Vol. 268, No. 11,lasue of April 15, pp. 7753”7758,1993 Printed in U.S.A.
THEJOURNAL OF BIOLOGICAL CHEMISTRY
0 1993 by The American Society for Biochemistry and Molecular Biology, Inc.
Activation by G Protein @r Subunits of @-Adrenergicand Muscarinic Receptor Kinase* (Received for publication, August 17, 1992)
Kimihiko Kameyamal, Kazuko Haga, Tatsuya Haga, Kenji Kontanig,Toshiaki Katadag, and Yoshitaka Fukadan From the Department of Biochemistry, Institute for Brain Research, Faculty of Medicine, the University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan, the §Department of Life Science, Tokyo Institute of Technology, Yokohama 227, Japan, and the VDepartment of Biophysics, Faculty of Science, Kyoto University, Kyoto606, Japan
We have shown previously that GTP-binding regulatory protein (G protein) B-y subunits stimulate the agonist- or light-dependent phosphorylation of muscarinic acetylcholine receptors (mAChRs) and rhodopsin by a protein kinase partially purified from porcine brain (mAChR kinase) but not the phosphorylation of rhodopsin by rhodopsin kinase (Haga, K., and Haga, T. (1992) J. Biol. Chem. 267,2222-2227). We report here that the mAChR kinase phosphorylates &adrenergic receptors (8-ARs) purified from bovine lung in an agonist-dependent manner, and the phosphorylation is also stimulated by G protein & subunits. We also report that recombinant &adrenergic receptor kinase 1 (B-ARKl)expressed in COS-7 cells phosphorylates mAChRs (humanm2 subtype) and rhodopsinin an agonist- or light-dependent manner, respectively, and that this phosphorylation is stimulated by G protein j3-y subunits. By contrast, the B-y subunits do not stimulate the phosphorylation of mAChRs or rhodopsin by a 8ARK1 mutant lacking a part of the carboxyl-terminal region which is present in &ARKS butnot in rhodopsin kinase. These results indicate that the &ARK1 is the same as or very similar to the mAChR kinase but is distinguished from the rhodopsin kinase with respect to activation by the /3-y subunits and that the extra carboxyl-terminal sequence in &ARKS is required for the stimulation by the 8-y subunits.
Rhodopsin and @-adrenergic receptors (P-ARs)’ are known to be phosphorylated ainlight- or agonist-dependent manner by rhodopsinkinaseand@-adrenergicreceptorkinase (@ARK), respectively, and the phosphorylationis thought to be involved in their homologous desensitization (1-3). cDNAs for @-ARK1 and2 (4, 5) and rhodopsin kinase (6) have been cloned and shown to be similar to each other except for an extra region encoding 127 or 128 amino acid residues in the carboxyl terminus of @-ARKS. The agonist-dependent phosphorylationof muscarinic acetylcholine receptors(mAChRs)hasbeendemonstrated by
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ To whom correspondence should be addressed. Tel.: 81-3-56897331; Fax: 81-3-3814-8154. ‘The abbreviations used are: @-ARs,@-adrenergic receptors; @ARK, @-adrenergicreceptor kinase; mAChRs, muscarinic acetylcholine receptors; G protein,GTP-binding regulatory protein; G., G protein that stimulates adenylylcyclase;CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonicacid.
Kwatra et al. (7) using purified @-ARKand by us (8,9) using a protein kinase (mAChR kinase) thatwas partially purified from porcine cerebrum. The @-ARK and mAChR kinase have some common properties, including the inhibitory effects of heparin and salts, the lack of stimulatory effects of calcium or cyclic AMP, the recognition of rhodopsin as a substrate, (8-11).It is possible, and similar behaviors during purification however, that the purifiedP-ARK preparation as well as the mAChR kinase preparation contain several kinds of kinases belonging to the @-ARK family,and it is not known if P-ARs and mAChRs are phosphorylated by the same kinase or by distinct kinases thathave similar properties. Recently, G protein @-y subunits were found to stimulate the agonist- or light-dependent phosphorylationof mAChRs and rhodopsin by mAChR kinase (9, 11).On the other hand, the light-dependent phosphorylation of rhodopsin by the rhodopsin kinase was not stimulated by the & subunits (11,12), and it has notbeen reported if the phosphorylationof (3-ARs by the @-ARKS is stimulated by the P-y subunits or not. It remainstobe shown whetherthestimulation by the @r subunits is the exclusive property of a specific kinase phosphorylating mAChRs or whether this property is shared by kinases phosphorylating p-ARs. Present experimentswere undertaken to answer these questions,and we present evidence thatthesubunits also stimulate the phosphorylation of @-ARsby the mAChR kinase and the phosphorylation of mAChRs and rhodopsin by recombinant P-ARK1. We alsoprovideevidence that the pyresponsive site is located in the carboxyl-terminalregion of the @-ARK1. EXPERIMENTALPROCEDURES
Materials-[y3*P]ATP was purchased from Du Pont-New England Nuclear. Heparin and alprenolol were purchased from Sigma. Restriction enzymes were purchased from Toyobo Corp. The P-ARK1 cDNA waskindly donated by Dr. R. J. Leflrowitz, the m2 baculovims and Sf9 cells by Dr. E. M. Ross, and mammalian expression vector pEF-BOS by Dr. S. Nagata and Dr. T. Shimizu. Purification of mAChRs,0-ARs,Rhodopsin, G Protein Subunits, and mAChR Kinase-mAChRs were purified by single step affinity chromatography from Sf9 cells expressing human m2 subtypes as described previously (13, 14). @-ARswere partially purified from bovine lung by a method modified from methods used by Caron et al. (15) and Benovic (16). The membrane preparation derived from 1.3-kg lung (350 ml, 4.8 nmol of @-ARand 8.8 g of protein) was extracted with 0.3% digitonin in a buffer solution (10 mM Tris-HC1 (pH 7.4), 0.1 M NaC1, 5 mM EDTA, 0.5 mM benzamidine, 2.5 pg/ml pepstatin, 0.25 mM phenylmethylsulfonyl fluoride; total volume, 1.5 liters), andthenthe pellet was reextracted with the same buffer solution containing 1%digitonin and 0.13% sodium cholate (total volume, 1.6 liters). Approximately 60% of @-ARsin membranes were recovered in the second supernatant fraction after centrifugation for 45 min at 40,000 rpm. The
7753
7754
Activation of &ARK by G Protein Pr Subunits
supernatant fraction was applied to an alprenolol affinity column (500 ml); after washing the alprenolol column with a buffer solution, a small column of phenyl-Sepharose (10 ml) was connected to the outlet of the alprenolol column. After elution with 0.1 mM alprenolol solution, the phenyl-Sepharose column was separated from the alprenolol column and eluted with a 10 mM Tris-HCI buffer solution containing 1%digitonin and 1 p~ alprenolol. The fraction with the [3H]dihydroalprenolo1 binding activity was applied to a column of DEAE-Sephacel(1 ml), and B-ARs were eluted from the column with a 10 mM Tris-HC1 buffer solution containing 0.05% digitonin and 0.5 M NaCl. The recovery of P-ARs was typically 15% (700 pmol). Rhodopsin-rich membranes were purified from bovine retina as described previously (11). G protein By subunits of G, (11)and transducin (17) were purified from porcine brain and bovine retina, respectively. ,971 and By11were purified from bovine brain and separated from each other by using phenyl 5PW chromatography; the PyI were eluted ahead of 0711 (18). mAChR kinase was partially purified from porcine brain as described (11).Approximately 0.4 unit of mAChR kinase was used per assay, where 1 unit was defined as the amount of enzyme which transfers 1 pmol of phosphate/min when assayed in the presence of 20 pM ATP, 30 nM mAChRs, 50 nM By subunits, and 1 mM carbamylcholine. Construction and Expression of Mammalian Expression Vector of @-ARK1and &ARK1 Mutants-The Hind111 fragment of pP-ARK3A (4) was first inserted into the Hind111 site of the plasmid pUCl19 (pUC P-ARKl), and the XbaI-SpeI fragment (2.7 kilobase pairs) of this plasmid was then transferred to theXbaI site of the expression vector pEF-BOS (19)(pEF-P-ARK1). Mutant &ARK1 expression vector pEF-S-ARK1 562-632D (deleted amino acid residues of 562632) was prepared by deleting a 213-base pair DNA segment between EcoT22I and PstI restriction sites within the carboxyl-terminal segment of the j3-ARK1 cDNA as follows. First, a 0.6-kilobase PstI restrictionfragment encoding the carboxyl terminus of P-ARK1 cDNA and partof the 3"nontranslated region was ligated to EcoT22Idigested @-ARK1cDNA in pUC119. A 1.7-kilobase KpnI-NheI fragment from the resulting plasmid was then isolated and ligated to pEF-b-ARK1 that had been previously digested with KpnI and NheI. Mutants pEF-P-ARK1563T and pEF-0-ARK1590T (truncated amino acid residues from 563 or 590, respectively) were constructed by inserting termination codons following the EcoT22I and XhoI sites as follows. Oligonucleotides with the sequences 5"TGACTAGTTGATCAC-3' and 5'-TCGAGTGATCAACTAGTCATGCA-3'were synthesized,phosphorylated,annealed, andthen ligated tothe EcoT22I or XhoI site of pUC-P-ARK1. COS-7 cells were transfected with the resultant vector (pEF-j3ARK1 or mutant expressionvectors)(20pg/dish) or the original vector pEF-BOS using the calcium phosphate method (20). The cells in a 10-cm dish were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetalbovine serum for 72 h after transfection and then suspended in 2.5 ml of ice-cold lysis buffer (20 mM HepesKOH (pH 8.0), 5 mM EDTA, 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 1 pg/ml pepstatin, 0.1 mM benzamidine). The suspension was homogenized with a Teflon homogenizer and then centrifuged for 30 min a t 300,000 X g. The supernatant fraction was used as thesource of &ARK1 (2 pl/tube ina typical assay). Phosphorylation Reaction-Phosphorylation of mAChRs by mAChR kinase or &ARK1 was carried outas follows. Purified mAChRs (100 pmol in 20 pl) were mixed with crude lipids (0.3 mg) in HEN buffer (20 mM Hepes-KOH buffer (pH 8), 1 mM EDTA, 160 mM NaC1; 0.2 ml) containing 0.09% sodium deoxycholate and 0.02% sodium cholate. The mixture was passed through a column of Sephadex G-50 fine (2ml) preequilibrated with HEN, and thevoid volume fraction (0.4 ml) was collected and used as a substrate for the phosphorylation reaction. In the standard experiment, an aliquot of the void volume fraction (5 p l ) was incubated with a mAChR kinase preparation or a cytosol fraction from COS-7 cells, 1 p~ [T-~*P]ATP (5-10 cpm/fmol), Gprotein by subunits (2.5 pmol), and 1 mM carbamylcholine in a buffer solution (20 mM Tris-HCI (pH 7.5), 2 mM EDTA, 5 mM MgCl,,0.5 mM EGTA; total volume, 50 pl). In some experiments, the Sephadex procedure was omitted, and purified mAChRs (0.2-0.5 pmol) were directly subjected to phosphorylation in thepresence of crude lipids (2-3 pgltube). After incubation for 60 min at 30 "C, 25 pl of 5% SDS solution was added to the incubation mixture, and analiquot (45 pl) was subjected to SDS-polyacrylamide gel electrophoresis followed by autoradiography. Relative incorporation of 32Pinto individual bands was estimated by cutting the bands and counting by use of Cerenkov's effect or by using an Image
Analyzer (Fuji BAS2000, Fuji Film Corp.). Phosphorylation of P-ARs by the mAChR kinase was carried out as described for the phosphorylation of mAChRs except that P-ARs reconstituted in crude lipid were precipitated before being subjected to phosphorylation. The 8-ARpreparation was mixed with lipid and then passed throughasmallSephadex column. The void volume fraction containing P-ARs (18pmol) was mixed with an HEN buffer solution containing 0.1 mM isoproterenol and 10 mM dithiothreitol (total volume, 400 pl) andthen with a 50% polyethylene glycol solution (120 pl). After incubation for 10 min at room temperature, 2 ml of ice-cold HEN solution was added, and the mixture was centrifuged for 30 min at 60,000 rpm. The pellet was resuspended in a buffer solution (20 mM Hepes-KOH buffer (pH 8.0) and 1 mM EDTA; 50 pl) and an aliquot (1.5 pllassay) was used as a substrate for the phosphorylation reactions.
a f3y -SUBUNIT
(-) Isoproterenol
-
-
+
+
+ -
+
-
67 K +
b
N
0,
-8
-7 -6 -5 LOG [Ligand] (M)
(-) Isoproterenol
-4
(-) Isoproterenol (10 pM)
+ Alprenolol 0 -7 -6 -5 -4 -3
0 -8 -7 -6 -5 -4
FIG. 1. Phosphorylation of &ARs by the mAChR kinase. Panel a, effects of isoproterenol and G protein by subunits. @ - A h were partially purified from bovine lung and subjected to phosphorylation by mAChR kinase partiallypurified from porcine brain. A broad band with an apparent molecular size of 60 kDa was identified as pARs. The concentrations of P-ARs, G-protein Py subunits, and isoproterenol were 14 nM, 25 nM and 10 pM, respectively. Panel b, effects of different concentrations of isoproterenol and alprenolol on the phosphorylation of 8-ARs. The concentrations of P-ARs and G protein fly subunits were 14 nM and 25 nM, respectively.
Activation of &ARK by G Protein P-y Subunits
7755
RK RK + Py 0’4’ 10’ 20’ 0’4’ 10’ 20’
mAChR-
\
1
3 10 30 Subunit (nM)
FIG. 3. Effects of G protein B7 subunits on the heat inactivation of the mAChR kinase. The mAChR kinase ( R K , 0.54 unit, FIG. 2. Effects of different species of 87 subunits on the 20 pl) in a 20 mM Tris-HCI and 1 mM EDTA solution was mixed phosphorylation of mAChRs, b-ARs, and rhodopsin by the with G protein 0-y subunits in a 10 mM Tris-HC1,l mM EDTA, 1 mM dithiothreitol, and 0.7% CHAPS solution (2 pmol, 2 pl; closed circles) mAChR kinase. &I and I1 were purified from bovine brain and transducin 0-y subunits (Gt-07) frombovine retina. Phosphorylation or thebuffer solution (2 pl; open circles), incubated for indicated time a t 45 “C, cooled, and mixed with the buffer or 0-y subunits, respecof mAChRs(25 nM) and 0-ARs (14 nM) was carried out in the tively, and then used for the phosphorylation of mAChRs (1 pmol) presence of 1 mM carbamylcholine and 10 p~ isoproterenol, respectively, as described under “Experimental Procedures,” and the phos- as described under “Experimental Procedures.” After incubation for phorylation of rhodopsin (150 nM) under fluorescent light was as 30 min, the incorporation of [32P]phosphate into themAChR bands was measured. described previously (11). By
unknown and may be the kinase or the receptor. If the P-y subunits interactwith and activate the kinase, the stimulatory effects of the @?subunits should not depend on the species RESULTSANDDISCUSSION of receptor. Wethereforeexamined the ability of several @-ARswere partially purified from bovine lung and sub- different species of the @rsubunits to stimulate the phosjected to phosphorylation by the mAChR kinase in the pres- phorylation of three G protein-coupled receptors, P-ARs, ence or absence of isoproterenol and G protein @?subunits mAChRs, and rhodopsin. Fig. 2 shows the effect of two different @ysubunits purified (Fig. la). Several bands were found to be phosphorylated under the present experimental conditions, but only a broad from bovine brain (18) and transducin @?subunits from band witha molecular size of approximately 60 kDa was bovine retina (17) on thephosphorylation of @-ARs,mAChRs phosphorylated in a manner dependent on the presence of (m2 subtype),and rhodopsin. The phosphorylation of all three isoproterenol. The purified P-ARs preparation is known to substrates was increased 5-10-fold by the increase of concenyield a broadband with an apparentmolecular size of 64 kDa trations of @?Iand 11from 1to 30 nM, whereas the stimulation on sodium dodecyl sulfate-polyacrylamide gel electrophoresis by transducin @r subunits was much less for the phosphoryl(15, 16), supporting the identification of this phosphorylated ation of all three substrates compared with &I and 11. This band
[email protected] phosphorylation of @-ARswas markedly result iscompatible with the assumption that the@-y subunits subunits, and this interact with and activate the mAChR kinase, although the increased in the presence of G protein @r effect of the @-y subunits was observed only in the presence of possibility remains that each of three different @y subunits with a similar affinity and isoproterenol but not in its absence. The effects of different interacts with the three substrates concentrations of isoproterenol and alprenolol on the phos- that the mAChR kinase phosphorylates the 8-y subunitsphorylation of @-ARsare shown in Fig. lb. A half-maximal bound forms of substrates more efficiently than their free effect was observed a t approximately 1p~ isoproterenol, and forms. It is important to determine if the phosphorylation of low the stimulation by 10 p~ isoproterenol was antagonized by alprenolol with a half-maximal effect a t 200 nM. A low con- molecular weight peptide substrates is stimulatedby G protein ) the phosphorylation of @r centration of heparin (1p ~ inhibited subunits. For example, Palczewski et al. (21) reported that @-ARs(data not shown). These results provide evidence that rhodopsin kinase can phosphorylate peptide C, a peptide the mAChR kinase phosphorylatesthe agonist-bound form of derived from the carboxyl terminus of rhodopsin. Since rhoP-ARs, and the phosphorylation is stimulated by G protein dopsin was phosphorylated in a light-dependent manner by @-y subunits. The targetof the @-y subunits, however, remains mAChR kinase, we attempted touse mAChR kinase to phosPhosphorylation of rhodopsin by mAChR kinase andD-ARKl was carried outas described (11).
Activation of @ARK by G Protein @-y Subunits a
phorylate peptide C. However, we were not able to detect appreciable phosphorylation under our experimental conditions. In the future, will it be necessaryto identify appropriate CCh + + + Air + peptides that can be used as substratesfor the mAChR kinase. Palczewski et al. (21) reported that the phosphorylation of peptide C by rhodopsin kinase is greatly stimulated using an illuminated rhodopsin preparation that lackscarboxyl-terminal phosphorylationsites. This resultsuggests that rhodopsin mayserve asanactivatoras well as a substrate for rhodopsin kinase. If the same principle applies to the phosmKhR phorylation of mAChRs by the mAChR kinase, phosphorylation may be stimulated by mAChR mutants that lack phosphorylation sites. Direct evidence forthe interactionbetween the Pr subunits and the mAChR kinase has been obtained from experiments showing the effect of Pr subunits on the heatinactivation of mAChR kinase. The inactivation rate of the mAChR kinase at 45 "C was decreasedfrom 0.19 to 0.078 min"by coincubating the mAChR kinase with the P-y subunits (Fig. 3). Kinetic analysis of the light-dependent phosphorylation of rhodopsin by the mAChR kinase alsosupports the assumption that the subunits and rhodopsinbind the mAChRkinase independentlyand inarandom order (11). These results, taken together, suggest that G protein Py subunits directly activate a kinase(s) that phosphorylate P-ARs and mAChRs and raise the question of whether the subunits also stimulate the kinase activities of @-ARK1and/or P-ARK2. To answer these questions, we first carried out experiments todeterminewhethernative@-ARK1kinase produced in COS-7 cells phosphorylates the mAChRs and whether the phosphorylation is stimulated by the subunits. The results of theseexperimentsare summarizedin Figs. 4 and 5. mAChRs (m2 subtype) were found to be phosphorylated by extracts of @-ARK1-expressing COS-7 cells but not by extracts of control COS-7 cells (Fig. 4a). The phosphorylation 0 was dependent on the presence of carbamylcholine and was 0 11 9 7 5 stimulated 3-6-fold by the @rsubunits of G proteins (Gs). - log I &subunit 1 Theconcentration of thesubunits giving ahalf-maximal 4 b ) . The phosphorylation effect was approximately 3 nM (Fig. C -Bysubunit +Bysubunit( 50nM ) of mAChRs was inhibited by heparin, irrespective of the -log[heparinI 0 9 8 7 6 0 9 8 7 6 presence or absenceof the Pr subunits (Fig. 4c). The concen" tration of heparin yielding a half-maximal effect was 10 nM, consistent with the previous results (22). The concentration of carbamylcholine yielding a half-maximal effect was approximately 10 PM, and the concentration of atropine which decreased the effect of 300 PM carbamylcholine to 50% was rnAChR -+ 0 approximately 0.3-1 VM (Fig. 5). Effective concentrations of carbamylcholine and atropine were not changed by the addition of the @rsubunits. The stimulatory effect of the subunits was not observed in theabsence of carbamylcholine or in the presence of atropine. These resultsprovide evidence that&ARK1phosphorylatesthe agonist-bound form of mAChRs and that the phosphorylation is stimulated by G proteinsubunitsand suggest that @-ARK1can be distinguished from the rhodopsin kinase with respect to interaction subunits. FIG. 4. Phosphorylation of mAChRs by &ARK1 and its ac- with the tivation by G protein subunits. Panel a, mAChRs (m2 subtype) Amino acid sequencesof P-ARKS and rhodopsin kinase are were subjected to phosphorylation by extracts prepared from COS-7 reported to be similar to each other except that the@-ARK1 cells transfected with pEF-BOS (lanes 1 and 2 ) or with pEF-BOS (689 amino acid residues) is 128 amino acid residues longer containing cDNA encoding P-ARK1 (pEF-@-ARK) (lanes 3 and 4 ) . than the rhodopsin kinase (561 amino acid residues) at the The concentrationsof mAChRs, carbamylcholine (CCh)and atropine (Atr) in the reaction mixture were 33 nM, 1 mM, and 10 pM, respec- carboxyl terminus (4-6). We therefore constructed mutants tively. G protein /3rsubunits were not added to the reaction mixture. of @-ARK1 lacking allor part of this sequence and expressed Panel b, effect of G protein /3-y subunits (GJ. The concentrations of these in COS-7 cells. @-ARK1563T and P-ARK1 590T lack, PEF-BOS 1 2
pEF-WRK 3 4 + +
0-
L
I
mAChRs and carbamylcholine were 10 nM and 1 mM, respectively. The inset shows the autoradiogram of phosphorylated mAChRs; the concentrations of added /3r subunits were 0 (lane I ), 0.5 PM (lane 2), 5 pM (lane 3 ) , 50 pM (lane 4 ) , 500 pM (lane 5), 5 nM (lane 6),50 nM (lane 7), and 500 nM (lane 8 ) . Panel c, inhibition by heparin of the
phosphorylation of mAChRs by 8-ARK1. The concentrations of mAChRs and carbamylcholine were 10 nM and 1 mM, respectively.
Activation of P-ARK by G Protein respectively, the last 127 and 100 amino acid residues of the carboxyl terminus, andP-ARK1562-632D contains an internal deletion of 71 amino acid residues (562-632) within the carboxyl-terminal end of the protein (Fig. 6a). We could not detect kinase activity in extracts from cells expressing the mutants @-ARK15631' and P-ARK1 590T but could detect the phosphorylating activityof mAChRs in the extract from cells expressing P-ARK1562-632D. As shown in Fig. 6, b and c, the kinase activityof mutant P-ARKl islower than thatof native P-ARK1. The kinase activityof @-ARK1562-632D in the extract was more labile than that of native P-ARK1; the formeractivity decreasedrapidlyina few days after the extraction, but the latter activity was stable a t least for a month. The @-ARK1 5631' and @-ARK1 590T kinases may betoo labilefor theiractivitytobemeasuredunderthe present experimental conditions. The phosphorylation of mAChRs by P-ARK1 562-632D was dependent on carbamylcholine but was not stimulatedby G protein @?subunits (Fig. 66). This result differs from the phosphorylation by native @-ARK1which is dependent upon carbamylcholine and is stimulated by the Py subunits. The light-dependentphosphorylation of rhodopsin by @-ARK1 subunits, but was also found tobe stimulated by G protein @r the phosphorylation of rhodopsin by the P-ARK1 562-632D mutant was not stimulated by G protein subunits (Fig. 6c). Preliminary data indicate that the affinityof the kinase for rhodopsin was not changed by the mutation. These results suggest that part of the carboxyl-terminalsequence found in P-ARK1 but absent in rhodopsin kinase isfor stimulation of the kinase activityby the @-y subunits. The present results indicate that the phosphorylation of P-
7757
a
catalytic domain
1 BARK 1 1 -
rhodopsinkinase
BARK1 562-632D
618 A.A.
pEF-WIARK
lane CCh (1 rnM) Atr(10pM)
subunit (50nM)
lane light
subunit C5OnH)
PEF-wARKl 562-632D
I 2
3 4 + + + +
5
- + - -
7 8
-
+
- + - +
+
"
1 2 3 4
+ - + - - + +
.
6
+ + + +
+
pEF-WRK
C
By
689 A.A.
I
b
py
561A.A.
L I
+ +
P E F - W R K l 562-632D 5
6
I 8
+ - + - - + +
r a m .
.Pv
.bv
carbamylcholine -1oglCChI 0 6 5.5 5
Subunits
4.5 4 3.5 3 2.5
carbamylcholine(300pM) +atropine -loglAtrl 0 7 6 5 4 """
mAChR 3
+bv (50nM)
carbamylcholine -1ogICChI
0
6 5.5 5 4.5 4 3.5 3 2.5
" "~
rnAChR 3
-
FIG. 6. Panel a, structures of rhodopsin kinase, native O-ARKl, and mutantP-ARK1562-632D. Panel b, phosphorylation of mAChRs by 8-ARK1 and the mutant (%ARK1 562-632D. mAChRs were subjected tophosphorylation by extractsprepared from COS-7 cells transfected with pEF-BOS containingcDNAencoding @-ARK1(pEFP-ARK, lanes 1-4) or pEF-BOS containing cDNA encoding mutant @-ARK1 lacking amino acidresidues 562-632 (pEF-@-ARKl 562632D, lanes 5-8). Experimental conditionswere the same as described in the legend to Fig. 4b. CCh, carbamylcholine; Atr, atropine. Panel c, phosphorylation of rhodopsin by pEF-@-ARK(lanes 1-4) or pEFP-ARK1 562-632D (lanes 5-8).The phosphorylation was carried out +by (50nM) carbamylchol1ne(300~M) as described in the legend to Fig. 2except that COS-7-expressed +atropine kinases were used as described in the legend to Fig. 4. -loglAlrl 0 7 6 5 4
mAChR
+
FIG.5. Effects of differentconcentrations of carbamylcholine and atropine on the phosphorylation of mAChRs by the 8-ARK1. mAChRs were phosphorylated by @-ARK1 asdescribed in the legend to Fig. 4 except that various concentrations of carbamylcholine (CCh) and atropine (Atr)were used as indicated.
ARs as well as mAChRs is stimulated by G-protein @-y subunits and that the @?subunits stimulate the activity of the @-ARK1probably by interacting with the carboxyl-terminal subunits are known region of the kinase. G protein CY and @r to influence the activities of several distinct protein kinases by regulating the production of second messengers (23) but havenot previouslybeenshown tointeract with protein kinases directly. This is the first report to provide evidence that protein kinasesmay be targets for direct regulation by G protein @y subunits. Protein kinases can, therefore, be considered tobe G protein effectors. G protein @?subunits have been reported to regulate adenylylcyclase (23, 24), phospholipase AP (25), and ion channels (26). The present results indicate that the @rsubunits play a role in the homologous
Activation of P-ARK by G Protein
7758
desensitization of G protein-coupled receptors by activating receptor kinases. Acknowledgments-We thank Dr. R. J. Lefkowitz for donation of the &ARK1 cDNA, Dr. M. D. Summers for permission to use the baculovirus vectors, Dr. E. M. Ross for donation of the m2 baculovirus and Sf9 cells, Dr. K. Palczewski for peptide C, Dr. S. Nagata and Dr. T. Shimizu for the mammalian expression vector pEF-BOS, and Dr. D. W. Saffen for comments and for editing the manuscript.
Subunits
7. 8. 9. 10. 11. 12. 13. 14. 15. 16.
Addendum-After submission of this manuscript, Pitcher et al. (27) reported that G protein P-y subunitsenhanced the @-ARKmediated phosphorylation of @-ARsand rhodopsin but not a peptide substrate and that a 222-amino acid residue peptide in the carboxyl subunits. terminus of b-ARK binds G protein REFERENCES 1. Benovic, J.L., Bouvier, M., Caron, M. G., and Lefkowitz, R. J. (1988) Annu. Rev. CeU Bwl. 4,405-428 2. Palczewski, K., and Benovic, J. L. (1991) Trends Biochem. Sci. 1 6 , 387,201 " . , I
3. Kuhn, H.(1987) Prog. RetinulRes. 3,123-156 4. Benovic, J. C., DeBlasi, A,, Stone, W. C., Caron, M. G., and Lefkowitz, R. J. (1989) Science 246. 235-240 5. Benovic, J. C., Onorato, J.J., Arriza,J. L.,Stone, W. C., Lohse, M., Jenkins, N. A., Gilbert, D.J., Copeland, N. G., Caron, M. G., and Lefkowitz, R. J. (1991) J. Biol. Chem. 266,14939-14946 6. Lorenz, W., Inglese, J., Palczewski, K., Onorato, J. J., Caron, M. G., and Lefkowitz, R. J. (1991) Proc. Natl. Acad. Sci. U. S. A. 88,8715-8719
17. 18. 19. 20. 21. 22. 23. 24. 25. 3623-3672 26. Lo othetis, D. E., Kurachi, Y., Galper, J., Neer, E. J., and Clapham, D.E. 8987) Nature 325,321-326 27. Pitcher, J. A Inglese, J., Hig 'ns J B Arriza, J. L. Casey, P. J., Kim, C., Benovic, J.'L., Kwatra, M. &., karo;. M. G.. and hfkowitz. R. J. (1992) Science 2 6 7 , 1264-1267
