Phosphorylation of Inositol 1,4,5-Trisphosphate Receptors by cAMP-dependent Protein Kinase
1998; Elsevier BV; Volume: 273; Issue: 10 Linguagem: Inglês
10.1074/jbc.273.10.5670
ISSN1083-351X
AutoresRichard J.H. Wojcikiewicz, Su Ge Luo,
Tópico(s)Phosphodiesterase function and regulation
ResumoThe ability of cAMP-dependent protein kinase (PKA) to phosphorylate type I, II, and III inositol 1,4,5-trisphosphate (InsP3) receptors was examined. The receptors either were immunopurified from cell lines and then phosphorylated with purified PKA or were phosphorylated in intact cells after activating intracellular cAMP formation. The former studies showed that the type I receptor was a good substrate for PKA (0.65 mol Pi incorporated/mol receptor), whereas type II and III receptors were phosphorylated relatively weakly. The latter studies showed that despite these differences, each of the receptors was phosphorylated in intact cells in response to forskolin or activation of neurohormone receptors. Detailed examination of SH-SY5Y neuroblastoma cells, which express ≥99% type I receptor, revealed that minor increases in cAMP concentration were sufficient to cause maximal phosphorylation. Thus, VIP and pituitary adenylyl cyclase activating peptide (acting through Gs-coupled pituitary adenylyl cyclase activating peptide-I receptors) were potent stimuli of type I receptor phosphorylation, and remarkably, even slight increases in cAMP concentration induced by carbachol (acting through Gq-coupled muscarinic receptors) or other Ca2+mobilizing agents were sufficient to cause phosphorylation. Finally, PKA enhanced InsP3-induced Ca2+ mobilization in a range of permeabilized cell types, irrespective of whether the type I, II, or III receptor was predominant. In summary, these data show that all InsP3 receptors are phosphorylated by PKA, albeit with marked differences in stoichiometry. The ability of both Gs- and Gq-coupled cell surface receptors to effect InsP3 receptor phosphorylation by PKA suggests that this process is widespread in mammalian cells and provides multiple routes by which the cAMP signaling pathway can influence Ca2+ mobilization. The ability of cAMP-dependent protein kinase (PKA) to phosphorylate type I, II, and III inositol 1,4,5-trisphosphate (InsP3) receptors was examined. The receptors either were immunopurified from cell lines and then phosphorylated with purified PKA or were phosphorylated in intact cells after activating intracellular cAMP formation. The former studies showed that the type I receptor was a good substrate for PKA (0.65 mol Pi incorporated/mol receptor), whereas type II and III receptors were phosphorylated relatively weakly. The latter studies showed that despite these differences, each of the receptors was phosphorylated in intact cells in response to forskolin or activation of neurohormone receptors. Detailed examination of SH-SY5Y neuroblastoma cells, which express ≥99% type I receptor, revealed that minor increases in cAMP concentration were sufficient to cause maximal phosphorylation. Thus, VIP and pituitary adenylyl cyclase activating peptide (acting through Gs-coupled pituitary adenylyl cyclase activating peptide-I receptors) were potent stimuli of type I receptor phosphorylation, and remarkably, even slight increases in cAMP concentration induced by carbachol (acting through Gq-coupled muscarinic receptors) or other Ca2+mobilizing agents were sufficient to cause phosphorylation. Finally, PKA enhanced InsP3-induced Ca2+ mobilization in a range of permeabilized cell types, irrespective of whether the type I, II, or III receptor was predominant. In summary, these data show that all InsP3 receptors are phosphorylated by PKA, albeit with marked differences in stoichiometry. The ability of both Gs- and Gq-coupled cell surface receptors to effect InsP3 receptor phosphorylation by PKA suggests that this process is widespread in mammalian cells and provides multiple routes by which the cAMP signaling pathway can influence Ca2+ mobilization. Inositol 1,4,5-trisphosphate (InsP3) 1The abbreviations used are: InsP3, inositol 1,4,5-trisphosphate; PKA, cAMP-dependent protein kinase; IBMX, 3-isobutyl-1-methylxanthine; VIP, vasoactive intestinal peptide; PACAP, pituitary adenylyl cyclase activating peptide. 1The abbreviations used are: InsP3, inositol 1,4,5-trisphosphate; PKA, cAMP-dependent protein kinase; IBMX, 3-isobutyl-1-methylxanthine; VIP, vasoactive intestinal peptide; PACAP, pituitary adenylyl cyclase activating peptide.receptors form tetrameric channels in endoplasmic reticulum membranes that conduct Ca2+ in an InsP3-sensitive manner (1Berridge M.J. Nature. 1993; 361: 315-325Crossref PubMed Scopus (6157) Google Scholar, 2Furuichi T. Mikoshiba K. J. Neurochem. 1995; 64: 953-960Crossref PubMed Scopus (180) Google Scholar, 3Joseph S.K. Cell. Signalling. 1996; 8: 1-7Crossref PubMed Scopus (120) Google Scholar). Thus, they link cell surface receptor-mediated increases in InsP3 formation to increases in cytoplasmic free Ca2+ concentration ([Ca2+]i). To date, the coding regions of three mammalian InsP3 receptor genes have been sequenced (4Furuichi T. Yoshikawa S. Miyawaki A. Wada K. Maeda N. Mikoshiba K. Nature. 1989; 342: 32-38Crossref PubMed Scopus (824) Google Scholar, 5Mignery G.A. Sudhof T.C. Takei K. De Camilli P. Nature. 1989; 342: 192-195Crossref PubMed Scopus (394) Google Scholar, 6Mignery G.A. Newton C.L. Archer III, B.T. Sudhof T.C. J. Biol. Chem. 1990; 265: 12679-12685Abstract Full Text PDF PubMed Google Scholar, 7Yamada M. Makino Y. Clark R.A. Pearson D.W. Mattei M.-G. Guenet J.-L. Ohama E. Fujino I. Miyawaki A. Furuichi T. Mikoshiba K. Biochem. J. 1994; 302: 781-790Crossref PubMed Scopus (107) Google Scholar, 8Sudhof T.C. Newton C.L. Archer III, B.T. Ushkaryov Y.A. Mignery G.A. EMBO J. 1991; 10: 3199-3206Crossref PubMed Scopus (319) Google Scholar, 9Blondel O. Takeda J. Janssen H. Seino S. Bell G.I. J. Biol. Chem. 1993; 268: 11356-11363Abstract Full Text PDF PubMed Google Scholar, 10Yamamoto-Hino M. Sugiyama T. Hikichi K. Mattei M.G. Hasegawa K. Sekine S. Sakurada K. Miyawaki A. Furuichi T. Hasegawa M. Mikoshiba K. Receptors Channels. 1994; 2: 9-22PubMed Google Scholar, 11Maranto A.R. J. Biol. Chem. 1994; 269: 1222-1230Abstract Full Text PDF PubMed Google Scholar). Their products, termed type I, II, and III receptors, are ∼2700 amino acids in length and are 60–70% identical at the amino acid level (2Furuichi T. Mikoshiba K. J. Neurochem. 1995; 64: 953-960Crossref PubMed Scopus (180) Google Scholar, 3Joseph S.K. Cell. Signalling. 1996; 8: 1-7Crossref PubMed Scopus (120) Google Scholar, 4Furuichi T. Yoshikawa S. Miyawaki A. Wada K. Maeda N. Mikoshiba K. Nature. 1989; 342: 32-38Crossref PubMed Scopus (824) Google Scholar, 5Mignery G.A. Sudhof T.C. Takei K. De Camilli P. Nature. 1989; 342: 192-195Crossref PubMed Scopus (394) Google Scholar, 6Mignery G.A. Newton C.L. Archer III, B.T. Sudhof T.C. J. Biol. Chem. 1990; 265: 12679-12685Abstract Full Text PDF PubMed Google Scholar, 7Yamada M. Makino Y. Clark R.A. Pearson D.W. Mattei M.-G. Guenet J.-L. Ohama E. Fujino I. Miyawaki A. Furuichi T. Mikoshiba K. Biochem. J. 1994; 302: 781-790Crossref PubMed Scopus (107) Google Scholar, 8Sudhof T.C. Newton C.L. Archer III, B.T. Ushkaryov Y.A. Mignery G.A. EMBO J. 1991; 10: 3199-3206Crossref PubMed Scopus (319) Google Scholar, 9Blondel O. Takeda J. Janssen H. Seino S. Bell G.I. J. Biol. Chem. 1993; 268: 11356-11363Abstract Full Text PDF PubMed Google Scholar, 10Yamamoto-Hino M. Sugiyama T. Hikichi K. Mattei M.G. Hasegawa K. Sekine S. Sakurada K. Miyawaki A. Furuichi T. Hasegawa M. Mikoshiba K. Receptors Channels. 1994; 2: 9-22PubMed Google Scholar, 11Maranto A.R. J. Biol. Chem. 1994; 269: 1222-1230Abstract Full Text PDF PubMed Google Scholar). Each receptor is thought to have the same overall structure, being divided into three domains: an N-terminal ligand binding domain, a C-terminal channel forming domain, and an intervening sequence, termed the coupling domain, that contains sites either known or hypothesized to be involved in receptor regulation (2Furuichi T. Mikoshiba K. J. Neurochem. 1995; 64: 953-960Crossref PubMed Scopus (180) Google Scholar, 3Joseph S.K. Cell. Signalling. 1996; 8: 1-7Crossref PubMed Scopus (120) Google Scholar, 4Furuichi T. Yoshikawa S. Miyawaki A. Wada K. Maeda N. Mikoshiba K. Nature. 1989; 342: 32-38Crossref PubMed Scopus (824) Google Scholar, 6Mignery G.A. Newton C.L. Archer III, B.T. Sudhof T.C. J. Biol. Chem. 1990; 265: 12679-12685Abstract Full Text PDF PubMed Google Scholar, 12Mignery G.A. Sudhof T.C. EMBO J. 1990; 9: 3893-3898Crossref PubMed Scopus (273) Google Scholar). Type I, II, and III receptors are expressed in different amounts in different cell types, and some cells co-express all three receptors (13De Smedt H. Missiaen L. Parys J.B. Bootman M.D. Mertens L. Van Den Bosch L. Casteels R. J. Biol. Chem. 1994; 269: 21691-21698Abstract Full Text PDF PubMed Google Scholar, 14Wojcikiewicz R.J.H. J. Biol. Chem. 1995; 270: 11678-11683Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar). InsP3 receptors form heterotetramers in intact cells, and such associations persist after receptor solubilization (14Wojcikiewicz R.J.H. J. Biol. Chem. 1995; 270: 11678-11683Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar, 15Monkawa T. Miyawaki A. Sugiyama T. Yoneshima H. Yamamoto-Hino M. Furuichi T. Saruta T. Hasegawa M. Mikoshiba K. J. Biol. Chem. 1995; 270: 14700-14704Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar, 16Wojcikiewicz R.J.H. He Y. Biochem. Biophys. Res Commun. 1995; 213: 334-341Crossref PubMed Scopus (86) Google Scholar, 17Joseph S.K. Lin C. Pierson S. Thomas A.P. Maranto A.R. J. Biol. Chem. 1995; 270: 23310-23316Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar). Characterization of differences between type I, II, and III receptors is at a preliminary stage, and it is not yet clear what properties are conferred upon a cell by the selective expression of a particular receptor. Recent studies have indicated, however, that type I, II, and III receptors bind InsP3 with different affinities (8Sudhof T.C. Newton C.L. Archer III, B.T. Ushkaryov Y.A. Mignery G.A. EMBO J. 1991; 10: 3199-3206Crossref PubMed Scopus (319) Google Scholar, 18Newton C.L. Mignery G.A. Sudhof T.C. J. Biol. Chem. 1994; 269: 28613-28619Abstract Full Text PDF PubMed Google Scholar,19Yoneshima H. Miyawaki A. Michikawa T. Furuichi T. Mikoshiba K. Biochem. J. 1997; 322: 591-596Crossref PubMed Scopus (87) Google Scholar), raising the possibility that this could influence the potency of InsP3 as a Ca2+ mobilizing agent. Also, from sequence analysis it is considered likely that the receptors will differ in other ways; for example, in their ability to be phosphorylated by cAMP-dependent protein kinase (PKA) (2Furuichi T. Mikoshiba K. J. Neurochem. 1995; 64: 953-960Crossref PubMed Scopus (180) Google Scholar,3Joseph S.K. Cell. Signalling. 1996; 8: 1-7Crossref PubMed Scopus (120) Google Scholar, 8Sudhof T.C. Newton C.L. Archer III, B.T. Ushkaryov Y.A. Mignery G.A. EMBO J. 1991; 10: 3199-3206Crossref PubMed Scopus (319) Google Scholar, 9Blondel O. Takeda J. Janssen H. Seino S. Bell G.I. J. Biol. Chem. 1993; 268: 11356-11363Abstract Full Text PDF PubMed Google Scholar, 10Yamamoto-Hino M. Sugiyama T. Hikichi K. Mattei M.G. Hasegawa K. Sekine S. Sakurada K. Miyawaki A. Furuichi T. Hasegawa M. Mikoshiba K. Receptors Channels. 1994; 2: 9-22PubMed Google Scholar, 11Maranto A.R. J. Biol. Chem. 1994; 269: 1222-1230Abstract Full Text PDF PubMed Google Scholar). Two serines within the PKA consensus sequence (R/K)(R/K)XS (20Walsh D.A. Van Patten S.M. FASEB J. 1994; 8: 1227-1236Crossref PubMed Scopus (214) Google Scholar) are present in the rat type I receptor coupling domain (serines 1589 and 1755 in the sequences RRDS and RRES, respectively), and in vitro studies on purified rat type I receptor (21Ferris C.D. Cameron A.M. Bredt D.S. Huganir R.L. Snyder S.H. Biochem. Biophys. Res. Commun. 1991; 175: 192-198Crossref PubMed Scopus (128) Google Scholar) have shown that both residues can be phosphorylated. These sites are also conserved in mouse and human type I receptors (4Furuichi T. Yoshikawa S. Miyawaki A. Wada K. Maeda N. Mikoshiba K. Nature. 1989; 342: 32-38Crossref PubMed Scopus (824) Google Scholar,7Yamada M. Makino Y. Clark R.A. Pearson D.W. Mattei M.-G. Guenet J.-L. Ohama E. Fujino I. Miyawaki A. Furuichi T. Mikoshiba K. Biochem. J. 1994; 302: 781-790Crossref PubMed Scopus (107) Google Scholar), testifying to their importance. In contrast, neither consensus sequence is conserved in type II and III receptors (8Sudhof T.C. Newton C.L. Archer III, B.T. Ushkaryov Y.A. Mignery G.A. EMBO J. 1991; 10: 3199-3206Crossref PubMed Scopus (319) Google Scholar, 9Blondel O. Takeda J. Janssen H. Seino S. Bell G.I. J. Biol. Chem. 1993; 268: 11356-11363Abstract Full Text PDF PubMed Google Scholar, 10Yamamoto-Hino M. Sugiyama T. Hikichi K. Mattei M.G. Hasegawa K. Sekine S. Sakurada K. Miyawaki A. Furuichi T. Hasegawa M. Mikoshiba K. Receptors Channels. 1994; 2: 9-22PubMed Google Scholar, 11Maranto A.R. J. Biol. Chem. 1994; 269: 1222-1230Abstract Full Text PDF PubMed Google Scholar), and although other serines in PKA consensus sequences are present elsewhere (8Sudhof T.C. Newton C.L. Archer III, B.T. Ushkaryov Y.A. Mignery G.A. EMBO J. 1991; 10: 3199-3206Crossref PubMed Scopus (319) Google Scholar, 9Blondel O. Takeda J. Janssen H. Seino S. Bell G.I. J. Biol. Chem. 1993; 268: 11356-11363Abstract Full Text PDF PubMed Google Scholar, 10Yamamoto-Hino M. Sugiyama T. Hikichi K. Mattei M.G. Hasegawa K. Sekine S. Sakurada K. Miyawaki A. Furuichi T. Hasegawa M. Mikoshiba K. Receptors Channels. 1994; 2: 9-22PubMed Google Scholar, 11Maranto A.R. J. Biol. Chem. 1994; 269: 1222-1230Abstract Full Text PDF PubMed Google Scholar), it is not yet known whether type II and III receptors are substrates for PKA. In the present study we have examined the ability of PKA to phosphorylate type I, II, and III receptors by analyzing PKA-induced phosphorylation of purified receptors and the ability of agents that raise cAMP to cause receptor phosphorylation in intact cells. SH-SY5Y human neuroblastoma cells, AR4–2J rat pancreatoma cells, and RINm5F rat insulinoma cells were obtained and cultured as monolayers in dishes (15 cm in diameter) as described (14Wojcikiewicz R.J.H. J. Biol. Chem. 1995; 270: 11678-11683Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar). Rabbit polyclonal antisera termed CT1, CT2, and CT3 were raised against the C termini of rat type I, II, and III receptors, respectively, and were affinity purified and shown to be type-specific (14Wojcikiewicz R.J.H. J. Biol. Chem. 1995; 270: 11678-11683Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar, 16Wojcikiewicz R.J.H. He Y. Biochem. Biophys. Res Commun. 1995; 213: 334-341Crossref PubMed Scopus (86) Google Scholar, 22Wojcikiewicz R.J.H. Furuichi T. Nakade S. Mikoshiba K. Nahorski S.R. J. Biol. Chem. 1994; 269: 7963-7969Abstract Full Text PDF PubMed Google Scholar). Receptors were immunoprecipitated from the three cell lines (14Wojcikiewicz R.J.H. J. Biol. Chem. 1995; 270: 11678-11683Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar, 16Wojcikiewicz R.J.H. He Y. Biochem. Biophys. Res Commun. 1995; 213: 334-341Crossref PubMed Scopus (86) Google Scholar) and were phosphorylated in a manner similar to that described previously (23Joseph S.K. Ryan S.V. J. Biol. Chem. 1993; 268: 23059-23065Abstract Full Text PDF PubMed Google Scholar). After removal of culture medium, cells were detached with 155 mm NaCl, 10 mm Hepes, 1 mm EDTA, pH 7.4 (HBSE), were centrifuged (500 ×g for 2 min), and were disrupted with 12 ml of ice-cold lysis buffer (50 mm Tris, 150 mm NaCl, 1% Triton X-100, 1 mm EDTA, 0.2 mmphenylmethylsulfonyl fluoride, 1 mm dithiothreitol, 10 μm leupeptin, 10 μm pepstatin, 0.2 μm soybean trypsin inhibitor, pH 8.0). After 30 min on ice, cells were centrifuged (38,000 × g for 10 min at 4 °C). Supernatants were then incubated at 4 °C with either CT1, CT2, or CT3 for 1 h and then for a further 1 h with protein A-Sepharose CL-4B. Immune complexes were then isolated by centrifugation (500 × g for 2 min), were washed twice with ice-cold phosphorylation buffer (120 mm KCl, 50 mm Tris, 0.1% Triton X-100, 0.3 mmMgCl2, pH 7.2), and were finally resuspended in phosphorylation buffer. Aliquots of washed beads were then placed in 1.5-ml microfuge tubes together with [γ-32P]ATP (∼5 μCi), 0–5 μm nonradioactive ATP, and 20 units of PKA catalytic subunit (final volume, 200 μl), were mixed gently, and were then incubated at 30 °C. Reactions were stopped by adding 1.3 ml of ice-cold phosphorylation buffer plus 1 mm ATP. Beads were then centrifuged (16,000 × g for 10 s), were washed twice with 1.5 ml of phosphorylation buffer plus ATP, and finally were resuspended in 2× gel loading buffer (14Wojcikiewicz R.J.H. J. Biol. Chem. 1995; 270: 11678-11683Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar). To assess the concentration and phosphorylation of purified type I, II, and III InsP3 receptors and the composition of immunoprecipitates, samples of washed beads were electrophoresed in 4% gels and were either silver-stained or immunoblotted as described (14Wojcikiewicz R.J.H. J. Biol. Chem. 1995; 270: 11678-11683Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar,16Wojcikiewicz R.J.H. He Y. Biochem. Biophys. Res Commun. 1995; 213: 334-341Crossref PubMed Scopus (86) Google Scholar), the molecular mass and concentration of receptors being established by comparison to standards of myosin (molecular mass, 205 kDa) and β-galactosidase (molecular mass, 116 kDa). Radioactivity associated with electrophoresed InsP3 receptors was assessed initially by autoradiography of dried gels and then quantitated by excision and scintillation counting of the ∼240–280-kDa region. Based on silver staining, equal amounts of phosphorylated type I, II, and III receptor were electrophoresed, and associated radioactivity was quantitated. The number of moles of phosphate incorporated was then calculated from the specific activity of the [γ-32P]ATP. The number of moles of InsP3 receptor loaded onto the gel was determined by measuring [3H]InsP3 binding to portions of the receptor preparations destined for phosphorylation. Briefly, washed beads in phosphorylation buffer were centrifuged (500 ×g for 2 min) and were washed and resuspended in 20 mm Tris, 1 mm EDTA, pH 8.0, and were then incubated with [3H]InsP3 as described (24Joseph S.K. Samanta S. J. Biol. Chem. 1993; 268: 6477-6486Abstract Full Text PDF PubMed Google Scholar). The number of specific binding sites was then determined and was assumed to equal the number of moles of InsP3 receptor. These experiments also confirmed that the equal amounts of type I, II, and III receptor defined by silver staining bound approximately equal amounts of InsP3. This was assessed either directly after labeling cells with32Pi or with a back-phosphorylation procedure (23Joseph S.K. Ryan S.V. J. Biol. Chem. 1993; 268: 23059-23065Abstract Full Text PDF PubMed Google Scholar, 25Komalavilas P. Lincoln T.M. J. Biol. Chem. 1996; 271: 21933-21938Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar), in which inhibition of PKA-catalyzed transfer of32P from [γ-32P]ATP to purified receptors reveals the degree to which PKA consensus sites are occupied by nonradioactive phosphate in intact cells. In the former procedure, cells were harvested in HBSE, were washed once with phosphate-free, 95% O2/5% CO2-gassed minimal essential medium, and were resuspended in the same medium, and 500-μl portions were incubated in microfuge tubes with ∼125 μCi of32Pi for 30–45 min at 37 °C. Cells were then stimulated, were centrifuged (16,000 × g for 10 s), and were resuspended in 1 ml of ice-cold lysis buffer plus 1 mm Na3VO4, 100 mmNaF, and 100 nm okadaic acid. InsP3 receptors were then purified by immunoprecipitation with CT1, CT2, or CT3 and protein A-Sepharose CL-4B and were electrophoresed. In the back-phosphorylation procedure, cells were harvested in HBSE, were washed once in gassed minimal essential medium, and were resuspended in the same medium, and 500-μl portions were stimulated. Cells were then centrifuged (16,000 × g for 10 s) and were resuspended in 1 ml of ice-cold lysis buffer plus 1 mmNa3VO4, 100 mm NaF, and 100 nm okadaic acid. InsP3 receptors were then purified by immunoprecipitation, were phosphorylated with [γ-32P]ATP and PKA as described above, and were electrophoresed. Cells were harvested in HBSE, were washed and resuspended in gassed minimal essential medium (containing 0.25 mm 3-isobutyl-1-methylxanthine (IBMX) in some experiments), and were aliquotted into 100-μl portions. After 10 min at 37 °C, cells were stimulated for 2 min, and 100 μl of ice-cold 1m trichloroacetic acid was added. After 15 min on ice, samples were centrifuged (16,000 × g for 3 min), and 160 μl of supernatant was removed and thoroughly mixed with 40 μl of 10 mm EDTA and 200 μl of freon/octylamine (1:1). After centrifugation (16,000 × g for 5 min), 100 μl of supernatant was neutralized with 50 μl of 25 mmNaHCO3, and cAMP content was measured by radioimmunoassay. Cells (1–2 dishes) were harvested in 10 ml of HBSE, were centrifuged (500 × gfor 2 min), were resuspended in 10 ml of ice-cold cytosol buffer (120 mm KCl, 2 mm KH2PO4, 2 mm MgCl2, 10 μm EGTA, 2 mm ATP, 20 mm Hepes, pH 7.0), were centrifuged again (500 × g for 2 min at 4 °C), and were resuspended in 0.8 ml of cytosol buffer. Cells were then permeabilized with nine discharges of a 3 microfarad capacitor (field strength, 3.75 kV/cm) as described (26Wojcikiewicz R.J.H. Nahorski S.R. J. Biol. Chem. 1991; 266: 22234-22241Abstract Full Text PDF PubMed Google Scholar), were diluted with cytosol buffer to 5 ml, were re-centrifuged, and were finally resuspended at 0.8–1 mg protein/ml in 1.3 ml of cytosol buffer. Suspensions were then incubated at 25 °C without or with 100 units/ml PKA for 10 min and then with ∼0.3 μCi of 45Ca2+/ml for 10 min. Aliquots of cell suspension (90 μl) were then added to tubes containing 10 μl of either InsP3 or ionomycin and were incubated at 25 °C for 1 or 2 min, respectively. Incubations were terminated by addition of 4 ml of ice-cold cytosol buffer and immediate filtration through Whatman GF/B filters; radioactivity bound to filters was assessed after addition of 4 ml of Ecoscint H and overnight extraction. Radioactivity (the amount of 45Ca2+ remaining sequestered in the permeabilized cells or “45Ca2+ content”) is expressed as a percentage of 45Ca2+ uptake (that remaining sequestered in the absence of stimulus). Importantly, uptake was unaffected by PKA; in the absence and the presence of PKA, respectively, 45Ca2+ uptake was 37 ± 5 and 38 ± 5 × 103 cpm for SH-SY5Y cells, 37 ± 1 and 35 ± 1 × 103 cpm for AR4–2J cells, and 34 ± 2 and 36 ± 2 × 103 cpm for RINm5F cells. In some experiments, control or PKA-treated permeabilized cells were centrifuged (16,000 × g for 10 s), and InsP3 receptors were immunoprecipitated and back-phosphorylated exactly as described for intact cells. Peroxidase-conjugated antibodies, molecular mass markers, dithiothreitol, protease inhibitors, IBMX, ionomycin, phorbol 12-myristate 13-acetate, and receptor agonists/antagonists were obtained from Sigma; forskolin was from Calbiochem; okadaic acid, thapsigargin, and NaF were from Alexis Corp.; [3H]InsP3 (21 Ci/mmol),45CaCl2 (10 Ci/g), and32Pi (H3PO4, carrier-free) were from NEN Life Science Products; [γ-32P]ATP (∼3000 Ci/mmol) was from Andotek; and PKA catalytic subunit was from Promega. Previous studies have shown that enrichment of type I, II, and III InsP3 receptors in SH-SY5Y, AR4–2J, and RINm5F cells, respectively, makes these cells convenient starting points for InsP3 receptor purification (14Wojcikiewicz R.J.H. J. Biol. Chem. 1995; 270: 11678-11683Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar, 16Wojcikiewicz R.J.H. He Y. Biochem. Biophys. Res Commun. 1995; 213: 334-341Crossref PubMed Scopus (86) Google Scholar). Because SH-SY5Y cells contain ≥99% type I receptor, a preparation composed solely of type I receptor can be immunoprecipitated from these cells with antiserum CT1 (14Wojcikiewicz R.J.H. J. Biol. Chem. 1995; 270: 11678-11683Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar, 16Wojcikiewicz R.J.H. He Y. Biochem. Biophys. Res Commun. 1995; 213: 334-341Crossref PubMed Scopus (86) Google Scholar). Type II and III receptor preparations immunoprecipitated from AR4–2J and RINm5F cells with antisera CT2 and CT3 are, in contrast, not homogeneous because they are “contaminated” with traces of co-immunoprecipitating type I receptor, which represents 12 and 4% of total receptor in these cell lines (14Wojcikiewicz R.J.H. J. Biol. Chem. 1995; 270: 11678-11683Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar, 16Wojcikiewicz R.J.H. He Y. Biochem. Biophys. Res Commun. 1995; 213: 334-341Crossref PubMed Scopus (86) Google Scholar). Fig. 1 A (lanes 1–9) reveals the extent to which the type I, II, and III InsP3 receptor preparations are phosphorylated by PKAin vitro. Because equal amounts of type I, II, and III receptors were loaded, it is clear that the type I receptor (lanes 1, 4, and 7) is phosphorylated more readily than type II (lanes 2, 5, and 8) or type III (lanes 3, 6, and 9) receptors. The phosphorylation seen is totally PKA-dependent, because none is detected if the kinase is omitted (lanes 10–12). Fig. 1 A also shows that two proteins are phosphorylated in the type II and III preparations, one of which (the upper band) co-migrates with type I receptor. Immunoblotting was performed to identify the different phosphoproteins (Fig. 1, B–D). Fig. 1 B (lanes 2 and 3) confirms the presence of type I receptor in the type II and III receptor preparations and that the upper phosphorylated band in the type II and III receptor preparations is indeed type I receptor. Faster migrating type II receptor (Fig. 1 C, lane 2) and type III receptor (Fig. 1 D, lane 3) correspond in size to the lower of the phosphorylated bands in the type II and III receptor preparations (Fig. 1 A). Regarding the efficiency of phosphorylation, it is remarkable that although type II receptor comprises ∼90% of the type II receptor preparation (14Wojcikiewicz R.J.H. J. Biol. Chem. 1995; 270: 11678-11683Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar,16Wojcikiewicz R.J.H. He Y. Biochem. Biophys. Res Commun. 1995; 213: 334-341Crossref PubMed Scopus (86) Google Scholar), it accounts for only ∼20% of the total 32P incorporated (Fig. 1 A, lanes 2, 5, and 8), showing that the type II receptor is phosphorylated very poorly. For the type III receptor preparation, however, ∼90% of32P incorporated is type III receptor-associated (Fig. 1 A, lanes 3, 6, and 9), confirming that the type III receptor is phosphorylated relatively well. For each receptor, phosphorylation was maximal at 15 min, at which point 0.65 ± 0.06, 0.04 ± 0.01, and 0.14 ± 0.03 mol of Pi were incorporated/mol type I, II, and III receptor, respectively (Fig. 2).Figure 2Stoichiometry of InsP3 receptor phosphorylation. The number of moles of Piincorporated into type I, II, or III receptors was calculated from the radioactive content of the ∼240–280-kDa region of gels and the estimate that 20 and 90% of 32Pi is type II and III receptor-associated in the type II and III receptor preparations. The number of moles of receptor loaded was calculated from the number of InsP3 binding sites in the preparations. Data shown are the means ± range of two independent experiments.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Because the type I receptor is a good substrate for PKAin vitro (Figs. 1 and 2), we examined whether it is phosphorylated in intact SH-SY5Y cells in response to cAMP elevation using either a back-phosphorylation procedure (23Joseph S.K. Ryan S.V. J. Biol. Chem. 1993; 268: 23059-23065Abstract Full Text PDF PubMed Google Scholar, 25Komalavilas P. Lincoln T.M. J. Biol. Chem. 1996; 271: 21933-21938Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar) or32Pi labeling of intact cells. Fig. 3 A shows that vasoactive intestinal peptide (VIP), which stimulates cAMP levels in SH-SY5Y cells (27Waschek J.A. Muller J.-M. Duan D.-S. Sadee W. FEBS Lett. 1989; 250: 611-614Crossref PubMed Scopus (31) Google Scholar), causes type I receptor phosphorylation in intact cells because back-phosphorylation was inhibited (lanes 1–5). Pituitary adenylyl cyclase activating peptide (PACAP), which belongs to the same neurohormone family as VIP (28Harmer A. Lutz E. Trends Pharmacol. Sci. 1994; 15: 97-99Abstract Full Text PDF PubMed Scopus (162) Google Scholar), had a similar effect (lanes 6–8). Because IC50 values for VIP and PACAP were 7 and 0.15 nm, respectively (Fig. 3 B), and PACAP-I receptors bind PACAP with ∼100-fold higher affinity than VIP (28Harmer A. Lutz E. Trends Pharmacol. Sci. 1994; 15: 97-99Abstract Full Text PDF PubMed Scopus (162) Google Scholar, 29Vertongen P. Devalck C. Sariban E. De Laet M.-H. Martelli H. Paraf F. Helardot P. Robberecht P. J. Cell. Physiol. 1996; 167: 36-46Crossref PubMed Scopus (38) Google Scholar) and are present in SH-SY5Y cells (29Vertongen P. Devalck C. Sariban E. De Laet M.-H. Martelli H. Paraf F. Helardot P. Robberecht P. J. Cell. Physiol. 1996; 167: 36-46Crossref PubMed Scopus (38) Google Scholar), it is likely that the effects of both PACAP and VIP are mediated by these receptors. Fig. 3 Cshows that forskolin, which also elevates cAMP levels in SH-SY5Y cells (30Baumgold J. Fishman P.H. Biochem. Biophys. Res Commun. 1988; 154: 1137-1143Crossref PubMed Scopus (52) Google Scholar, 31Hirst R.A. Lambert D.G. Biochem. Pharmacol. 1995; 49: 1633-1640Crossref PubMed Scopus (26) Google Scholar), similarly inhibited back-phosphorylati
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