Use of cell fusion techniques to probe the mechanism of catecholamine-induced desensitization of adenylate cyclase in frog erythrocytes

The catecholamine-sensitive adenylate cyclase system appears to be comprised of at least three components; the beta-adrenergic receptor (R component), the catalytic unit of adenylate cyclase (C component) and a nucleotide regulatory protein (N component), responsible for mediating the effects of guanine nucleotides on the system. Cell fusion techniques were used to investigate the role of these three components in the process of homologous desensitization in the frog erythrocyte. Dicyclohexylcarbodiimide (DCCD) was used to inhibit beta-receptor function in one population of frog erythrocytes, whilst phenyl glyoxal was employed to inactivate the N and C components in a second population of frog erythrocytes. Using Sendai virus to fuse the two types of modified cell, heterologous beta-adrenergic receptor-adenylate cyclase systems were constructed which contained components from each cell type. When beta receptors from cells previously desensitized to catecholamines were coupled to N-C components derived from fresh erythrocytes, the resulting hybrid exhibited a densitized response to isoproterenol. By contrast, when beta-adrenergic receptors from fresh cells were coupled to N-C components derived from desensitized erythrocytes, no decreased responsiveness to isoproterenol was apparent in the hybrid. That this resensitization was the result of the addition of fresh beta-adrenergic receptors was demonstrated in a control experiment. Frog erythrocytes were desensitized simultaneously to catecholamines and prostaglandin E1 and modified with DCCD which inactivates the beta-adrenergic receptor but not the prostaglandin receptor. When fresh beta-adrenergic receptors were supplied by cell fusion to these doubly desensitized erythrocytes, only the beta-adrenergic response was restored to control levels. The response to prostaglandin remained desensitized in the hybrids, indicating that the observed resensitization of catecholamine-stimulated adenylate cyclase activity was specific and was due to the addition of fresh beta-adrenergic receptors. These data suggest that in the frog erythrocyte, homologous desensitization is primarily the result of receptor-related alterations.     

Association of sequestered beta-adrenergic receptors with the plasma membrane: a novel mechanism for receptor down regulation

Chronic exposure of frog erythrocytes to beta-adrenergic agonists leads to desensitization of the responsiveness of adenylate cyclase to isoproterenol and is accompanied by "down-regulation", a decrease in the number of beta-adrenergic receptors on the cell surface. When frog erythrocyte plasma membranes are prepared by osmotic lysis of cells, the receptors lost from the cell surface during desensitization can be recovered in a "light membrane fraction", obtained by centrifuging the cell cytosol at 158,000 X g for 1 hr. These receptors are sequestered away from the plasma membrane fraction which contains the adenylate cyclase and the guanine nucleotide regulatory protein. If desensitized frog erythrocytes are disrupted by gentler freeze/thaw procedures, however, the sequestered beta-adrenergic receptors can be demonstrated to be physically associated with the plasma membrane. Typically, plasma membranes prepared in this fashion do not demonstrate a significant down regulation despite attenuation of isoproterenol-stimulated adenylate cyclase activity. Under these conditions, beta-adrenergic receptors from control and desensitized preparations co-migrate on sucrose density gradients in exactly the same place as the plasma membrane marker, adenylate cyclase. In contrast, when membranes from osmotically lysed desensitized cells are fractionated on sucrose gradients the down regulated receptors are sequestered in a light membrane fraction which barely enters the gradient and which is physically separated from adenylate cyclase activity. The data are consistent with a novel mechanism of receptor down-regulation which appears to involve the sequestration of the beta-adrenergic receptors away from the cell surface into a membrane compartment which remains physically associated with the plasma membrane.     

Biochemical characterization of phosphorylated beta-adrenergic receptors from catecholamine-desensitized turkey erythrocytes

Isoproterenol-induced desensitization of turkey erythrocyte adenylate cyclase is accompanied (1) by a decrease in the mobility of beta-adrenergic receptor proteins, specifically photoaffinity labeled with 125I-(p-azidobenzyl)carazolol (125I-PABC), on sodium dodecyl sulfate (SDS)-polyacrylamide gels and (2) by a 2-3-fold increase in phosphate incorporation into the beta receptor [Stadel, J.M., Nambi, P., Shorr, R. G. L., Sawyer, D. F., Caron, M. G., & Lefkowitz, R. J. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 3173]. Analysis of 32P-labeled beta receptors partially purified by affinity chromatography and subsequently hydrolyzed in 6 N HCl revealed that the beta receptor from control erythrocytes contained only phosphoserine and that agonist-promoted phosphorylation of the receptor in desensitized cells occurred on serine residues. Comparison of limited-digest peptide maps of 125I-PABC-labeled beta receptors from control and desensitized erythrocytes reveals distinctly different sensitivities of the two beta receptors to cleavage by chymotrypsin and Staphylococcus aureus protease. The altered mobility of the 125I-PABC-labeled beta receptor from desensitized erythrocytes was eliminated when 5 M urea was included in the SDS-polyacrylamide gels. Limited-digest peptide mapping of 32P-labeled beta receptors from control and desensitized cells with the protease papain identified a unique phosphorylated peptide in desensitized preparations. Our results are consistent with the hypothesis that the altered mobility of beta-receptor proteins on SDS gels following desensitization is due to changes in conformation promoted by prolonged exposure to agonists.     

Desensitization of the beta-adrenergic receptor-coupled adenylate cyclase in cultured mammalian cells. Receptor sequestration versus receptor function

Human A431 and rat glioma C6 cells exposed to isoproterenol underwent a time- and dose-dependent loss of isoproterenol-stimulated adenylate cyclase activity. Desensitization was accompanied by sequestration of beta-adrenergic receptors, which became less accessible to the hydrophilic antagonist 3H-labeled 4-(3-tert-butylamino-2-hydroxypropoxy)benzimidazole-2-one hydrochloride ([3H]CGP-12177) and redistributed from the heavier density plasma membrane fraction to a lighter density membrane fraction. Prior treatment of the cells with concanavalin A or phenylarsine oxide blocked sequestration of the receptors but not desensitization of the agonist-stimulated adenylate cyclase. The membranes from such pretreated cells were exposed to alkali to inactivate adenylate cyclase, and the receptors were transferred to a foreign adenylate cyclase by membrane fusion with polyethylene glycol. beta receptors from desensitized cells exhibited a reduced ability to maximally stimulate the foreign adenylate cyclase, but remained accessible to [3H]CGP-12177 in the fused membranes. When isoproterenol-treated cells were washed free of agonist, there was a time-dependent recovery of agonist responsiveness and [3H]CGP-12177-binding sites. Using the fusion technique, the receptors recovered their functional activity in the resensitized cells. In concanavalin A-treated cells, desensitization and resensitization appeared to occur in the absence of receptor sequestration. Finally, membranes from desensitized cells pretreated with concanavalin A were fused with polyethylene glycol and assayed for agonist-stimulated adenylate cyclase. There was no reversal of the desensitized state. Thus, the primary, essential step in the desensitization process is a reduction in functional activity of the beta-adrenergic receptor. In contrast, sequestration of the receptors is not a prerequisite, but a secondary event during desensitization.     

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.     

Functional modification of the guanine nucleotide regulatory protein after desensitization of turkey erythrocytes by catecholamines

Densensitization of turkey erythrocytes by exposure to the beta-adrenergic agonist (-)isoproterenol leads to decreased activation of adenylate cyclase by agonist, NaF, and guanyl-5'-yl imido diphosphate, with no reduction in the number of beta-adrenergic receptors. Interactions between the receptor and the guanine nucleotide regulatory protein (N protein) also seem to be impaired. These observations suggest that a component distal to the beta-adrenergic receptor may be a locus of modification. Accordingly we examined the N protein to determine whether it was altered by desensitization. The rate at which (-)isoproterenol stimulated the release of [3H]GDP from the N protein was substantially lower in membranes prepared from desensitized cells, providing further evidence for uncoupling of the receptor and the N protein. The amount of N protein in membranes from control and desensitized cells was compared by labeling the 42,000 Mr component of the N protein with [32P]NAD+ and cholera toxin; no significant difference was found. However, significantly more N protein (p less than .001) was solubilized by cholate extraction of desensitized membranes, suggesting an altered association of the N protein with the membrane after desensitization. The functional activity of the N protein was measured by reconstitution of cholate extracts of turkey erythrocyte membranes into S49 lymphoma cyc- membranes. Reconstitution of (-)isoproterenol stimulation of adenylate cyclase activity was reduced significantly (p less than .05) after desensitization. These observations suggest that desensitization of the turkey erythrocyte by (-)isoproterenol results in functional modifications of the guanine nucleotide regulatory protein, leading to impaired interactions with the beta-adrenergic receptor and reduced activation of adenylate cyclase.     

Functional activity and regulation of human beta 2-adrenergic receptors expressed in Xenopus oocytes

The recently cloned human beta-adrenergic cDNA and several mutated forms have been expressed in Xenopus laevis oocytes by injection of RNA made from the cDNA under the control of the bacteriophage SP6 promoter. The cDNA and gene of the beta 2-adrenergic receptor possess the unusual feature of having a second upstream ATG (-101 base pairs) and a 19-codon open reading frame 5' to the initiator methionine codon of the receptor (Kobilka, B. K., Dixon, R. A. F., Frielle, T., Dohlman, H. G., Bolanowski, M., Sigal, I. S., Yang-Feng, T. L., Francke, U., Caron, M. G., and Lefkowitz, R. J. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 46-50). RNA lacking this upstream AUG and open reading frame was translated approximately 10-fold more efficiently both in an in vitro rabbit reticulocyte system and in oocytes. Injected oocytes but not water injected controls expressed typical beta 2-adrenergic receptors as assessed by ligand binding (450 fmol/mg membrane protein) and catecholamine-stimulated adenylate cyclase (approximately 20 fold). Moreover, these receptors displayed typical agonist-induced homologous desensitization when oocytes were incubated with isoproterenol at room temperature for 3-24 h. Among a series of mutations, truncations of the membrane-anchored core of the receptor eliminated receptor binding and cyclase stimulating activity. In contrast, disruption of one of the cAMP-dependent protein kinase phosphorylation sites or removal of the serine/threonine-rich carboxyl terminus had little or no effect on these functions or on the extent of agonist-induced desensitization relative to that observed with native receptor. These studies validate the beta 2-adrenergic nature of the cloned human beta-adrenergic cDNA, document the utility of the Xenopus oocyte system for studying functional and regulatory properties of receptors coupled to adenylate cyclase, and suggest the possibility that elements in the 5' untranslated region of the beta 2-adrenergic receptor RNA may regulate its translation in vivo.     

Alkaline phosphatase relieves desensitization of adenylate cyclase-coupled beta-adrenergic receptors in avian erythrocyte membranes.

Desensitization of adenylate cyclase-coupled beta-adrenergic receptors in avian erythrocytes results in a 40-65% decrease in agonist-stimulated adenylate cyclase activity and correlates with increased phosphorylation of beta-adrenergic receptors. To assess the role of phosphorylation in desensitization, membranes from isoprenaline- and dibutyryl cyclic AMP-desensitized turkey erythrocytes were incubated with alkaline phosphatase for 30 min at 37 degrees C, pH 8.0. In both preparations alkaline phosphatase treatment significantly decreased desensitization of agonist-stimulated adenylate cyclase activity by 40-75% (P less than 0.05). Similar results were obtained after alkaline phosphatase treatment of membranes from isoprenaline- and dibutyryl cyclic AMP-desensitized duck erythrocytes. Moreover, alkaline phosphatase treatment of membranes from duck erythrocytes desensitized with 12-O-tetradecanoylphorbol 13-acetate returned agonist-stimulated adenylate cyclase activity to near control values. In all experiments, inclusion of 20 mM-sodium phosphate to inhibit alkaline phosphatase during treatment of membranes attenuated the enzyme's effect on agonist-stimulated adenylate cyclase activity. In addition, alkaline phosphatase treatment of membranes from control and isoprenaline-desensitized turkey erythrocytes increased the mobility of beta-adrenergic-receptor proteins, specifically photoaffinity-labelled with [125I]iodocyanopindolol-diazirine, on SDS/polyacrylamide-gel electrophoresis. The increased mobility of the beta-adrenergic-receptor proteins after alkaline phosphatase treatment of membranes was again inhibited by 20 mM-phosphate. These results provide additional evidence for a direct role for phosphorylation in desensitization of adenylate cyclase-coupled beta-adrenergic receptors in avian erythrocytes.     

Role of glycosylation for beta 2-adrenoceptor function in A431 cells

A431 cells incubated with tunicamycin (0.15 micrograms/ml) for 40 h under conditions where incorporation of [3H] leucine into protein was inhibited less than 10% expressed mainly a beta-receptor species of about Mr 40,000 which was ascribed to the nonglycosylated form of the beta-receptor of about Mr 75,000 found in normal A431 cells by photoaffinity labeling. However, the tunicamycin-treated cells expressed the same number of specific beta 2-receptor-binding sites as untreated cells. Moreover, the aglycoreceptors had the same ligand binding properties as beta-adrenoceptors from control cells; but, functional tests of the receptor from tunicamycin-treated cells in reconstituted lipid vesicles showed that receptors from tunicamycin-treated cells had lost coupling efficiency. The coupling defect was at the receptor level since control experiments indicated that the other components of the signal transmission chain from beta-adrenoceptor to adenylate cyclase, the stimulatory regulatory GTP-binding protein of adenylate cyclase and adenylate cyclase, were fully functional. Homologous desensitization in tunicamycin-treated cells was characterized by export from the cell surface and sequestration of about the same number of beta-adrenoceptors as in normal desensitized cells but without further reduction of hormonally stimulated adenylate cyclase below the low level already attained in nondesensitized tunicamycin-treated cells. This was explained by assuming that the receptors removed in the course of homologous desensitization from the surface of tunicamycin-treated cells were already nonfunctional. Thus, beta-adrenergic desensitization in tunicamycin-treated cells is characterized by the functional disengagement of receptor removal and loss of adenylate cyclase activity.     

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.     

Differences between agonist and antagonist binding following beta-adrenergic receptor desensitization.

The specific beta-adrenergic agonist radioligand (+/-)-[3H]hydroxybenzylisoproterenol ([3H]HBI) was used to investigate alterations in the beta-adrenergic receptors of frog erythrocytes occurring during the process of agonist-induced, receptor-specific desensitization. There was close agreement between the percentage fall in [3H]HBI binding and that in catecholamine-stimulated adenylate cyclase activity following periods of preincubation of up to 7 h with 0.1 mM (-)-isoproterenol. Desensitization was maximal by 5 h, resulting in a 69% reduction in [3H]HBI binding and a 67% reduction in isoproterenol-stimulated adenylate cyclase activity. In contrast, binding of the beta-adrenergic antagonist (-)-[3H]dihydroalprenolol was significantly less affected by desensitization (p is less than 0.05 at 2 1/2, 5, and 7 h), showing a maximum reduction in binding of only 35% in these experiments. The consistent close agreement of reduction in agonist binding with that in hormone-stimulated adenylate cyclase activity, together with the significant difference observed between agonist and antagonist binding, implies that an alteration occurs during desensitization which preferentially interferes with agonist binding, while antagonist binding is less affected. The locus of this agonist-specific alteration may be the receptor binding site or a site involved in receptor-enzyme coupling. Agonist binding studies may now be used to assess more completely the desensitized state of beta-adrenergic receptors in systems in which marked desensitization of beta-adrenergic responses is associated with little or no reduction in antagonist binding.     

Functional desensitisation of beta-adrenergic receptors of avian erythrocytes by catecholamines and adenosine 3',5'-phosphate

Prolonged exposure to beta-adrenergic agonists of pigeon erythrocytes causes a reversible loss (70%) of catecholamine-stimulated adenylate cyclase activity without reduction in the number of beta-adrenergic receptors. In addition a less pronounced decrease in non-stimulated and NaF-stimulated adenylate cyclase activity (15-22%) is observed, appearing at different agonist concentrations and at a different rate. Dibutyryladenosine 3',5'-phosphate and the phosphodiesterase inhibitor methylisobutylxanthine partially mimick the action of the beta-adrenergic agonist, thus pointing to a possible role of adenosine 3',5'-phosphate in establishing desensitization. When adenylate cyclase from desensitized cells is stimulated with 5'-guanylyl-imidodiphosphate in the presence or absence of catecholamines the lag period preceding the attainment of maximal activity is extended. Likewise the rate of reversal by GTP or GTP of persistent activation of adenylate cyclase is slowed down. This is therefore interpreted to mean that the loss in hormonal stimulation on treatment of pigeon red blood cells with beta-adrenergic agonists is due to a delayed exchange of GDP against GTP on the regulatory GTP-binding protein. Furthermore, we conclude that events causing the refractory state in avian erythrocytes should occur at a site distal to the beta-adrenergic receptor.     

On the mechanism of isoproterenol-induced desensitization of adenylate cyclase in cultured differentiated hepatocytes

The adenylate cyclase of cultured differentiated RL-PR-C hepatocytes is desensitized to 1-isoproterenol by exposure to this beta-agonist. Virtually complete desensitization occurred by 60 min (intact cells) or 30 min (isolated plasma membranes). Isoproterenol was maximally effective at 10 micrometers, although substantial desensitization occurred at isoproterenol concentrations as low as 10 nM. Protein synthesis was not required for desensitization. Recovery from desensitization under tissue culture conditions was only 25% complete by 24 h. Maximum desensitization was accompanied by only a modest 35% decrease in binding sites (as determined by binding assays with [3H]dihydroalprenolol), with no change in binding affinity. Adenylate cyclase desensitized to 1-isoproterenol responded normally to guanine nucleotides and to fluoride, suggesting that the regulatory and catalytic proteins were not the sites of the desensitization "defect'. Using N-ethylmaleiimide to inactive the regulatory and catalytic proteins, and dicyclohexylcarbodiimide to inactivate the beta-adrenergic receptor, of intact hepatocytes, various heterologous cell fusion hybrids were produced, and their adenylate cyclases tested for responsiveness to 1-isoproterenol; only hybrids containing "desensitized' receptor failed to respond to isoproterenol. These results suggest that the mechanism of desensitization to isoproterenol involves only the receptor component of the receptor-regulatory protein(s)-adenylate cyclase complex, and that the receptors are reduced in number and/or ability to interact with the regulatory protein as a result of the desensitization process.     

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.     

Homologous desensitization of beta-adrenergic receptor coupled adenylate cyclase. Resensitization by polyethylene glycol treatment

Brief (approximately 20-min) exposure of S49 lymphoma cells to beta-agonists such as isoproterenol leads to a homologous form of desensitization in which beta-agonist but not prostaglandin E1-sensitive or NaF-sensitive adenylate cyclase is reduced. The desensitized receptors (R) appear to be sequestered away from the effector system (guanine nucleotide regulatory protein (Ns) and adenylate cyclase (C)). Membrane perturbants such as polyethylene glycol are known to reorient membrane proteins and lipids. Thus, we fused agonist-desensitized S49 lymphoma cells to each other, using polyethylene glycol as fusogen, in an attempt to functionally reunite the R, N, and C components which might have become sequestered in microdomains of the plasma membrane during desensitization. Such treatment completely restored isoproterenol-stimulated adenylate cyclase to normal and re-established the ability of R and N to functionally couple as assessed by the ability to form a high affinity, guanine nucleotide-sensitive state of the receptor. These results support the concept that agonist-promoted sequestration plays a functionally significant role in the homologous desensitization of the beta-adrenergic receptor.     

Homologous desensitization of adenylate cyclase is associated with phosphorylation of the beta-adrenergic receptor

We recently demonstrated that heterologous desensitization of adenylate cyclase in turkey erythrocytes is highly correlated with phosphorylation of the beta-adrenergic receptor. In contrast, little is known of the biochemical mechanisms underlying the homologous form of beta-adrenergic receptor desensitization, which is agonist-specific and not cAMP-mediated. Accordingly, the present studies were undertaken to examine if phosphorylation of the beta-adrenergic receptor is also associated with this form of desensitization in a well studied model system, the frog erythrocyte. Preincubation of these cells with the beta-adrenergic agonist isoproterenol leads to a 45% decline in isoproterenol-stimulated adenylate cyclase activity without significant changes in basal, prostaglandin E1-, NaF-, guanyl-5'-yl-imidodiphosphate-, forskolin-, or MnCl2-stimulated enzyme activities. There is also a 48% decline in [125I]iodocyanopindolol membrane binding sites. Conversely, preincubation of the cells with prostaglandin E1 attenuates only the prostaglandin E1-stimulated enzyme activity and does not affect [125I]iodocyanopindolol binding. Phosphorylation of the beta-adrenergic receptor was assessed by preincubating the cells with 32Pi and desensitizing them, and subsequently purifying the receptors by affinity chromatography. Under basal conditions there is about 0.62 mol of phosphate/mol of receptor whereas after desensitization with isoproterenol this increases to 1.9 mol/mol. This isoproterenol-induced receptor phosphorylation exhibits stereospecificity and is blocked by the beta-adrenergic antagonist propranolol. In addition, preincubation with prostaglandin E1 does not promote beta-adrenergic receptor phosphorylation. These data suggest that receptor phosphorylation is involved in homologous as well as heterologous forms of desensitization and may provide a unifying mechanism for desensitization of adenylate cyclase-coupled hormone receptors.     

Alpha 2-adrenergic receptor stimulation mobilizes intracellular Ca2+ in human erythroleukemia cells

Human erythroleukemia cells are a model system for studies of alpha 2-adrenergic receptors and their coupling to inhibition of adenylate cyclase (McKernan, R. M., Howard, M. J., Motulsky, H. J., and Insel, P. A. (1987) Mol. Pharmacol. 32, 258-265). Using Fura-2, we show that alpha 2-adrenergic receptor stimulation also increases intracellular Ca2+ in these cells by 80-250 nM. Although epinephrine only inhibited forskolin-stimulated cAMP generation when beta-adrenergic receptors were blocked, the Ca2+ increase was not affected by beta-adrenergic receptor blockade. The Ca2+ increase was not affected by forskolin or 8-bromo-cAMP. Thus, alpha 2-adrenergic receptors independently couple to elevation of intracellular Ca2+ and adenylate cyclase inhibition. Chelating all extracellular Ca2+ did not reduce the response, demonstrating mobilization of intracellular, rather than influx of extracellular Ca2+. The epinephrine-stimulated Ca2+ mobilization occurred prior to any detectable increase in inositol-(1,4,5)-trisphosphate. It was abolished by pretreatment with pertussis toxin (which blocks some G protein-mediated processes), but not by aspirin and indomethacin (which inhibit cyclooxygenase), nordihydroguaiaretic acid (which inhibits lipoxygenase), or Na+-free buffer (to block any Na+H+ exchange). We conclude, therefore, that alpha 2-adrenergic receptors on human erythroleukemia cells couple to mobilization of intracellular Ca2+ via a (pertussis toxin-sensitive) G protein-mediated mechanism that is independent of inhibition of adenylate cyclase.     

Catecholamine-induced desensitization in turkey erythrocytes: cAMP mediated impairment of high affinity agonist binding without alteration in receptor number

Desensitization of turkey erythrocyte adenylate cyclase by exposure of these cells to the beta-adrenergic agonist isoproterenol leads to a decrease in subsequent adenylate cyclase stimulation by isoproterenol, F-, or Gpp(NH)p without any apparent loss or down regulation of receptors (B.B. Hoffman et al. J. Cyclic Nucl. Res. 5: 363-366, 1979). We now report that the desensitization is associated with a functional "uncoupling" of the beta-adrenergic receptor. This is evidenced by an impaired ability of receptors to form a high affinity, guanine nucleotide sensitive complex with agonist as assessed by computer analysis of radioligand binding data. The changes in adenylate cyclase responsiveness as well as the alterations in receptor affinity for agonists are reproduced by incubation of turkey erythrocytes with the cAMP analog 8-Bromo-adenosine 3':5'- cyclic monophosphate. These findings suggest that one possible mechanism for the development of desensitization in adenylate cyclase systems may be a cAMP mediated alteration of a component(s) of the beta-adrenergic receptor-adenylate cyclase complex which results in impaired receptor-cyclase coupling.     

Regulation of adenylyl cyclase activity by beta-adrenergic agonists in a desensitization-resistant mutant cell line.

Mutant clones resistant to ACTH-induced desensitization of adenylyl cyclase (Y1DR) were previously isolated from the Y1 mouse adrenocortical tumor cell line. In this study, both parental Y1 cells (Y1DS) and a Y1DR mutant were transfected with a gene encoding the mouse beta 2-adrenergic receptor, and transfectants isolated from both Y1DS and Y1DR cells were shown to express beta 2-adrenergic receptors. These transfectants responded to the beta-adrenergic agonist isoproterenol with increases in adenylyl cyclase activity and steroidogenesis and changes in cell shape. The transfectants were analyzed to determine whether the Y1DR mutation was specific for ACTH-induced desensitization of adenylyl cyclase or also affected desensitization of adenylyl cyclase via the beta 2-adrenergic receptor. Treatment of intact Y1DS transfectants with isoproterenol caused a rapid desensitization of the adenylyl cyclase system to further stimulation by the beta-adrenergic agonist. Treatment of intact cells with isoproterenol did not affect ACTH-stimulated adenylyl cyclase activity, indicating that desensitization was agonist specific or homologous. Y1DR transfectants were resistant to the desensitizing effects of isoproterenol in intact cells as well as in cell homogenates. These results indicate that the mutation in Y1DR transfectants affects a component that is common to the pathways of isoproterenol-induced desensitization and ACTH-induced desensitization of adenylyl cyclase. As determined using the hydrophilic beta-receptor antagonist CGP-12177, isoproterenol caused a rapid sequestration of cell surface receptors in both Y1DS and Y1DR transfectants. From these results we infer that the DR phenotype does not arise from mutations affecting receptor sequestration and that receptor number does not limit the response to isoproterenol in these transfectants.     

Activation of the inhibitory GTP-binding protein of adenylate cyclase, Gi, by beta-adrenergic receptors in reconstituted phospholipid vesicles

beta-Adrenergic receptors and the inhibitory GTP-binding protein, Gi of the adenylate cyclase system were reconstituted into phospholipid vesicles by the method described previously for reconstituting receptors and the stimulatory GTP-binding protein, Gs (Brandt, D. R., Asano, T., Pedersen, S. E., and Ross, E. M. (1983) Biochemistry 22, 4357-4362). In the receptor-Gi vesicles, beta-adrenergic agonists stimulated both the high-affinity binding of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) to Gi and GTPase activity to an extent similar to that observed in vesicles containing beta-adrenergic receptors and Gs. Stimulation required receptors and displayed appropriate beta-adrenergic specificity. The prior treatment of receptor-Gi vesicles with islet-activating protein (pertussis toxin) plus NAD markedly inhibited both the isoproterenol-stimulated binding of GTP gamma S and the isoproterenol-stimulated GTPase activity. No contamination of Gi by Gs was apparent. These data suggest that receptors that typically stimulate adenylate cyclase activity may also activate the inhibitory system, perhaps as one mechanism of desensitization.