(Received for publication, September 1, 1994; and in revised form, November 10, 1994)
From the
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.
The second messenger inositol 1,4,5-trisphosphate (IP3) ()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.
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
100.
Figure 5:
Immunocytochemistry of IP3R in human T
lymphocytes (Jurkat). Permeabilized (100% methanol, 5 min) cells were
stained with anti-IP3R antibody (-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
100.
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),
-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.
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.
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 (-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.
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).
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 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) 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.
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].