©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Human Type 1 Inositol 1,4,5-Trisphosphate Receptor from T Lymphocytes
STRUCTURE, LOCALIZATION, AND TYROSINE PHOSPHORYLATION (*)

(Received for publication, September 1, 1994; and in revised form, November 10, 1994)

David J. Harnick (§) Thottala Jayaraman Yongsheng Ma Philip Mulieri Loewe O. Go (¶) Andrew R. Marks (**)

From the Cardiovascular Institute, Molecular Medicine Program, Department of Medicine, and Brookdale Center for Molecular Biology, Mount Sinai School of Medicine, New York, New York 10029

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Inositol 1,4,5-trisphosphate receptors (IP3R) are intracellular calcium release channels involved in diverse signaling pathways. An IP3R is thought to play a role in mobilizing calcium required for activation of T lymphocytes. The IP3R is a tetrameric structure comprised of four 300-kDa subunits encoded by a 10-kilobase mRNA. In the present study we determined the structure of the human type 1 IP3R expressed in T lymphocytes (Jurkats). The IP3R in human T cells had a predicted molecular mass of 308 kDa and was most similar to the non-neuronal form of the rodent type 1 IP3R. Two putative tyrosine phosphorylation sites were identified, one near the amino terminus and one near the putative channel pore at the carboxyl terminus. During T cell activation the IP3R was tyrosine phosphorylated. A site-specific anti-IP3R antibody was used to localize the carboxyl terminus of the IP3R to the cytoplasm in T cells.


INTRODUCTION

The second messenger inositol 1,4,5-trisphosphate (IP3) (^1)triggers intracellular calcium release by activating IP3 receptor (IP3R)/calcium release channels on the endoplasmic reticulum of many types of cells. During T cell activation there is a rapid early rise in cytoplasmic calcium due to intracellular release. It has been proposed that this intracellular calcium release in T cells is triggered by IP3, reviewed in (1) . Presumably this IP3-induced calcium release occurs via an IP3R in the T cell endoplasmic reticulum. However, to date the complete structure of a T cell IP3R has not been reported.

Three forms of IP3R have been identified in other tissues. The type 1 IP3R has been biochemically characterized in murine brain (cerebellum), vas deferens, and aortic smooth muscle(2, 3, 4, 5) . The type 1 IP3R, purified from bovine aortic smooth muscle, has a molecular mass of approximately 240 kDa, based on polyacrylamide gel electrophoresis (5) and a molecular mass of 313 kDa based on cDNA cloning(6) . Examination of the single channel properties of the IP3R reconstituted into planar lipid bilayers has shown that it forms an IP3-gated cation channel(7, 8) . The type 2 IP3R has been cloned from rat cerebellum (9) and human endothelial cells(10) . The type 2 IP3R shares 69% amino acid identity with the type 1 IP3R. A third form (IP3R type 3) shares 64% identity with the amino acid sequence of the type 1 IP3R(10, 11, 12) .

A model for the transmembrane topography of the IP3R has been proposed based on hydropathy analyses of its deduced amino acid sequence(6, 13) . Hydrophobic sequences forming the putative pore are clustered in the carboxyl-terminal 25% of the linear sequence, similar to the calcium release channels/ryanodine receptors (RyR) of the sarcoplasmic reticulum(14, 15, 16) . Three domains have been proposed for the IP3R: a ligand binding domain near the amino terminus(17) , a coupling domain in the middle of the molecule that may link IP3 binding to calcium release, and the putative pore region at the carboxyl terminus(18) . Alternative splicing of the type 1 IP3R transcript (18, 19) defines neuronal and non-neuronal forms(20) .

Recently Khan et al.(21) reported that a polyclonal antibody directed against the complete IP3R protein recognized a molecule on the plasma membrane of T lymphocytes. Other groups have found IP3R protein on intracellular membranes corresponding to the endoplasmic reticulum(22, 23, 24, 25) .

The present study was designed to determine the primary structure of the type 1 IP3R in human T cells by cDNA cloning. Analysis of this primary structure revealed two consensus tyrosine phosphorylation sites, one located adjacent to the putative channel pore and the other near the IP3 binding region. To assess whether these tyrosine phosphorylation sites could potentially play a role in regulating calcium release channel function, tyrosine phosphorylation of the IP3R was examined during T cell activation. After activation of human T cells (Jurkat) by anti-CD3 antibody, the IP3R was phosphorylated on tyrosine residues. Elucidation of the primary structure of the human type 1 IP3R also provided the sequence necessary to synthesize a peptide to serve as the antigen for site-specific polyclonal antibodies. These site-specific anti-IP3R antibodies were used to localize the carboxyl terminus of the IP3R to the cytoplasm and possibly to the perinuclear region.


MATERIALS AND METHODS

Cell Culture

Jurkat cells were grown in RPMI 1640 medium containing 5% fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin. Medium was changed every 48 h.

cDNA Library Screening

To isolate the IP3R cDNA, 1 times 10^6 recombinants from a human leukemic T cell line cDNA library were screened at high stringency (final wash, 0.2 times SSC, 55 °C) as described previously(26) . The gt11 human leukemic T cell line cDNA library was screened with the following cDNA probes: 1) a 946-bp mouse aortic smooth muscle IP3R cDNA corresponding to nucleotides 817-1763 of the mouse brain type 1 IP3R sequence(27) ; 2) a 497-bp human T cell type 1 IP3R cDNA corresponding to nucleotides 5270-5766 of the mouse brain IP3R sequence(^2); 3) a second 500-bp human T cell type 1 IP3R cDNA corresponding to nucleotides 4200-4700 of the mouse brain IP3R sequence; 4) a 600-bp type 1 IP3R cDNA was amplified from human T cell total RNA by polymerase chain reaction to fill a gap between nucleotides 6500 and 7000. Human T cell type 1 IP3R cDNAs used as probes to screen the cDNA library were amplified from total RNA using reverse transcriptase followed by the polymerase chain reaction as described previously(27) , using primers based on the published mouse brain type 1 IP3R sequence(6) . After tertiary screening, 26 IP3R cDNA clones were isolated and sequenced using the dideoxy chain termination methodology(28) . The sequence reported in this study was obtained entirely from cDNAs isolated from the gt11 cDNA library with the exception of the 500 nucleotides between bp 6500 and 7000 that were sequenced from three separate amplified cDNAs.

RNA Isolation and Northern Hybridization

Total RNA was isolated from Jurkat T cells using the guanidinium isothiocyanate method as described previously(29) . RNA samples (20 µg) were size fractionated on a 1% formaldehyde-agarose gel and transferred to nitrocellulose. Hybridization was at 42 °C overnight in 50% formamide, 50 mM sodium phosphate, 5 times Denhardt's solution and calf thymus DNA. The final washing was with 0.2 times SSC, at 55 °C for 10 min. The probe used for Northern hybridization was the 5` 1.3 kilobases of the human T lymphocyte IP3R cDNA labeled with [alpha-P]dCTP to a specific activity of 1 times 10^9 cpm/µg. Autoradiography was performed with a single intensifying screen at -80 °C for 48 h.

Immunoblotting

For both immunoblotting and immunocytochemistry we used a site-specific affinity purified polyclonal anti-IP3R antibody that we had previously shown to be specific for the IP3R both by immunoblotting and immunocytochemistry (30) . This antibody was directed against a synthetic peptide, IP3R2652, based on the deduced amino acid sequence of the human T cell type 1 IP3R amino acid residues 2652-2663 ((Cys)-Glu-Gln-Asn-Glu-Leu-Arg-Asn-Leu-Gln-Glu-Lys-Leu, with an amino-terminal cysteine added for coupling to keyhole lympet hemacyanin prior to immunization in rabbits as described previously(30) . Jurkat T cell membranes were prepared using a sucrose gradient essentially as described elsewhere(31) . Fifty µg of membrane protein was size fractionated on 6% SDS-polyacrylamide gels. Proteins were transferred to polyvinylidene difluoride membranes using a Semidry apparatus (Bio-Rad) at 15 V for 1 h followed by 25 V for 1 h, both at 4 °C as described previously(30) . Filters were incubated with blocking buffer (80 mM Na(2)HP0(4), 20 mM NaH(2)P0(4), 100 mM NaCl, 0.05% Tween 20, 5% dry milk) for 1 h. Affinity purified rabbit anti-IP3R antibody was added at 1/100 dilution. Filters were washed with phosphate-buffered saline with 0.1% Tween and incubated for 1 h with goat anti-rabbit IgG-horseradish peroxidase conjugate at a dilution of 1/7500 in blocking buffer. Signals were detected using the ECL detection reagents (Amersham International Inc.) followed by autoradiography.

Immunocytochemistry

Permeabilized or unpermeabilized Jurkat cells were treated with affinity purified anti-IP3R antibody at 1/50 dilution. Cells were washed three times in phosphate-buffered saline containing 2% fetal bovine serum followed by incubation with donkey anti-rabbit IgG conjugated to biotin. Cells were washed and avidin-rhodamine conjugate was added at a 1/1000 dilution, followed by washing and fixation in 2% paraformaldehyde. For permeabilization, cells were treated with 100% methanol at room temperature for 5 min, washed, and stained as above. Cells were also stained using a rabbit antiprotein disulfide isomerase antibody (SPA-890, StressGen Biotechnologies Corp., Victoria, BC, Canada) used to stain the endoplasmic reticulum. As a negative control T cells were stained using anti-IP3R antibody that had been preabsorbed with 1 mg/ml antigenic peptide at 37 °C for 45 min (Fig. 6f) to demonstrate the specificity of the staining as described previously(30) . Incubation at 37 °C for 45 min without the antigenic peptide did not block the anti-IP3R staining of permeabilized T cells.


Figure 6: Co-localization of the IP3R and endoplasmic reticulum in human T lymphocytes (Jurkat). Cells were permeabilized and stained as in Fig. 5using either anti-IP3R antibody (panels a and c) or an antiprotein disulfide isomerase antibody used to localize the endoplasmic reticulum (panels b and d). Preabsorbed anti-IP3R antibody was used to stain the cell shown in panel e and no signal was detected (panel f). Magnification is times 100.




Figure 5: Immunocytochemistry of IP3R in human T lymphocytes (Jurkat). Permeabilized (100% methanol, 5 min) cells were stained with anti-IP3R antibody (alpha-IP3R-1) and a rhodamine-conjugated secondary antibody. Serial sections through a representative T lymphocyte are shown at 10-Å intervals. IP3R is detected inside the cell and on the endoplasmic reticulum adjacent to the inner surface of the plasma membrane. Staining of the perinuclear membrane is seen in panels c and d. Magnification is times 100.



Fluorescence Activated Cell Sorting Analysis

Affinity purified rabbit anti-IP3R antibody was added to 0.5 times 10^6 Jurkat cells at a 1/50 dilution in phosphate-buffered saline containing 2% fetal bovine serum for 30 min on ice followed by incubation with secondary antibody (donkey anti-rabbit IgG conjugated to fluorescein isothiocyanate) for an additional 30 min. Cells were either immediately analyzed using a fluorescence activated cell sorter (FACS) analyzer (Coulter) or fixed in 2% paraformaldehyde and then analyzed within 2 days. Cells were incubated with either phosphate-buffered saline or secondary antibody alone as negative controls. Permeabilization of Jurkat cells was with 100% methanol for 5 min followed by antibody staining as described above. Three thousand cells were analyzed for each sample.

Tyrosine Phosphorylation

Jurkat cells (2.5 times 10^8) were stimulated by incubating with anti-CD3 (1% culture supernatant, kindly provided by Dr. Asit Panja) at 37 °C for 3 min. Control cells were treated under identical conditions except that phosphate-buffered saline was added instead of the anti-CD3 supernatant. Cells were harvested and lysed in 0.5% Brij 96, 20 mM Tris, pH 7.5, 150 mM NaCl, 10 mM NaF, 1 mM sodium orthovanadate, 1 µg/ml leupeptin, 1 mM phenylmethylsulfonyl fluoride, 1 µg/ml pepstatin, 1.8 mg/ml iodoacetamide. Insoluble material was removed by centrifugation for 10 min at 4 °C. The antiphosphotyrosine antibody 4G10 was used for immunoprecipitation and for Western blotting as described(32, 33, 34) .


RESULTS

Primary Structure of the Human Type 1 Inositol 1,4,5-Trisphosphate Receptor

Fig. 1shows the deduced amino acid sequence of the human type 1 IP3R. A consensus sequence for eukaryotic translational start sites (CCGCCATG) (35) was present before the first in-frame ATG. The open reading frame encoded 2713 amino acid residues with a predicted molecular mass of 308 kDa. This value is consistent with the size of the IP3R protein based on immunoblot data (Fig. 3). There is 98% amino acid identity between the human and rat type 1 IP3R sequences. The neuronal specific SII exon (19) is excluded from the human T cell type 1 IP3R form, and the alternatively spliced SI exon is retained (Fig. 1). Three consensus sequences for nucleotide-binding sites (G-X-G-X-X-G-(nX)-K, n = 17-21;(36) ) are conserved at amino acid residues 1689-1694, 1737-1742, and 1978-1983 (Fig. 1). Two putative protein kinase A phosphorylation sites (R/K-R/K-X-S/T;(37) ) are conserved at serine residues 1589 and 1717 (Fig. 1). Although the transmembrane topography of the type 1 IP3R has not been fully established experimentally, the preferred model identifies six putative transmembrane segments, designated M1 through M6 (Fig. 1), that may form the channel pore clustered near the carboxyl terminus(38) .



Figure 1: Deduced amino acid sequence of the human type I inositol 1,4,5-trisphosphate receptor. Comparison of the human and rat (18) type 1 IP3R sequences reveals 98% identity. The first line is the human sequence, the second line indicates the amino acid residue where there are differences with the rat form. The alternatively spliced exon denoted SI (underlined sequence) is preferentially expressed in the thymus and spleen. The alternatively spliced SII exon (underlined sequence) is excluded from the present form of the IP3R consistent with the non-neuronal splicing pattern. The amino acid sequence of the human type 1 IP3R was deduced from cDNA cloning as described under ``Materials and Methods.'' Two putative tyrosine phosphorylation sites are denoted by the small asterisks (*) above residues 482-486 and 2617-2621. The large asterisks (*) at serine residues 1589 and 1717 denote putative protein kinase A phosphorylation sites. Six putative transmembrane sequences are overlined and labeled M1 through M6. The double underlining identifies consensus sequences for nucleotide-binding sites at amino acid residues 1689-1694, 1737-1742, and 1978-1983. Two stretches of 10 amino acids that are 90% conserved between the IP3R and RyR channels are denoted by under- and overlines: residues 2001-2010, SLTEYCQGPC, with only one mismatch (I for C in the RyR); and residues 1931-1940, ILRFLQLLCE, with only one mismatch (F for L in the RyR). The exclamation marks (!) at amino acid residues 2652-2663 identifies the sequence of the synthetic peptide used to raise site-specific anti-IP3R antibodies.




Figure 3: Immunoblot of IP3R in human T lymphocytes and in rat brain. Rat brain homogenate (50 µg of total protein) and crude membrane preparation from Jurkat were size fractionated on a 6% SDS-polyacrylamide gel, blotted to a polyvinylidene difluoride (PVDF) membrane, and probed with affinity-purified anti-IP3R antibody. A single band migrating at 300,000 Da is seen in both human T lymphocytes (Jurkat) and rat brain, a nonspecific lower molecular band is seen in rat brain, but not in human T lymphocytes. Primary antibody was used at a 1:100 dilution. Position of molecular weight markers are indicated: myosin (205 kDa), beta-galactosidase (116.5 kDa), bovine serum albumin (prestained, migrates at 80 kDa), and ovalbumin (49.5 kDa).



The type 1 IP3R is related to the RyR/calcium release channel from the sarcoplasmic reticulum. These two molecules are members of the intracellular calcium release channel family that is distinct from other known ion channel structures. Several regions of significant homology exist between the IP3R and the RyR. Two stretches of 10 amino acids are 90% conserved between the two channels: residues 2001-2010, SLTEYCQGPC, with only one mismatch (I for C in the RyR); and residues 1931-1940, ILRFLQLLCE, with only one mismatch (F for L in the RyR). In both channels these sequences are located in the putative cytoplasmic domains and could serve as binding sites for molecules that regulate both channels. Several such agents exist including calcium and caffeine although the modulatory effects of each agent differs markedly between the two channels. For example, the channels have differential sensitivities to calcium (7) and caffeine activates the RyR but inhibits the IP3R(39) . In addition to these small regions of sequence identity it has been previously observed that there is approximately 40% homology between the IP3R and RyR at the carboxyl-terminal region encoding the putative transmembrane pore forming segments(40) .

Two putative tyrosine phosphorylation sites are present at residues 482 (EDLvY) and 2617 (DsTEY) of the human type 1 IP3R. Interestingly, the site at residue 482 is not conserved in human type 2 and 3 receptors (10) , whereas the site at amino acid 2617 is conserved in the human type 2 but not the type 3 receptor. The putative tyrosine phosphorylation site at amino acid 482 is near the IP3 binding region identified by Mignery and Sudhof(17) . The putative tyrosine phosphorylation site at amino acid 2617 is near the predicted channel pore region.

Identification of the IP3R mRNA and Protein in T Lymphocytes

A single 10-kb mRNA species was identified by Northern hybridization of Jurkat total RNA using a human T lymphocyte cDNA probe (Fig. 2). IP3R mRNA levels determined by Northern hybridization analyses were constant during T cell activation by anti-CD3 (Fig. 2). Immunoblot analysis was performed using crude homogenates from rat brain and from human T cell (Jurkat) membranes to determine the specificity of the anti-IP3R antibody. The anti-IP3R antibody recognized a single high molecular mass band (300 kDa) in these crude homogenates (Fig. 3). The IP3R protein in brain and T cells had similar mobilities. The sequence of the synthetic peptide used as the antigen for the anti-IP3R antibody production is 90% identical to the type 2 and 70% identical to the type 3 IP3R. Therefore, we cannot exclude the possibility that the IP3R protein that we detected represents a mixture of types 1, 2, and 3 IP3R.


Figure 2: Northern hybridization analysis of IP3R mRNA in human T lymphocytes. A 1.3-kilobase human IP3R cDNA was hybridized to 20 µg of total RNA isolated from: lane 1, rat brain; lane 2, rat heart; lane 3, Jurkat; lane 4, PMA stimulated Jurkat; lane 5, anti-CD3 activated Jurkat. A 10-kilobase mRNA is detected in each lane. Phorbol 12-myristate 13-acetate and anti-CD3 had no effect on IP3R mRNA level in Jurkat lymphocytes. Ethidium bromide staining of the 28 S and 18 S ribosomal RNAs (after transfer) is shown to indicate that equal amounts of RNA were loaded in each lane.



Localization of the Type 1 Inositol 1,4,5-Trisphosphate Receptor in Human Lymphocytes

We used the anti-IP3R antibody to determine the cellular localization of the IP3R in human T lymphocytes (Jurkats) using two approaches. First, we analyzed immunostained cells using a FACS. Cells were stained with either preimmune serum or affinity purified anti-IP3R antibody, fixed, and fluorescence intensity was assessed by FACS analysis. Fluorescence signal was observed only on permeabilized cells (Fig. 4), nonpermeabilized cells gave no signal. These results indicated that the IP3R epitope recognized by our anti-IP3R antibody was cytoplasmic.


Figure 4: Fluorescence activated cell sorter analysis of human T lymphocyte (Jurkat) IP3R. Top panel, nonpermeabilized T cells (Jurkat); bottom panel, permeabilized T cells (Jurkat). Cells were stained with a polyclonal site-specific anti-IP3R antibody (alpha-IP3R-1) and a fluorescein isothiocyanate-conjugated secondary antibody. IP3R was detected only in permeabilized cells. Normal rabbit serum and secondary antibody alone gave no significant signal.



Using a second approach, cells were fixed and stained with the same anti-IP3R antibody and analyzed using confocal microscopy (Fig. 5). Immunofluorescence signals were observed only in permeabilized cells (Fig. 5, panels a-f) but not in non-permeabilized cells (not shown) indicating that the epitope recognized by this antibody was intracellular. Clumps of signal appeared to be associated with the plasma membrane. However, these signals were observed only in permeabilized cells, therefore we concluded that they must be recognizing an epitope inside the cell, rather than on the outside of the plasma membrane. In some confocal planes IP3R signal was observed in the perinuclear region (panels c and d). An antiprotein disulfide isomerase antibody was used to stain T cells to localize the endoplasmic reticulum (Fig. 6, a-d). The localization of the endoplasmic reticulum was similar to that of the IP3R. A preabsorbed anti-IP3R antibody did not stain T cells (Fig. 6, e and f) demonstrating the specificity of this reaction.

Tyrosine Phosphorylation

Tyrosine phosphorylation of the IP3R was examined 3 min after T cell activation by anti-CD3. Jurkat cell lysates were immunoprecipitated using an antiphosphotyrosine monoclonal antibody. These immunoprecipitates were size fractionated on SDS-polyacrylamide gels and immunoblotted using the antiphosphotyrosine antibody. Several proteins were observed to be tyrosine phosphorylated in activated T cells but not in nonactivated cells (Fig. 7, lanes 3 and 4). Another group of proteins were tyrosine phosphorylated in both nonactivated and in activated T cells but the level of phosphorylation increased following T cell activation (Fig. 7). One of the high molecular weight phosphotyrosine proteins co-migrated with the IP3R as determined by subsequent immunoblotting of the same filter with an anti-IP3R antibody (Fig. 7, lanes 5 and 6). Phosphorylated IP3R was only detected in activated T cells (Fig. 7, lane 6). To demonstrate that this high molecular weight protein was the IP3R, the reverse experiment was performed in which the IP3R was immunoprecipitated from unactivated and activated T cells using the anti-IP3R antibody. This immunoprecipitate was size fractionated and immunoblotted using the antiphosphotyrosine antibody, demonstrating that the IP3R was tyrosine phosphorylated in the activated T cells (Fig. 7, lanes 7 and 8).


Figure 7: Tyrosine phosphorylation of the human type 1 inositol 1,4,5-trisphosphate receptor. Lane 1 is an immunoblot of T cell (Jurkat) lysates using preimmune serum. Lane 2 is an immunoblot of a lysate from unactivated T cells using an anti-IP3R antibody showing the 308-kDa IP3R. Lanes 3 and 4 were immunoblotted with antiphosphotyrosine antibody. Lane 3 contains the antiphosphotyrosine antibody immunoprecipitate from unactivated T cells, lane 4 contains similar immunoprecipitate from cells activated with anti-CD3 antibody. The same filter was subsequently immunoblotted with the anti-IP3R antibody demonstrating IP3R in the activated (lane 6) but not in the unactivated (lane 5) T cells immunoprecipitated with antiphosphotyrosine antibody. The same lysates were immunoprecipitated with anti-IP3R antibody, size fractionated, and immunoblotted using antiphosphotyrosine antibody (lanes 7 and 8). Tyrosine-phosphorylated IP3R was detected only in activated T cell lysate (lane 8).




DISCUSSION

In the present study we cloned the human type 1 IP3R cDNA from T lymphocytes, demonstrated its cellular localization and its phosphorylation at tyrosine. The human type 1 IP3R is structurally similar to the type 1 receptors from rodent. Based on sequence analysis, the type 1 IP3R expressed in human T lymphocytes corresponds to the non-neuronal form of the type I IP3R. IP3R heterogeneity is created by alternative splicing, and distinct areas of the brain in rats and mice express different IP3Rs(19) . A 15-amino acid sequence near the NH(2) terminus and a 40-amino acid sequence located between two putative cytoplasmic phosphorylation sites determine the brain and non-brain forms of the type 1 IP3R (expressed predominately in brain and aortic smooth muscle)(19, 41) . The human T cell type 1 IP3R form that we have sequenced includes the alternatively spliced SI exon (amino acid residues 318-332), but not the larger 40-amino acid splice, SII, at residue 1698. Interestingly, the SI exon is expressed at highest relative levels in tissues that contain T cells and/or hematopoetic cells, thymus and spleen; whereas the SII exon is expressed almost exclusively in cerebellum(19) .

The type 1 IP3R in human T lymphocytes is most likely the intracellular calcium release channel required for T cell activation. As such we would expect it to be constitutively expressed. Indeed we did not observe regulation of IP3R mRNA during mitogenic activation of T lymphocytes (Fig. 2).

In human T lymphocytes the type 1 IP3R is expressed predominantly in the periphery of the cytoplasm near the plasma membrane, and possibly in the perinuclear membrane (Fig. 5). The site-specific antibody used in the present study for both FACS analysis and immunolocalization allows us to assign the location of the carboxyl terminus of the IP3R to an intracellular site in T lymphocytes. No signal was seen using FACS or immunocytochemistry in nonpermeabilized cells, whereas permeabilized cells reproducibly gave a strong signal ( Fig. 4and Fig. 5). Therefore, the epitope recognized by our anti-IP3R antibody must be cytoplasmic. The intense staining apparently at the inner surface of the plasma membrane suggests that the T lymphocyte IP3R might also be localized to plasmalemma caveolae, as has been reported in endothelial cells(22) , and/or to endoplasmic reticulum near the plasmalemma.

Khan et al.(21) reported that an IP3R was found in the plasma membrane of human T lymphocytes using a polyclonal antibody raised against the entire protein. The present study adds information regarding the topography of the IP3R in the membrane because it places the carboxyl terminus in the cytoplasm. Bourguignon et al.(25) showed that a monoclonal anti-IP3R antibody stained permeabilized but not nonpermeabilized mouse T lymphoma cells(25) . However, the location of the epitope for the monoclonal antibody was not identified. The finding that our anti-IP3R antibody does not recognize nonpermeabilized cells either by FACS or immunofluorescence staining excludes the possibility that the receptor could be on the plasma membrane facing outward. Moreover, if the IP3R were in the plasma membrane facing outward the IP3 binding site would be extracellular, a localization that is inconsistent with the fact that IP3 is an intracellular second messenger. Thus, two possible transmembrane configurations of the channel are consistent with existing data: 1) the IP3R is on the ER; 2) the IP3R is on the plasma membrane with the bulk of the protein including the IP3 binding site in the cytoplasm. The latter configuration would make the IP3R a calcium influx channel. We believe that this configuration is unlikely because the IP3R is a relatively nonspecific cation channel(42) . On the ER (which under physiological conditions has no gradient for sodium or potassium across its membrane(43) ), the IP3R functions as a calcium release channel due to the large electrochemical gradient for calcium. If an IP3R does exist on the plasma membrane, as has been proposed(21, 22, 44) , it could be another form of IP3R that is more selective for divalent cations than the IP3R on the ER.

Our conclusion regarding the subcellular localization of the human type 1 IP3R also agrees with functional data from Mikoshiba and colleagues (45) who demonstrated that an antibody which recognizes nearly the same carboxyl-terminal epitope as our antibody was capable of inhibiting IP3-induced intracellular calcium release in Xenopus oocytes. Again, these functional results would place the IP3R on the ER. Of interest, however, is the fact that the epitope for the monoclonal antibody used by Mikoshiba and colleagues (45) to block calcium release also overlaps the putative tyrosine phosphorylation site at amino acid residue 2617. Therefore, the possibility exists that the interference with calcium release was due to inhibition of tyrosine phosphorylation.

In T cells, activation of the T cell receptor (TCR)-CD3 complex results in recruitment of tyrosine kinases that are members of the src family including fyn and lck. It has been proposed that the src family of tyrosine kinases activate a phospholipase C isoform (PLC(1)) which in turn stimulates phosphoinositide hydrolysis leading to the generation of IP3 and subsequent activation of the IP3R. Our data showing that the IP3R is tyrosine phosphorylated during T cell activation suggest that it might also be possible that the type 1 IP3R in T cells could be a substrate for tyrosine kinases during T cell activation. Of interest, our data also agrees with that of Khan et al.(21) who showed that the IP3R co-caps with the T cell receptor during T cell activation. Co-capping would place the IP3R near the TCR during T cell activation. This association with the TCR would facilitate phosphorylation of the IP3R by tyrosine kinases that are activated during T cell activation.

One putative tyrosine phosphorylation site was located near the IP3 binding site. Tyrosine phosphorylation of this site could positively or negatively modulate the affinity of the IP3R for IP3. A decrease in the affinity of the IP3R for the negatively charged IP3 could be induced by the presence of an added negative charge of a phosphate group near the IP3 binding site. Alternatively, tyrosine phosphorylation could increase access to the binding site via a conformational change. A second site is near the putative channel pore region. Phosphorylation at this site could modify channel gating perhaps by inducing a conformational change in the IP3R, or by increasing the affinity of the channel for calcium due to the added negative charge of a phosphate group located near the channel pore. Thus, IP3 and tyrosine phosphorylation could be co-activators of the IP3R. Alternatively, negative regulation of the IP3R by tyrosine phosphorylation could be play a role in shutting off intracellular calcium release via the IP3R after anti-CD3 activation of the T cell receptor. The release of intracellular calcium during T cell activation occurs rapidly during the first few minutes after T cell activation. Activation is mediated by IP3, inactivation could be due to the subsequent phosphorylation of the IP3R. Both the IP3 generation and the tyrosine phosphorylation could be triggered by activation of the TCR. Thus, T cell activation via the TCR could signal both the activation and the inactivation of the IP3R/calcium release channel.

Localization of the IP3R to the perinuclear region (Fig. 5) is of potential significance in T cells. Early events during T cell activation are calcium dependent(1) . For example, translocation of NF-AT to the nucleus where it triggers interleukin 2 transcription is dependent on the activity of the calcium/calmodulin-dependent protein phosphatase calcineurin which is also a target for the immunosuppressant drugs FK506 and cyclosporin A(46) . Phosphoinositide signaling has been localized to the nucleus(47, 48) , and the IP3R has been reported to be present in the perinuclear region of Xenopus laevis oocytes(23) . Localization of the IP3R to the perinuclear region in Jurkats suggests that the IP3R could be involved in regulating calcium flux to the nucleus of human T cells. Moreover, cyclic changes in IP3 levels have been linked to cell-cycle changes in calcium transients and inositol polyphosphate levels have recently been shown to vary in a cell-cycle dependent manner(49, 50) , suggesting a possible role for an IP3R-mediated signaling pathway in the regulation of cell cycle progression.

The present study establishes that the non-neuronal form of the type 1 IP3R is expressed in human T cells. Moreover, there is now evidence that tyrosine phosphorylation of an IP3R in T cells occurs during T cell activation via the TCR. Finally, IP3R protein appears to be expressed in the perinuclear region and most probably in the ER of human T cells. It remains to be determined whether distinct forms of the IP3R are expressed on separate membranes in T cells.


FOOTNOTES

*
This work was supported in part by grants from the National Institutes of Health (NS29814) and the American Heart Association (to A. R. M.). The first two authors contributed equally to this work. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with the accession number(s) L38019[GenBank].

§
Howard Hughes Medical Student Fellow.

ACC/Merck Fellow.

**
Bristol-Meyers Squibb Established Investigator of the American Heart Association. To whom correspondence should be addressed: Box 1269, Mount Sinai School of Medicine, One Gustave L. Levy Place, New York, NY 10029. Tel.: 212-241-0309; Fax: 212-996-4498.

(^1)
The abbreviations used are: IP3, inositol 1,4,5-trisphosphate; IP3R, inositol 1,4,5-trisphosphate receptor; RyR, ryanodine receptor; bp, base pair(s); ER, endoplasmic reticulum; FACS, fluorescence activated cell sorter; TCR, T cell receptor.

(^2)
A. R. Marks, unpublished data.


ACKNOWLEDGEMENTS

We thank Drs. Barbara Ehrlich and James Watras for helpful discussions and for reading this manuscript.


REFERENCES

  1. Gardner, P. (1989) Cell 59, 15-20 [Medline] [Order article via Infotrieve]
  2. Ross, C. A., Meldolesi, J., Milner, T. A., Saloh, T., Supattapone, S., and Snyder, S. H. (1989) Nature 339, 468-470 [CrossRef][Medline] [Order article via Infotrieve]
  3. Ferris, C. D., Huganir, R. L., Supattapone, S., and Snyder, S. H. (1989) Nature 342, 87-89 [CrossRef][Medline] [Order article via Infotrieve]
  4. Mourey, R. J., Verma, A., Supattapone, S., and Snyder, S. H. (1990) Biochem. J. 272, 383-389 [Medline] [Order article via Infotrieve]
  5. Chadwick, C. C., Saito, A., and Fleischer, S. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 2132-2136 [Abstract]
  6. Furuichi, T., Yoshikawa, S., Miyawaki, A., Wada, K., Maeda, N., and Mikoshiba, K. (1989) Nature 342, 32-38 [CrossRef][Medline] [Order article via Infotrieve]
  7. Bezprozvanny, I., Watras, J., and Ehrlich, B. (1991) Nature 351, 751-754 [CrossRef][Medline] [Order article via Infotrieve]
  8. MayrLeitner, M., Chadwick, C., Timerman, A., Fleischer, S., and Schindler, S. (1991) Cell Calcium 12, 505-514 [Medline] [Order article via Infotrieve]
  9. Sudhof, T., Newton, C., Archer, B., III, Ushkaryov, Y., and Mignery, G. (1991) EMBO J. 11, 3199-3206
  10. Yamamoto-Hino, M., Sugiyama, T., Hikichi, K., Mattel, M., Hasegawa, K., Sekine, S., Sakurada, K., Miyakawa, A., Furuichi, T., Hasegawa, M., and Mikoshiba, K. (1994) Receptors and Channels 2, 9-22 [Medline] [Order article via Infotrieve]
  11. Blondel, O., Takeda, J., Janssen, H., Seino, S., and Bell, G. (1993) J. Biol. Chem. 268, 11356-11363 [Abstract/Free Full Text]
  12. Maranto, A. (1994) J. Biol. Chem. 269, 1222-1230
  13. Miyawaki, A., Furuichi, T., Maeda, N., and Mikoshiba, K. (1990) Neuron 5, 11-18 [CrossRef][Medline] [Order article via Infotrieve]
  14. Takeshima, H., Nishimura, S., Matsumoto, T., Ishida, H., Kangawa, K., Minamino, N., Matsuo, H., Ueda, M., Hanaoka, M., Hirose, T., and Numa, S. (1989) Nature 339, 439-445 [CrossRef][Medline] [Order article via Infotrieve]
  15. Zorzato, F., Fujii, J., Otsu, K., Phillips, M., Green, N. M., Lai, F. A., Meissner, G., and MacLennan, D. H. (1990) J. Biol. Chem. 265, 2244-2256 [Abstract/Free Full Text]
  16. Marks, A. R., Fleischer, S., and Tempst, P. (1990) J. Biol. Chem. 265, 13143-13149 [Abstract/Free Full Text]
  17. Mignery, G. A., and Sudhof, T. C. (1990) EMBO J. 9, 3893-3898 [Abstract]
  18. Mignery, G. A., Newton, C. L., Archer, B. T., III, and Sudhof, T. C. (1990) J. Biol. Chem. 265, 12679-12685 [Abstract/Free Full Text]
  19. Nakagawa, T., Okano, H., Furuichi, T., Aruga, J., and Mikoshiba, K. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 6244-6248 [Abstract]
  20. Danoff, S., Ferris, C., Donath, C., Fischer, G., Munemitsu, S., Ullrich, A., Snyder, S., and Ross, C. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 2951-2955 [Abstract]
  21. Khan, A., Steiner, J., Klein, M., Schneider, M., and Snyder, S. (1992) Science 257, 815-818 [Medline] [Order article via Infotrieve]
  22. Fujimoto, T., Nakade, S., Miyawaki, A., Mikoshiba, K., and Ogawa, K. (1992) J. Cell Biol. 119, 1507-1513 [Abstract]
  23. Kume, S., Muto, A., Aruga, J., Nakagawa, T., Michikawa, T., Furuichi, T., Nakade, S., Okano, H., and Mikoshiba, K. (1993) Cell 73, 555-570 [Medline] [Order article via Infotrieve]
  24. Kijima, Y., Saito, A., Jetton, T., Magnuson, M., and Fleischer, S. (1993) J. Biol. Chem. 268, 3499-3506 [Abstract/Free Full Text]
  25. Bourguignon, L., Jin, H., Iida, N., Brandt, N., and Zhang, S. (1993) J. Biol. Chem. 268, 7290-7297 [Abstract/Free Full Text]
  26. Marks, A., Tempst, P., Hwang, K., Taubman, M., Inui, M., Chadwick, C., Fleischer, S., and Nadal-Ginard, B. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 8683-8687 [Abstract]
  27. Marks, A. R., Tempst, P., Chadwick, C. C., Riviere, L., Fleischer, S., and Nadal-Ginard, B. (1990) J. Biol. Chem. 265, 20719-20722 [Abstract/Free Full Text]
  28. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
  29. Marks, A. R., Taubman, M., Saito, A., Dai, Y., and Fleischer, S. (1991) J. Cell Biol. 114, 303-312 [Abstract]
  30. Moschella, M. C., and Marks, A. R. (1993) J. Cell Biol. 120, 1137-1146 [Abstract]
  31. Peterson, R., and Biedler, J. (1978) J. Supramol. Struct. 9, 289-298 [Medline] [Order article via Infotrieve]
  32. Letourneuer, F., and Klausner, R. (1992) Science 255, 79-83 [Medline] [Order article via Infotrieve]
  33. June, C. H., Fletcher, M., Ledbetter, J., and Samelson, L. (1990) J. Immunol. 144, 1591-1598 [Abstract/Free Full Text]
  34. Strauss, D., and Weiss, A. (1992) Cell 70, 585-591 [Medline] [Order article via Infotrieve]
  35. Kozak, M. (1984) Nucleic Acids Res. 12, 857-872 [Abstract]
  36. Wierenga, R., and Hol, W. (1983) Nature 302, 842-844 [Medline] [Order article via Infotrieve]
  37. Huganir, R., Miles, K., and Greengard, P. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 6968-6972 [Abstract]
  38. Michikawa, T., Hamanaka, H., Otsu, H., Yamamoto, A., Miyawaki, A., Furuichi, F., Tashiro, Y., and Mikoshiba, K. (1994) J. Biol. Chem. 269, 9184-9189 [Abstract/Free Full Text]
  39. Bezprozvanny, I., Bezprozvannaya, S., and Ehrlich, B. (1994) Mol. Biol. Cell. 5, 97-103 [Abstract]
  40. Mignery, G., Sudhof, T., Takei, K., and Camilli, P. (1989) Nature 342, 192-195 [CrossRef][Medline] [Order article via Infotrieve]
  41. Miyawaki, A., Furuichi, T., Ryou, Y., Yoshikawa, S., Nakagawa, T., Saitoh, T., and Mikoshiba, K. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 4911-4915 [Abstract]
  42. Bezprozvanny, I., and Ehrlich, B. (1994) J. Gen. Physiol. 104, 821-856 [Abstract]
  43. Somlyo, A., Shuman, H., and Somlyo, A. (1977) J. Cell Biol. 74, 828-857 [Abstract/Free Full Text]
  44. Kuno, N., and Gardner, P. (1987) Nature 326, 301-304 [CrossRef][Medline] [Order article via Infotrieve]
  45. Miyazaki, S., Yuzaki, M., Nakade, K., Shirakawa, H., Nakanishi, S., Nakade, S., and Mikoshiba, K. (1992) Science 257, 251-255 [Medline] [Order article via Infotrieve]
  46. Liu, J., Farmer, J., Jr., Lane, W., Friedman, J., Weissman, I., and Schreiber, S. (1991) Cell 66, 807-815 [Medline] [Order article via Infotrieve]
  47. Divecha, N., Banfic, H., and Irvine, R. (1991) EMBO J. 10, 3207-3214 [Abstract]
  48. Martelli, A., Gilmour, R., Bertagnolo, V., Neri, L., Marzoll, L., and Cocco, L. (1992) Nature 358, 242-245 [CrossRef][Medline] [Order article via Infotrieve]
  49. Ciapa, B., Pesando, D., Wilding, M., and Whitaker, M. (1994) Nature 368, 875-878 [CrossRef][Medline] [Order article via Infotrieve]
  50. Balla, T., Sim, S. S., Baukal, A. J., Rhee, S. G., and Catt, K. J. (1994) Mol. Biol. Cell 5, 17-27 [Abstract]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.