enzyme reconstituted with turkey erythrocyte ghosts.

A receptor and G-protein-regulated polyphosphoinositide-specific phospholipase C from turkey erythrocytes. II. P2Y-purinergic receptor and G-protein-mediated regulation of the purified enzyme reconstituted with turkey erythrocyte ghosts.

Guanine Nucleotide-sensitive Interaction of a Radiolabeled Agonist with a Phospholipase C-linked P2y-purinergic Receptor

Analogs of ATP and ADP produce a guanine nucleotide-dependent activation of phospholipase C in turkey erythrocyte membranes with pharmacological properties consistent with those of a P2y-purinergic receptor (Boyer, J. L., Downes, C. P., and Harden, T.K. (1989) J. Biol. Chem. 264, 884-890). This study describes the interaction of adenosine-5'-O-2-thio[35S] diphosphate ([35S]ADP beta S) with this putative P2y-purinergic receptor on purified plasma membranes prepared from turkey erythrocytes. In binding assays performed at 30 degrees C, the association rate constant of [35S] was 1.1 x 10(7) M-1 min-1 and the dissociation rate constant was 3.8 x 10(-2) min-1. [35S]ADP beta S bound with high affinity (Kd = 6-10 nM) to an apparently homogeneous population of sites (Bmax = 2-4 pmol/mg protein). ATP and ADP analogs (2-methylthio ATP, ADP beta S, ATP, ADP, 5'-adenylyl imidodiphosphate, alpha, beta-methylene adenosine-5'-triphosphate, and beta, gamma-methylene adenosine 5'-triphosphate) inhibited the binding of [35S]ADP beta S with properties consistent with ligand interaction by simple law of mass action kinetics at a single site. The rank order of potency for inhibition of [35S]ADP beta S binding was identical to the potency order observed for these same agonists for stimulation of phospholipase C in turkey erythrocyte ghosts. Guanine nucleotides inhibited [35S]ADP beta S binding in a noncompetitive manner with the following potency order: guanosine 5'-O-(3-thiotriphosphate) greater than 5'-guanylyl imidodiphosphate greater than GTP = GDP greater than guanosine 5'-O-2-(thiodiphosphate). The data are consistent with the idea that [35S]ADP beta S may be used to radiolabel the P2y-purinergic receptor linked to activation of phospholipase C in turkey erythrocyte membranes. In addition, interaction of radiolabeled agonist with the receptor is modified by guanine nucleotides, providing evidence that an agonist-induced receptor/guanine nucleotide regulatory protein complex may be involved in P2y-receptor action.

Short Chain Alcohols Activate Guanine Nucleotide-dependent Phosphoinositidase C in Turkey Erythrocyte Membranes

The ability of alcohols to regulate inositol lipid-specific phospholipase C (phosphoinositidase C) was examined in turkey erythrocyte ghosts prepared by cell lysis of erythrocytes which were prelabeled with [3H] inositol. Guanosine 5'-[gamma-thiotriphosphate] GTP[S] stimulated the production of both [3H]inositol bisphosphate (18-fold) and [3H]inositol trisphosphate (6-fold) in this system. The accumulation of [3H]inositol bisphosphate and [3H]inositol trisphosphate was linear up to 8 min following an initial lag period of 1-2 min. Ethanol (300 mM) reduced the lag period for [3H]inositol phosphate accumulation at submaximal GTP[S] concentrations and caused a shift to the left (3-fold) in the dose-response curve. Other short chain alcohols, methanol (300 mM), 1-propanol (200 mM), and 1-butanol (50 mM) also enhanced the accumulation of [3H] inositol phosphates in the presence of submaximal GTP[S] concentrations. Receptor activation by the purinergic agonist adenosine 5'-[beta-thio]disphosphate (ADP[S]) (10 microM) also reduced the lag period for [3H] inositol phosphate formation and shifted the GTP[S] dose response to the left (10-fold). In addition, ADP[S] increased the response to maximal GTP[S] concentrations. The formation of [3H]inositol phosphates induced by GTP[S] was associated with a concomitant decrease in labeling of both [3H]phosphatidylinositol monophosphate and [3H]phosphatidylinositol bisphosphate, but no decrease in [3H]phosphatidylinositol was observed. All of the alcohols tested enhanced the breakdown of [3H]polyphosphoinositides in the presence of GTP[S]. The dose response to guanosine 5'-[beta gamma-imino]triphosphate for [3H]inositol phosphate formation was displaced to the left by ethanol (300 mM) and ADP[S] (10 microM) (2- and 7-fold), respectively. ADP[S] also enhanced the maximal response to guanosine 5'-[beta gamma-imino]triphosphate. The [3H]inositol phosphate formation produced in response to NaF was unaffected by either ethanol or receptor activation. These results indicate that alcohols initiate an activation of phosphoinositidase C, mediated at the level of the regulatory guanine nucleotide-binding protein.

Agonist-mediated conformational changes of beta-adrenoceptors could occur independent of functional coupling to Ns.

Agonist and antagonist binding characteristics of beta-adrenoceptors in turkey erythrocyte ghosts were determined at different temperatures ranging between 7 degrees C and 42 degrees C. [3H]-DHA saturation binding experiments revealed that the antagonist-receptor interaction is entropy-driven with a small enthalpic contribution. Isoproterenol/[3H]-DHA competition binding followed the law of mass action at all the investigated temperatures. The agonist-receptor interaction is enthalpy driven with a small unfavorable decrease in entropy. This is consistent with the agonist's ability to favor an endoenergetic transconformation of the receptors. Only part of the agonist-bound receptors can undergo functional coupling to the stimulatory component of the adenylate cyclase system (Ns). This coupling process is associated with "locking-in" of the agonist and becomes persistent in the presence of the alkylating reagent N-ethylmaleimide. The number of agonist/N-ethylmaleimide-sensitive sites (i.e. coupling-prone receptors) increases with the temperature until it reaches a plateau value of 50% between 27-32 degrees C. Qualitatively similar data were obtained for rat lung and turkey erythrocyte membranes. These observations suggest that the whole receptor population can undergo agonist-mediated conformational changes but that only part of them can couple to Ns.

Reconstitution of a hormone-sensitive adenylate cyclase with membrane extracts from Neurospora and avian erythrocytes.

Adenylate cyclase activity associated to wild type Neurospora membranes is highly dependent on Mn2+ and insensitive to fluoride, guanyl nucleotides, and cholera toxin. These membranes are able to interact with components of detergent extracts from turkey erythrocyte ghosts. The reconstituted cyclase system is catalytically active in the presence of Mg2+ and it is activated by guanyl-5'-yl imidodiphosphate plus isoproterenol and fluoride. When detergent extracts were prepared from avian erythrocyte membranes treated with cholera toxin, the reconstituted system was stimulated by guanyl-5'-yl imidodiphosphate in the absence of isoproterenol and cyclase activities were higher than those observed with extracts from membranes not treated with the toxin. Dose-response curves for isoproterenol and fluoride in the reconstituted system were similar to those reported for avian erythrocyte and liver membranes, respectively.

CGP-12177. A hydrophilic beta-adrenergic receptor radioligand reveals high affinity binding of agonists to intact cells.

A new, hydrophilic beta-adrenergic receptor radioligand, (+/-)-[3H]CGP-12177 (4-(3-tertiarybutylamino-2-hydroxypropoxy)-benzimidazole-2-on hydrochloride), was synthesized and radiolabeled to 40 Ci/mmol. The nonspecific binding of this compound to turkey erythrocyte ghosts and C6 glioma cell membranes was less than 5% of the total binding at five times the appropriate KD. Binding assays of intact C6 glioma cells also showed low nonspecific binding, less than 20% of the total binding at five times the appropriate KD. The affinities of the antagonists (-)- and (+)-propranolol as well as of the agonist (-)-isoproterenol were examined by their potency to displace various radioligands from intact C6 glioma cell and membrane preparations. With membrane preparations, both [3H] CGP-12177 and [3H]dihydroalprenolol (DHA) were displaced stereospecifically by the antagonists (-)- and (+)-propranolol and the agonist (-)-isoproterenol. With whole cells, [3H]CGP-12177 and [3H]DHA behaved differently. [3H]DHA and [3H]carazolol could be stereospecifically displaced by antagonists but only partially displaced by agonists, while [3H]CGP-12177 could be completely displaced by both antagonists and agonists as in membranes. In contrast to [3H]CGP-12177, the lipophilic ligand [3H]DHA is actually taken up by cells. The inability of agonists to displace lipophilic radioligands from receptors on intact cells may not be due to a low affinity of agonists for receptors on cells, but to an agonist-induced change in the receptors which renders them inaccessible to hydrophilic agonists and antagonists. This change is likely to be their internalization into the cell.

Lateral mobility of phospholipids in turkey erythrocytes. Implications for adenylate cyclase activation.

Hormone activation of turkey erythrocyte adenylate cyclase is affected by temperature and by cis-vaccenic acid incorporation. This was correlated with changes in membrane "fluidity" which were previously assumed to affect lateral diffusion of the beta-adrenergic receptor (Rimon, G., Hanski, E., and Levitzki, A. (1980) Biochemistry 19, 4451-4460; Briggs, M. M., and Lefkowitz, R. J. (1980) Biochemistry 19, 4461-4466). To test this hypothesis, we measured the lateral mobility of the fluorescent phospholipid N-4-nitrobenzo-2-oxa-1,3-diazolyl-phosphatidylethanolamine (NBD-PE in turkey erythrocyte ghosts, using fluorescence photobleaching recovery. Ghosts labeled only in the external leaflet exhibited a linear Arrhenius plot of the lateral diffusion coefficient (D) with little change in the mobile fraction of NBD-PE between 45 and 5 degrees C. Labeling from both sides, however, yielded a break point (approximately 30 degrees C) in the Arrhenius plot and a 30% decrease in the mobile fraction of NBD-PE from 24 to 10 degrees C, indicating differences between the external and inner monolayers. The observed discontinuities are not due to gel-phase formation, since fluorescence depolarization of cis- and transparinaric acid did not reveal discontinuities at this temperature range. cis-Vaccenic acid incorporation did not markedly affect either D or the mobile fraction of NBD-PE at 25 degrees C. These results are discussed in view of the effects of temperature and cis-vaccenic acid incorporation on adenylate cyclase activation.

Preparation and characterization of hormone-sensitive, resealed erythrocyte ghosts.

A method for preparing resealed turkey erythrocyte ghosts is described which utilizes hypotonic lysis and resealing following restoration of isotonicity. The resealed ghosts are isolated above 55% sucrose. The resealed ghosts are shown to be capable of maintaining high intracellular K+ concentrations in the presence of a low K+ extracellular environment. When ATP and an ATP-regenerating system are included during the resealing stage, (R)-(-)-epinephrine- and NaF-stimulated cyclic AMP accumulation, which is linear for 20 min, can be demonstrated. The concentration of (R)-(-)-epinephrine producing a half-maximal response in resealed ghosts is 1.0 +/- 0.4 X 10(-6) M. This is the same as that for (R)-(-)-epinephrine in the intact erythrocyte. The resealed ghosts are impermeable to Ca2+, but Ca2+ inhibition of cyclic AMP accumulation is noted if the divalent cation ionophore. A-23187, is present or if Ca2+ is included during the resealing stage.

Binding of [ 3H]guanylylimidodiphosphate to membranes: Lack of correlation with adenylate cyclase activation

The binding of tritiated guanylylimidodiphosphate ([ 3H]GMP-P(NH)P) to turkey erythrocyte ghosts was studied in parallel with the activation by GMP-P(NH)P of adenylate cyclase. The high affinity binding capacity for GMP-P(NH)P, 50 pmoles per mg protein, exceeds the estimated quantity of adenylate cyclase of 1 pmole per mg of protein. The rate of nucleotide binding is not affected by isoproterenol. Further, in the presence of the hormone the rate of binding is much slower than the rate of activation. Although the rate of dissociation of bound [ 3H]GMP-P(NH)P is negligible at 37°, it is increased dramatically by unlabeled GMP-P(NH)P, GTP, EDTA, ATP, AMP-P(CH 2)P, or p-aminophenylmercuric acetate. In contrast, the rate of decay of the GMP-P(NH)P-simulated state is not altered by these agents. Thus, the major fraction of GMP-P(NH)P binding to membranes is not relevant to cyclase activation.

The interaction of catecholamines, Ca 2+ and adenylate cyclase in the intact turkey erythrocyte

Epinephrine stimulated adenylate cyclase in turkey erythrocyte ghosts is inhibited by calcium. The inhibition of adenylate cyclase is not apparent when intact erythrocytes are incubated with calcium and epinephrine. However, in the presence of the specific cation ionophore A 23187 and 5 m m Ca 2+, a 90% inhibition of epinephrine stimulated 3′,5′-adenosine monophosphate formation is found. The effect of catecholamines on calcium transport in the intact turkey erythrocyte was studied. Epinephrine causes a small but significant increase in Ca 2+ efflux. This effect is inhibited by propranolol. No effect of epinephrine on Ca 2+ uptake was observed. However, a 22% increase in Ca 2+ uptake in the presence of propranolol could be detected. The propranolol effect was found to possess high statistical significance ( p < .001). The absence of an epinephrine effect on influx probably reflects the presence of endogenous catecholamines in the control samples. It is proposed that the activation of adenylate cyclase by catecholamines occurs in two phases. The first phase is the increase of net Ca 2+ efflux from a crucial Ca 2+ pool, thus removing Ca 2+ from its inhibitory sites on the adenylate cyclase complex. The second phase is the activation of the deinhibited adenylate cyclase by the hormone.

The control of adenylate cyclase by calcium in turkey erythrocyte ghosts.

The adenylate cyclase of turkey erythrocytes is inhibited by low concentrations of calcium. Calcium binds to the enzyme system so tightly that the enzyme can compete with ethylene glycol bis(beta-aminoethyl ether)-N, N1-tetraacetic acid (EGTA) for the metal. The calcium binding site is shown to be distinct from the magnesium binding sites required for activity. Thus Ca2+ functions as a negative allosteric effector. Calcium decreases dramatically the V max of the catecholamine-stimulated activity without affecting the affinity for the hormone or for the substrate ATP. The cooperativity in the response toward Mg2+ dependence (Hill coefficient, nH equals 3) is also unaffected by Ca2+ where as the S0.5 (concentration yielding one-half V max) for Mg2+ is affected only slightly. The Ca2+ effect is cooperative (nH equals 2) and therefore brought about by a cluster of Ca2+ binding sites. Mn2+ can substitute for Mg2+ as the enzyme activator but the Mn2+-activated enzyme is no longer inhibited by Ca2+. The possible physiological significance of the Ca2+ effect is discussed.

Stereospecific binding of propranolol and catecholamines to the beta-adrenergic receptor.

The stereospecificity of the beta-adrenergic receptor of turkey erythrocyte ghosts was studied. Binding of the specific beta-adrenergic antagonist, propranolol, was found to occur exclusively with the l-stereoisomer. The stereospecificity of catecholamine binding was determined by assaying the ability of the catecholamines to displace [(3)H]propranolol. Binding of the catecholamines was also found to be stereospecific for the l-stereoisomers. Furthermore, the d-stereoisomers do not compete with the l form for binding. Using this displacement technique, we were able to calculate the dissociation constants for l-epinephrine, l-norepinephrine, and l-isoproterenol from the beta-receptor and compare these values to the apparent dissociation constants obtained from the direct activation of adenylate cyclase (EC 4.6.1.1) by these catecholamines.

The binding characteristics and number of beta-adrenergic receptors on the turkey erythrocyte.

Turkey erythrocyte ghosts (empty membranes) possess a class of receptors that can bind both L-[(3)H]isoproterenol and DL-[(3)H]propranolol. The binding of [(3)H]isoproterenol to these receptors occurs with a dissociation constant of 0.15 muM and can be fully inhibited by 1 muM propranolol. The binding of [(3)H]propranolol occurs with a dissociation constant of 2.5 nM and can be fully inhibited by 0.2 mM DL-isoproterenol. Ligand binding is sensitive to sonication, boiling, and 8 M urea. The cells possess 500 to 1000 beta-adrenergic receptors per cell. Binding of propranolol to the beta-receptor was found to be stereospecific for the L stereoisomer. If one assumed a 1:1 relationship between beta-adrenergic receptors and adenylate cyclase, the turnover number of this adenylate cyclase would be close to 100 min(-1).

β-adrenergic receptors: Stereospecificity and lack of affinity for catechols

Studies of adenylate cyclase activity in rat liver, heart and fat cell microsomal preparations and in turkey and rat erythrocyte ghosts indicate that β-adrenergic receptors exhibit very strict stereospecificity for (−)-catecholamines. (+)-Isomers of active catecholamines and inactive catechol compounds do not inhibit the β-adrenergic-mediated stimulation of adenylate cyclase and thus do not interact with specific receptors. However, very high concentrations (above 10 −4 M) of (−)- and (+)-isomers, as well as of biologically inactive non-catecholamine catechols (e.g., pyrocatechol, dihydroxymandelic acid), inhibit in a nonspecific manner the basal, hormone (catecholamine, glucagon)- and NaF-stimulated adenylate cyclase activity. Studies with propranolol suggest that the low activity (0.1 to 1%) of (+)-isomers of norepinephrine can be explained by contamination with the (−)-isomer. The activity of soterenol, a potent non-catechol β-adrenergic agonist, is uninfluenced by (+)-catecholamines or catechols. It is concluded that the binding of 3H-labeled catecholamines to a variety of cells, microsomes and membranes as described in various previous studies cannot represent specific receptor interactions. Binding to receptors must demonstrate strict stereospecificity and must not be affected by unrelated catechol substances.