Solubilization of Receptors for the Novel Ca2+-mobilizing Messenger, Nicotinic Acid Adenine Dinucleotide Phosphate
2002; Elsevier BV; Volume: 277; Issue: 46 Linguagem: Inglês
10.1074/jbc.m203224200
ISSN1083-351X
AutoresG. Berridge, George D. Dickinson, John Parrington, Antony Galione, Sandip Patel,
Tópico(s)Piperaceae Chemical and Biological Studies
ResumoNicotinic acid adenine dinucleotide phosphate (NAADP) is a potent Ca2+ mobilizing agent in a variety of broken and intact cell preparations. In sea urchin egg homogenates, NAADP releases Ca2+ independently of inositol trisphosphate or ryanodine receptor activation. Little, however, is known concerning the molecular target for NAADP. Here we report for the first time solubilization of NAADP receptors from sea urchin egg homogenates. Supernatant fractions, prepared following Triton X-100 treatment, bound [32P]NAADP with similar affinity and selectivity as membrane preparations. Furthermore, the unusual non-dissociating nature of NAADP binding to its receptor was preserved upon solubilization. NAADP receptors could also be released into supernatant fractions upon detergent treatment of membranes prelabeled with [32P]NAADP. Tagged receptors prepared in this way, were readily resolved by native gel electrophoresis as a single protein target. Gel filtration and sucrose density gradient centrifugation analysis indicates that NAADP receptors are substantially smaller than inositol trisphosphate or ryanodine receptors, providing further biochemical evidence that NAADP activates a novel intracellular Ca2+ release channel. Nicotinic acid adenine dinucleotide phosphate (NAADP) is a potent Ca2+ mobilizing agent in a variety of broken and intact cell preparations. In sea urchin egg homogenates, NAADP releases Ca2+ independently of inositol trisphosphate or ryanodine receptor activation. Little, however, is known concerning the molecular target for NAADP. Here we report for the first time solubilization of NAADP receptors from sea urchin egg homogenates. Supernatant fractions, prepared following Triton X-100 treatment, bound [32P]NAADP with similar affinity and selectivity as membrane preparations. Furthermore, the unusual non-dissociating nature of NAADP binding to its receptor was preserved upon solubilization. NAADP receptors could also be released into supernatant fractions upon detergent treatment of membranes prelabeled with [32P]NAADP. Tagged receptors prepared in this way, were readily resolved by native gel electrophoresis as a single protein target. Gel filtration and sucrose density gradient centrifugation analysis indicates that NAADP receptors are substantially smaller than inositol trisphosphate or ryanodine receptors, providing further biochemical evidence that NAADP activates a novel intracellular Ca2+ release channel. Changes in cytosolic Ca2+ are indispensable for normal cell function (1Berridge M.J. Lipp P. Bootman M.D. Nat. Rev. Mol. Cell. Biol. 2000; 1: 11-21Crossref PubMed Scopus (4451) Google Scholar). A multitude of cell surface Ca2+ channels have been characterized that mediate influx of Ca2+ from the extracellular space upon activation. These include voltage-operated Ca2+ channels, ligand-gated Ca2+ channels that are regulated directly by neurotransmitters and Ca2+ channels coupled to depletion of intracellular Ca2+ stores (1Berridge M.J. Lipp P. Bootman M.D. Nat. Rev. Mol. Cell. Biol. 2000; 1: 11-21Crossref PubMed Scopus (4451) Google Scholar). In contrast, only two types of intracellular Ca2+ channels have been described to date: receptors for the second messenger inositol 1,4,5-trisphosphate (IP3) 1The abbreviations used are: IP3, inositol trisphosphate; cADPR, cyclic ADP-ribose; NAADP, nicotinic acid adenine dinucleotide phosphate; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid and ryanodine receptors (2Taylor C.W. Biochim. Biophys. Acta. 1998; 1436: 19-33Crossref PubMed Scopus (145) Google Scholar, 3Patel S. Joseph S.K. Thomas A.P. Cell Calcium. 1999; 25: 247-264Crossref PubMed Scopus (371) Google Scholar, 4Sorrentino V. Adv. Pharm. 1995; 33: 67-90Crossref PubMed Scopus (71) Google Scholar). The latter are modulated by the NAD metabolite, cyclic ADP-ribose (cADPR) (5Lee H.C. Annu. Rev. Pharmacol. Toxicol. 2001; 41: 317-345Crossref PubMed Scopus (387) Google Scholar, 6Galione A. Mol. Cell. Endocrinol. 1994; 98: 125-131Crossref PubMed Scopus (111) Google Scholar). Activation of these two pathways by a range of diverse extracellular stimuli evokes increases in cytosolic Ca2+ from intracellular Ca2+stores that are largely independent of extracellular Ca2+. Although the subunit size of ryanodine receptors (∼560 kDa) is almost twice that of IP3 receptors (∼300 kDa), these ubiquitous intracellular Ca2+ channels display several similarities in their structure and function. Multiple hydrophobic stretches of amino acids at the extreme C terminus of both receptors form the transmembrane region encompassing the Ca2+ channel, and it is within this region that the highest degree of sequence similarity between these proteins is found (7Mignery G.A. Südhof T.C. Takei K. De Camilli P. Nature. 1989; 342: 192-195Crossref PubMed Scopus (395) Google Scholar, 8Furuichi T. Yoshikawa S. Miyawaki A. Wada K. Maeda M. Mikoshiba K. Nature. 1989; 342: 32-38Crossref PubMed Scopus (825) Google Scholar). Both proteins assemble as tetrameric complexes with native molecular weights in excess of 1000 kDa (7Mignery G.A. Südhof T.C. Takei K. De Camilli P. Nature. 1989; 342: 192-195Crossref PubMed Scopus (395) Google Scholar, 9Lai F.A. Erickson H.P. Rousseau E. Liu Q.-Y. Meissner G. Nature. 1988; 331: 315-319Crossref PubMed Scopus (68) Google Scholar). 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Chem. 1992; 267: 9474-9477Abstract Full Text PDF PubMed Google Scholar) and the cytoskeletal protein, ankyrin (15Joseph S.K. Samanta S. J. Biol. Chem. 1993; 268: 6477-6486Abstract Full Text PDF PubMed Google Scholar, 16Bourguignon L.Y.W. Jin H. J. Biol. Chem. 1995; 270: 7257-7960Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 17Bourguignon L.Y.W. Chu A. Jin H. Brandt N.R. J. Biol. Chem. 1995; 270: 17917-17922Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Perhaps the most important regulator of these Ca2+ channels is Ca2+ itself. Modest elevations in cytosolic Ca2+ are stimulatory resulting in Ca2+-induced Ca2+ release whereas higher concentrations of Ca2+ inhibit Ca2+ release (18Iino M. Mol. Cell. Biochem. 1999; 190: 185-190Crossref PubMed Google Scholar). Dual regulation of IP3 and ryanodine receptors by their respective ligand and Ca2+ provides intricate control of channel function and is central to the generation of complex spatio-temporal Ca2+ signals (19Thomas A.P. Bird G.St.J. Hajnóczky G. Robb-Gaspers L.D. Putney Jr., J.W. FASEB J. 1996; 10: 1505-1517Crossref PubMed Scopus (422) Google Scholar). Sea urchin egg homogenates contain readily accessible functional Ca2+ stores providing an ideal experimental tool for the study of intracellular Ca2+ release pathways (20Clapper D.L. Walseth T.F. Dargie P.J. Lee H.C. J. Biol. Chem. 1987; 262: 9561-9568Abstract Full Text PDF PubMed Google Scholar). In this preparation, both IP3 and cADPR effect robust release of sequestered Ca2+ through activation of IP3 and ryanodine receptors, respectively. In addition, recent studies have uncovered a third intracellular Ca2+ release pathway activated by nicotinic acid adenine dinucleotide phosphate (NAADP) (21Lee H.C. Aarhus R. J. Biol. Chem. 1995; 270: 2152-2157Abstract Full Text Full Text PDF PubMed Scopus (401) Google Scholar,22Chini E.N. Beers K.W. Dousa T.P. J. Biol. Chem. 1995; 270: 3216-3223Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). That this pathway is not modulated by changes in cytosolic Ca2+ clearly distinguishes it from IP3- and cADPR-mediated Ca2+ release (23Chini E.N. Dousa T.P. Biochem. J. 1996; 316: 709-711Crossref PubMed Scopus (91) Google Scholar, 24Genazzani A.A. Galione A. Biochem. J. 1996; 315: 721-725Crossref PubMed Scopus (162) Google Scholar). Indeed, NAADP-induced Ca2+ mobilization is demonstrable in the presence of specific IP3/cADPR antagonists (21Lee H.C. Aarhus R. J. Biol. Chem. 1995; 270: 2152-2157Abstract Full Text Full Text PDF PubMed Scopus (401) Google Scholar, 22Chini E.N. Beers K.W. Dousa T.P. J. Biol. Chem. 1995; 270: 3216-3223Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar), after depletion of IP3 and ryanodine/cADPR-sensitive Ca2+ stores with the Ca2+ ATPase inhibitor, thapsigargin (24Genazzani A.A. Galione A. Biochem. J. 1996; 315: 721-725Crossref PubMed Scopus (162) Google Scholar) and also in subcellular fractions insensitive to IP3 and cADPR (21Lee H.C. Aarhus R. J. Biol. Chem. 1995; 270: 2152-2157Abstract Full Text Full Text PDF PubMed Scopus (401) Google Scholar,25Lee H.C. Aarhus R. J. Cell Sci. 2000; 113: 4413-4420Crossref PubMed Google Scholar). The inactivation properties of this pathway are also somewhat unique. Concentrations of NAADP that are below the threshold for Ca2+ release are able to effect complete block of subsequent normally maximal NAADP challenge (26Aarhus R. Dickey D.M. Graeff R. Gee K.R. Walseth T.F. Lee H.C. J. Biol. Chem. 1996; 271: 8513-8516Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 27Genazzani A.A. Empson R.M. Galione A. J. Biol. Chem. 1996; 271: 1159911602Abstract Full Text Full Text PDF Scopus (149) Google Scholar). From radioligand experiments, NAADP appears to bind its receptor irreversibly (26Aarhus R. Dickey D.M. Graeff R. Gee K.R. Walseth T.F. Lee H.C. J. Biol. Chem. 1996; 271: 8513-8516Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 28Patel S. Churchill G.C. Galione A. Biochem. J. 2000; 352: 725-729Crossref PubMed Scopus (51) Google Scholar,29Billington R.A. Genazzani A.A. Biochem. Biophys. Res. Commun. 2000; 276: 112-116Crossref PubMed Scopus (51) Google Scholar), and this unusual property may underlie the unique inactivation of this pathway. These independent lines of evidence from broken cell preparations support the notion that NAADP targets a novel intracellular Ca2+ channel. Despite such discrete properties of NAADP sensitive Ca2+release in broken cell preparations, the pharmacology of NAADP-induced Ca2+ signals in intact cells is much more complex. In sea urchin eggs (30Churchill G.C. Galione A. J. Biol. Chem. 2000; 275: 38687-38692Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar) and starfish oocytes (31Santella L. Kyozuka K. Genazzani A.A., De Riso L. Carafoli E. J. Biol. Chem. 2000; 275: 8301-8306Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar), NAADP-mediated Ca2+ signals can be inhibited by a combination of heparin and 8-amino-cADPR, antagonists of IP3 and cADPR responses, respectively. Similarly, in pancreatic acinar cells, NAADP induces Ca2+ oscillations, reminiscent of those evoked by the brain-gut peptide, cholecystokinin, which are again sensitive to inhibitors of IP3 and cADPR (32Cancela J.M. Churchill G.C. Galione A. Nature. 1999; 398: 74-76Crossref PubMed Scopus (319) Google Scholar). This apparent discrepancy in the pharmacology of NAADP-induced Ca2+ release in broken and intact cells can be explained if NAADP initiates a “trigger” Ca2+ increase in the intact cell that is then amplified by Ca2+-induced Ca2+ release via IP3and cADPR receptors. This effect is lost in biochemical experiments where normal cellular architecture is disrupted. Additionally, Ca2+ release from NAADP-sensitive Ca2+ stores in intact sea urchin eggs may also be shuttled to thapsigargin-sensitive Ca2+ stores resulting in sensitization of IP3 and ryanodine receptors and the generation of Ca2+ oscillations (33Churchill G.C. Galione A. EMBO J. 2001; 20: 1-6Crossref PubMed Scopus (138) Google Scholar). Thus, this novel Ca2+-mobilizing agent may serve to coordinate Ca2+ signals via interaction with other intracellular Ca2+ release channels (34Patel S. Churchill G.C. Galione A. Trends Biochem. Sci. 2001; 26: 482-489Abstract Full Text Full Text PDF PubMed Scopus (131) Google Scholar). Indeed, blockade of NAADP receptors attenuates Ca2+ signals initiated by cholecystokinin in acinar cells (32Cancela J.M. Churchill G.C. Galione A. Nature. 1999; 398: 74-76Crossref PubMed Scopus (319) Google Scholar), T-cell receptor activation in T-lymphocytes (35Berg I. Potter V.L. Mayr G.W. Guse A.H. J. Cell Biol. 2000; 150: 581-588Crossref PubMed Scopus (149) Google Scholar) and sperm in ascidian eggs (36Albrieux M. Lee H.C. Villaz M. J. Biol. Chem. 1998; 273: 14566-14574Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar), events that require functional IP3 and/or ryanodine receptors. That metabolism of NAADP is regulated by Ca2+ in brain membranes (37Berridge G. Cramer R. Galione A. Patel S. Biochem. J. 2002; 365: 295-301Crossref PubMed Scopus (39) Google Scholar) adds an additional point of regulation for the fine-tuning of Ca2+ dynamics. Although the biochemical and molecular properties of IP3and ryanodine receptors are well defined, little is known concerning the nature of NAADP receptors. Here, we report for the first time, solubilization, and characterization of NAADP receptors from sea urchin eggs. Our data provide evidence that NAADP targets a novel protein that is distinct from known intracellular Ca2+ release channels. Sea urchin (Lytechinus pictus) egg homogenates (50% v/v) were prepared as described previously (27Genazzani A.A. Empson R.M. Galione A. J. Biol. Chem. 1996; 271: 1159911602Abstract Full Text Full Text PDF Scopus (149) Google Scholar), washed twice by centrifugation (20,000 ×g, 10 min) at 4 °C in binding medium composed of 20 mm HEPES (pH 7.2), 250 mm potassium gluconate, 250 mm N-methyl d-glucamine and 1 mm MgCl2. Washed homogenates (17% v/v) were solubilized by incubation with either Triton X-100, CHAPS, or SDS (1%) for 60 min and centrifuged at 100,000 × g for 60 min. Supernatants were analyzed for [32P]NAADP binding (see below). Solubilization and centrifugation were performed at either 4 °C (for Triton X-100 and CHAPS) or room temperature (for SDS). [32P]NAADP was prepared enzymatically from [32P]NAD (1000 Ci/mmol, Amersham Biosciences) as described previously (28Patel S. Churchill G.C. Galione A. Biochem. J. 2000; 352: 725-729Crossref PubMed Scopus (51) Google Scholar, 38Patel S. Churchill G.C. Sharp T. Galione A. J. Biol. Chem. 2000; 275: 36495-36497Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar). Solubilized extracts were incubated in binding medium supplemented with [32P]NAADP (0.2 nm) together with the indicated concentrations of unlabeled NAADP or the NAADP analogues, 2′,3′-cyclic NAADP and 3′-NAADP (38Patel S. Churchill G.C. Sharp T. Galione A. J. Biol. Chem. 2000; 275: 36495-36497Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 39Lee H.C. Aarhus R. J. Biol. Chem. 1997; 272: 20378-20383Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Incubations were performed at room temperature for 1–3 h (final detergent concentration = 0.2% w/v). γ-globulin (400 μg) was then added to samples and protein precipitated by incubation with 15% (w/v) polyethylene glycol (average molecular weight = 8000) for 30–90 min. Samples were then centrifuged at 12000 × g for 5 min, the resulting pellets washed with 15% (w/v) polyethylene glycol and dissolved in H2O for Cerenkov counting. Specific binding (1000–2000 cpm/incubation) was typically 70% of total binding. Sea urchin egg homogenates (0.1–25% v/v) were incubated for 1–16 h at room temperature in binding medium supplemented with 1–2 nm [32P]NAADP. Samples were subsequently washed twice in binding medium by centrifugation (2 min, 100,000 × g, 4 °C). Washed membranes (17% v/v) were then solubilized with the appropriate detergent as described for unlabeled homogenates. Homogenates (25% v/v) were prelabeled with [32P]NAADP in binding buffer (as described above) and subsequently washed and solubilized with Triton X-100 (1%) in a modified buffer composed of 20 mm HEPES (pH 7.2). This step was necessary since we found that binding buffer interfered with electrophoresis (not shown). Solubilized samples were separated on native 7.5% polyacrylamide gels (pH 8.8) at 4 °C according to standard procedures (40Shi Q. Jackowski G. Hames B.D. Gel electrophoresis of proteins. Oxford University Press, Oxford1998Google Scholar). Buffers were supplemented with 0.2% Triton X-100 to prevent protein precipitation. Samples were also subject to electrophoresis on 3–10 pH gradient gels (Bio-Rad) according to the manufacturer's instructions. Gels were dried and apposed to Hyperfilm (Amersham Biosciences) at −80 °C for ∼16 h prior to developing. Prelabeled NAADP receptors solubilized with Triton X-100 or CHAPS in binding medium (100–500 μl) were injected onto a Superdex 200 HR 10/30 column linked to an AKTA FPLC system (Amersham Biosciences) equilibrated with binding buffer supplemented with the appropriate detergent (1%). Fractions (0.5–1 ml) were then collected (flow rate of 0.5 ml/min) and analyzed directly for radioactivity. Unlabeled solubilized samples were also separated under identical conditions and individual fractions assayed for [32P]NAADP binding using polyethylene glycol (see above). NAADP receptor migration was compared with the migration of the molecular mass markers, cytochrome c (12.5 kDa), bovine serum albumin (66 kDa), alcohol dehydrogenase (150 kDa), β-amylase (200 kDa), apoferritin (443 kDa), and thyroglobulin (669 kDa). Triton X-100-solubilized receptors prelabeled with their ligand (200 μl) were layered onto a 1.8-ml 5–20% (w/v) sucrose density gradient (prepared in 3% increments) in binding medium supplemented with 1% Triton X-100. Samples were centrifuged in a swing-out rotor at 166,000 × g for 3.5 h at 4 °C. Fractions (195 μl) were collected from the top of the gradient and analyzed for radioactivity. Unlabeled NAADP receptors were separated under identical conditions. In order to improve resolution of lower molecular weight proteins, prelabeled receptors (200 μl) were also layered onto larger 3.6-ml 5–20% (w/v) sucrose density gradients, centrifuged at 164,000 × g for 17 h at 4 °C and fractionated as with smaller gradients. We and others have previously reported binding of [32P]NAADP to membrane preparations derived from sea urchin eggs (26Aarhus R. Dickey D.M. Graeff R. Gee K.R. Walseth T.F. Lee H.C. J. Biol. Chem. 1996; 271: 8513-8516Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 28Patel S. Churchill G.C. Galione A. Biochem. J. 2000; 352: 725-729Crossref PubMed Scopus (51) Google Scholar, 29Billington R.A. Genazzani A.A. Biochem. Biophys. Res. Commun. 2000; 276: 112-116Crossref PubMed Scopus (51) Google Scholar). In the present study, washed sea urchin egg homogenates were treated with the non-ionic detergent Triton X-100 and supernatants, following ultracentrifugation, analyzed for [32P]NAADP binding. Binding of [32P]NAADP was readily detected following Triton X-100 solubilization (yield = 153 ± 31%,n = 3). From isotope dilution experiments, [32P]NAADP binding to solubilized receptors was inhibited by NAADP (IC50 = 1 ± 0.3 nm,n = 8) and the NAADP analogues, 2′,3′-cyclic NAADP (IC50 = 3 ± 1 nm, n = 3) and 3′-NAADP (IC50 = 8 ± 4 nm,n = 3; Fig.1 A). The rank order of potency for solubilized NAADP receptors (NAADP>2′,3′-cyclic NAADP>3′-NAADP) is therefore the same as that reported previously for membrane-bound NAADP receptors (38Patel S. Churchill G.C. Sharp T. Galione A. J. Biol. Chem. 2000; 275: 36495-36497Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar) and correlates closely with the rank order of potency of NAADP and its analogues in stimulating Ca2+ mobilization from sea urchin egg homogenates (39Lee H.C. Aarhus R. J. Biol. Chem. 1997; 272: 20378-20383Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). An intriguing feature of [32P]NAADP binding to sea urchin egg homogenates, but notably not mammalian brain (38Patel S. Churchill G.C. Sharp T. Galione A. J. Biol. Chem. 2000; 275: 36495-36497Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar) or heart (41Bak J. Billington R.A. Timar G. Dutton A.C. Genazzani A.A. Curr. Biol. 2001; 11: 987-990Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar) membranes, is its apparent non-dissociating nature (26Aarhus R. Dickey D.M. Graeff R. Gee K.R. Walseth T.F. Lee H.C. J. Biol. Chem. 1996; 271: 8513-8516Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 28Patel S. Churchill G.C. Galione A. Biochem. J. 2000; 352: 725-729Crossref PubMed Scopus (51) Google Scholar, 29Billington R.A. Genazzani A.A. Biochem. Biophys. Res. Commun. 2000; 276: 112-116Crossref PubMed Scopus (51) Google Scholar). As originally described by Lee and colleagues (26Aarhus R. Dickey D.M. Graeff R. Gee K.R. Walseth T.F. Lee H.C. J. Biol. Chem. 1996; 271: 8513-8516Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar), an excess of unlabeled NAADP competes with [32P]NAADP if added simultaneously with the radioligand but has no effect once association is initiated (28Patel S. Churchill G.C. Galione A. Biochem. J. 2000; 352: 725-729Crossref PubMed Scopus (51) Google Scholar, 29Billington R.A. Genazzani A.A. Biochem. Biophys. Res. Commun. 2000; 276: 112-116Crossref PubMed Scopus (51) Google Scholar). In converse experiments, we have shown that if sea urchin egg homogenates are incubated first with unlabeled NAADP prior to addition of radioligand, an apparent leftward-shift in competition curves is observed compared with experiments in which labeled and unlabeled NAADP are added together (28Patel S. Churchill G.C. Galione A. Biochem. J. 2000; 352: 725-729Crossref PubMed Scopus (51) Google Scholar). These data are again inconsistent with a reversible interaction. As shown in Fig. 1 B, addition of unlabeled NAADP (1 μm) following prior incubation of solubilized receptors with radioligand was without effect on binding of [32P]NAADP over the time course of the experiment (90 min). Thus, sea urchin egg NAADP receptors, whether membrane-bound or soluble, appear to bind their ligand in an essentially irreversible manner. The data from Fig. 1 show that the binding characteristics of membrane-bound and solubilized NAADP receptors are similar. Indeed, binding of [32P]NAADP to Triton X-100 extracts was inhibited by high concentrations (1 m) of NaCl but unaffected by pH in the range 6–8 (data not shown), again consistent with previous studies using membrane preparations (28Patel S. Churchill G.C. Galione A. Biochem. J. 2000; 352: 725-729Crossref PubMed Scopus (51) Google Scholar). Taken together, our data indicate that detergent treatment of sea urchin egg homogenates results in solubilization of functional NAADP receptors. Since binding of NAADP to both membrane-bound and solubilized receptors does not readily dissociate, we determined whether membrane-bound NAADP receptors could be first tagged with their ligand and then solubilized as a receptor-ligand complex. Sea urchin egg homogenates were incubated with [32P]NAADP and membranes washed by centrifugation in order to remove unbound radioligand. [32P]NAADP binding to sea urchin egg homogenates was similar whether determined by this centrifugation method (14.8 ± 1 fmol/μl of homogenate,n = 3) or filtration (10.3 fmol/μl of homogenate,n = 2). These data indicate that washing does not induce dissociation of radioligand validating the use of this assay for the preparation of labeled membranes. Simultaneous addition of unlabeled NAADP together with radioligand during prelabeling of sea urchin egg homogenates (0.1% v/v) reduced the levels of tagged receptors following subsequent centrifugation in a concentration-dependent manner (Fig.2 A). The IC50value for NAADP in these protection assays (2 ± 0.6,n = 3) is similar to the apparent affinity of membrane-bound (28Patel S. Churchill G.C. Galione A. Biochem. J. 2000; 352: 725-729Crossref PubMed Scopus (51) Google Scholar) and solubilized (Fig. 1 A) NAADP receptors. At higher homogenate concentrations (25% v/v), we noted that the apparent affinity of NAADP receptors determined by centrifugation (16 ± 2 nm, n = 3) and also by filtration (data not shown) was somewhat reduced (Fig.2 A). Accordingly, reduced levels of [32P]NAADP binding to a fixed amount of sea urchin egg homogenate was evident when incubations were performed at lower dilutions (Fig. 2 B). Clearly then, binding of NAADP versus homogenate concentration is non-linear (Fig. 2 B, inset), an effect that may be due to increased NAADP metabolism at higher protein concentrations. Preincubation of sea urchin egg homogenates with a low concentration of NAADP prior to addition of radioligand reduced subsequent tagging to a greater extent than when the same concentration of NAADP was incubated together with the radioligand (Fig.2 C). In addition, incubation of a normally maximal concentration of NAADP was without effect on tagging if added after the radioligand during preincubation. These effects, which were similar at low and high concentrations of sea urchin egg homogenates during labeling (Fig. 2 C), further indicate that NAADP does not dissociate from its receptor following washing by centrifugation confirming again our results obtained by conventional filtration assays (28Patel S. Churchill G.C. Galione A. Biochem. J. 2000; 352: 725-729Crossref PubMed Scopus (51) Google Scholar). In the next set of experiments, we treated membranes labeled with [32P]NAADP with the Triton X-100, the zwitterionic detergent CHAPS, and the ionic detergent, SDS. All three detergents resulted in efficient release of radioligand from prelabeled membranes into supernatant fractions following ultracentrifugation whereas buffer alone was without effect (Fig. 3,A and B). In order to determine whether released radioligand following solubilization of prelabeled membrane remained bound to its receptor or had dissociated due to detergent treatment, we precipitated fractions with polyethylene glycol and analyzed the resulting pellets for radioactivity (Fig. 3, A andC). Following solubilization with Triton X-100 and CHAPS, 63 ± 13% and 74 ± 11% (n = 3) of the radioactivity in supernatant fractions could be precipitated, respectively, indicating that binding of [32P]NAADP to its receptor is preserved. In contrast, recovery after SDS treatment was minimal (4 ± 1%, n = 3, Fig. 3 C). Thus, under the latter denaturing conditions, supernatant radioactivity is likely free, unbound radioligand. These data, together with the finding that high salt concentrations initiate partial dissociation of bound [32P]NAADP to membrane preparations (28Patel S. Churchill G.C. Galione A. Biochem. J. 2000; 352: 725-729Crossref PubMed Scopus (51) Google Scholar), provide evidence that irreversible binding of [32P]NAADP to NAADP receptors is not due to covalent modification of the target protein. Having defined solubilization conditions that retain binding of [32P]NAADP to its receptor, we analyzed prelabeled NAADP receptors by native polyacrylamide gel electrophoresis. Autoradiograms following separation of labeled membranes solubilized with Triton X-100 revealed that a significant proportion of the radioactivity migrated as a band distinct from the dye-front (Fig.4 A). These data further suggest that [32P]NAADP remains bound to its receptor following solubilization of labeled membranes with mild detergents. Additionally, [32P]NAADP appears to bind only a single protein under these conditions. However, dissociation of [32P]NAADP from its receptor was also evident (Fig.4 A). We also analyzed NAADP receptor migration by electrophoresis on pH gradient gels (Fig. 4 B). From these experiments the isoelectric point of prelabeled NAADP receptors was 5.9 ± 0.3 (n = 3). Although NAADP receptor-ligand complexes could be resolved by native polyacrylamide gel electrophoresis (Fig. 4), separation of proteins by this method is dependent not solely on size but also net charge and is therefore not well suited for determining molecular size. SDS (which eliminates the endogenous charge of proteins) could not be included during electrophoresis since it induced ligand dissociation (Fig3 C). The molecular size of NAADP receptors was therefore determined by gel filtration. With this method, previous studies have demonstrated that both IP3 and ryanodine receptors, (which assemble as large tetrameric complexes) migrate to fractions corresponding to molecular weights of ∼1000 and 2000 kDa, respectively (42Supattapone S. Worley P.F. Baraban J.M. Snyder S.H. J. Biol. Chem. 1988; 263: 1530-1534Abstract Full Text PDF PubMed Google Scholar, 43Pessah I.N. Francini A.O. Scales D.J. Waterhouse A.L. Casida J.E. J. Biol. Chem. 1986; 261: 8643-8648Abstract Full Text PDF PubMed Google Scholar). In contrast, we provide evidence that NAADP receptors are substantially smaller. Fractionation of prelabeled NAADP receptors solubilized in the presence of Triton X-100 on Superdex 200 indicated a molecular weight of 471 kDa (n = 3, Fig.5 A, closed circles). Similar results were obtained following fractionation of unlabeled homogenates (Fig. 5 A, inset). In these postlabeling experiments, NAADP receptor migration was determined conventionally by analyzing the individual fractions for [32P]NAADP binding as in Fig. 1. Similar results were also obtained when gel filtration was performed in the presence of a protease inhibitor mixture (data not shown). Migration of prelabeled NAADP receptors solubilized with CHAPS (Fig. 5 B) revealed that the molecular weight was consistently smaller (408 kDa,n = 3) than that determined in the presence of Triton X-100 (471 kDa, n = 3). Gel filtration of the NAADP receptor in the presence of CHAPS, where total protein distribution could be determined in parallel (Fig. 5 B, dotted line), indicated a 9.3 ± 0.4-fold (n = 3) enrichment of NAADP receptors in the peak fraction. We next determined the molecular weight of NAADP receptors by sucrose density gradient centrifugation (Fig. 6). As with gel filtration, both IP3 and ryanodine receptors migrate as high molecular weight complexes and are accordingly recovered in heavy fractions with this technique (7Mignery G.A. Südhof T.C. Takei K. De Camilli P. Nature. 1989; 342: 192-195Crossref PubMed Scopus (395) Google Scholar, 9Lai F.A. Erickson H.P. Rousseau E. Liu Q.-Y. Meissner G. Nature. 1988; 331: 315-319Crossref PubMed Scopus (68) Google Scholar). Prelabeled NAADP receptors were therefore layered onto a 5–20% sucrose gradient and fractions analyzed for radioactivity following ultracentrifugation (Fig 6 A). In contrast to the results from gel filtration experiments, where NAADP receptors coeluted with apoferretin (molecular size 443 kDa, Fig. 5), the estimated molecular size of prelabeled NAADP receptors with this method was substantially smaller. Thus, whereas apoferretin migrated to fraction 5.2 ± 0.2 (n = 7), NAADP receptors prelabeled with [32P]NAADP at a homogenate concentration of either 25% v/v or 0.1% v/v migrated earlier to fractions 2.4 ± 0.3 (n = 5) and 2.7 ± 0.4 (n = 3), respectively (Fig. 6 A, closed circles, pooled data from all eight experiments). This distribution is more similar to that of alcohol dehydrogenase (2.7 ± 0.4, n = 3; molecular size 150 kDa). Again, similar results were obtained when NAADP receptor migration was determined by postlabeling of the collected fractions with [32P]NAADP following separation of unlabeled NAADP receptors (peak fraction = 2.5, n = 2; Fig.6 A, open circles). These data further highlight the usefulness of the prelabeling method for tracking NAADP receptors during purification. Migration of prelabeled NAADP receptors on glycerol density gradients was also the same as sucrose density gradients (data not shown). In order to obtain a more accurate molecular weight by sucrose density gradient centrifugation, we performed experiments on larger gradients following prolonged centrifugation (Fig. 6 B). From these experiments, the molecular mass of prelabeled NAADP receptors was estimated to be 120 ± 2 kDa. (n = 3). Inclusion of an excess of unlabeled NAADP during incubation of homogenates with [32P]NAADP, resulted in substantial reduction in radioactivity following solubilization and fractionation, confirming the specificity of binding (Fig. 6 B, open circles). The surprising discrepancy between the calculated molecular weight of NAADP receptors by gel filtration (Fig. 5) and sucrose density gradient centrifugation (Fig. 6) was further analyzed by combining the two techniques in sequence (Fig. 7,insets). Separation of partially purified NAADP receptors following gel filtration on sucrose gradients, indicated that prelabeled NAADP receptors again coeluted with alcohol dehydrogenase (Fig. 7 A, n = 3) and were thus substantially smaller than that estimated by gel filtration (Fig. 5). Conversely, prior separation of NAADP receptors on sucrose density gradients followed by purification of peak fractions by gel filtration, indicated that NAADP receptors were substantially larger than when analyzed on sucrose density gradients (Fig. 6). In these experiments, NAADP receptors comigrated with apoferritin (Fig. 7 B,n = 3) as did crude solubilized samples (Fig.5 A). One possibility to reconcile the results from gel filtration and sucrose density gradient experiments is that NAADP receptors form oligomeric complexes that are dissociated by the latter technique in a reversible manner. Although the mechanism underlying this effect is not known at present, this feature is not shared by known intracellular Ca2+ channels, suggesting that NAADP binds to a distinct, smaller target. Indeed, under the present conditions optimized for solubilization of NAADP receptors, we were unable to solubilize ryanodine receptors with Triton X-100 following prelabeling of homogenates with [3H]ryanodine (data not shown). In summary, we have for the first time characterized the binding properties of NAADP receptors solubilized from sea urchin eggs. Solubilized NAADP receptors bound their ligand with the appropriate affinity and selectivity. Furthermore, binding of [32P]NAADP to detergent extracts is not readily reversible indicating that this unusual property is likely to be intrinsic to the NAADP receptor. We also show that NAADP receptors can be tagged with their ligand and solubilized intact thereby providing a convenient means of tracking NAADP receptors during purification. Results from gel filtration and sucrose density gradient centrifugation experiments indicate that NAADP receptors are significantly smaller than IP3 and ryanodine receptors. Our data provide further evidence that NAADP mediates Ca2+ mobilization via a novel pathway. We thank Rachel Ashworth, Steve Bolsover, Philippe Champiel, Grant Churchill, F. Anthony Lai, Chi Li, Roser Masgrau, and Anthony Morgan for useful discussions.
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