Vol. 121, No. 3, 1984 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS June 29, 1984 Pages 973-979
PHORBOL DIESTERS PROMOTE %-ADRENERGIC RECEPTOR PHOSPHORYLATION AND ADENYLATE CYCLASE DESENSITIZATION IN DUCK ERYTHROCYTES
David R. Sibley, Ponnal Nambi, Jack R. Peters and Robert J. Lefkowitz
Howard Hughes Medical Institute Research Laboratories Departments of Medicine (Cardiology) and Biochemistry
Duke University Medical Center Durham, North Carolina 27710
Received May 21, 1984
Preincubation of duck erythrocytes with tumor promoting phorbol diesters or catecholamines leads to attenuation of adenylate cyclase activity. 12-O-Tetradecanoyl phorbol-13-acetate (TPA) and phorbol 12,13-dibutyrate treatment induced a 38% and 30% desensitization of isoproterenol-stimulated adenylate cyclase activity, respectively. In contrast, the inactive phorbol diester, 4a-phorbol 12,13-didecanoate, was without effect in promoting adenylate cyclase desensitization. 51% desensitization. Incubation of
35' catecholamine isoproterenol induced a Pi labeled erythrocytes with TPA
promoted a 3- to O-fold increase in phosphorylation of the %-adrenergic receptor as did incubation with isoproterenol. Treatment of the cells with both TPA and isoproterenol together resulted in desensitization and receptor phosphorylation which were no greater than those observed with either agent alone. These data suggest a potential role for protein kinase C in regulating %-adrenergic receptor function.
The tumor-promoting phorbol diesters have been shown to directly affect a
variety of cellular functions when administered in vivo or in vitro. These
actions of phorbol diesters appear to be mediated by a Ca 2+
dependent protein kinase identified by Nishizuka and coworkers (1,2) and
termed protein kinase C. In the presence of concentrations of Ca 2+ comparable
to those found in most cells , protein kinase C also requires diacylglycerol
which is produced from phosphatidylinositol turnover in a receptor-dependent
fashion (2,3). Phorbol diesters can substitute for diacylglycerol by directly
binding to and activating protein kinase C (4-6).
Recently, treatment with the potent phorbol diester, 12-O-tetradecanoyl
phorbol-13-acetate (TPA)' has been shown to decrease %-adrenergic
responsiveness in mouse epidermis (7,8) and in cultured epidermal (9) and
astrocytoma (10) cells. In the mouse epidermis system, this TPA-induced
desensitization was shown to be due to uncoupling of the %-adrenergic
1 The abbreviations used are: TPA, 12-O-tetradecanoyl phorbol-13-acetate; PDB, phorbol 12,13-dibutyrate; 4a-PDD, 4a-phorbol 12,13-didecanoate; CYP, cyanopindolol; pABC, para-azidobenzylcarazolol; SDS, sodium dodecyl sulfate; ISO, isoproterenol; ALP, alprenolol; BSA, bovine serum albumin.
973 Copyright 0 1984 by Acudemic Press, Inc.
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Vol. 121, No. 3, 1984 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATtONS
receptors from adenylate cyclase with no change in receptor number (7,8).
This form of desensitization is similar to that seen in avian erythrocytes
where incubation with catecholamines promotes desensitization of adenylate
cyclase and uncoupling of the B-adrenergic receptors (11,12). Recently, we
have shown that, in turkey erythrocytes, the S-adrenergic receptor is a
phosphoprotein and that the degree of receptor phosphorylation is highly
correlated with the extent of catecholamine-induced desensitization of
adenylate cyclase (13,14). We now report that in duck erythrocytes phorbol
diesters can also promote desensitization of adenylate cyclase activity in
conjunction with phosphorylation of the 6-adrenergic receptor.
32 P Incorporation into Duck Erythrocyte S-Adrenergic Receptors: Washed
duck erythrocytes were suspended to 20% hematocrit in 157.5 mM NaCl, 2.5 mM KCl, 11.1 mM glucose, 10 mM HEPES, pH 7.4 (HEPES buffer) with 300 U/ml penicillin and 1 mgfml streptomycin ag2described by Sibley et al. (14). The 1&1s were incubated with 0.5 mCi/ml Pi at 41C for 20 hr. to allow maximal
P incorporation into the intracellular ATP. After the 20 hr. preincubation, the cells were further incubated with various phorbol diesters and/or isoproterenol for 3 hr. The cells were then washed three times with HEPES buffer and lysed by hypotonic shock in 5 mM MgCl , 5 mM Tris-HCl, pH 7.5. The membranes were isolated by centrifugation at 30,600 x g and washed three times by resuspension and centrifugation. Adenylate cyclase assays were performed using these membranes as previously described (14). The membranes were then solubilized with 2% digitonin, 100 mM NaCl, 10 mM Tris-HCl, pH 7.2 and the solubilized $-adrenergic receptors purified by alprenolol-Sepharose affinity chromatography as described (14). The affinFty purified receptor fractions were concentrated by ultrafiltration, desalted on Sephadex w 0 columns and assayed for maximal receptor binding activity with 300 pM [ IICYP (2,200 Ci/mmol, New England Nuclear Corp.) as previously described (14). Each sample was next lyophilized to dryness and resuspended in electrophoresis sample buffer containing 25 mM Tris-HCl, pH 6.8, 10% SDS, 10% glycerol and 5% B-mercaptoethanol.
Photoaffinity Labeling of Duck Erythrocyte Membranes: Duck erythrocyte membranes were diluted in 75 mM Tris-HCl, pH 7.5, 12.5 mM MerCl,, 1.5 mM EDTA (g$,fer A) to a receptor concentration of 30-50 pM and incuba& with 30-50 pM t I]pABC (2,200 Ci/mmol, New England Nuclear Corp.) for 90 min. at 25C in the dark. The incubation mixture was then diluted with Buffer A containing 0.5% fatty acid free bovine serum albumin (BSA) and centrifuged at 40,000 x g for 10 min. The washing with the BSA buffer was repeated twice and one final wash was performed without BSA. The membranes were then suspended in 15 ml of Buffer A and irradiated as described (15). The irradiated samples were pelleted by centrifugation and dissolved in electrophoresis sample buffer.
SDS Polyacrylamide Gel Electrophoresis: Gel electrophoresis was performed according to the method of Laemmli (16) as previously described (14) using 8% homogeneous slab gels. Upon completion of the electrbphoresis run, gels were dried prior to autoradiography at -70C for 24-36 hr. After visualization of the phosphorylated receptor bands, the gel was rehydrated, permeabilized with protosol gel solubilizer and the quantitated by liquid scintillation spectroscopy.
When duck erythrocytes are preincubated with the potent phorbol diester,
TPA, basal adenylate cyclase activity is unchanged (data not shown) whereas
Vol. 121, No. 3, 1984 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Figure 1. Desensitization of isoproterenol-sensitive adenylate cyclase of duck erythrocytes. The cells were preincubated with the indicated concentrations of desensitizing agents followed by preparation of membranes and assessment of basal, fluoride and isoproterenol-stimulated adenylate cyclase activity as described in ref. 14. Typical control enzyme activities were : basal, 4.5 pm01 CAMP/assay; isoproterenol-stimulated, 30 pmol CAMP/assay; fluoride-stimulated, 230 pmol cAi+/assay. The data represent the mean + SEM percent desensitization of the isoproterenol-stimulated activity from between 3-10 experiments.
isoproterenol-stimulated activity is attenuated by 38 + 2% (Figure 1).
Exposure of the cells to phorbol 12,13-dibutyrate (PDB) also desensitizes
isoproterenol-stimulated enzyme activity by 30 + 6% (Figure 1). In contrast,
preincubation with 4cc-phorbol 12,13-didecanoate, which is devoid of
tumor-promoting activity and which does not activate protein kinase C, fails
to promote desensitization in these cells (Figure 1). Incubation with the
catecholamine, isoproterenol, also promotes a 51 ? 7% desensitization of the
isoproterenol-stimulated response (Figure 1). Interestingly, when the cells
are exposed to both TPA and isoproterenol, the desensitization seen (49 + 5%)
is essentially nonadditive, never being greater than that observed with
isoproterenol alone (Figure 1). Both TPA and isoproterenol also promote
desensitization of fluoride-stimulated adenylate cyclase activity to a smaller
but still significant extent (14.5 + 3%, n = 10 and 12.5 ? 5%, n = 5,
Since the phorbol diesters promote desensitization of isoproterenol-
stimulated adenylate cyclase, we wished to examine their effects on
phosphorylation of the B-adrenergic receptor. This is tested by first
incubating the erythrocytes with 32 Pi in order to prelabel the intracellular
ATP pool followed by incubation with the desensitizing agent(s). Membranes
are then prepared and the receptors are solubilized with digitonin and
Vol. 121, No. 3, 1984 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
I 32P INCORPORATION  p ABC
Figure 2. SDS polyacrylamide gel electrophoresis of lz51]pABC labeled B-adrenergic receptor peptidfj?
3'P labeled and
52 from duck erythrocytes. For P labeling, the cells were preincubated with Pi prior to desensitization with the indicated agents as described in Methods. The percent desensitization of isoproterenol-stimulated adenylate cyclase activity in each sample was: 1 !JM TPA, 35%; 10 nM isoproterenol, 31%; 1 uM TPA + 10 UM isoproterenol, 39%.
the amount of receptor binding was . The amounts