Phorbol esters promote a1-adrenergic receptor phosphorylation and

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1 Pro. Natl. Aad. Si. USA Vol. 82, pp , September 1985 Biohemistry Phorbol esters promote a1-adrenergi reeptor phosphorylation and reeptor unoupling from inositol phospholipid metabolism (smooth musle/ddt, MF-2 ell line/ateholamines/afflinity hromatography/photoaffinity labeling) L. M. FREDRK LEEB-LUNDBERG*, SUSANNA COTECCHA*, JON W. LOMASNEY*, JOHN F. DEBERNARDSt, ROBERT J. LEFKOWTZ*t, AND MARC G. CARON* *Howard Hughes Medial nstitute, Departments of Mediine (Cardiology), tbiohemistry, and Physiology, Duke University Medial Center, Durham, NC 2771; and tdivision of Cardiovasular Researh, Abbott Laboratories, Abbott Park, North Chiago, L 664 Communiated by Joseph L. Goldstein, May 2, 1985 ABSTRACT DDT, MF-2 ells, whih are derived from hamster vas deferens smooth musle, ontain a1-adrenergi reeptors (54,8 ± 27 sites per ell) that are oupled to stimulation of inositol phospholipid metabolism. nubation of these ells with tumor-promoting phorbol esters, whih stimulate alium- and phospholipid-dependent protein kinase, leads to a marked attenuation of the ability of a,-reeptor agonists suh as norepinephrine to stimulate the turnover of inositol phospholipids. This turnover was measured by determining the 32p ontent of phosphatidylinositol and phosphatidi aid after prelabeling of the ellular ATP pool with 32p;. These phorbol ester-treated ells also displayed a derease in binding affinity of ellular al reeptors for agonists with no hange in antagonist affinity. By using affinity hromatography on the affinity resin Affi-Gel-A55414, the a, reeptors were purified =3-fold from ontrol and phorbol ester-treated 32P1-prelabeled ells. As assessed by NaDodSO4/polyarylamide gel eletrophoresis, the Mr 8, a,-reeptor ligandbinding subunit is a phosphopeptide ontaining 1.2 mol of phosphate per mol of a, reeptor. After phorbol ester treatment this inreased to 3.6 mol of phosphate per mol of a, reeptor. The effet of phorbol esters on norepinephrinestimulated inositol phospholipid turnover and a,-reeptor phosphorylation showed the same rapid time ourse with a til2 < 2 min. These results indiate that alium- and phospholipid-dependent protein kinase may play an important role in regulating the funtion of reeptors that are oupled to the inositol phospholipid yle by phosphorylating and deativating them. The mehanisms whereby hormones regulate ellular metabolism are of great urrent biohemial interest. n addition to the well-delineated, seond messenger-generating adenylate ylase system, another potentially quite general pathway has been demonstrated reently. This pathway involves reeptor-mediated stimulation of the breakdown of membrane polyphosphoinositides (phosphatidylinositol phosphates) to diaylglyerol and inositol trisphosphate (1-5). Both produts are potential seond messengers serving to stimulate alium- and phospholipid-dependent protein kinase (protein kinase C) (6, 7) and to mobilize intraellular alium (8, 9), respetively. Fators known to stimulate this pathway inlude agonists suh as aetylholine (1), angiotensin (11), and ateholamines ating via a1-adrenergi reeptors (1, 12-16). A powerful approah to the study of this signal transfer pathway has been the use of the tumor-promoting phorbol esters, whih appear to mimi endogenously produed diaylglyerol in ativating protein kinase C (17, 18), thus The publiation osts of this artile were defrayed in part by page harge payment. This artile must therefore be hereby marked "advertisement" in aordane with 18 U.S.C solely to indiate this fat bypassing the reeptor signal. By using this approah it has been demonstrated reently that reeptors for epidermal growth fator (19, 2), insulin (21, 22), and somatomedin C (21) are either diretly or indiretly phosphorylated by protein kinase C. Although in a few of these systems reeptor phosphorylation has been shown to orrelate with alterations in ligand binding (23-26), almost nothing is known about the possible relationship of these phosphorylation events to alterations in physiologial responsiveness. Moreover, the biohemial signal transfer pathways for these reeptors have not yet been learly eluidated. The adenylate ylaseoupled,3-adrenergi reeptors have also been shown to be a target of protein kinase C-mediated phosphorylation (27, 28). This ovalent modifiation of the f3 reeptor appears to unouple it from adenylate ylase stimulation, thus leading to "desensitization" of the reeptor response. Protein kinase C is presumably ativated subsequent to the stimulation of reeptors that lead to the breakdown of polyphosphoinositides. Thus, agonist-promoted protein kinase C-mediated phosphorylation of suh reeptors might serve as a mehanism for feedbak regulation of the funtions of those reeptors. n the present studies we sought to test whether the ativation of protein kinase C by phorbol esters leads to alterations of a1-adrenergi reeptor funtion. Our results indiate a striking phorbol ester-indued unoupling of a1-reeptor-mediated stimulation of phosphatidylinositol (Ptdns) turnover paralleled by phosphorylation of the a,- adrenergi reeptor protein in ultured DDT1 MF-2 smooth musle ells. MATERALS AND METHODS A55414 {4-amino-6,7-dimethoxy-2[4'-(4"-aminobenzyl)1- piperazinyliquinazoline; see also Fig. 3}, a prazosin analogue and a potent a1-adrenergi antagonist, was prepared at Abbott Laboratories, and its synthesis and biologial ativity will be desribed separately. Cell Growth and Labeling. DDT1 MF-2 ells were grown in 3-liter suspension ultures as desribed by Cornett and Norris (3). Cells (19) were then inubated in phosphate-free Dulbeo's modified Eagle's medium (DMEM) with 6 mci (1 Ci = 37 GBq) of 32P, in a total volume of 6 ml for 1 hr at 37 C. Aliquots of ells were inubated for various lengths of times with or without phorbol esters as indiated in the figure legends. nubations were terminated by diluting with 3 vol Abbreviations: HEAT, 2-[3-(4-hydroxyphenyl)ethylaminoethyl]tetralone; NE, norepinephrine; Ptdlns-P, phosphatidylinositol-4-phosphate; Ptdns-P2, phosphatidylinositol-4,5-bisphosphate; Ptdlns, phosphatidylinositol; PtdOH, phosphatidi aid; DMF, dimethylformamide; EDAC, 1-ethyl-3-(3-dimethylaminopropyl)arbodiimide; PMA, 4,3-phorbol 12f3-myristate 13a-aetate; apdd, 4a-phorbol 12,8,13p-dideanoate; BPDD, 4f3-phorbol 12,8,13a-dideanoate; ['25]APDQ, 4-amino-6,7-dimethoxy-2-{4-[5-(4-azido-3-[1251]iodophenyl)pentanoyl]-1-piperazinyl}quinazoline.

2 5652 Biohemistry: Leeb-Lundberg et al. of ie-old buffer (5 mm Tris HCl/1 mm NaF, ph 7.4) and entrifugation. The ells were washed one in the same buffer. Labeling of Phospholipids. Cells were washed with phosphate-free DMEM and inubated in the same buffer ontaining 32p; (2,uCi/ml) for 1 hr at 37C. Cells were washed and treated with or without phorbol esters. The ells were then inubated in the absene or presene of 1,M norepinephrine (NE) at 37TC in a total volume of.9 ml (3 x 16 ells). n the first series of experiments (Fig. 1), ells were washed one with ie-old buffer after treatment with phorbol esters and were further stimulated with 1 AM NE for 1 min. The same results were obtained when NE was oinubated with the phorbol esters. n the seond series of experiments (Fig. 5), in whih the effets of short-time phorbol ester treatment were investigated, ells were stimulated with 1 AM NE for 1 min following phorbol ester treatment. At 1 min of NE stimulation, phosphatidi aid (PtdOH) 32p labeling was the most onvenient parameter to follow. Phospholipid 32p labeling was stable up to 2 min after 32Pi removal. nubation with NE was terminated with 2 ml of ie-old methanol/hcl (1:1) and phospholipids were extrated aording to Agranoff et al. (31) and measured aording to Jolles et al. (32). The migration of the labeled phospholipids was verified with authenti phospholipids visualized with iodine vapor. Following thin-layer hromatography and autoradiography, the bands were ut and the 32p was quantitated. Membrane and Reeptor Purifiation. Whole ells (19) were lysed by resuspension in hypotoni buffer (5 mm Tris HCl/1 mm NaF/5 mm EDTA, ph 7.4) and membranes were pelleted at 45, x g for 2 min. The pellet was resuspended in 1 ml of buffer (5 mm Tris HCl/1 mm NaF, ph 7.4) and soniated by six 5-se bursts with 1 min between the bursts at 4 C. The membrane suspension was'diluted to 4 ml and entrifuged at 6 x g for 1 min. This supernatant was entrifuged at 45, x g for 15 min. The pellet was washed twie in the same buffer. The membranes were then solubilized by soniation for 9 se with 3.5 ml of 1% digitonin in 5 mm Tris'HC/48% glyerol/15 mm NaCl/5 mm EDTA, ph 7.4, inluding leupeptin (1,tg/ml), soybean trypsin inhibitor (1,ug/ml), and baitrain (2,ug/ml). The resulting suspension was diluted 1:2 with the above buffer laking digitonin and glyerol and was entrifuged at 45, x g for 9 min. The supernatant was frozen in liquid N2 and stored at -7 C. The soluble extrat (7 ml) was thawed and applied to 9 ml of Affi-Gel-A55414 (see below) preequilibrated with 3 olumn vol of 5 mm Tris HC/15 mm NaC/5 mm EDTA/1 mm NaF/.5% digitonin/24% glyerol, ph 7.4, inluding the above protease inhibitors. After entering the resin, the soluble extrat was allowed to equilibrate with the resin for 3 min at 25 C and then for 15 min at 4 C. The resin was then washed with 3 olumn vol of ie-old equilibration buffer laking glyerol but inluding 3 mm NaCl. After the temperature of the resin returned to 25 C ('15 min), the a1 reeptor was eluted with 3 olumn vol of the same buffer but inluding 2,M phentolamine. The phentolamine eluate was then onentrated to -1 ml on an Amion Centriflo ultrafiltration membrane'one by entrifugation at 6 x g. The onentrate was then desalted on a Sephadex G-5 olumn (.5 x 12 m). An aliquot of the eluate was used for determination of the reeptor number by 125-labeled 2-[3-(4-hydroxyphenyl)ethylaminoethyl]tetralone (125_ HEAT) binding and the rest was lyophilized to dryness and redissolved in.1 ml of NaDodSO4/PAGE sample buffer. Proteins were measured by the method of Lowry et al. (33) and the amido shwartz method of ref. 34. Radioligand Binding Assays. Soluble binding assays were performed by inubating 5 gl of reeptor preparation with a saturating onentration of 125-HEAT (29) (1.5-2 nm) in 5 mm Tris HC/15 mm NaCl/5 mm EDTA/.5% digitonin in Pro. Natl. Aad. Si. USA 82 (1985) a volume of 1,4 for 6 min at 25C. Nonspeifi binding was assessed in the presene of 1 jim prazosin. After inubation, bound 125-HEAT was separated from free HEAT by Sephadex G-5 hromatography. Whole ell reeptor binding assays were performed by inubating ells with 125-HEAT with and without ompetitor in phosphatebuffered saline inluding.1% bovine serum albumin in a volume of.4 ml for 3 min at 37C. The assays were terminated with ie-old buffer and were filtered on Whatman GF/C filters and filters washed with an additional 2 ml of buffer. Binding data were analyzed by nonlinear leastsquares omputer urve fitting and the signifiane of differenes between fitted parameters was tested as desribed (35). Photoaffinity Labeling. Photoaffinity labeling of purified plasma membranes was performed as desribed (36). Purified a,-reeptor preparations were photoaffinity labeled by inubation with 1 nm 4-amino-6,7-dimethoxy-2-{4-[5-(4-azido-3- [1251]iodophenyl)pentanoyl]-1-piperazinyl}quinazoline ([125]APDQ) in a volume of.5 ml overnight at 4C. The mixture was desalted on Sephadex G-5 olumns (.5 x 12 m) into 5 mm Tris HCl/.5% digitonin, ph 6.8. The eluate was irradiated for 9 se (36). The photolyzed preparation was lyophilized and then dissolved in.1 ml of NaDodSO4/ PAGE sample buffer. NaDodSO4/PAGE. Gel eletrophoresis was performed aording to the method of Laemmli (37) with 1% slab gels. Gels were dried and autoradiographed at -8 C with Kodak XAR-5 films and intensifying sreens. After autoradiographi visualization the gel was slied and rehydrated and the 32P was quantitated by liquid sintillation spetrosopy. Preparation of Affi-Ge-A Affi-Gel 22 (a suinylaminoalkyl derivative of rosslinked Bio-Gel A) was inubated with A55414 by using a ratio of 12 g (1.86 mmol of suinylaminoalkyl groups) of wet gel ake to 24 mg (.48 mmol) of A55414 in 12 ml of 5% dimethylformamide (DMF)/H2, ph 4.5, in the presene of 1-ethyl-3-(3-dimethylaminopropyl)arbodiimide (EDAC) (2.4 mmol). The same amount of EDAC was added again after 5 min and after 7 min. After 18 hr of inubation at 22 C the gel was allowed to reat for an additional 2 hr with glyine (2.4 mmol) and EDAC (2.4 mmol) to blok any unreated suinylaminoalkyl groups. The gel was then washed extensively with 1 liter of 5% DMF/H2, 2 liters of H2, 1 liter of 6 M guanidine HCl, 12 liters of H2, and 4 liters of 25 mm Tris HCl (ph 7.4). ATP Determination. The speifi ativity of the labeled ATP was determined as desribed (38) and was found to be.18 Ci/mmol. There was no hange in the speifi ativity of the ATP after phorbol ester treatment under the onditions of the experiments. RESULTS One of the early ellular events resulting from agonist oupany of a1 reeptors is a breakdown of membrane polyphosphoinositides [phosphatidylinositol-4-phosphate (Ptdns-P); phosphatidylinositol-4,5-bisphosphate (Ptdns- P2)] to diaylglyerol and inositol trisphosphate (1-5). Beause of rapid reeptor-mediated metabolism, this proess an be effiiently followed in ells in whih the intraellular ATP pool is labeled by 32P by monitoring the resynthesis of Ptdns and PtdOH. As shown in Fig. 1, inubation 4fDDT1 MF-2 ells preequilibrated with 32P, with the a,-reeptor agonist NE at 1,M resulted in a signifiant inrease (mean + SEM; n = 3) in 32p labeling of both Ptdns (34% + 1%) and PtdOH (121% + 12%). A small but signifiant inrease in the 32p labeling of Ptdns-P and Ptdns-P2 (1% ± 5%) also ourred, whereas the labeling of the other phospholipids was not altered. This stimulation by NE was ompletely bloked by 1 gm prazosin, indiating that the NE effet is a1-adrenergi in nature (data not shown).

3 Biohemistry: Leeb-Lundberg et al. Pro. Natl. Aad. Si. USA 82 (1985) 5653 C o 22 C 2 EC' 18 (O,' -u o 16 -= n.,14- E 12 n- D- Q ' ) 5o ) a, Q Z 1. Control. 1 JUM 1 gm 1 t~m PMA apdd PPDD Fi. 1. NE stimulation of phospholipid 32p labeling in ontrol and phorbol ester-treated DDT1 MF-2 ells. Cells were inubated with 32P, for 6 min, washed, and then treated with phorbol ester for 1 min. Ptdlns-P and Ptdns-P2, lear bars; Ptdlns, hathed bars; PtdOH, filled bars. The results represent the NE-stimulated inrease in phospholipid 32p labeling expressed as the perent of the labeling observed in the absene of NE (basal). Results are mean ± SEM of at least three experiments performed in tripliate. Under basal onditions Ptdlns-P and Ptdns-P2 = 14,565 ± 2883 pm per assay, Ptdlns pm per assay, and PtdOH = pm per assay (mean ± SEM) for ontrol ells. Phorbol ester treatment for up to 1 min did not alter these numbers signifiantly. Different from ontrol by two-tailed paired Student's t test: ***, P <.1; **, P <.1; *, P < C 4-. U, n V HEAT, pm 7 8 Preinubation of ells with ative phorbol esters for 1 min resulted in a drasti attenuation of the NE-promoted stimulation of Ptdns and PtdOH labeling. Fig. 1 shows that this attenuation ourred after preinubation with.1,m 4fphorbol 12f3-myristate 13a-aetate (PMA) or with 1,M 4,-phorbol 12,B,13a-dideanoate (,3PDD). By ontrast, the nontumor-promoting 4a-phorbol 12,B,13a-dideanoate (apdd) had no effet. ntat DDT1 MF-2 ells in suspension ulture and at a density of -3, ells per ml ontain 54,8 ± 27 (mean ± SEM; n = 8) a1 reeptors per ell, as determined by speifi 1251-HEAT binding. nubation of intat ells with tumor-promoting phorbol esters for 1 min resulted in apparent hange in the total number of a1 reeptors per ell or antagonist affinity for the antagonist 125-HEAT (Fig. 2 Upper). On the other hand, the poteny of the agonist NE in ompetition for 125-HEAT binding was redued, as shown in Fig. 2 Lower (see Fig. 2 Lower legend for EC5 and nh values). This effet of PMA was mimiked by 1,M f3pdd but not by apdd (data not shown). Sine phorbol esters ativate protein kinase C, we hypothesized that phorbol esters unouple a, reeptors from ellular metaboli events by ovalent phosphorylation of the a,- reeptor protein. To test this hypothesis, an affinity hromatography proedure for the purifiation of the solubilized a1 reeptor was developed. After disruption of DDT1 MF-2 ells and preparation of reeptor-rih membrane fragments (.8 pmol/mg), a, reeptors were solubilized with 1% digitonin with a yield of -3% (2.8 pmol/mg). Soluble a, reeptors were purified by affinity hromatography on the a,-reeptor affinity resin Affi-Gel-A As seen in Fig. 3, passage of a solubilized preparation over Affi-Gel-A55414 followed by a high-salt buffer wash resulted in -4% retention of the reeptors. Approximately 7% of the adsorbed reeptors were biospeifially eluted by 2 AM phentolamine, resulting in an inrease in the speifi ativity of a,-reeptor binding of -3-fold. The binding of 125-HEAT to the purified a1-reeptor preparations displayed appropriate a, pharmaologial speifiity (data not shown). As seen no 6 5 -logo[(-)-ne] FG. 2. Charateristis of a1-adrenergi reeptor binding in ontrol (o) and PMA-treated (o) DDT, MF-2 ells. (Upper) Saturation binding isotherm of 1251-HEAT. (Lower) Competition of NE for 1251-HEAT binding. The ells were treated with (o) or without (o) 1 nm PMA for 1 min. (Upper) The results shown are typial of three experiments with eah assay performed in tripliate. The omputerdrawn urves represent the best fit to the experimental data (35). The Kd values are 96 and 78 pm and the Bmax values are 94 and 85 fmol per 16 ells in ontrol and PMA-treated ells, respetively. (Lower) Binding assays were performed by using 1-2 pm 1251-HEAT. The results are the average of three experiments with eah assay performed in tripliate and are normalized to perentage of maximal speifi binding, whih represents 45 ± 6 and 43 ± 4 fmol per 16 ells (mean ± SEM; n = 3) in ontrol and PMA-treated ells, respetively. The omputer-drawn urves represent the best fit to the experimental data (35). The EC" values (mean ± SEM; n = 3) are 36 ± 11,uM and ,uM (P <.5) and the slope fator (nh) values are and.63 ±.7 (P <.1) in ontrol and PMA-treated ells, respetively. in Fig. 3 nset, the major a1-reeptor peptide labeled by [1251]APDQ in the purified preparations was of Mr 8,. Fig. 4A shows the results when the a1 reeptor was purified by affinity hromatography from 32P-labeled ells pretreated or not pretreated with ative phorbol esters and suh preparations were subjeted to NaDodSO4/PAGE. n the absene of phorbol esters (ontrol), purifiation yielded a phosphorylated peptide Of Mr 8,. Exposure of ells to 1 nm PMA resulted in an inrease in the 32p ontent of the M, 8, peptide. The ative 8PDD, but not the inative apdd, at 5 nm also inreased the 32p ontent of the same peptide. Fig. 4B shows the eletrophoreti pattern of the a1-reeptor photoaffinity labeled in purified DDT, MF-2 plasma membranes with the a1-reeptor probe [1251]APDQ in the absene (ontrol) and presene of 1,uM prazosin. Clearly, the majom speifi peptide labeled by [1251]APDQ omigrated with the

4 t-( H.;? 5654 Biohemistry: Leeb-Lundberg et al. i HGCOH 2-.-r)H 2 OC.-CNih N- - C 2 rh A,-.i,ds- -- "O Matrix A So C_ 75 <' 5 - u- 25 Crude soluble 67 -ow 43-- T i.Ṗass- High-salt Phentolamine through wash eluate FG. 3. Affinity hromatography of digitonin-solubilized a, reeptors from purified DDT1 MF-2 ell membranes on Affi-Gel- A The results are the average (mean ± SEM) of nine experiments. The speifi ativity of the starting material was in general 2.8 pmol/mg of protein, as determined from speifi l25i-heat binding and the amido shwartz protein assay. (nset) An example of a NaDodSO4/PAGE pattern of the phentolamine eluate photoaffinity labeled with [125]APDQ. Arrows on the left indiate moleular weights (shown as Mr x 1-3) of known proteins used as standards. Arrows on the right indiate the major a,-reeptor peptides. The upper part of the figure shows the hemial struture of the affinity resin. major phosphorylated peptide purified by the a1-reeptorspeifi affinity hromatography. nterestingly, the minor phosphorylated peptide at Mr = 35,-4, omigrated with the major proteolyti produt of the a, reeptor in these membranes. The phorbol ester-promoted phosphorylation of the Mr 8,Q a,-reeptor peptide resulted in an inrease in the 32p ontent ofthe reeptorfrom to mol of 32p per mol of a, reeptor (mean SEM; n = 4). The e 43 3 A İ. B otl FG. 4. Effet of phorbol esters on the phosphorylation of the a,-adrenergi reeptor peptides of DDT, MF-2 ells. (A) NaDodSO4/PAGE of3p-labeled reeptor peptides from DDT, MF-2 ells before and after phorbol ester treatment. For 32p labeling the ells were preinulbated with 32p; for 6 min prior to inubation with phorbol esters for 15 min. The amount of reeptor loaded on eah gel lane was.7 pmol. (B) NaDodSO4/PAGE of [1211]APDQ-labeled a, reeptor. Purified DDT, MF-2 ell membranes were inubated with ["2]APDQ in the absene (ontrol) or presene of 1 AuM prazosin and were proessed. Moleular weights of protein standards are shown as M, x 1-3. The experiment shown was performed twie with similar results m!c fo ;ZT Pro. Natl. Aad. Si. USA 82 (1985) i 67 TT T T o - t) C) mw i_ +-- a-ar Time of PMA treatment, min 1 -a ) )._ (a 5 t T ~ -o 25 E FG. 5. Time ourse of PMA-promoted inrease in a1-adrenergi reeptor (a1-ar) 32P labeling and attenuation of NE-stimulated PtdOH 32P labeling in DDT1 MF-2 ells. Data regarding the 32p labeling of the a, reeptor were obtained by proedures desribed in the legend to Fig. 4. (nset) An example of the NaDodSO4/PAGE autoradiogram of a 32P-labeling time ourse that was used to loalize the M, 8, a,-reeptor peptide and quantify its 32p ontent. The M, 67, protein standard is indiated. Data points are the average of at least two determinations with the PtdOH 32p labeling done in tripliate. reovery of reeptor ativity was similar for ontrol and PMA-treated ells. Fig. 5 shows the perent inrease in the 32p ontent of the Mr 8, reeptor peptide and the attenuation of NEstimulated PtdOH 32p labeling as a funtion of the time of PMA treatment. The time ourses of the two events losely paralleled eah other, with the half-maximal effet ourring within 2 min and the maximal effet beoming evident after =1 min. DSCUSSON Treatment of DDT1 MF-2 smooth musle ells with tumorpromoting phorbol esters leads to a striking and rapid alteration of a,-reeptor funtion, as evidened by the dereased NE stimulation of inositol lipid metabolism and a derease in the affinity of agonist binding to the a, reeptors. This altered funtion is losely paralleled by phosphorylation of the a1-adrenergi reeptor protein. The former effet is onsistent with a reent report that phorbol ester treatment leads to inhibition of a1-reeptor-mediated stimulation of hepati glyogen metabolism (39). The mehanisms by whih phosphorylation of the a1- reeptor protein leads to its unoupling from polyphosphoinositide metabolism and the agonist-speifi hanges in binding affinity are unlear. This may be due to ritial onformational hanges indued in the reeptor, whih hange its interation with its effetor system. Another possibility might be the triggering of internalization or sequestration of the reeptor protein away from its normal plasma membrane loation. Although diret ligand binding " () w z

5 Biohemistry: Leeb-Lundberg et al. with '25-HEAT showed no derease in the number of reeptors in intat ells, it is possible that this hydrophobi ligand penetrates the ell membrane and is able to bind to both surfae and sequestered reeptors. By ontrast, the hydrophili agonist may be unable to gain aess to sequestered reeptors, and, hene, its binding affinity in suh whole ell assays might be diminished. Just suh a mehanism has been demonstrated reently for the adenylate ylase-oupled f3-adrenergi reeptors (4). The hoie between these two possibilities will require further experimentation. Protein kinase C-mediated phosphorylation of the reeptor for transferrin appears to trigger its internalization, albeit perhaps for very different reasons (41). There is an obvious analogy in the ations of phorbol esters on a,- and f3-adrenergi reeptors, although these reeptors are oupled to different effetor systems (Ptdns turnover versus adenylate ylase). n eah ase the phosphorylated reeptor appears to be funtionally unoupled from its biologial effetor system (27, 28). However, for a, reeptors the protein kinase C is presumably stimulated physiologially as a result of diaylglyerol generated onsequent to Ptdns- P2 hydrolysis. Thus, this pathway represents a potential route for feedbak regulation of a,-reeptor ations just as the yli AMP-dependent protein kinase appears to regulate j3-adrenergi reeptors (42, 43). Sine numerous other agonists an also ativate the inositol phospholipid yle there are also many other possible pathways for regulating a,- reeptor funtion via the ation of these agonists. Moreover, these results raise the possibility that the physiologial funtion of many reeptors oupled to this pathway may be ontrolled by protein kinase C-mediated phosphorylation. Note. After submission of this manusript, two reports (44, 45) doumented phorbol ester-indued attenuation of an a1-adrenergi response in hepatoytes. We thank Dr. Per-Otto Hagen, in whose laboratories the phospholipid labeling experiments were performed, supported by National nstitutes of Health Grant HL15448; Dr. Ruth Strasser, for determining the speifi ativity of the ATP; and Lynn Tilley and Donna Addison, for their expert seretarial assistane. DDT, MF-2 ells were generously supplied by Dr. J. S. Norris of the University of Arkansas for Medial Sienes. S.C. is supported by a fellowship from Mario Negri nstitute, Milan, taly, and J.W.L. is supported by the Stanley J. Sarnoff Endowment for Cardiovasular Siene, n. 1. Mihell, R. H. (1975) Biohim. Biophys. Ata 415, Nishizuka, Y. 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