Phosphorylation of human m1 muscarinic acetylcholine receptors by G protein-coupled receptor kinase 2 and protein kinase C.

Human muscarinic acetylcholine receptor m1 subtypes (m1 receptors) were expressed in and purified from insect Sf9 cells and then subjected to phosphorylation by G protein-coupled receptor kinase 2 (GRK2) expressed in and purified from Sf9 cells and by protein kinase C purified from rat brain (a mixture of α, β, and types, PKC). The m1 receptor was phosphorylated by either GRK2 or PKC in an agonist-dependent or independent manner, respectively. G protein β subunits stimulated the phosphorylation by GRK2 but did not affect the phosphorylation by PKC. The number of incorporated phosphates was 4.6 and 2.8 mol/mol of receptor for phosphorylation by GRK2 and PKC, respectively. The number of incorporated phosphates was 7.5 mol/mol receptor for phosphorylation by GRK2 followed by PKC, but was 5.8 mol/mol of receptor for the phosphorylation by PKC followed by GRK2. Major sites phosphorylated by GRK2 and PKC were located in the third intracellular loop and the carboxyl-terminal tail, respectively. These results indicate that GRK2 and PKC phosphorylate different sites of m1 receptors and that the phosphorylation by PKC partially inhibits the phosphorylation by GRK2, probably by affecting activation of GRK2 by agonist-bound receptors.

Muscarinic acetylcholine receptors consist of five subtypes. The m1, m3, and m5 subtypes are linked to G q family G proteins and the m2 and m4 subtypes to G i /G o family G proteins (for reviews, see Refs. [1][2][3]. Muscarinic receptors as well as other G protein-coupled receptors are known to undergo desensitization following exposure to agonists (4 -6). Desensitization of receptors is generally believed to be mediated by receptor phosphorylation. Homologous desensitization is thought to be linked to agonist-dependent phosphorylation of receptors by G protein-coupled receptor kinase (GRK) 1 and heterologous desensitization to agonist-independent phosphorylation of receptors by second messenger-activated protein kinases such as cAMP-dependent protein kinase and protein kinase C (PKC).
GRK is a subfamily of serine and threonine kinases and is characterized by phosphorylation of only the stimulated forms of G protein-coupled receptors (for reviews, see Refs. [7][8][9]. The GRK family includes rhodopsin kinase (GRK1), ␤-adrenergic receptor kinases 1 and 2 (GRK2, GRK3) and GRK4, GRK5, GRK6. GRK1 and GRK2/3 have been characterized much more extensively than the other members. GRK1 phosphorylates rhodopsin in a light-dependent manner, and the light dependence is at least partly due to the activation of GRK1 by lightabsorbed rhodopsin (10,11). GRK2/3 are different from other GRKs in that GRK2/3 are activated by G protein ␤␥ subunits and have longer carboxyl termini, which are the sites that interact with ␤␥ subunits (12)(13)(14)(15). GRK2 is synergistically activated by ␤␥ subunits and mastoparan (16) and by ␤␥ subunits and agonist-bound receptors (17). Recently GRK2/3 have been reported to be activated by phospholipids as well as ␤␥ subunits, although there are some discrepancies among authors (18,19). GRK2 was originally isolated as a kinase that phosphorylates ␤-adrenergic receptors in an agonist-dependent manner, but is now known to phosphorylate different kinds of G protein-coupled receptors, including muscarinic m2 (17, 20 -22) and m3 (23), ␣2-adrenergic (␣2A and ␣2B) (24), and substance P (25) receptors. On the other hand, ␣1- (26) and ␣2C (24)-adrenergic receptors are reported not to be phosphorylated by GRK2. It remains to be known how the substrate specificity of GRK2 is determined.
Some contradictory results have been reported concerning the phosphorylation of m1 receptors. Muscarinic receptors purified from porcine brain, which contain m1 receptors as a major component, were phosphorylated in an agonist-dependent manner by a muscarinic receptor kinase that was purified from porcine brain and had properties similar to GRK2 (27). On the other hand, human m1 receptors expressed in and purified from insect Sf9 cells were not phosphorylated in an agonistdependent manner by either the muscarinic receptor kinase or GRK2 under the same conditions where m2 receptors purified from SF9 cells were phosphorylated in an agonist-dependent manner by the muscarinic receptor kinase or GRK2 (28). Richardson et al. (29) also reported that human m2 receptors expressed in Sf9 cells were phosphorylated in an agonist-dependent manner by an endogenous kinase, but human m1 receptors were not phosphorylated under the same conditions.
The m1 receptor as well as the m2 and ␣2 receptors have a long third intracellular loop that contains sequences similar to the putative phosphorylation sites in m2 (30) and ␣2 receptors (31). In fact, a peptide corresponding to the homologous se-quence in m1 receptors can be phosphorylated by GRK2, and the phosphorylation was markedly stimulated by G protein ␤␥ subunits and mastoparan (6). In addition, human m1 receptors were found to be phosphorylated by GRK2 in an agonistdependent manner in the presence of ␤␥ subunits and m2 or phosphorylation site-deleted m2 receptors (6). These results indicate that the phosphorylation of m1 receptors by GRK2 is enhanced when GRK2 is activated by ␤␥ subunits and mastoparan or by ␤␥ subunits and agonist-bound m2 receptors. This raises the question whether m1 receptors are unable to activate GRK2 even in the presence of agonist or the ability was impaired during the preparation of m1 receptors from Sf9 cells. We have extensively examined the purification procedure of m1 receptors and found that m1 receptors can be phosphorylated in an agonist-dependent manner by GRK2 after removing unknown factor(s) that copurify with m1 receptors.
PKC are known to phosphorylate muscarinic receptors purified from porcine brain (32), human m1 receptors expressed in and purified from Sf9 cells (33), and muscarinic receptors purified from chick heart (34). The present experiments were undertaken to determine whether GRK2 and PKC phosphorylation sites in human m1 receptors are independent or shared and whether the phosphorylation by one kinase affects the phosphorylation by another. We report here that different sites in m1 receptors are phosphorylated in an agonist-dependent and -independent manner by GRK2 and PKC, respectively, and that the phosphorylation by PKC reduces subsequent phosphorylation by GRK2.

EXPERIMENTAL PROCEDURES
Materials-Human m1 muscarinic receptors were expressed in and purified from Sf9 cells by single step affinity chromatography as described previously (35,36). Yields of m1 receptors in a typical experiment were 15 nmol of membrane receptors and 3-4 nmol of purified receptors starting from 10 liters of culture. GRK2 was expressed in and purified from Sf9 cells, as described previously (17). Antibody against a synthetic peptide corresponding to the carboxyl terminus of m1 receptors (residue 436 -460) was prepared as described (37). A fusion protein (I3-GST) of glutathione S-transferase and a peptide corresponding to the central part of the third intracellular loop of human m1 receptors (residues 226 -320) was expressed in and purified from Escherichia coli and was used as an antigen to produce rabbit antibodies. PKC was purified from rat or porcine brain as described previously (38), and a mixture of ␣, ␤, and ␥ types was used in the present experiments. Cholesteryl hemisuccinate (Tris salt), egg L-␣-phosphatidylcholine (type XV-E), and L-␣-phosphatidylinositol (ammonium salt) were purchased from Sigma and polyethylene glycol 6000 from Nakarai Chemicals.
Phosphorylation Reaction-Purified m1 receptors and G protein G o were reconstituted in lipid vesicles as follows (39,40). Cholesteryl hemisuccinate (10 mg/ml methanol, 16 l), phosphatidylcholine (20 mg/ml chloroform, 96 l), and phosphatidylinositol (20 mg/ml chloroform, 96 l) were mixed, dried under nitrogen, and suspended in 1 ml of HEN solution (20 mM Hepes-KOH buffer (pH 8.0), 1 mM EDTA, and 160 mM NaCl) containing 1% sodium cholate by sonication for 20 min at 4°C in a bath-type sonicator. The lipid suspension (100 l) was mixed with purified m1 receptors (72-90 pmol), G protein G o (360 -900 pmol), bovine serum albumin (25 g), carbamylcholine (1 mM), dithiothreitol (10 mM), MgCl 2 (10 mM) (total volume 200 l), and the mixture was passed through a small column of Sephadex G50 (fine) (2 ml). The void volume fraction (400 l) was used for phosphorylation of m1 receptors in some experiments. Recovery of m1 receptors and G o in the void volume fraction was 40 -70%. The void volume fraction was mixed with 1.1 ml of HEN solution containing 5 mM dithiothreitol and 1 mM carbamylcholine and then with 0.5 ml of 50% (w/v) polyethylene glycol 6000 (PEG), kept for 10 min at room temperature, and then centrifuged for 30 min at 50,000 rpm. The pellet was resuspended in 400 l of 20 mM Hepes buffer containing 1 mM EDTA and used as substrates for phosphorylation reactions by GRK2 and PKC. The m1 receptor was quantitatively recovered in the pellet by precipitation with PEG.
A standard assay tube for phosphorylation of m1 receptors by GRK2 contained the reconstituted vesicle (1 l; 1.8 -4.0 nM m1 receptors and 9 -40 nM G o in final concentrations), 1 mM carbamylcholine or 10 M atropine, 100 M GTP, 1 or 10 M [␥-32 P]ATP (2 ϫ 10 5 cpm/tube, 1-10 cpm/fmol), and purified GRK2 (13 nM) in a medium of 20 mM Tris-HCl (pH 7.5), 5 mM MgCl 2 , 2 mM EDTA, 0.5 mM EGTA (total volume, 40 l). G o and GTP were added to supply G protein ␤␥ subunits and were substituted by ␤␥ subunits in some experiments. A standard assay tube for phosphorylation of m1 receptors by PKC contained the same components as the above except that 2 mM EDTA and 0.5 mM EGTA were replaced by 0.2 mM CaCl 2 and GRK2 was replaced by PKC (4.2 nM). The phosphorylation reaction was carried out at 30°C and was terminated by addition of 20 l of 5% sodium dodecyl sulfate (SDS) solution containing medium for SDS-polyacrylamide gel electrophoresis (SDS-PAGE), followed by autoradiography. The band of m1 receptors was cut out and counted by the use of Cerenkov's effect.
Immunological Analysis-The m1 receptors phosphorylated with GRK2 or PKC were treated with different concentrations of trypsin at 30°C for 5 min, followed by addition of excess trypsin inhibitor. After addition of a 2.5% SDS solution, an aliquot was subjected to SDS-PAGE: the acrylamide concentration was 18% and 4.2 M urea was included in the gel. Electroblotting onto transfer membranes (FLUO-ROTRANS, PVM020C3R, Japan Genetics) was carried out for 1 h at 1.5-2 mA/cm 2 , as described previously (30). The blotted sheet was incubated with anti-i3 (10 l) or anti-C tail (30 l) antiserum in a phosphate-buffered saline containing 0.5% skim milk and 0.1% Tween 20 (10 ml) at 4°C overnight. The incubation with the secondary antibody (horseradish peroxidase-conjugated anti-rabbit IgG) and the detection with diaminobenzidine were carried out as described (22). In some experiments, phosphorylated receptors and peptides were precipitated with anti-i3 or anti-C tail antiserum before analysis by SDS-PAGE. After treatment with trypsin, digitonin was added to the reaction mixture to a final concentration of 0.1% and then the suspension (200 l) was mixed with anti-i3 (10 l) or anti-C tail serum (20 l) followed by incubation at 4°C overnight. After addition of Pansorbin suspension (5% w/v, 50 l) and incubation at 4°C for 2 h, the suspension was centrifuged for 10 min at 15,000 rpm, and the pellet was resuspended in a 2.5% SDS solution (50 l). An aliquot of the suspension was subjected to SDS-PAGE in an 18% acrylamide gel containing 4.2 M urea, followed by autoradiography. Molecular weights of small peptides were estimated from the mobility of marker proteins and a phosphorylated synthetic peptide corresponding to the carboxyl terminus of m1 receptors (residue 435-460).

RESULTS
Phosphorylation by GRK2-Human m1 receptors purified from Sf9 cells were reconstituted with G proteins in lipid vesicles and then subjected to phosphorylation by GRK2. Phosphorylation of m1 receptors was detected but the phosphorylation was not affected by the presence of carbamylcholine or atropine (Fig. 1, the upper figure). This phosphorylation was inhibited by 0.1 M heparin, a potent inhibitor of GRK2 (41), indicating that the phosphorylation was due to GRK2 and not to contaminating kinases (data not shown). The agonist-independent phosphorylation of m1 receptors by GRK2 contrasts with the agonist-dependent phosphorylation of m2 receptors by GRK2 under the same experimental conditions (14). We have attempted to remove putative contaminants that may inhibit the agonist-dependent phosphorylation of m1 receptors from m1 and GRK2 preparations. In one of such trials, we found that m1 receptors became to be phosphorylated in an agonist-dependent manner when m1 receptors were precipitated with PEG after reconstitution into lipid vesicles (Fig. 1, the lower panel). The stimulation by carbamylcholine was dose-dependent and antagonized by atropine, confirming that the effect of carbamylcholine was mediated by its binding to m1 receptors (Fig.  2). The agonist-dependent phosphorylation of m1 receptors was markedly stimulated by G protein ␤␥ subunits (Fig. 4), as was the case of the agonist-dependent phosphorylation of m2 receptors. In some experiments, m1 receptors were reconstituted in lipid vesicles containing 10% phosphatidylinositol bisphosphate and then subjected to phosphorylation by GRK2, but no appreciable stimulation by phosphatidylinositol bisphosphate was detected. The rate of phosphorylation of m1 receptors was approximately half of the rate of phosphorylation of m2 receptors under the same experimental conditions (data not shown).
Phosphorylation by PKC-Phosphorylation of m1 receptors by PKC was not increased by addition of agonist, whether or not the m1 receptors in vesicles were subjected to precipitation with PEG (Fig. 3). A slightly higher level of phosphorylation of m1 receptors was reproducibly observed in the presence of atropine compared with the phosphorylation in the presence of carbamylcholine, although detailed statistical analysis was not performed. The phosphorylation by PKC of m1 receptors was not affected by G protein ␤␥ subunits, in contrast to the phosphorylation by GRK2 (Fig. 4).
Phosphorylation by GRK2 and PKC- Fig. 5 shows the time courses of phosphorylation of m1 receptors which were incu- bated with GRK2 or PKC alone, with GRK2 and then PKC or with PKC and then GRK2. The amounts of phosphates incorporated in m1 receptors were significantly increased by addition of another kinase, indicating that the phosphorylation sites by GRK2 and PKC are different from each other. Unexpectedly, the amounts of phosphates incorporated into m1 receptors were dependent on the order of addition of the two kinases and were apparently greater when m1 receptors were phosphorylated by GRK2 and then PKC, compared with when m1 receptors were phosphorylated by PKC and then GRK2 (Fig. 5). This observation was further confirmed by phosphorylation of m1 receptors in the presence of different concentrations of ATP (Fig. 6). Amounts of phosphates incorporated in m1 receptors were greater when phosphorylated in the order GRK2 then PKC, compared with the reverse order. Table I summarizes data concerning the amount of phosphates incorporated into m1 receptors (moles of [ 32 P]P i /mol of [ 3 H]quinuclidinyl benzylate binding site), when m1 receptors were subjected to phosphorylation in the presence of 50 M ATP at 30°C for 120 min with a single kind of kinase or with two kinases applied in different orders. The amount of phosphates incorporated into m1 receptors was estimated to be 4.6, 2.8, or 7.6, for GRK2 alone, PKC alone, and for GRK2 and then PKC, respectively. The value of 7.6 is virtually the same as the sum (7.4) of 4.6 and 2.8, indicating that the phosphorylation sites by GRK2 and PKC are mutually exclusive. On the other hand, the amount of phosphate incorporated into m1 receptors incubated with PKC and then GRK2 was estimated to be 5.8 and significantly lower than the value of 7.4. This result indicates that the phosphorylation by PKC suppresses the further phosphorylation by GRK2.
Very recently, Tsu et al. (42) reported that GRK2 is phosphorylated by PKC and that the phosphorylated GRK2 has a 31% lower K m and 10% higher V max for phosphorylation of rhodopsin. We have also examined if the activity of GRK2 to phosphorylate m1 receptors is affected by preincubation of GRK2 with PKC, but have not found significant increase of the activity of GRK2. It remains to be determined if this is due to the difference in substrates or experimental conditions.
Location of Phosphorylation Sites-To locate phosphoryla-

FIG. 3. Phosphorylation of m1 receptors by PKC.
Purified m1 receptors were reconstituted with G o , precipitated with PEG, and then subjected to phosphorylation by PKC in the presence of 0.1 mM GTP and 1 mM carbamylcholine or 10 M atropine. Procedures for preparation of m1 receptors and phosphorylation assay were the same as described in the legend to Fig. 1, except that 2 mM EDTA and 0.5 mM EGTA in the reaction medium were replaced by 0.2 mM CaCl 2 . In some experiments, phosphatidylserine and diolein were also included in the incubation medium in addition to the lipid mixture, but no significant difference was found in the level of phosphorylation.

FIG. 4. Effect of G protein ␤␥ subunits on the phosphorylation of m1 receptors by GRK2 and PKC.
Purified m1 receptors were reconstituted in a lipid vesicle without G proteins, precipitated with PEG, and then subjected to phosphorylation by GRK2 or PKC in the presence or absence of 1 mM carbamylcholine and 66 nM G protein ␤␥ subunits. Counts of 32 P in the band of m1 receptors in SDS-PAGE were measured and those for m1 receptors phosphorylated in the presence of both carbamylcholine and ␤␥ subunits were taken as 100%.

FIG. 5. Phosphorylation of m1 receptors by GRK2 and PKC.
Purified m1 receptors were reconstituted with G o , precipitated with PEG, and then subjected to phosphorylation by GRK2 and then PKC (the upper figure) or by PKC and then GRK2 (the lower figure). The assay medium for GRK2 contained 10 M [ 32 P]ATP, 1 mM carbamylcholine, 2 mM EDTA, and 0.5 mM EGTA. To this assay medium, 5 mM CaCl 2 was added together with PKC (the upper figure). The assay medium for PKC contained 10 M [ 32 P]ATP, 1 mM carbamylcholine, and 0.1 mM CaCl 2 . To this medium, 2 mM EGTA and 1 mM EGTA were added together with GRK2 (the lower figure). The sum of phosphorylation by GRK2 alone and that by PKC alone is shown as GRK2 ϩ PKC (calc.) in both figures. tion sites, m1 receptors phosphorylated by GRK2 or PKC were partially hydrolyzed with trypsin and then analyzed with immunological methods. We detected only a single major band with an apparent molecular size of 38 kDa after mild trypsin treatment of m1 receptors phosphorylated by GRK2, whereas a single major band with 14 kDa was detected after the same trypsin treatment of m1 receptors phosphorylated by PKC (Fig.  7). The 38-kDa band was stained with an antibody against the third intracellular loop peptide (anti-i3) but not with antibody against the carboxyl-terminal tail peptide (anti-C tail). In contrast, the 14-kDa band was stained with anti-C tail but not with anti-i3 (Fig. 7). This result indicates that major GRK2 phosphorylation sites reside in the 38-kDa fragment, which includes the third intracellular loop but not the carboxyl-terminal part, and that the major PKC phosphorylation sites are located in the 14-kDa carboxyl-terminal fragment.
By treatment of m1 receptors phosphorylated by GRK2 with higher concentrations of trypsin, molecular sizes of 32 P-labeled bands decreased from 38 to 13 and finally to 3 and 2 kDa (Fig.  8). All of these phosphorylated bands were immunoprecipitated with anti-i3 but not with anti-C tail antibodies. Bands with 3 and 2 kDa were resistant to treatment with higher concentrations of trypsin. The recovery of radioactivity in the 3-and 2-kDa bands obtained by treatment of the receptor with 50 g/ml trypsin was 50 -70% of the initial amount in the intact receptor. This result indicates that these bands contain the major phosphorylation sites, although they may not be the sole phosphorylation sites. In contrast, when m1 receptors phosphorylated by PKC were treated with higher concentrations of trypsin, the 14-kDa peptide with phosphorylation sites by PKC was broken down to smaller fragments, and no 32 P-labeled bands were detected on SDS-PAGE (data not shown). Bands immunoprecipitated with anti-C tail antibodies also became undectable following treatment with higher concentrations of trypsin under conditions where bands precipitated with anti-i3 antibodies could still be detected. These results indicate that major PKC phosphorylation sites are located in the carboxylterminal portion, which is easily broken down by treatment with trypsin, and that major GRK2 phosphorylation sites are in peptides which are resistant to trypsin treatment and contain at least part of the third intracellular loop. pmol as original amount in total volume of 300 l) were phosphorylated by GRK2, then treated with indicated concentrations of trypsin for 5 min at 30°C. An aliquot of the reaction mixture (33 l) was directly subjected to SDS-PAGE followed by autoradiography. Digitonin (0.1% in a final concentration) and then anti-i3 or anti-C tail antibodies (10 l of anti-serum) were added to a portion of the reaction mixture (100 l). After incubation at 4°C overnight, Pansorbin was added and the suspension was centrifuged. The pellet was subjected to SDS-PAGE followed by autoradiography. FIG. 6. Effect of ATP concentrations on phosphorylation of m1 receptors by GRK2 and PKC. Experimental procedures were the same as described in the legend to Fig. 5, except that m1 receptors were subjected to phosphorylation in the presence of different concentrations of [ 32 P]ATP (2.35 cpm/fmol) for 120 min with GRK2 alone (q) or PKC alone (f) or for 60 min with GRK2 and then for another 60 min after addition of PKC (GRK2 then PKC) (E) or for 60 min with PKC and then for 60 min after addition of GRK2 (PKC then GRK2) (Ⅺ). The sum of phosphorylation by GRK2 alone and that by PKC alone is shown as "Calc. (PKC ϩ GRK2)" ( ).

TABLE I The number of GRK2 and PKC phosphorylation sites in m1 receptors
The m1 receptor was phosphorylated in the presence of 50 M [␥-32 P]ATP, 100 M GTP, and 20 nM G protein G o for 120 min by 1) GRK2 alone, 2) PKC alone, or 5) both GRK2 and PKC. As shown in Fig.  4, the ␤␥ subunits derived from G o stimulate the phosphorylation by GRK2 but do not affect the phosphorylation by PKC. In the experiment (3), the m1 receptor was incubated with GRK2 for 60 min and then for another 60 min after addition of PKC. In the experiment (4), the m1 receptor was incubated with PKC and then GRK2 was added.

Kinases
Number

DISCUSSION
In the present paper we have shown that human m1 receptors are phosphorylated in an agonist-dependent manner by GRK2 and independent manner by PKC. The number of phosphorylation sites were estimated to be 4 -5 for phosphorylation by GRK2 and 2-3 for phosphorylation by PKC. These phosphorylation sites appear to be different from each other, because the sum of sites phosphorylated by GRK2 and PKC was not significantly different from the number of sites phosphorylated following sequential phosphorylation by GRK2 and PKC. This conclusion was further supported by the finding that major phosphorylation bands obtained by trypsin treatment of m1 receptors phosphorylated by GRK2 are different from those obtained by the same treatment of m1 receptors phosphorylated by PKC. This conclusion is consistent with the fact that the consensus sequence for phosphorylation by PKC should include basic amino acids around serine and threonine residues (43) but phosphorylation sites by GRK2 should be franked by acidic amino acids rather than basic amino acids (44).
Major phosphorylation sites by PKC have been located in a sequence within 14 kDa from the carboxyl-terminal segment, consistent with previous results using muscarinic receptors purified from porcine brain (37). The 14-kDa peptide is thought to contain residues 333-460 based on the assumption that the peptide ends at the carboxyl terminus, and the average molecular mass of each residue is 110 Da. The 14-kDa peptide labeled with 32 P was sensitive to trypsin treatment and was broken down to small peptides undectable following SDS-PAGE, consistent with the fact that there are numerous lysine and arginine residues in the carboxyl-terminal portion. There are two serine and two threonine residues in the carboxylterminal tail (R*DT 428 FR*, K*R*PGS 451 VHR*T 455 D-S 457 R* QC-OH) and one serine and two threonine residues in the carboxyl-terminal portion of the third intracellular loop (K*R*PT 330 R*K*, K*R*K*T 354 FS 356 LVK*EK*K*K*), which are franked by basic amino acids and are therefore good candidates for PKC phosphorylation sites. In fact, peptides corresponding to the carboxyl-terminal tail (sequence 435-460) and to the carboxyl-terminal part of the third intracellular loop (sequence 346 -365) were phosphorylated by protein kinase C, although a peptide corresponding to the sequence 422-434 was not phosphorylated (6). Thus, Thr 354 , Ser 356 , Ser 451 , Thr 455 , and Ser 457 are likely candidates for sites phosphorylated by PKC.
Major GRK2 phosphorylation sites have been recovered in 3and 2-kDa bands, which interact with anti-i3 and are resistant to treatment with trypsin. It is interesting to note that the amino-terminal half of the third intracellular loop contains fewer basic and many more acidic amino acid residues than the carboxyl-terminal half. Serine and threonine residues in the amino-terminal half of the third intracellular loop are not flanked by basic amino acid residues, in contrast with serine and threonine residues in the carboxyl-terminal tail, the carboxyl-terminal half of the third intracellular loop, and the second intracellular loop. In particular, serine and threonine residues in the sequence from 275 to 303, K*E # E # E # E # E # D # E # G S 2 8 4 M E # S 2 8 7 L T 2 8 9 S 2 9 0 S 2 9 1 E # G E # E # D # GS 298 E # VVIK*, are most likely to be GRK2 phosphorylation sites, because 1) the sequence contains basic amino acid residues only at both ends and the corresponding peptide with a molecular mass of 3 kDa is expected to be resistant to trypsin treatment, 2) a peptide corresponding to the sequence 279 -293 was phosphorylated in vitro by GRK2 (6), and 3) sequence 286 -291 fits to a consensus sequence for phosphorylation in vivo by GRK2, which was proposed from studies on ␣2-adrenergic receptors (31). In addition, the replacement by alanine of serine and threonine residues in the sequence 284 -291 is reported to cause the attenuation of sequestration of m1 receptors (45). The kinase involved in the sequestration of m1 receptors has not been identified, but may be GRK2 or related kinases, because the phosphorylation by GRK2 of serine and threonine residues in the third intracellular region of m2 receptors is known to facilitate their sequestration (22). These results taken together suggest that the primary GRK2 phosphorylation sites reside in the 276 -303 amino acid sequence of the third intracellular loop, although this does not exclude the possibility that the serine and threonine residues in the other parts of intracellular loops might be phosphorylated by GRK2.
The number of sites phosphorylated by GRK2 was estimated to be 2.9 when m1 receptors were subjected to phosphorylation by GRK2 following phosphorylation by PKC, a value significantly lower than the value of 4.6 obtained following phosphorylation with GRK2 alone. This result indicates that the phosphorylation of m1 receptors by GRK2 is inhibited by prior phosphorylation by PKC. GRK2 is known to be synergistically activated by G protein ␤␥ subunits and mastoparan or related peptides (16) and by ␤␥ subunits and agonist-bound receptors (17). Peptides corresponding to the second intracellular loop, the carboxyl-terminal end of the third intracellular loop and the carboxyl-terminal tail of m2 receptors activated GRK2 (16). GRK2 was also strongly activated by a peptide corresponding to the carboxyl-terminal tail of m1 receptors and weakly activated by a peptide corresponding to the carboxyl-terminal end of the third intracellular loop of m1 receptors. 2 All these peptides contain many basic amino acid residues. It is tempting to speculate that the phosphorylation by PKC of serine and threonine residues in the carboxyl-terminal tail or in the carboxylterminal end of the third intracellular loop of m1 receptors reduces the ability of the receptor to interact with and activate GRK2. Another possibility is that the interaction between ␤␥ subunits and m1 receptors is impaired by phosphorylation of m1 receptors by PKC. The carboxyl-terminal tail of rhodopsin has been reported to be the site for interaction with ␤␥ subunits (46), although no direct evidence is available for m1 receptors.
The agonist-dependent phosphorylation of m1 receptors was first detected when m1 receptors reconstituted into lipid vesicles were precipitated with PEG prior to the phosphorylation reaction. The PEG treatment reproducibly caused both a decrease in the agonist-independent phosphorylation and an increase in the agonist-dependent phosphorylation. One of the simplest explanation is that inhibitory and/or stimulatory factors were removed upon precipitation with PEG. The putative factors are not likely to be residual detergents or agonists, because the concentrations of these compounds in the reaction mixture are too low to affect the phosphorylation. We have detected a band of 3 kDa in the m1 preparations, which was well phosphorylated by GRK2, and the amount of the band was markedly reduced with the precipitation procedure with PEG. Such a band was not detected in the m2 preparation, which are phosphorylated by GRK2 in an agonist-dependent manner even in the absence of the precipitation procedure. The nature of the band remains to be identified.
In the present paper, we have shown that m1 receptors undergo the phosphorylation by GRK2 and PKC, which may provide the molecular basis of homologous and heterologous desensitization, respectively. A novel type of cross-talk between phosphorylation of m1 receptors by PKC and GRK2 was suggested from the finding that the phosphorylation of m1 receptors by GRK2 is attenuated by the preceding phosphorylation with PKC.