Download pdf - Phorbol diesters promote β-adrenergic receptor phosphorylation and adenylate cyclase desensitization in duck erythrocytes

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+

and phospholipid

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.

0006-291X/84 $1.50

973 Copyright 0 1984 by Acudemic Press, Inc.

All ri,qhts of reproducfion in any form rexenaed.

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.

METHODS

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 41°C 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 25°C 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 -70°C 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.

RESULTS

When duck erythrocytes are preincubated with the potent phorbol diester,

TPA, basal adenylate cyclase activity is unchanged (data not shown) whereas

974

Vol. 121, No. 3, 1984 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

601

10’6M TPA

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,

desensitization, respectively).

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

975


94K-+

67K -

43K -

30K-e

I 32P INCORPORATION [“51] 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 of receptor loaded on the gel

were: control, 1.42 pmol; 1 PM TPA, 1.57 pmol; 10 UM ISO, 1.6 pmol; 32 1 !JM TPA + 10 UM ISO, 1.34 pmol. Subsequent to electrophoresis, the P incorporation into the receptor peptides ~2 s quantitated as described in Methods. The specific activities of the P labeled receptors in the samples were : control, 85 cpm/pmol; 1 PM TPA, 349l~~m/pmol; 1 UM TPA + 10 UM ISO, 339 cpm/p~s&. For [

10 PM ISO, 261 cpm/pmol~ I]pABC labeling, duck erythrocyte

membranes were incubated with [ IlpABC in the absence (control) or presence of 10 PM alprenolol (+ 10 PM ALP) and processed as described in Methods. The experiment shown was performed twice with similar results.

purified by affinity chromatography prior to characterization on SDS-PAGE.

Figure 2 shows the results of such an experiment. There is little 32P

incorporation into the receptor under control conditions, whereas after

exposure to TPA or isoproterenol two major phosphorylated peptides of

molecular weight Mr = 48,000 and Mr = 40,000 are clearly detectable. TPA and

isoproterenol increase the 32 P content of these peptides by 3-4 fold (Figure

2) * These peptides are identical to those observed when the photoaffinity

probe [ 125 I]pABC is used to visualize the receptors by covalent incorporation

in membranes (Figure 2). The labeling of these peptides by [ 125 I]pABC is

completely blocked by the B-adrenergic antagonist alprenolol indicating the

B-adrenergic receptor nature of these peptides (Figure 2). These

photoaffinity labeling results agree well with those previously reported for

duck erythrocyte B-adrenergic receptors (17).

976


In addition to the major Mr = 48,000 and Mr = 40,000 peptides, there is a

minor phosphopeptide seen at Mr = 34,000 which seems to be selectively

phosphorylated in response to TPA, and one at Mr = 30,000 whose

phosphorylation is promoted by both TPA and isoproterenol (Figure 2). Whether

or not these peptides represent receptor degradation products or are simply

nonreceptor contaminants is currently not known. Strikingly, when the cells

are exposed to both TPA and isoproterenol, the phosphorylation of the receptor

is not additive being no greater than that observed with either agent alone

(Figure 2). DISCUSSION

The major findings of the present study are: 1) that phorbol diesters

promote desensitization of isoproterenol-stimulated adenylate cyclase

activity; 2) that this desensitization is associated with phosphorylation of

S-adrenergic receptors and 3) that phorbol diesters and catecholamines are

nonadditive with respect to promoting adenylate cyclase desensitization and

receptor phosphorylation.

Several lines of evidence suggest that the phorbol diester-induced

desensitization is specific in nature. Firstly, the phorbol diesters display

the expected order of potency with TPA being more potent than PDB, and 4o-PDD,

which does not activate protein kinase C, being inactive (Figure 1).

Secondly, addition of TPA to the membranes during the adenylate cyclase assay

does not affect enzymatic activity (data not shown). Additionally, the

phorbol diester effects are seen primarily with the isoproterenol-stimulated

enzyme activity with either no or little desensitization of the basal and

fluoride activities, respectively. This is similar to the pattern observed

with catecholamine-induced desensitization (12). Finally, the desensitization

of adenylate cyclase by TPA is associated with a specific phosphorylation of

the S-adrenergic receptors.

It should be noted that both TPA and isoproterenol promote

phosphorylation of both B-adrenergic receptor peptides in proportion to their

relative preponderance as identified by [ 125 I]pABC labeling (Figure 2). This

suggests that neither peptide is selectively phosphorylated. Using turkey

erythrocytes, we have recently investigated the relationship between the two

S-adrenergic receptor peptides which are characteristically seen in avian

erythrocytes and found that the smaller peptide is derived from the larger

(18). We have also previously attempted to elicit phorbol diester-induced

desensitization in intact turkey erythrocytes with little success (14).

However, using a turkey erythrocyte lysate system (19), TPA does promote

desensitization similar to that seen in this study (Nambi, P. et al., in -- preparation). This suggests that intact turkey erythrocytes may be relatively

impermeable to phorbol diesters which must partition into intracellular

compartments in order to activate protein kinase C.

977


The fact that phorbol diesters, which activate protein kinase C, and

catecholamines, which activate CAMP-dependent protein kinase, promote

%-adrenergic receptor phosphorylation in a nonadditive fashion suggests a

common mechanism or pathway of action. One possibility is that both enzymes

phosphorylate the receptor on the same site(s). Another possibility is that

both enzymes phosphorylate the receptor at separate sites but that

phosphorylation of one set of sites inhibits the phosphorylation of the other

sites. Alternatively, both protein kinase C and CAMP-dependent protein kinase

might activate a third or additional kinase(s) which in turn phosphorylate the

receptor.

Phorbol diesters have now been shown to decrease the affinity and/or

responsiveness of a number of membrane receptors including those for EGF (ZO),

insulin (21) and al-adrenergic receptors (22). Recently, phorbol diesters

were demonstrated to promote phosphorylation of insulin (23) and EGF (24,25)

receptors which in the latter case resulted in diminished receptor tyrosine

kinase activity. The data in this study clearly demonstrate that phorbol

diesters can promote phosphorylation of the %-adrenergic receptor in

association with desensitization of adenylate cyclase. An exciting hypothesis

is that these phorbol diester promoted receptor phosphorylations represent a

newly recognized mechanism for heterologous desensitization which is

physiologically elicited through hormone-stimulated diacylglycerol production

and activation of protein kinase C.

ACKNOWLEDGEMENTS

We wish to thank Dr. James E. Niedel for providing the PDB and 4a-PDD, Dr. Marc G. Caron for helpful discussions and Donna Addison and Lynn Tilley for expert secretarial assistance. grants HL16037 and HL20339.

This work was partially supported by NIH D.R. Sibley is a recipient of NIH Postdoctoral

Fellowship HL06631. Fellowship, 1982-83.

J.R. Peters is a recipient of Peel Travelling Research

1.

2.

3.

4.

5.

6.

7. 8. 9.

10.

REFERENCES

Takai, Y., Kishimoto, A., Iwasa, Y., Kawahara, Y., Mori, T. and Nishizuka, Y. (1979) J. Biol. Chem. 254, 3692-3695. Kishimoto, A., Takai, Y., Mori, T., Kikkawa, U. and Nishizuka, Y. (1980) J. Biol. Chem. 255, 2273-2276. Sano, K., Takai, Y., Yamanishi, J. and Nishizuka, Y. (1983) J. Biol. Chem. 258, 2010-2013. Castagna, M., Takai, Y., Kaibuchi, K., Sano, K., Kikkawa, U. and Nishizuka, Y. (1982) J. Biol. Chem. 257, 7847-7851. Niedel, J.E., Kuhn, L.J. and Vandenbark, G.R. (1983) Proc. Natl. Acad. Sci. USA 80, 36-40. Leach, K.L., James, M.L. and Blumberg, P.M. (1983) Proc. Natl. Acad. Sci. USA 80, 4208-4212. Belman, S. and Garte, S.J. (1980) Cancer Res. 40, 240-244. Garte, S.J. and Belman, S. (1980) Nature 284, 171-173. Garte, S.J., Currie, D. and Belman, S. (1983) Carcinogenisis 4, 939-940. Mallorga, P., Tallman, J.F., Hennebury, R.C., Hirata, F., Strittmatter, W.T. and Axelrod, J. (1980) Proc. Natl. Acad. Sci. USA 77, 1341-1345.

978


11.

12.

13.

14.

15.

16. 17.

18.

19.

20. 21. 22.

23.

24.

25.

Hoffman, B.B., Mullikin-Kilpatrick, B. and Lefkowitz, R.J. (1979) J. - Cyclic Nucleotide Res. 5, 355-366. Stadel, J.M., De Lean, A., Mullikin-Kilpatrick, D., Sawyer, D.D. and Lefkowitz, R.J. (1981) J. Cyclic Nucleotide Res. 7, 37-47. Stadel, J.M., Nambi, P., Shorr, R.G.L., Sawyer, D.F., Caron, M.G. and Lefkowitz, R.J. (1983) Proc. Natl. Acad. Sci. USA 80, 3173-3177. Sibley, D.R., Peters, J.R., Nambi, P., Caron, M.G. and Lefkowitz, R.J. (1984) J. Biol. Chem., in press. Lavin, T.N., Nambi, P., Heald, S.L., Jeffs, P.W., Lefkowitz, R.J. and Caron, M.G. (1982) J. Biol. Chem. 257, 12332-12340. Laemmli, U.K. (1970) Nature 227, 680-685. Rashidbaigi, A. and Ruoho, A.E. (1981) Proc. Natl. Acad. Sci. USA 78, 1609-1613. Sibley, D.R., Peters, J.R., Nambi, P., Caron, M.G. and Lefkowitz, R.J. (1984) Biochem. Biophys. Res. Commun. 119, 458-464. Nambi, P., Sibley, D.R., Stadel, J.M., Michel, T., Peters, J.R. and Lefkowitz, R.J. (1984) J. Biol. Chem., 259, 4629-4633. Shoyab, M., De Larco, J.E. and Todaro, G.J. (1979) Nature 279, 387-391. Grunberger, G. and Gordon, P. (1982) Am. J. Physiol. 243, E319-E324. Corvera, S. and Garcia-Sainz, J.A. (1984) Biochem. Biophys. Res. Commun. 119, 1128-1133. Jacobs, S., Sahyoun, N.E., Saltiel, A.R. and Cuatrecasas, P. (1983) Proc. Natl. Acad. Sci. USA 80, 6211-6213. Cachet, C., Gill, G.N., Meisenhelder, J., Cooper, J.A. and Hunter, T. (1984) J. Biol. Chem. 259, 2553-2558. Iwashita, S. and Fox, C.F. (1984) J. Biol. Chem. 259, 2559-2567.

979