Reconstitution of turkey erythrocyte beta-adrenergic receptors into human erythrocyte acceptor membranes. Demonstration of guanine nucleotide regulation of agonist affinity

Digitonin-solubilized turkey erythrocyte beta-adrenergic receptors were reconstituted by dialysis into human erythrocyte acceptor membranes which lack beta receptors. Incorporation of turkey beta receptors into acceptor membranes was directly proportional to the quantity of soluble protein added to the reconstitution system. Reconstituted beta receptors demonstrate saturable [125I]iodohydroxybenzylpindolol binding (Bmax = 11.1 +/- 0.8 fmol/mg, K = 77.8 +/- 8.6 pM) and stereospecificity ((-)-propranolol, K = 11.0 nM; (+)-propranolol, K = 2000 nM; (-)-isoproterenol, K = 250 nM; (+)-isoproterenol, K = 82 micro M). Reconstituted beta receptors appear to be incorporated into acceptor membranes as integral proteins. Reconstituted beta receptors cannot be extracted by high salt or pH (3 to 11); detergent is required for resolubilization of reconstituted beta receptors. Adenylate cyclase stimulation was not obtained in reconstituted membranes since acceptor membranes lack a catalytic subunit. However, guanine nucleotide regulation of agonist affinity was observed indicating a functional reconstitution. GTP (100 micro M) produces a 5-fold decrease in the affinity of isoproterenol for reconstituted beta receptors. Experiments with sulfhydryl reagents indicate that the reconstituted beta receptor couples with the guanine nucleotide regulatory protein of the acceptor membranes. These data describe the successful reconstitution of a beta receptor and indicate that the reconstituted beta receptor can interact with the GTP binding protein of human erythrocyte acceptor membranes.     

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.     

Catecholamine-stimulated GTPase activity in turkey erythrocyte membranes.

Determination of specific GTPase (EC 3.6.1.--) activity in turkey erythrocyte membranes was achieved using low concentration of GTP (0.25 muM), inhibition of nonspecific nucleoside triphosphatases by adenosine 5'(beta,gamma-imino-triphosphate (App(NH)p) and suppression of the transfer of gamma-32P from GTP to ADP with an ATP regeneration system. Under these conditions catacholamines caused a 30--70% increase in GTP hydrolysis. The stimulation of GTPase activity by catecholamines required the presence of Mg2+ or Mn2+. DIfferent batches of membranes revealed the following specific activities (pmol 32Pi/mg protein min): basal GTPase (determined in the absence of catecholamine), 6-- 11; catecholamine-stimulated TTPase, 3--7; and residual non-specific NTPase 3--5. The stimulation of GTPase activity by catecholamines fulfilled the stereospecific requirements of the beta-adrenergic receptor, and was inhibited by propranolol. The concentrations of DL-isoproterenol which half-maximally activated the GTPase and adenylate cyclase were 1 and 1.2 muM, respectively. The following findings indicate that the catecholamine-stimulated GTPase is independent of the catalytic production of cyclic AMP by the adenylate cyclase. Addition of cyclic AMP to the GTPase assay did not change the rate of GTP hydrolysis. Furthermore, treatment of the membrane with N-ethylmaleimide (MalNEt) at 0 degrees C which caused 98% inhibition of the adenylate cyclase, had no effect on the catecholamine-stimulated GTPase. The affinity and specificity for GTP in the GTPase reactions are similar to those previously reported for the stimulation of the adenylate cyclase. The apparent Km for GTP in the basal and the catecholamine-stimulated GTPase reaction was 0.1 muM. These GTPase activities were inhibited by ITP but not by CTP and UTP. It is proposed that a catecholamine-stimulated GTPase is a component of the turkey erythrocyte adenylate cyclase system.     

The regulation of adenylate cyclase activity in turkey erythrocyte membranes by forskolin

The mechanisms by which forskolin stimulates adenylate cyclase activity in turkey erythrocyte membranes and is influenced by manganese and Gpp(NH)p were studied. Forskolin-dependent adenylate cyclase activity in particulate turkey erythrocyte membranes is enhanced following preincubation of membranes with isoproterenol and GMP (cleared membranes). In contrast, solubilization of turkey erythrocyte membranes, previously cleared, renders them relatively refractory to forskolin but not to Gpp(NH)p. Whereas adenylate cyclase activity due to the simultaneous presence of forskolin and Mn2+ in particulate turkey erythrocyte membranes is additive, their copresence becomes synergistic after solubilization. The apparent Kact for forskolin activation of adenylate cyclase is not influenced by clearance or by the presence of Mn2+ in particulate turkey erythrocyte membranes. Following solubilization, the Vmax for forskolin-dependent adenylate cyclase activation determined in the presence of Mn2+ is also independent of clearance. Forskolin activation of turkey erythrocyte adenylate cyclase appears to be influenced at sites in addition to the catalytic unit.     

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.     

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.     

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.     

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 the turkey erythrocyte beta-adrenergic receptor in a cell-free system. Evidence that multiple protein kinases can phosphorylate and desensitize the receptor

We have used a recently developed cell-free system (cell lysate) derived from turkey erythrocytes to explore the potential role of cAMP-activated and other protein kinase systems in desensitizing the adenylate cyclase-coupled beta-adrenergic receptor. Desensitization by the agonist isoproterenol required more than simple occupancy of the receptor by the agonist since under conditions where adenylate cyclase was not activated, no desensitization occurred. As in whole cells, addition of cyclic nucleotides to the cell lysate produced only approximately 50% of the maximal isoproterenol-induced desensitization obtainable. Addition of the purified cAMP-dependent protein kinase holoenzyme plus isoproterenol to isolated turkey erythrocyte plasma membranes mimicked the submaximal desensitization induced in lysates by cAMP. This effect was entirely blocked by the specific inhibitor of the cAMP-dependent protein kinase. By contrast, maximal desensitization induced in lysates by isoproterenol was only approximately 50% attenuated by the protein kinase inhibitor. In the lysate preparations, isoproterenol was also shown to induce, in a stereospecific fashion, phosphorylation of the beta-adrenergic receptor. Phosphorylation promoted by isoproterenol was attenuated by cAMP-dependent protein kinase inhibitor to the same extent as desensitization (i.e. approximately 50%). Phorbol diesters also promoted receptor desensitization and phosphorylation in cell lysates. The desensitization was mimicked by incubation of isolated turkey erythrocyte membranes with partially purified preparations of protein kinase C plus phorbol diesters. In the cell lysate, calmodulin also promoted receptor phosphorylation and desensitization which was blocked by EGTA. Desensitization of adenylate cyclase by isoproterenol, phorbol diesters, and calmodulin was not observed to be additive. These findings suggest that: (a) multiple protein kinase systems, including cAMP-dependent, protein kinase C-dependent, and Ca2+/calmodulin-dependent kinases, are capable of regulating beta-adrenergic receptor function via phosphorylation reactions and that (b) cAMP may not be the sole mediator of isoproterenol-induced phosphorylation and desensitization in these cells.     

Adenosine receptor permanently coupled to turkey erythrocyte adenylate cyclase.

The mode of coupling of the adenosine receptor to adenylate cyclase in turkey erythrocyte membranes was probed by two independent approaches. The progressive inactivation of the adenosine receptor by an adenosine receptor affinity label resulted in the proportional reduction in the adenosine plus GppNHp dependent specific activity. In contrast, the intrinsic rate constant (k3), characterizing the process of adenylate cyclase activation by the adenosine-adenosine receptor complex, is independent of the extent of receptor inactivation. This behavior favors the precoupled mechanism, A + R.E: formula: (see text), where the receptor R and the enzyme E are permanently coupled to each other and the adenosine A binds to the receptor and induces the first-order process of cyclase activation to its active form ARE'. The finding that adenosine receptor is permanently coupled to the cyclase catalytic unit is corroborated by the observation that the progressive increase in membrane fluidity has no effect on the rate constant (k3) of adenylate cyclase activation by the adenosine-adenosine receptor complex and that the dose-response curve for adenosine is noncooperative.     

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.