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J. Biol. Chem., Vol. 281, Issue 25, 17410-17419, June 23, 2006

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ATP Binding to a Unique Site in the Type-1 S2^- Inositol 1,4,5-Trisphosphate Receptor Defines Susceptibility to Phosphorylation by Protein Kinase A^*

From the Department of Pharmacology and Physiology, School of Medicine and Dentistry, University of Rochester Medical Center, Rochester, New York 14642

Received for publication, February 10, 2006 , and in revised form, March 29, 2006.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
The subtype- and splice variant-specific modulation of inositol 1,4,5-trisphosphate receptors (InsP₃R) by interaction with cellular factors plays a fundamental role in defining the characteristics of Ca²⁺ release in individual cell types. In this study, we investigate the binding properties and functional consequences of the expression of a putative nucleotide binding fold (referred to as the ATPC site) unique to the S2^- splice variant of the type-1 InsP₃R (InsP₃R-1), the predominant splice variant in peripheral tissue. A glutathione S-transferase fusion protein encompassing amino acids 1574-1765 of the S2^- InsP₃R-1 and including the glycine-rich motif Gly-Tyr-Gly-Glu-Lys-Gly bound ATP specifically as measured by fluorescent trinitrophenyl-ATP binding. This binding was completely abrogated by a point mutation (G1690A) in the nucleotide binding fold. The functional sensitivity of S2^- InsP₃R-1 constructs was evaluated in DT40-3KO-M3 cells, a null background for InsP₃R, engineered to express muscarinic M3 receptors. The S2^- InsP₃R-1 containing the G1690A mutation was markedly less sensitive to agonist stimulation than wild type S2^- InsP₃R-1 or receptors containing a similar (Gly Ala) mutation in the established nucleotide binding sites in InsP₃R-1 (the ATPA and ATPB sites). The ATP sensitivity of InsP₃-induced Ca²⁺ release, however, was not altered by the G1690A mutation when measured in permeabilized DT40-3KO cells, suggesting a unique role for the ATPC site. Ca²⁺ release was dramatically potentiated following activation of cAMP-dependent protein kinase in DT40-3KO cells transiently expressing wild type S2^- InsP₃R or Gly Ala mutations in the ATPA and ATPB sites, but phosphorylation of the receptor and the potentiation of Ca²⁺ release were absent in cells expressing the G1690A mutation in S2^- InsP₃R. These data indicate that ATP binding specifically to the ATPC site in S2^- InsP₃R-1 controls the susceptibility of the receptor to protein kinase A-mediated phosphorylation, contributes to the functional sensitivity of the S2^- InsP₃R-1 and ultimately the sensitivity of cells to agonist stimulation.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
The release of Ca²⁺ from intracellular stores mediated by inositol 1,4,5-trisphosphate binding to inositol 1,4,5-trisphosphate receptors is a ubiquitous process important for control over a diverse array of physiological processes (1-3). The inositol 1,4,5-trisphosphate receptor (InsP₃R)³ family is encoded by three genes, resulting in the expression of three distinct proteins with molecular mass of 300 kDa, named InsP₃R-1, InsP₃R-2, and InsP₃R-3 (4-6). The pivotal importance of individual InsP₃R subtypes, in particular physiological processes, is highlighted by the genetic ablation of individual InsP₃R types (7-9). Additional diversity exists at the protein level by the expression of several splice variants, particularly of the InsP₃R-1 and InsP₃R-2 (10-12).

The functional protein consists of four individual InsP₃R monomers and can exist in both homo- and heterotetrameric forms (13-15). The domain organization for the InsP₃R family is well established, with the linear structure divided into three general regions. The InsP₃ binding core is toward the N terminus and was originally identified by mutagenesis studies to consist of a stretch of dispersed, positively charged amino acids critical for InsP₃ binding (16, 17). This observation indicated the importance of the three-dimensional structure of the binding pocket in coordinating the negatively charged phosphate groups of InsP₃, a proposal that was recently supported by solving the crystal structure of the InsP₃R binding core (18).

The ion-conducting pore of the InsP₃R is toward the extreme C terminus of the protein. This region, termed the "channel domain," is predicted to span the endoplasmic reticulum membrane six times (19, 20) and contains a motif between the fifth and sixth putative transmembrane span, which is conserved in potassium and calcium channels (GVGD) and has subsequently been demonstrated to constitute the ion-conducting pore of the InsP₃R (20). Whereas the InsP₃R binding core and channel domain are highly conserved between InsP₃R family members, the intervening 1700-amino acid region is appreciably more variable and has been called the "modulatory" or "regulatory and coupling domain."

This designation is based on the presence of numerous loci for interaction with regulatory factors that influence the Ca²⁺ release properties of the receptor. Moreover, because of the variability in sequence in this region, modulation can potentially occur in an InsP₃R-specific manner. The most important regulator of InsP₃R activity is undoubtedly Ca²⁺ itself. Ca²⁺ acts as a co-agonist to facilitate Ca²⁺ release at low concentrations and inhibit channel activity at high concentrations (21, 22). Numerous additional factors, such as the binding of proteins, interaction with adenine nucleotides, and phosphorylation by numerous kinases, can also significantly influence the activity of the receptor (for reviews, see Refs. 1 and 2). For example, our laboratory has recently demonstrated that phosphorylation by cyclic nucleotide-dependent protein kinases can greatly augment the activity of the InsP₃R-1 (23, 24) (see Refs. 25-29). This event results in enhanced Ca²⁺ release and serves as a potentially important point of interaction between Ca²⁺ and cAMP-mediated signaling. Interestingly, whereas both the peripherally expressed S2^- and neuronal S2⁺InsP₃R-1 variant contain two identical consensus motifs for phosphorylation by cAMP-dependent protein kinase (PKA) (10, 30) (at Ser-1589 and Ser-1755), one of which is also a motif predicted to be phosphorylated by cGMP-dependent protein kinase (Ser-1755), mutagenesis studies indicated that the particular functionally important phosphorylation sites were different in the individual splice variants. Specifically, PKA-dependent phosphorylation of only Ser-1755 led to enhanced Ca²⁺ release in the S2⁺ form, whereas phosphorylation of both sites was functionally relevant in the S2⁺ form was modulated following phosphorylation by cGMP-dependent protein kinase (23). The S2^- (23, 24). Furthermore, only the S2⁺ InsP₃R differs from the S2⁺ form by the excision of 39 amino acids (Glu-1693 to Arg-1731) between the two phosphorylation sites (see Fig. 1A). The structural or mechanistic grounds for this and other differences between the two splice variants, however, are presently unknown. Of interest, sequence analysis of the S2^- InsP₃R-1 indicates that the only striking difference from the S2⁺ InsP₃R-1 is the presence of a glycinerich motif (Gly-X-Gly-X-X-Gly) at aa 1688-1732 (Fig. 1A) (31-33). This motif could potentially serve as a nucleotide binding fold and thus is suggestive of a role for S2 ^--specific effects mediated by the binding of adenine nucleotides.

View larger version (17K): [in this window] [in a new window]   FIGURE 1. Schematic diagram of a portion of the regulatory domain of S2+ InsP3R-1. A, schematic diagram of the region surrounding the S2 splice site in InsP3R-1. Ser-1589 and Ser-1755 are PKA phosphorylation sites that flank the S2 splice site. In the rat S2- InsP3R-1 splice variant, 39 amino acids are excised between Lys-1692 and Gly-1732. This event introduces a glycine-rich motif (Gly-X-Gly-X-X-Gly), which often forms a nucleotide binding motif referred to as ATPC. Mutation of the second Gly in the motif would be predicted to disrupt the nucleotide binding (see "Results and Discussion"). This mutation is subsequently referred to as change in ATPC (ATPC). B,a schematic diagram of additional nucleotide binding sites in InsP3R-1. The ATPA site spans amino acids 1773-1780, and the ATPB site is between amino acids 2016 and 2021. To disrupt nucleotide binding in the ATPA site, Gly-1775 and Gly-1777 were mutated to Ala (ATPA). Similarly, Gly-2018 was mutated to Ala to create ATPB.

In this study, we have constructed a GST fusion protein encompassing the putative nucleotide binding site corresponding to the ATPC site and confirmed that the motif indeed binds ATP with high affinity. In addition, by constructing a mutation within this motif, which fails to bind ATP, we have investigated any role this unique site plays in the specific properties of the peripheral S2^- InsP₃R-1 splice variant. Our studies demonstrate that binding of ATP to this site results in enhanced sensitivity of the InsP₃R to InsP₃. Surprisingly, our studies also demonstrate that the ATPC site plays a crucial role in defining the susceptibility of the S2^-InsP₃R-1 to phosphorylation by PKA. Thus, binding of ATP to the ATPC site in S2^-InsP₃R-1 is important in defining the functional sensitivity of the receptor when the InsP₃R pathway is activated concurrently with PKA activation.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Mutagenesis—potential ATP binding sites of the rat InsP R-1 S1^-3/S2^- isoform were mutated by sequential PCR. All numbering is based on the S2⁺InsP₃R-1. The addition of an amino acid with a side chain on the second Gly of the ATP binding recognition motif Gly-X-Gly-X-X-ly is predicted to prevent the pyrophosphate moiety of ATP from interacting with the binding site. Mutation of the ATPC site in S2^- InsP₃R-1 (amino acids 1688-1732) was created by mutation of Gly-1690 to Ala (construct ATPC). ATPA was altered by mutating both Gly-1775 and Gly-1777 to Ala (construct ATPA). The ATPB site in S2^-InsP₃R-1 (aa 2016-2021) was altered by mutating Gly-2018 to Ala (construct ATPB).

Creation of GST Fusion Proteins—GST fusion proteins were created using the pFN2A (GST) Flexi Vector (Promega, Madison, WI). Nucleotides corresponding to amino acids 1574-1765 (ATPC), 1756-1850 (ATPA), and 1944-2040 (ATPB) of the rat InsP₃ R-1 S2^- splice variant were amplified by PCR. SgfI and PmeI restriction sites were incorporated into the oligonucleotides used for PCR amplification. The PCR products were restriction enzyme-digested and ligated into pFN2A at the SgfI and PmeI sites. This creates a fusion construct with GST at the N terminus of the InsP₃R-1 and a TEV protease recognition sequence in between to allow cleavage and removal of GST. WT S2^-InsP₃R-1 and a construct with G1690A mutation in this region were created and were verified by sequencing. BL21 (DE3) pLysS cells (Promega, Madison, WI) were transformed with each DNA construct. Individual colonies were picked and grown overnight in 10 ml of Luria broth supplemented with ampicillin. The overnight cultures were added to 990 ml of Luria broth. The 1000-ml cultures were grown for 2 h at 37°C and200rpm. Protein production was induced by the addition of 1 ml of 0.1 M isopropyl 1-thio--D-galactopyranoside (EMD Biosciences, San Diego, CA), and cultures were further incubated for 2 h. Cells were pelleted and lysed, and GST fusion protein was purified using a GST purification kit (BD Biosciences). This purified protein was then cleaved with TEV protease (Invitrogen), and GST was removed by running through a GST purification column. The GST fraction was bound to the glutathione column, whereas the InsP₃R-1 protein immediately eluted off. Purified InsP₃R-1 protein was concentrated using an Amicon Ultra-15 centrifugal filter device (Millipore Corp., Bedford, MA) and brought up to a final con centration of 0.5 mg/ml in 50 mM Tris buffer. Purified proteins were separated on 15% polyacrylamide gels and were either detected by Bio-Safe Coomassie Blue staining (Bio-Rad) or by immunoblotting with a monoclonal -GST (Rockland, Gilbertsville, PA) antibody or a polyclonal antibody directed against the nonphosphorylated sequence flanking Ser-1755 (43) (kindly provided by Dr. S. Snyder).

ATP Binding Assay—The fluorescent ATP analogue TNP-ATP (Molecular Probes, Inc., Carlsbad, CA) was used to measure binding of ATP to the purified InsP₃R-1 protein fragments. TNP-ATP fluorescence increases upon binding to protein (44, 45) (excitation, 403 nm; emission, 546 nm). 1 mg of protein in 2 ml of 50 mM Tris-HCl was used for each assay. Increases in TNP-ATP fluorescence were detected using a PerkinElmer LS-5B luminescence spectrometer (Wellesley, MA). Increasing concentrations of TNP-ATP were added sequentially to cuvettes containing protein, and fluorescence was measured every second. Background fluorescence was determined by measuring TNP-ATP emission at various concentrations in Tris buffer alone. Net fluorescence for binding to protein was determined by subtracting background fluorescence. Normalized concentration-response relationships were created and fit with a logistic equation, and apparent EC₅₀ values were calculated using OriginPro software (OriginLab, Northampton, MA). ATP competition assays were performed identically to the standard TNP-ATP binding assay, except protein-induced fluorescence was measured in the presence of 10 mM Na-ATP.

Creation of Stable Muscarinic M3 Receptor-expressing DT40-3KO Cells—3x HA-tagged human M3R cDNA in pCDNA3.1 was obtained from the UMR cDNA resource center (available on the World Wide Web at www.cdna.org). MfeI-digested plasmid was introduced into DT40 cells lacking expression of all InsP₃R types (38, 46) (DT40-3K0 cells) by nucleofection using program B23 and solution T as per the manufacturer's instructions (Amaxa, Inc.) to create the DT40-3KO-M3 stable cell line. After nucleofection, the cells were incubated in growth medium for 24 h prior to dilution in selection medium containing 2 mg/ml Geneticin (Invitrogen). Cells were then seeded into 96-well tissue culture plates at 1000 cells/well and incubated in selection medium for at least 7 days. Wells exhibiting growth after the selection period were picked for expansion.

Transient Transfection of DT40-3KO Cells—For permeabilized cell studies, DT40-3KO cells were transfected by nucleofection, as described above. For concentration-response relationships in intact DT40-3KO-M3 cells, cells were transfected by electroporation at 350 V and 950 microfarads (4-mm gap cuvette). 2 x 10⁷ cells were co-transfected with 25 µg of the InsP₃R-1 cDNA, and 4 µg of the red fluorescent protein plasmid pHcRed1-N1 (BD Biosciences). For forskolin-induced PKA potentiation assays, DT40-3KO cells were co-transfected with 25 µg of the InsP₃R-1 cDNA, 25 µg of the mouse type 3 muscarinic receptor, and 4 µg of the red fluorescent protein plasmid pHcRed1-N1 (BD Biosciences). Cells were incubated with DNA in 500 µl of Opti-MEM medium (Invitrogen) on ice for 10 min. The cell/DNA mixture was electroporated, incubated on ice for 30 min, increased to 5 ml with Opti-MEM, and placed in a 5% CO₂ incubator at 39 °C for 5 h. The cells were then centrifuged and resuspended in 12 ml of complete RPMI medium (Invitrogen). Transfection efficiency was typically 20%. Experiments were performed within 32 h of transfection.

Transfection of HEK-293 Cells and Assessment of Phosphorylation— HEK-293 cells were plated onto 25-cm² culture flasks and allowed to grow to near confluence. Cells were transfected with 5 µg of each S2^- InsP₃R-1 DNA construct by using the Lipofectamine reagent (Invitrogen) as per the manufacturer's instructions. The following day, batches of cells were treated in the presence or absence of 20 µM forskolin for 10 min, aspirated from flasks, lysed in a buffer containing 50 mM Tris-HCl, 250 mM NaCl, 50 mM NaF, 5 mM EDTA, 0.1% Triton, and 1 mM Complete Protease inhibitor tablet (Roche Applied Science) at pH 7.4. InsP₃R were immunoprecipitated with a polyclonal -InsP₃R-1 antibody that recognizes amino acids 2731-2749 of InsP₃R-1 (-InsP₃R-1). Immunoprecipitates were separated on 5% SDS gels, transferred to nitrocellulose, and then probed with either a polyclonal antibody raised against the sequence flanking Ser-1755 of InsP₃R-1 (-S1755) or a polyclonal antibody raised against a similar region that specifically recognizes the phosphorylated state of Ser-1755 (-S1755P). Blots that were probed with -S1755P were stripped and reprobed with the -S1755 antiserum to confirm the presence and relative quantity of the InsP₃R-1.

Digital Imaging of [Ca²⁺]_i in Intact Cells—Transfected DT40-3KO cells were washed once in a HEPES-buffered physiological saline solution (HEPES-PSS) containing 5.5 mM glucose, 137 mM NaCl, 0.56 mM MgCl₂, 4.7 mM KCl, 1 mM Na₂HPO₄, 10 mM HEPES (pH 7.4), 1.2 mM CaCl₂, and 1% (w/v) bovine serum albumin. Cells were then resuspended in bovine serum albumin HEPES-PSS with 1 µM Fura-2 (AM) (Teflabs Inc., Austin, TX), placed on a 15-mm glass coverslip in a low volume perfusion chamber, and allowed to adhere for 30 min at room temperature. Cells were perfused continuously for 10 min with HEPES-PSS before experimentation to allow complete Fura-2 de-esterification. A field of cells for each experiment was chosen that contained a wide range of transfection efficiency based upon the intensity of red fluorescence emitted when excited at 560 nm. Individual cells that had emission gray levels between 1500 and 2500 were subsequently chosen to standardize expression levels. [Ca²⁺]_i imaging was performed essentially as described previously, using an inverted epifluorescence Nikon microscope with a x40 oil immersion objective lens (numerical aperture, 1.3). Cells were excited alternately with light at 340-nm and 380 ± 10-nm bandpass filters (Chroma, Rockingham, VT) using a monochrometer (TILL Photonics, Pleasanton, CA). Fluorescence images were captured and digitized with a digital camera driven by TILL Photonics software. Images were captured every 2 s withan exposure of 10 ms and 4 x 4 binning. 340/380 ratio images were calculated online and stored immediately to a hard disk.

Permeabilized Cell Ca²⁺ Measurements—InsP₃R expression constructs along with a nuclear targeted HcRed expression construct (pHcRed-nuc; BD Biosciences, Palo Alto, CA) were introduced into DT40-3KO cells using nucleofector protocol B23 and solution T (Amaxa Biosystems, Gaithersburg, MD). 8 µg of InsP₃R DNA and 2 µg of pHcRed-nuc DNA were used in each nucleofection. Cells were loaded with 20 µM Furaptra-AM (Teflabs, Austin, TX) at 39 °C for1hin HEPES-PSS supplemented with 1% bovine serum albumin 16-20 h after nucleofection. Furaptra-loaded cells were permeabilized by superfusion for 1-2 min with 40 µM -escin in intracellular medium (ICM) containing 125 mM KCl, 19 mM NaCl, 1.4 mM MgCl₂, 0.33 mM CaCl₂,10 mM HEPES, 3 mM ATP, 1 mM EGTA (pH 7.3). The free [Ca²⁺] was estimated to be 50 nM (MaxChelator). Permeabilized cells were then washed in ICM without -escin for 5 min to facilitate removal of cytosolic dye. Transfected cells were identified by the presence of nuclear localized red fluorescence. The cells were then superfused in ICM containing 0.650 mM CaCl₂ (free [Ca²⁺] of 200 nM) to load the intracellular stores. The experimental recordings presented are taken after establishment of a new stable base line following loading of the stores. The free [Ca²⁺] was subsequently maintained at a constant 200 nM throughout all experimental maneuvers. Prior to application of InsP₃, the cells were superfused in ICM without MgCl₂ for 1 min to disable SERCA activity (38). The unidirectional flux of Ca²⁺ upon InsP₃ application was then monitored in the same solution containing various concentrations of InsP₃ and ATP by monitoring the emission of the dye above 505 nm following excitation at 340 and 380 nm (exposure for 20 ms, once per second). Following removal of InsP₃, refilling of the stores to allow repeated stimulations was accomplished by superfusion of ICM containing MgCl₂. Rates of Ca²⁺ release were estimated by fitting the initial 20-s period of decreasing fluorescence to a single exponential function (GraphPad Prism, San Diego, CA).

Concentration-response Relationships and Statistical Analysis—The effects of treatment were determined by normalizing the peak change in fluorescence ratio by stimulation following forskolin exposure to that of stimulation in control HEPES-PSS. Thus, pooled data represent a normalized-fold increase over control for the treated trial. Multiple cells were analyzed in each experiment. This mean value from an individual experiment was pooled and averaged with multiple experimental runs from at least three different batches of transfected cells. Where statistical significance is indicated, two-tailed heteroscedastic t tests were performed. p values of <0.05 were considered to indicate statistical significance and are denoted by an asterisk in the figures.

Normalized F concentration-response relationships were fit with the following logistic Equation 1,

(Eq. 1)

where F represents the change in fluorescence normalized to the maximal response, C is agent concentration; EC₅₀ is the concentration where the response is half of maximum, and SlopeF is a slope factor related to the Hill coefficient.

	RESULTS AND DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS AND DISCUSSION REFERENCES

 
Binding of ATP to the "ATPC" Site in S2^-InsP₃R-1—cDNA encoding fusion proteins of GST and aa 1574-1765 of the S2^-InsP₃R-1 were constructed, and the proteins were expressed and purified from Escherichia coli. The cDNAs were designed to include the putative ATPC site (aa 1688-1693, Gly-Tyr-Gly-Glu-Lys-Gly) unique to S2^-InsP₃R-1 and include the Ser-1755 phosphorylation site as well as sufficient flanking sequence to obtain stable proteins. The latter consideration was based on previous observations by Maes et al. (33) that small receptor fragments bound nucleotides poorly. The tertiary structure of nucleotide binding proteins predicts that the Gly-X-Gly-X-X-Gly motif facilitates binding of ATP by forming a pocket that allows a favorable interface for hydrogen bonding and further favors interaction of the negatively charged pyrophosphate moiety of ATP with protein. The second glycine residue is especially important in this regard, and substitution of this residue with any amino acid with a side chain is predicted to interfere with this interaction (41). Indeed, several naturally occurring mutations in this motif lead to disease states. These include mutations in the insulin receptor, p21, and H-Ras-1 (41, 47, 48). We therefore also designed a construct encoding a GST fusion of the same region of the S2^-InsP₃R but encoding a mutation of the second glycine in the Gly-X-Gly-X-X-Gly motif to alanine (G1690A; Gly-Tyr-Ala-Glu-Lys-Gly), which we referred to as GST-ATPC.

Following purification by affinity chromatography and further purification by size exclusion, the fusion proteins were separated by SDS-PAGE and either transferred to nitrocellulose for Western analysis or stained with Coomassie Blue. Fig. 2A (top) shows a sample of GST-ATPC (lane 1) or GST-ATPC (lane 2) stained with Coomassie Blue and confirms that the proteins migrate at the predicted molecular mass of 44.5 kDa. The middle panel shows that antiserum raised against the sequence flanking Ser-1755 recognizes the GST-ATPC and GST-ATPC. Similarly, -GST antiserum also recognizes proteins of identical weight. Following cleavage with TEV protease, the protein fraction retained on the column from either construct migrates in a fashion predicted for GST alone (27 kDa; lanes 3 and 4; top) and is recognized by -GST antiserum (lanes 3 and 4, bottom) but not -S1755 (lanes 3 and 4, middle). Conversely, the cleaved, eluted fraction runs at a mass predicted for the ATPC site protein (17.5 kDa; lanes 5, top)or ATPC (lane 6, top) and is recognized by -S1755 antiserum (lanes 5 and 6; ATPC or ATPC, respectively; middle) but not -GST antiserum (lanes 5 and 6, bottom).

Next, experiments were performed to judge nucleotide binding to ATPC following cleavage and purification from GST. This was assessed by monitoring the binding of TNP-ATP, a derivative of ATP that exhibits enhanced fluorescence and a spectral shift in maximal fluorescence when bound to peptides (44, 45). Increasing concentrations of TNP-ATP in the presence of 1 mg of ATPC resulted in an increase in fluorescence as shown in Fig. 2B (filled circles) (corrected for the increase in fluorescence in the absence of protein in all cases). The EC₅₀ for the enhanced fluorescence was 300 ± 10 nM TNP-ATP (n = 11 preparations of protein performed in triplicate). The increase in TNP-ATP fluorescence was completely abrogated by competition with 10 mM ATP incubated prior to the addition of the TNP-ATP as shown in the filled triangles in Fig. 2B (n = 4). These data establish that the ATPC site is indeed capable of binding ATP with relatively high affinity and presumably indicate that in situ, the motif binds ATP in the S2^-InsP₃R. Importantly, as shown in Fig. 2B (open circles), the single point mutation in the ATPC site construct (ATPC) completely negates binding of TNP-ATP to ATPC.

Experiments were also performed to confirm ATP binding to fragments of the InsP₃R-1 containing the ATPA and ATPB sites and importantly to determine if similar Gly Ala mutations abrogated binding to these sites. As shown in Fig. 2, C and D, for the ATPA and ATPB sites, respectively, TNP-ATP bound to both fragments with high affinity (EC₅₀ = 53 ± 4nM for ATPA; EC₅₀ 71 ± 4nM for ATPB (n = 3). Protein fragments were again constructed containing Gly Ala mutations within the respective nucleotide binding folds. In the case of the ATPA site, which contains two putative binding folds, both Gly-1775 and Gly-1777 were mutated to Ala, whereas in the ATPB site, a Gly-2018 Ala construct was made. As shown in Fig. 2, C and D, respectively, the Gly Ala mutation in either site completely abolished binding of TNP-ATP.

It should be noted, however, that it is difficult to make a definitive conclusion regarding the affinity of the ATPC site in vivo or to make comparisons with the ATPA and ATPB sites. This is primarily because the apparent affinity observed is probably only relevant in the structural context of this particular fragment and secondarily because the affinity of TNP-ATP for nucleotides has been reported to be higher than ATP itself (44).

View larger version (33K): [in this window] [in a new window]   FIGURE 2. Assessment of ATP binding to ATPC. A, GST fusion proteins consisting of GST and amino acids 1574-1765 of S2- InsP3R-1 either WT or ATPC were expressed and purified from E. coli. Fusion proteins or proteins cleaved following TEV protease incubation were separated by SDS-PAGE and either Coomassie Blue-stained (top) or transferred to nitrocellulose for immunoblotting (IB) (middle and bottom). The fusion proteins ran at the expected size of 44 kDa (lanes 1 and 2) and were recognized by antiserum recognizing InsP3R-1 or GST (middle and bottom, respectively). Following cleavage of GST, the fraction retained on the column (lanes 3 and 4) migrates as expected for GST and is recognized by -GST (bottom) but not -S1755 (middle). Conversely, the flow through from the column (lanes 5 and 6) migrates as expected for the ATPC/ATPC fragment. This fragment was not recognized by-GST (middle) but was recognized by-S1755 (bottom). B, the binding of ATP to ATPC was assessed by monitoring TNP-ATP fluorescence as described under "Materials and Methods." TNP-ATP bound specifically to ATPC with an EC50 = 300 ± 10 nM (filled circles). This specific binding was completely inhibited by prior incubation with 10 mM ATP (filled triangles). TNP-ATP did not bind to ATPC (open circles). C, TNP-ATP bound with high affinity to the ATPA site consisting of a fragment of S2- InsP3R-1 corresponding to amino acids 1756-1850 (filled squares; EC50 = 53 ± 4 nM). TNP-ATP failed to bind to ATPA (open squares). D, TNP-ATP bound with high affinity to the ATPB site consisting of a fragment of S2- InsP3R-1 corresponding to amino acids 1944-2040 (filled inverted triangles; EC50 = 71 ± 5 nM). TNP-ATP failed to bind to ATPB (open inverted triangles).

View larger version (24K): [in this window] [in a new window]   FIGURE 3. Assessment of the functional sensitivity of ATP binding site mutants. Concentration-response relationships were generated in DT40-3KO-M3 cells transiently expressing the indicated construct. A, a representative trace from WT S2- InsP3R-1-expressing cells stimulated with a range of CCh concentrations (0.3-100 µM). B, a representative trace for cells expressing the ATPC S2- InsP3R-1 construct, markedly less sensitive to CCh stimulation. C, a representative trace from cells expressing the ATPA construct, which, in contrast to ATPC, shows a concentration versus response relationship indistinguishable from WT. D, a representative trace from cells expressing ATPB; similarly, the concentration versus response relationship is similar to WT. E, the initial peak of each response (as an indication of Ca2+ release) was normalized to maximal response attained in a particular cell and pooled data used to create a concentration versus response relationship for each construct. The fit for each data set is shown and illustrates that only ATPC is altered when compared with WT. F, the maximal change in fluorescence attained with each construct, which was not different, indicating a change in sensitivity of release but not efficacy. G, the oscillation frequency for each construct through the range of CCh concentrations used. In WT, ATPA, and ATPB, the oscillation frequency is not markedly modulated by CCh concentration. In ATPC, although the threshold for the generation of regular oscillations is higher than WT, the profile of the response is not markedly different.

View larger version (37K): [in this window] [in a new window]   FIGURE 4. Assessment of effects of ATP on Ca2+ release in ATPC. A, bright field image of DT40-3KO cells transiently expressing S2- InsP3R-1. B, a fluorescent image of the same field of cells loaded with Furaptra (excitation, 340 nm; emission, 505 nm). C, a fluorescent image following permeabilization with -escin, showing perinuclear fluorescence of Furaptra. D, a fluorescent image of retained nuclear HcRed-nuc fluorescence as a successful transfection marker (excitation, 560 nm; emission, 585 nm). E, DT40-3KO cells expressing WT S2- InsP3R-1. Following permeabilization, the remaining Furaptra fluorescence was monitored as an indication of the luminal [Ca2+]. A saturating [ATP] enhances the rate of Ca2+ release following stimulation with a maximal [InsP3]. F, a similar enhancement is observed in cells expressing ATPC. G, in cells expressing WT S2- InsP3R-1, Ca2+ release in response to a submaximal [InsP3] is dramatically enhanced in the presence of 5 mM ATP. H, a similar enhancement is observed in cells expressing ATPC.

Mutation of the ATPC Site Abrogates the Functional Effect of PKA-dependent Phosphorylation of S2^- InsP₃R-1—We have previously reported unique characteristics of the S2^- InsP₃R-1 in regard to its phosphorylation by cyclic nucleotide-dependent kinases (23, 24). Because ATP binding to the ATPC site represents a specific, major structural and potentially mechanistic difference between the S2^- and S2⁺InsP₃R-1, any potential role this site may play in defining the specific characteristics of PKA-mediated phosphorylation of the S2^- InsP₃R-1 was next investigated. Agents that raise cAMP, such as forskolin, result in a dramatic potentiation of Ca²⁺ signals stimulated by InsP₃ as a result of phosphorylation at either Ser-1589 or Ser-1755 in S2^-InsP₃R-1 (23, 24). Since the major effect of InsP₃R-1 phosphorylation is to increase the sensitivity of the receptor to InsP₃, the phosphorylation status of InsP₃R can also contribute to the sensitivity of a cell to stimulation by InsP₃-mobilizing agonists. In support of this idea, DT40-3KO cells expressing phosphomimetic (Ser Glu) InsP₃R mutants are more sensitive to stimulation than cells expressing WT S2^-InsP₃R-1 (24).

View larger version (20K): [in this window] [in a new window]   FIGURE 5. Functional effects of PKA activation in ATPC. A, a representative trace from a DT40-3KO cell transiently expressing both M3R and WT S2- InsP3R-1. Application of a threshold concentration of CCh results in a small increase in [Ca2+]i. Following incubation with forskolin to increase cAMP, subsequent stimulation with an identical concentration of CCh resulted in a dramatic potentiation of the [Ca2+]i signal. B, a representative trace from a DT40-3KO cell expressing M3R and ATPC. The potentiating effect of raising cAMP is lost with this construct. C, raising cAMP results in an enhanced CCh-stimulated Ca2+ signal in ATPA/M3R-expressing cells. D, raising cAMP results in an enhanced CCh-stimulated Ca2+ signal in ATPB/M3R-expressing cells. Pooled data from each construct expressed as -fold enhancement of the CCh response in the presence of forskolin. The potentiation was also lost in cells expressing ATPC constructs in which individual PKA phosphorylation sites were mutated from Ser to Ala (A-ATPC-S, S1589A/G1690A/Ser-1755; S-ATPC-A, Ser-1589/G1690A/S1755A).

Foremost, these data demonstrate that nucleotide binding to the ATPC site plays an important role in defining the susceptibility of the S2-InsP₃R-1 to regulation by PKA. Secondarily, however, this striking lack of PKA regulation in the ATPC mutant InsP₃R may also, at least in part, explain the decreased sensitivity of the ATPC mutant observed in Fig. 3B. To support this idea, the assumptions must be made that any constitutive functional effect of PKA phosphorylation under resting conditions is not present in the ATPC mutant and also absent in permeabilized cells. The absence of effects in the Ca²⁺ release assay could occur because permeabilization results in loss of PKA or, alternatively, because the effective infinite volume of the cytoplasm created by permeabilization results in insufficient levels of cAMP to activate any remaining anchored kinase. The decreased sensitivity of the ATPC mutant is consistent with data indicating that a S2^-InsP₃R-1 construct containing mutations at S1589A and S1755A (referred to in Ref. 24 as the "AA" construct), which also is not subject to phosphoregulation by PKA, is characterized by an 8-fold right shift in the sensitivity of agonist-induced Ca²⁺ release when compared with WT S2^- InsP₃R-1-expressing cells (24). This difference in sensitivity is consistent with a portion of the WT InsP₃R-1 but not the AA mutant being phosphorylated at these sites under basal conditions (24). Notwithstanding this idea, it should, however, be noted that the absence of regulation by PKA of ATPC under basal conditions may not account in full for the decrease in the sensitivity of Ca²⁺ release observed in cells expressing this construct. Indeed, these data do not necessarily exclude the possibility that the decrease in sensitivity and loss of phosphoregulation in ATPC mutants under basal conditions are not, in fact, causally associated. In this scenario, the increase in sensitivity of the receptor may simply result from the modulation by ATP binding to the ATPC site of additional unrelated and, to this point, undefined processes that impact the regulation of Ca²⁺ release through the S2^- InsP₃R-1.

View larger version (33K): [in this window] [in a new window]   FIGURE 6. PKA fails to phosphorylate ATPC. The phosphorylation status of individual constructs transiently expressed in HEK-293 was assessed as described under "Materials and Methods" using an antibody that specifically recognizes InsP3R-1 phosphorylated at Ser-1755. Incubation with forskolin resulted in the increased phosphorylation of WT S2- InsP3R-1 (compare lanes 1 and 2) but not of ATPC (lanes 3 and 4) or A-ATPC-S (lanes 5 and 6). In the lower panel, the blot was reprobed with antiserum that recognizes unphosphorylated InsP3R-1 and shows the presence of InsP3R in each lane. WB, Western blot.

In total, these data strongly suggest that the ATPC mutant is not subject to regulation by PKA and that this occurs as a result of the failure of PKA to phosphorylate the ATPC mutant. A reasonable correlate of this idea is that binding of ATP to the ATPC site in S2^-InsP₃R-1 controls the susceptibility of the receptor to modulation by PKA-dependent phosphorylation. Because this pathway is ubiquitous and is often activated appreciably under basal conditions (49), this regulation may contribute to defining the sensitivity of the receptor to InsP₃ and thus the absolute sensitivity of cells to agonist stimulation, especially under conditions of concurrent activation of both signal transduction pathways. In the absence of information regarding the in vivo affinity of the ATPC site for ATP, it is difficult to predict whether this regulation occurs in a graded or all-or-nothing fashion. The functional sensitivity of the InsP₃R-1 to ATP measured as single channel activity has been estimated at 130 µM (34, 37, 42). If the affinity of the ATPC site in vivo is within this range, ATP binding may simply establish a conformation of the receptor that is constantly susceptible to regulation by PKA. Conversely, however, the functional sensitivity to ATP in the context of the InsP₃R-3 is much lower, in the range of 2 mM (34, 35). If in vivo the ATPC site has an affinity in this range, the site in the S2^-InsP₃R-1 might be tuned to sense dynamic changes in ATP over the physiological cellular range. In this case, regulation by PKA and thus ultimately the sensitivity of the cells to agonist could be tuned to sense the metabolic status of the cell.

	FOOTNOTES

 
^* This work was supported by National Institutes of Health (NIH) Grants RO1-DK54568, R01-DE14756, and R01-DE16999 (to D. I. Y.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported by NIDCR, NIH, Grant T32-DE07202.

² To whom correspondence should be addressed. Tel.: 585-273-2154; E-mail: David_Yule{at}urmc.rochester.edu.

³ The abbreviations used are: InsP₃R, inositol 1,4,5-trisphosphate receptor; InsP₃, inositol 1,4,5-trisphosphate; CCh, carbamylcholine (carbachol); PKA, cAMP-dependent protein kinase; aa, amino acids; GST, glutathione S-transferase; TNP-ATP, trinitrophenyl-ATP; WT, wild type; TEV, tobacco etch virus.

	ACKNOWLEDGMENTS

 
We thank Drs. T. Shuttleworth and D. Brown for helpful discussion throughout this study, J. Thompson for thorough proofreading of the manuscript, and L. Bilodeau for excellent technical assistance throughout the study.

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