The G-Protein-Coupled Receptor Phosphatase: a Protein Phosphatase Type 2A with a Distinct Subcellular Distribution and Substrate Specificity JULIE A

Proc. Natl. Acad. Sci. USA Vol. 92, pp. 8343-8347, August 1995 Biochemistry

The G-protein-coupled receptor phosphatase: A protein phosphatase type 2A with a distinct subcellular distribution and substrate specificity JULIE A. PITCHER*, E. STURGIS PAYNE*, CSILLA CSORTOSt, ANNA A. DEPAOLI-ROACHt, AND ROBERT J. LEFKOWITZ*t *Howard Hughes Medical Research Institute, Departments of Medicine and Biochemistry, Duke University Medical Center, Box 3821, Durham, NC 27710; and tDepartment of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, IN 46202 Contributed by Robert J. Lefkowitz, May 17, 1995

ABSTRACT Phosphorylation of G-protein-coupled re- MATERIALS AND METHODS ceptors plays an important role in regulating their function. In receptor phosphatase Preparation of 32P-Labeled Substrates. Purified reconsti- this study the G-protein-coupled tuted P3AR (200 nM) (4-6) was phosphorylated (500-,ul reac- (GRP) capable of dephosphorylating G-protein-coupled re- tion volume) with purified PARK, PAR kinase 2 (I3ARK-2), ceptor kinase-phosphorylated receptors is described. The RK (7), or cAMP-dependent protein kinase (PKA) (125 nM) GRP activity of bovine brain is a latent oligomeric form of as described (8). Reactions were terminated after incubation protein phosphatase type 2A (PP-2A) exclusively associated at 30°C for 20 min by addition of 1 ml of ice-cold 50 mM with the particulate fraction. GRP activity is observed only Tris-HCl, pH 7.0/0.1 mM EDTA/50 mM 2-mercaptoethanol when assayed in the presence of protamine or when phos- (buffer A). Labeled receptor was centrifuged (350,000 x g for phatase-containing fractions are subjected to freeze/thaw 15 min), washed three times, and resuspended in buffer A. treatment under reducing conditions. Consistent with its Stoichiometries of phosphorylation were 2-4 mol of Pi per mol identification as a member of the PP-2A family, the GRP is of J3AR for P3ARK or ,3ARK-2, 1-2 mol of Pi per mol of ,BAR potently inhibited by okadaic acid but not by 1-2, the specific for RK, and 1.5-1.7 mol of Pi per mol of ,BAR for PKA. inhibitor of protein phosphatase type 1. Solubilization of the Purified Sf9 cell membranes containing human PAR or membrane-associated GRP followed by gel filtration in the a2C2AR and rod outer segment membranes (9, 10) were absence of detergent yields a 150-kDa peak of latent receptor phosphorylated as described for the purified P3AR. The ,BAR phosphatase activity. Western blot analysis of this phos- and a2C2AR were phosphorylated by using the ,BARK and phatase reveals a likely subunit composition of ABaC. PP-2A rhodopsin was phosphorylated by using RK to stoichiom- of this subunit composition has previously been characterized etries of 3.0, 3.2, and 4.0 mol of Pi per mol of receptor, as a soluble enzyme, yet negligible soluble GRP activity was respectively. Phosphorylase a was prepared as in ref. 11. observed. The subcellular distribution and substrate speci- Phosphatase Assays. To assess GRP activity, 32P-labeled fi'city of the GRP suggests significant differences between it P3AR (1.0 pmol) was incubated with a source of phosphatase and previously characterized forms of PP-2A. in buffer A in 30 ,lI. Incubations were performed at 30°C for the times indicated. Dephosphorylation reactions were termi- Exposure of the f32-adrenergic receptor (,BAR) receptor to nated by the addition of SDS sample loading buffer and agonists results in a rapid decline in receptor responsiveness, proteins were separated on SDS/10% polyacrylamide gels. Phosphorylated P'AR was visualized by autoradiography and a process that appears to involve receptor phosphorylation (1, quantified by phospho imager analysis. Okadaic acid (OA) (5 2). In addition to the second messenger-dependent protein ,uM) was included in a control incubation to eliminate the kinases (1), agonist-specific phosphorylation of this receptor possibility that proteolysis contributes to the loss of32P-labeled can also be effected by the P3AR kinase (PARK), a member of receptor. the family of second messenger-independent G-protein- Phosphorylase a phosphatase assays were performed as coupled receptor kinases (GRKs). described in ref. 12. To determine protein phosphatase type 1 Despite the considerable progress that has been made in (PP-1) and PP-2A activities, 1 nM OA was used to inhibit identifying and characterizing the mechanism of action of PP-2A and 100 nM I-2, the recombinant rabbit skeletal muscle ,BARK, little is known of the phosphatases responsible for inhibitor 2 (13), was used to inhibit PP-1. 1-2 at 100 nM and OA reversing this phosphorylation event. Resensitization, presum- at 1 nM or OA at 5 ,uM were used to inhibit both PP-1 and ably due to dephosphorylation of the ,BAR, has been shown to PP-2A. Where indicated phosphatase-containing fractions occur rapidly upon removal of agonists (3). Regulation of were subjected to freeze/thaw treatment in the presence of 0.2 receptor phosphatase activity, therefore, represents an impor- M 2-mercaptoethanol prior to assay. tant potential mechanism for modulating receptor function. Sources of Protein Phosphatases. Frozen bovine cerebral In this study, we characterize the f3ARK-phosphorylated P3AR cortex (100 g) was homogenized in 500 ml of 50 mM Tris HCl, phosphatase present in extracts ofbovine brain. This phosphatase pH 7.0/5 mM EDTA/50 mM 2-mercaptoethanol (buffer B) at activity, which is also capable of dephosphorylating ,BARK- 4°C and passed through a double layer of cheesecloth. This phosphorylated a2C2-adrenergic receptors (a2C2ARs) and rho- fraction, the homogenate, was centrifuged at 300,000 x g for dopsin kinase (RK)-phosphorylated rhodopsin, is termed the 45 min yielding a supernatant containing soluble phosphatases G-protein-coupled receptor phosphatase (GRP). The enzyme, an oligomeric form of protein phosphatase type 2A (PP-2A), is Abbreviations: GRP, G-protein-coupled receptor phosphatase; ,BAR, latent and specifically associated with the particulate fraction. 132-adrenergic receptor; a2C2AR, a2C2-adrenergic receptor; GRK, G-protein-coupled receptor kinase; f3ARK, PAR kinase; RK, rhodopsin kinase; PKA, cAMP-dependent protein kinase; PP-2A, protein The publication costs of this article were defrayed in part by page charge phosphatase type 2A; PP-1, protein phosphatase type 1; OA, okadaic payment. This article must therefore be hereby marked "advertisement" in acid; munit(s), unit(s) x 10-3. accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 8343 Downloaded by guest on September 29, 2021 8344 Biochemistry: Pitcher et al. Proc. Natl. Acad. Sci. USA 92 (1995) (the soluble fraction) and a pellet of membrane-enriched A particulate matter. The particulate material was rehomog- 3.0 cc enized in buffer B containing 500 mM NaCl and extracted on 4: ice for 30 min. Centrifugation at 300,000 x g for 45 min yielded a soluble salt-wash fraction that was concentrated and de- 0 -5 2.0- salted. The remaining particulate material was resuspended in E 25 ml of buffer B and is termed the particulate fraction. Gel Filtration ofGRP Activity. The particulate fraction from E U F/T+Prot. 1.0 *F/T bovine brain was incubated with 0.25% dodecyl ,3-maltoside in oD-6 EJ F/T+I1-2 C') buffer B at 4°C for 45 min. Soluble proteins were recovered cu A + Prot. after centrifugation at 350,000 x g for 10 min. Where indi- OD o no additions cated, detergent was removed from this preparation by using Extracti-Gel (Pierce) (5). Gel filtration was performed on a 0 10 20 30 Superose-12 column (10 x 300 mm) in 20 mM Tris-HCl, pH Time, min 7.0/2 mM EDTA/5% (vol/vol) glycerol/100 mM NaCl in the presence or absence of 0.05% dodecyl ,B-maltoside. The col- B *@*@ umn was developed at a flow rate of 0.3 ml/min and 0.3-ml fractions were collected. e.A\# xAX X,. Immunoblot Analysis. Western blot analysis of GRP- k x 'x P containing fractions was performed by using rabbit antisera x C, directed against the catalytic subunit (14), the structural A x subunit (15) and regulatory B subunits [Ba (16), Bp (16), and xIa B' (17)] of PP-2A, and the ECL kit (Amersham). Purified PP-2A (16) and a lysate of Sf9 cells overexpressing the Bp FIG. 1. GRP activity of a bovine brain homogenate. (A) GRP subunit were used as positive controls. assays were performed with no additions (0) or in the presence of protamine sulfate (1 mg/ml) (A). After freeze/thaw treatment (F/T) of the homogenate, assays were performed in the presence (a) or RESULTS AND DISCUSSION absence (0) of protamine sulfate (Prot., 1 mg/ml) or in the presence The Principle GRP Activity of Bovine Brain Is a Latent of 100 nM 1-2 (o). Assays were performed at a phosphorylase a Form of PP-2A. A homogenate of bovine brain was prepared phosphatase activity of 0.1 munit/ml. The results shown represent the mean ± SEM for three experiments. (B) Autoradiograph showing the and assayed for its ability to dephosphorylate ,BARK- effects of various treatments on GRP activity. Samples were incubated phosphorylated ,3AR. Surprisingly, negligible spontaneous for 30 min at 30°C and subsequently electrophoresed on SDS/10% receptor phosphatase activity was observed (Fig. 1), although polyacrylamide gels. The band corresponding to phosphorylated P3AR both PP-1 [0.28 x 10-3 unit (munit)/mg of protein] and PP-2A is shown. Assays contained the following additions, where indicated: (0.5 munit/mg of protein) activities could be detected by using protamine, 1 mg/ml; 1-2, 100 nM; OA, 5 ,iM; Mg2+, 5 mM; Ca2+, 0.1 phosphorylase a as a substrate. Similarly, negligible receptor mM; Ca2+/calmodulin, 0.1 mM/0.2 ,LM. The effect of freeze/thaw phosphatase activity was detected when assays were per- treatment of the homogenate is also shown (F/T). formed in the presence of 5 mM Mg2+, 0.1 mM Ca2+, or Ca2+/calmodulin (0.1 mM/0.2 ,uM), indicating that the cation- localization of the GRP distinguish this enzyme from the dependent phosphatases PP-2C and PP-2B do not play a role spontaneously active soluble PP-2A reported to dephospho- in dephosphorylating this substrate (Fig. 1B). Potent GRP rylate rhodopsin (20-22) and the cholecystokinin receptors activity was, however, observed upon addition of protamine or (23). However, previous studies failed to assess the receptor after a freeze/thaw treatment of the extract in the presence of phosphatase activity of the salt-washed particulate fraction. 2-mercaptoethanol (Fig. 1). These treatments have been Additionally, when assessing cholecystokinin receptor dephos- shown to potently activate PP-2A (18). The initial rate of dephosphorylation of the ,BAR when assayed in the presence of protamine after a freeze/thaw treatment was -0.4 mol of 2.0 0 Particulate fraction cc * Homogenate Pi released per mol of ,BAR per min with 75% of the substrate 4: A being dephosphorylated after 10 min. Addition of I-2 (100 Salt wash nM), a potent and selective inhibitor of PP-1, inhibited 40% of 1.50 Soluble fraction the total phosphorylase a phosphatase activity but had no E effect on either the protamine or freeze/thaw-stimulated receptor phosphatase activity (Fig. 1). Furthermore, OA potently inhibited freeze/thaw-activated GRP activity with an 0 IC50 of 0.1 nM (data not shown), consistent with reported values for the catalytic subunit of PP-2A (19). The GRP o0.5- activity of bovine brain would, thus, appear to be a low-activity oligomeric form of PP-2A. The GRP Activity Is Membrane-Associated. To assess the 0 20 40- 60 intracellular distribution of the receptor phosphatase, soluble, Time, min particulate, and salt-wash fractions were prepared from the bovine brain homogenate and assayed for GRP activity. The FIG. 2. Receptor phosphatase activity is associated with the par- receptor phosphatase activities of the fractions were assayed at ticulate fraction. The GRP activities of a bovine brain homogenate (0) approximately equivalent phosphorylase a phosphatase activ- and three fractions derived from this extract, a soluble fraction (LI), a ities (0.05 munit/ml). As described (Fig. 1) negligible receptor salt-wash fraction (A), and a particulate fraction (a), were compared. Fractions were subjected to freeze/thaw treatment in the presence of phosphatase activity was observed in any fraction in the 0.2 M 2-mercaptoethanol prior to assay. The extract, soluble, and absence of activators of PP-2A. Freeze/thaw treatment in the salt-extracted fractions were assayed at a spontaneous phosphorylase presence of 2-mercaptoethanol revealed significant GRP ac- a phosphatase activity of 0.1 munit/ml, and the particulate fraction tivity in the homogenate, which was exclusively associated with was assayed at a phosphorylase a phosphatase activity of 0.05 munit/ the particulate fraction (Fig. 2). The latency and membrane ml. The data shown represent the mean ± SEM of three experiments. Downloaded by guest on September 29, 2021 Biochemistry: Pitcher et aL Proc. Natl. Acad. Sci. USA 92 (1995) 8345 phorylation, pancreatic cells were lysed and phosphatase assays were performed in the presence of detergent, conditions A , favoring dissociation of the latent GRP (23). A role for the latent membrane-associated GRP in the dephosphorylation of these receptors (rhodopsin and cholecystokinin) cannot, there- cr fore, be discounted. The particulate localization of the GRP was a somewhat 0 surprising finding since PP-2A is generally considered to be a -6 soluble enzyme. That PP-2A phosphatase activity was indeed present in the particulate fraction was confirmed by using E phosphorylase a as a substrate. OA and I-2 were used to + okadaic acid determine the relative activities of PP-1 and PP-2A in each of C] no additions the fractiohs assayed for GRP (Table 1). As expected, the 0.0 freeze/thaw bovine brain homogenate cottained approximately equal !Z freezelthaw + 1-2 amounts of spontaneously active PP-1 and PP-2A phosphatase + protamine activity. The soluble fraction was predominantly PP-2A (85%) P + protamine + 1-2 and the salt-extracted fraction PP-1 (85%). The particulate fraction cqntained little spontaneous phosphatase activity (0.25 munit/mg of protein). B ARK pARK-2 Freeze/thaw treatment under reducing conditions dissoci- 4 ates oligomeric forms of PP-2A and leads to an activation of this enzyme (18). This treatment thus activates the PP-2A in both the homogenate and the soluble fractions (Table 1). .0 2.0 Interestingly, the particulate fraction, which contains the GRP activity, but very little spontaneously active PP-2A, displays the largest fold increase in PP-2A-like activity upon freeze/thaw E 1 L7| treatment. After freeze/thaw activation, the phosphorylase a phosphatase activity of this fraction increases -9-fold. Thus a t-5 PKA pool of latent PP-2A, including the GRP, is tightly associated 0 with the particulate fraction. 13 The presence of latent PP-2A and PP-1 in a crude synaptic 0 plasma membrane preparation derived from rat forebrain was ._ recently reported (24). Extraction of the membranes with 500 mM NaCl and 0.3% Triton X-100 resulted in the elution and activation of both enzymes (24). In this study, treatment of the membrane fraction with 500 mM NaCl alone released mem- FIG. 3. Substrate specificity of the GRP. (A) The GRP activity of brane-associated PP-1 but not PP-2A activity (Table 1), sug- the particulate fraction assayed against ,BARK-phosphorylated P3AR, gesting different mechanisms of membrane association for f3ARK-phosphorylated a2C2AR, and RK-phosphorylated rhodopsin. these two classes of serine/threonine protein phosphatases. Assays were performed by using untreated particulate material in the Substrate Specificity of GRP. To investigate the receptor presence or absence of 5 IuM OA (+ okadaic acid, no additions) or specificity of the GRP activity, the ability of this enzyme to using freeze/thaw-treated particulate material in the presence or dephosphorylate three GRK-phosphorylated G-protein-coupled absence of 100 nM I-2. The receptor substrates are indicated. All receptors,was investigated. Purified Sf9 cell membranes contain- assays were performed at a phosphorylase a phosphatase activity of ing ,ARK-phosphorylated or a2C2AR or rod outer seg- 0.05 munit/ml. The data shown represent the mean ± SEM for three PAR experiments. (B) GRP activity of a salt-washed particulate fraction ment membranes containing RK-phosphorylated Fhodopsin were assessed by using as substrates purified reconstituted phos- utilized as substrates for the GRP. All three GRK-phosphory- P3AR phorylated with P3ARK (2.7 ± 0.3 mol of Pi per mol of ,BAR), ,BARK-2 lated receptors were dephosphorylated in an 1-2-resistant fashion (3.1 ± 0.5 mol of Pi per mol of P3AR), RK (1.5 ± 0.5 mol of Pi per mol after freeze/thaw activation of the particulate fraction (Fig. 3A). of PAR), or PKA (1.6 ± 0.1 mol of Pi per mol of PAR). Assays were The GRP would thus appear to have a broad specificity for performed at a phosphorylase a phosphatase activity of 0.05 munit/ GRK-phosphorylated G-protein-coupled receptors. ml by using untreated particulate material in the presence or absence Does the GRP exhibit specificity for particular phosphory- of 5 ,uM OA or after freeze/thaw treatment of the phosphatase lation sites? To examine this question, the ability of the preparation, in the presence of protamine (1 mg/ml), or in the salt-washed particulate fraction to dephosphorylate GRK- or presence of protamine (1 mg/ml) and 100 nM I-2. The data shown represent the mean ± SEM for three experiments. Table 1. Distribution of PP-1 and PP-2A enzyme activities in bovine brain extracts PKA-phosphorylated PAR substrates was examined. ,ARK- 1,2 and RK but not PKA-phosphorylated PAR served as Phosphorylase a phosphatase activity, substrates for the activated GRP (Fig. 3B). Dephosphorylation munits/mg of protein was unaffected by 1-2 addition. Thus the membrane-associated Freshly Freeze/thaw GRP dephosphorylates a number of G-protein-coupled recep- prepared activated tors; moreover, for the P3AR, those sites phosphorylated by GRKs rather than PKA appear to be the preferred substrates Extract PP-2A PP-1 PP-2A PP-1 for this enzyme. Homogenate 0.5 0.281 2.75 0.35 Resolution of Two Forms of the GRP by Gel Filtration. To Soluble 1.13 0.198 2.1 0.3 gain information concerning the structure of the GRP, deter- Salt wash 0.495 2.81 0.46 1.89 gent-solubilized preparations derived from the particulate Particulate 0.125 0.125 2.08 0.17 fraction of bovine brain were subjected to gel filtration on Samples were assayed before or after freeze/thaw treatment. The Superose-12 (Fig. 4). The solubilized preparation exhibited results shown represent the mean of six determinations performed on spontaneous GRP activity when assayed in the presence of three bovine brain preparations. detergent (Fig. 44). Removal of detergent, however, resulted Downloaded by guest on September 29, 2021 8346 Biochemistry: Pitcher et al. Proc. Natl. Acad. Sci. USA 92 (1995)

148 4325 not the absence of detergent (Fig. 4). This apparent difference A in GRP latency is specific to the PAR substrate since the 8.0 phosphorylase a phosphatase activity of the 35-kDa GRP is 1.0 similar in either the presence or absence of detergent. Thus receptor conformation appears to play a role in regulating receptor dephosphorylation. The latency of the GRP may thus 6.0 potentially be overcome by direct activation of this enzyme or, alternatively, by alteration of the conformation of its receptor substrates. E 4.0 In a previous study (25), the ability of various purified Cl)E 0.4 E preparations of phosphatase catalytic subunits to dephosphor- C c CL0. ylate the P3AR was investigated. Of the purified phosphatase E 2.0 catalytic subunits utilized, PP-2A, PP-1, PP-2B, and the cata- -0.2 E 0 lytic subunit of a latent high molecular weight phosphatase Cu a: LP-2, only the latter was capable of dephosphorylating ,ARK- a4: phosphorylated P3AR. Although LP-2 shares some properties ._ a0 with the GRP-namely, its latency-it is a soluble enzyme with 158 43 25 15E a subunit molecular mass of 49 kDa and, thus, seems unlikely E0 to account for the GRP activity of bovine brain homogenates. Immunological Analysis of the GRP. PP-2A holoenzymes 0. 8.0 L- GRP activity IF have been demonstrated to exist as a heterotrimeric complex co ECo consisting of a 36-kDa catalytic subunit (C), a 65-kDa struc- 0 o-o + protamine 4I b 0 t -0.8 tural subunit (A), and a third regulatory subunit ranging in CL I 19 4.0 -13- o - Ii protamine co molecular mass between 52 and 74 kDa (26). In an attempt to 0 I I 0 gain information concerning the between the 6.0 I I relationship 20 It -0.6 2 GRP and previously characterized forms of PP-2A, fractions containing the 150-kDa GRP activity, (Fig. 4B, fractions -0.4 36-38) were pooled and subjected to immunoblot analysis. II Polyclonal antibodies specific for the catalytic (36 kDa), A (65 11 kDa), and three distinct B subunits [Ba (55 kDa), Bp (55 kDa), 0.2 and B' (53 kDa)] of PP-2A were utilized. As shown in Fig. 5, immunoreactive species of appropriate molecular masses were observed in the GRP fraction blotted with either the catalytic 0 10 20 30 40 50 60 subunit (Fig. SA) or A subunit (Fig. SB) antibodies. Of the Fraction three B-subunit antibodies utilized, only that raised against a peptide corresponding to residues 14-26 of the human PP-2A FIG. 4. Gel filtration of the receptor phosphatase activity. The detergent-solubilized particulate fraction was subjected to gel filtra- A B tion on a Superose-12 column in the presence of detergent (0.05% dodecyl f3-maltoside) (A). Alternatively, prior to gel filtration, deter- 143- gent was removed from the solubilized fraction and gel filtration was performed in the absence of detergent (B). Fractions were assayed for 97- phosphorylase a phosphatase (0) and GRP activity. GRP activity was 50- measured in the presence (0) or absence (o) of protamine sulfate (1 The elution of aldolase mg/ml). positions (158 kDa), ovalbumin (43 35- _ kDa), and chymotrypsinogen A (25 kDa) are indicated. - 29.7- in the formation of a GRP activity with properties similar to those of the native enzyme; i.e., the enzyme was inactive in the GRP GRP absence of protamine (Fig. 4B). Gel filtration, in the presence of detergent, revealed a major peak of both phosphorylase a C D E phosphatase and GRP activities. The receptor phosphatase activity was spontaneous, inhibited by 2 nM OA, and stimu- 143- lated by protamine. Furthermore, by gel filtration, it had an 97- apparent molecular mass of 35 kDa, observations consistent _...*.rn with the identification of the catalytic subunit of the GRP as 50-- cobHS_ the catalytic subunit of PP-2A (Fig. 4A4). Gel filtration of the solubilized latent form of the GRP (i.e., 35- in the absence of detergent) revealed no spontaneous activity 29.7 - (Fig. 4B). However, two peaks of GRP activity, with apparent molecular masses of 150 kDa and 35 kDa, were observed when GRP GRP GRP the fractions were assayed in the presence of protamine (Fig. FIG. 5. Immunoblot analysis of the 150-kDa GRP activity. Frac- 4B). Thus removing detergent appears to promote the reas- tions containing the large molecular mass form of the GRP activity sociation of the catalytic subunit of the GRP with one or more (Fig. 4B, fractions 36-38) were pooled and subjected to Western blot regulatory subunits. At equivalent phosphorylase a phos- analysis with rabbit antisera directed against the catalytic subunit phatase activities, the receptor phosphatase activity of the (anti-C) (A), the structural A subunit (anti-A) (B), and three regulatory B subunits, Ba (anti-Ba) (C), Bp (anti-Bp) (D), and B' (anti-B') oligomeric enzyme is "4-fold higher than that of the free (E) of PP-2A. Each panel contains two lanes, a control lane (unla- catalytic subunit. beled) for the antisera containing the protein against which the The free catalytic subunit of the GRP displays spontaneous antibody is directed and a lane containing the pooled GRP fractions receptor phosphatase activitywhen assayed in the presence but (labeled GRP). Downloaded by guest on September 29, 2021 Biochemistry: Pitcher et al. Proc. Natl. Acad. Sci. USA 92 (1995) 8347 subunit Bc. displayed immunoreactivity with a protein of 6. Gierschik, P., Simons, C., Woodward, C., Somers, R. & Spiegal, appropriate molecular mass in the GRP-containing fraction A. M. (1984) FEBS Leu. 172, 321-325. (Fig. SC). These results support the identification of the GRP 7. Kim, C. M., Dion, S. B., Onorato, J. J. & Benovic, J. L. (1993) as an oligomeric form of PP-2A and suggest a potential subunit Receptor 3, 39-55. 8. Pitcher, J. A., Inglese, J., Higgins, J. B., Arriza, J. L., Casey, P. J., structure of ABaC. Interestingly, PP-2A of this subunit com- Kim, C., Benovic, J. L., Kwatra, M. M., Caron, M. G. & Lefko- position, the most prevalent soluble form of PP-2A in bovine witz, R. J. (1992) Science 257, 1254-1267. brain extracts (27), was recently identified as the principle 9. Pei, G., Tiberi, M., Caron, M. G. & Lefkowitz, R. J. (1994) Proc. soluble rhodopsin phosphatase of rod outer segments (28). Natl. Acad. Sci. USA 91, 3633-3636. However, we observe negligible soluble GRP activity. Thus 10. Papermaster, D. S. & Dreyer, W. J. (1974) Biochemistry 13, although apparently composed of immunologically similar 2438-2444. subunits, two prominent characteristics distinguish the GRP 11. Shenolikar, S. & Ingebritsen, T. S. (1984) Methods Enzymol. 107, from soluble bovine brain PP-2A-namely, its particulate 102-129. localization and substrate specificity. What potentially could 12. Cohen, P., Alemany, S., Hemmings, B. A., Resink, T. J., Stralfors, account for these differences? The Ba subunit antibodies P. & Tung, H. Y. L. (1988) Methods Enzymol. 159, 390-408. 13. Park, I. K., Roach, P., Bondor, J., Fox, S. P. & DePaoli-Roach, utilized in this study were raised against a short peptide A. A. (1994) J. Biol. Chem. 269, 944-954. sequence derived from this protein (16). The GRP may thus 14. Kamibayashi, C., Lickteig, R. L., Estes, R., Walter, G. & Mumby, contain a regulatory B subunit highly related to but distinct M. C. (1992) J. Biol. Chem. 267, 21864-21872. from Ba. Alternatively, since two isoforms (a and 13) of the C 15. Walter, G., Ruediger, R., Slaughter, C. & Mumby, M. C. (1990) and A subunits of PP-2A have been identified (29-31), the Proc. Natl. Acad. Sci. USA 87, 2521-2525. GRP and soluble PP-2A may have different isoform composi- 16. Zolnierowicz, S., Csortos, C., Bondor, J. A., Verin, A., Mumby, tions. Finally, covalent modifications of the catalytic subunit of M. C. & DePaoli-Roach, A. A. (1994) Biochemistry, 33, 11858- PP-2A have been reported including phosphorylation (32, 33) 11867. and carboxyl methylation (34). Differential post-translational 17. Mumby, M. C., Russell, K. L., Garrard, L. J. & Green, D. D. (1987) J. Biol. Chem. 262, 6257-6265. modification of one or more of the constituent subunits of the 18. Cohen, P. (1989) Annu. Rev. Biochem. 58, 453-508. GRP may account for the altered properties of this enzyme. 19. Cohen, P. (1991) Methods Enzymol. 201, 389-398. In conclusion, GRP, a latent membrane-associated form of 20. Palczewski, K., Hargrave, P. A., McDowell, J. H. & Ingebritsen, PP-2A representing the principle PAR phosphatase activity of T. S. (1989) Biochemistry 28, 415-419. bovine brain, has been identified. Immunological analysis 21. Fowles, C., Akhtar, M. & Cohen, P. (1989) Biochemistry 28, suggests a potential subunit structure for the GRP of ABaC; 9385-9391. however, significant differences exist between the GRP and 22. Palczewski, K., McDowell, J. H., Jakes, S., Ingebritsen, T. S. & the previously characterized soluble PP-2A of this composi- Hargrave, P. A. (1989) J. Biol. Chem. 264, 15770-15773. tion. The reasons for the particulate localization of the GRP 23. Lutz, M. P., Pinon, D. I., Gates, L. K, Shenolikar, S. & Miller, L. J. (1993) J. Biol. Chem. 268, 12136-12142. remain obscure; however, the latency and localization of this 24. Sim, A. T. R., Ratcliff, E., Mumby, M. C., Villa-Moruzzi, E. & enzyme suggest that it may represent an important regulatory Rostas, J. A. P. (1994) J. Neurochem. 62, 1552-1559. target for modulating the phosphorylation state of membrane- 25. Yang, S.-D., Fong, Y.-L., Benovic, J. L., Sibley, D. R., Caron, associated receptors. M. G. & Lefkowitz, R. J. (1988) J. Biol. Chem. 263, 8856-8858. 26. Mayer-Jaekel, R. E. & Hemmings, B. A. (1994) Trends Cell Biol. We thank Dr. Marc C. Mumby (University of Texas, Dallas) for 4, 287-291. PP-2A antibodies; Darrell Capel for purified ,3ARK-1, 3ARK-2, and 27. Kamibayashi, C., Estes, R., Lickteig, R. L., Yang, S.-I., Craft, C. RK; and Grace Irons for virus and cell culture. This work was & Mumby, M. C. (1994) J. Biol. Chem. 269, 20139-20148. supported in part by grants from the National Institutes of Health, 28. King, A. J., Andjelkovic, N., Hemmings, B. A. & Akhtar, M. Grants HL16037 to R.J.L. and DK36569 and P01 HL06308, subpro- (1994) Eur. J. Biochem. 225, 383-394. gram 4, to A.A.D.-R. 29. Stone, S. R., Hofsteenge, J. & Hemmings, B. A. (1987) Biochem- istry 26, 7215-7220. 1. Hausdorff, W. P., Caron, M. G. & Lefkowitz, R. J. (1990) 30. Arino, J., Woon, C. W., Brautigan, D. L., Miller, T. B., Jr., & FASEB J. 4, 2881-2889. Johnson, G. L. (1988) Proc. Natl. Acad. Sci. USA 85, 4252-4256. 2. Palczewski, K. & Benovic, J. L. (1991) Trends Biochem. Sci. 16, 31. Hemmings, B. A., Adams-Pearson, C., Maurer, F., Muller, P., 387-391. Goris, J., Merlevede, W., Hofsteenge, J. & Stone, S. R. (1990) 3. Yu, S. S., Lefkowitz, R. J. & Hausdorf, W. P. (1993) J. Bio. Biochemistry 29, 3166-3173. Chem. 268, 337-341. 32. Chen, J., Martin, B. L. & Brautigan, D. L. (1992) Science 257, 4. Pitcher, J. A., Touhara, K., Payne, E. S. & Lefkowitz, R. J. (1995) 1261-1264. J. Bio. Chem. 270, 11707-11710. 33. Guo, H. & Damuni, Z. (1993) Proc. Natl. Acad. Sci. USA 90, 5. Phillips, W. J., Trukawinski, S. & Cerione, R. A. (1989) J. Bio. 2500-2504. Chem. 264, 16679-16688. 34. Xie, H. & Clarke, S. (1994) J. Biol. Chem. 269, 1981-1984. Downloaded by guest on September 29, 2021