By
From the Department of Molecular Genetics, Institute for Liver Research, Kansai Medical University, Moriguchi 570-8506, Japan
![]() |
Abstract |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Paired immunoglobulin-like receptor (PIR)-A and PIR-B possess similar ectodomains with six
immunoglobulin-like loops, but have distinct transmembrane and cytoplasmic domains. PIR-B
bears immunoreceptor tyrosine-based inhibitory motif (ITIM) sequences in its cytoplasmic domain that recruit Src homology (SH)2 domain-containing tyrosine phosphatases SHP-1 and
SHP-2, leading to inhibition of B and mast cell activation. In contrast, the PIR-A protein has a
charged Arg residue in its transmembrane region and a short cytoplasmic domain that lacks
ITIM sequences. Here we show that Fc receptor chain, containing an immunoreceptor tyrosine-based activation motif (ITAM), associates with PIR-A. Cross-linking of this PIR-A
complex results in mast cell activation such as calcium mobilization in an ITAM-dependent
manner. Thus, our data provide evidence for the existence of two opposite signaling pathways upon PIR aggregation. PIR-A induces the stimulatory signal by using ITAM in the associated
chain, whereas PIR-B mediates the inhibitory signal through its ITIMs.
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cross-linking of immune receptors such as BCR, TCR, or FcR on a variety of cells leads to their activation through the sequential activation of protein tyrosine kinases (PTKs) (1). Several features have emerged that are common to these activating receptors. They are all oligomeric complexes in which ligand binding and signal transduction are compartmentalized into distinct receptor subunits. Hence, these receptors comprise one or more immunoreceptor tyrosine-based activation motif (ITAM)- containing subunits. When the ligand-binding subunit(s) of the receptor is engaged, the cytoplasmic ITAMs are tyrosine phosphorylated by src-family PTKs. This leads to the recruitment of syk-family PTKs, which trigger a cascade of intracellular phosphorylations that result in cellular activation.
Balancing these activation responses are the inhibitory receptors and their associated signaling molecules, which are responsible for setting threshold levels for activation signals as well as terminating activation responses. These inhibitory receptors also share several common features. They have one or more immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in their cytoplasmic domains, tyrosine phosphorylation of which leads to the recruitment of signaling molecules capable of inhibiting cell activation (4- 7). Indeed, NK inhibitory receptors recruit tyrosine phosphatase SHP-1 to inhibit NK cell activation (8). Certain isoforms of NK receptors that lack ITIM sequences have been proposed to function as activation receptors rather than inhibitory receptors (11). A conspicuous feature of these noninhibitory NK receptors is the presence of a basic amino acid in the transmembrane domain, which may allow their association with signal-transducing proteins such as DAP12, as demonstrated recently for killer-activating receptors (12, 13).
The paired Ig-like receptors (PIRs) recently identified
on B cells and myeloid lineage cells include PIR-A molecule, which has a short cytoplasmic domain, and PIR-B
molecule, which bears four potential ITIMs in its cytoplasmic domain (14, 15). In contrast to the unique transmembrane and cytoplasmic domains between PIR-A and PIR-B,
extracellular regions of these molecules are very homologous, suggesting that both molecules bind the putative common ligand. We and others have recently demonstrated that PIR-B functions as an inhibitory receptor in B
and mast cells (16, 17). Here, we show that PIR-A functions as an activation receptor as a consequence of its association with the ITAM-bearing FcR chain in mast cells.
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cells, Expression Constructs, and Abs.
Chicken DT40 cells and mouse A20 IIA1.6 cells (FcImmunoprecipitation and Western Blotting Analysis.
To detect association ofNorthern Analysis.
RNA was prepared from A20 IIA1.6 and RBL-2H3 cells using the guanidium thiocyanate method. Total RNA (20 µg) was separated in a 1.2% formaldehyde gel, transferred to Hybond-N+ nylon membrane (Amersham Pharmacia Biotech), and probed with 32P-labeled FcRCalcium Measurements.
Cells (5 × 106) were suspended in PBS containing 20 mM Hepes (pH 7.2), 5 mM glucose, 0.025% BSA, and 1 mM CaCl2, and loaded with 3 µM Fura-2/AM at 37°C for 45 min. Cells were washed twice and adjusted to 106 cells/ml. Continuous monitoring of fluorescence from the cell suspension was performed using Hitachi F-2000 fluorescence spectrophotometer (Hitachi Limited, Tokyo, Japan) at an excitation wavelength of 340 nm and an emission wavelength of 510 nm. Calibration and calculation of calcium levels were done as previously described (24). Fc ![]() |
Results and Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Structural features of PIR-A have prompted us to test whether PIR-A is
able to transmit activation signals. For this purpose, a chimeric molecule with the transmembrane and cytoplasmic
domains of PIR-A and the extracellular domain of human
FcRIII was constructed (Fc
RIII-PIR-A), and transfected
into mouse A20 IIA1.6 B cells and rat RBL-2H3 basophilic
leukemia cells in order to obtain transformants. Expression
level of this chimeric receptor was assessed by flow cytometric analysis using anti-human Fc
RIII mAb, 3G8 (Fig.
1 A, insets). As shown in Fig. 1 A, stimulation of Fc
RIII-
PIR-A chimeric receptor on RBL-2H3 cells with 3G8 and
subsequent cross-linking resulted in a transient rise in
[Ca2+]i, whereas this [Ca2+]i increase could not be detected
in the A20 IIA1.6 transformant expressing Fc
RIII-PIR-A, demonstrating that Fc
RIII-PIR-A is able to evoke the
activation signal only in RBL-2H3 cells. Thus, these data
suggest that Fc
RIII-PIR-A is not capable of transmitting the activation signal by itself and that the responsible molecule(s) for the activation signal is expressed in RBL-2H3
cell but not in A20 IIA1.6 cells. One such candidate is the
FcR
chain, which is one component of the Fc
RIII and
Fc
RI complexes in mast cells (25). Indeed, the
chain is
expressed in RBL-2H3 cells but not A20 IIA1.6 cells, as
revealed by Northern (Fig. 1 B) and Western blotting analysis (data not shown).
|
To demonstrate that chain is associated with Fc
RIII-PIR-A,
RBL-2H3 cells expressing this chimeric receptor were lysed
by digitonin buffer, followed by immunoprecipitation with
3G8. The immune complexes were separated by SDS-PAGE gel and blotted with anti-
chain Ab, demonstrating
the association of
chain with Fc
RIII-PIR-A in RBL-2H3 cells (Fig. 1 C). This association was also shown by
transfection experiments using 293 T cells. Fc
RIII-PIR-A by itself can be expressed on the cell surface of 293 T cells to some extent, whereas cotransfection of
with Fc
RIII-
PIR-A enhanced cell surface expression of this chimeric
receptor (Fig. 2 A), suggesting that
chain is involved in
transport of Fc
RIII-PIR-A to cell surface in 293 T cells.
Moreover, the interaction of
chain with Fc
RIII-PIR-A
was reconstituted in 293 T cells, as demonstrated by coimmunoprecipitation experiments using digitonin buffer (Fig.
2 B). This association was also observed by cotransfection of
with HA-tagged PIR-A (Fig. 2 B), indicating that native PIR-A can be associated with FcR
chain.
|
To show that the association of chain with Fc
RIII-
PIR-A is critical for transmitting activation signals, we used
the DT40 B cell system, since this cell line does not express
chain like A20 cells. After obtaining DT40 transformant
expressing Fc
RIII-PIR-A (Fig. 3 A), we transfected
wild-type
chain or mutated
chain into this DT40 transformant using another drug selection marker to isolate
DT40 cells expressing both Fc
RIII-PIR-A and
chain. Mutation of the
chain (Tyr65 and Tyr76 to Phe) was designed to test whether the activation signal is dependent on
the ITAM sequence located in the cytoplasmic domain of
chain. Expression level of
chain was assessed by Western blotting analysis with anti-
chain Ab, and clones expressing comparable levels of wild-type
and mutated
were selected (Fig. 3 B) and analyzed. Although Fc
RIII-
PIR-A alone was expressed on the cell surface of DT40 B
cells, introduction of either wild-type
chain or mutated
chain enhanced the level of cell surface expression of the
chimeric receptor about fivefold, as demonstrated by flow
cytometric analysis (Fig. 3 A). These observations strengthen our previous conclusion that
chain contributes to cell
surface expression of Fc
RIII-PIR-A. However, this
chain function does not require phosphorylation of tyrosine residues in its ITAM sequence.
|
Stimulation of FcRIII-PIR-A complex with wild-type
chain evoked calcium mobilization upon receptor engagement, whereas this [Ca2+]i increase could not be detected in DT40 cells expressing Fc
RIII-PIR-A alone nor
in the receptor with mutated
chain (Fig. 4 A), indicating that ITAM of
chain is essential for transmitting the activation signal. This conclusion was supported further by the
observation that
chain, but not mutated
chain, is tyrosine phosphorylated by Fc
RIII-PIR-A stimulation (Fig.
4 B). Taken together, our results demonstrate that Fc
RIII-
PIR-A is complexed with FcR
chain, the presence of
which is essential for transmitting activation signals.
|
Coexistence of both activation and inhibitory pathways
upon receptor aggregation allows immune cells to generate
graded responses under different ligand conditions. Assuming that PIR-A and PIR-B recognize the same ligand, the
data presented here together with previous observations
(16, 17), support the model that cell surface receptors with
same or related ligand-binding specificity transmit both activation and inhibitory signals through ITAM and ITIM
sequences, respectively (26). Reminiscent of this type of
regulation is Fc receptors; FcRIIIA transmits the activation signal, whereas Fc
RIIB inhibits the ITAM-induced
activation signal despite the same ligand specificity between
these two receptors (25).
FcR chain is physically and functionally associated with
Fc
RIII-PIR-A. Since the
chain has a very short extracellular domain (18), this association is presumably mediated by transmembrane-transmembrane interactions. Supporting this notion, the transmembrane region of another
chain-interacting receptor, Fc
R, is very similar to that of
PIR-A in that positively charged Arg residue is located in
the same position, although their extracellular and cytoplasmic domains are divergent (27). Given the evidence that
HA-tagged PIR-A is also associated with
chain (Fig. 2
B), our data strongly suggest that native PIR-A associates
with FcR
chain in mast cells.
Functional interaction was demonstrated by requirement
of chain in Fc
RIII-PIR-A-induced calcium mobilization. More importantly, ITAM of the
chain was tyrosine
phosphorylated upon receptor aggregation (Fig. 4 B), indicating that the PIR-A complex transmits the activation signal through an ITAM-dependent mechanism. Thus, PIR-A uses src- and syk-family PTKs to transmit the positive signal, in a similar manner to the situation with other
ITAM-bearing receptors such as TCR, BCR, or FcRs,
whereas PIR-B mediates the negative signal through recruitment of SHP-1 and SHP-2 to phosphorylated ITIMs
in its cytoplasmic domain.
Both PIR-A and PIR-B appear to be expressed in B
cells and myeloid lineage cells, as assessed by reverse transcriptase PCR (15). The RNA content of PIR-A and PIR-B
may not necessarily reflect the cell surface expression of
each molecule, because associated molecules such as chain may enhance the transport of PIR-A to cell surface,
as seen in this study. Since the relative expression level of
PIR-A and PIR-B on cell surface is one of the critical determinants for stimulatory or inhibitory responses, associated chains such as
participate in PIR functions by not only conferring the stimulatory capability on PIR-A but
also promoting its cell surface expression.
![]() |
Footnotes |
---|
Address correspondence to Tomohiro Kurosaki, Department of Molecular Genetics, Institute for Liver Research, Kansai Medical University, Moriguchi 570-8506, Japan. Phone: 81-6-993-9445; Fax: 81-6-994-6099; E-mail: kurosaki{at}mxr.meshnet.or.jp
Received for publication 4 May 1998 and in revised form 19 June 1998.
Note added in proof. The results that PIR-A is able to transmit a stimulatory signal have been obtained independently (Yamashita, Y., M. Ono, and T. Takai. J. Immunol. In press).We would like to thank N. Yamamoto and T. Yasuda for providing us with RBL-2H3 cells and Dr. T. Takai (Tohoku University, Sendai, Japan) for communicating his unpublished results.
This work was supported by grants to T. Kurosaki from the Ministry of Education, Science, Sports, and Culture of Japan, the Sumitomo Foundation, and the Uehara Memorial Foundation.
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Weiss, A., and D.R. Littman. 1994. Signal transduction by lymphocyte antigen receptors. Cell. 76: 263-274 [Medline]. |
2. | Kurosaki, T.. 1997. Molecular mechanisms in B cell antigen receptor signaling. Curr. Opin. Immunol. 9: 309-318 [Medline]. |
3. | DeFranco, A.L.. 1997. The complexity of signaling pathways activated by the BCR. Curr. Opin. Immunol. 9: 296-308 [Medline]. |
4. |
Cambier, J.C..
1997.
Inhibitory receptors abound?
Proc. Natl.
Acad. Sci. USA.
94:
5993-5995
|
5. |
Yokoyama, W.M..
1997.
What goes up must come down: the
emerging spectrum of inhibitory receptors.
J. Exp. Med.
186:
1803-1808
|
6. | Unkeless, J.C., and J. Jin. 1997. Inhibitory receptors, ITIM sequences and phosphatases. Curr. Opin. Immunol. 9: 338-343 [Medline]. |
7. | Vivier, E., and M. Daëron. 1997. Immunoreceptor tyrosine-based inhibition motifs. Immunol. Today. 18: 286-291 [Medline]. |
8. | Burshtyn, D.N., A.M. Scharenberg, N. Wagtmann, S. Rajagopalan, K. Berrada, T. Yi, J.P. Kinet, and E.O. Long. 1996. Recruitment of tyrosine phosphatase HCP by the killer cell inhibitory receptor. Immunity. 4: 77-85 [Medline]. |
9. | Lanier, L.L.. 1997. Natural killer cell receptors and MHC class I interactions. Curr. Opin. Immunol. 9: 126-131 [Medline]. |
10. | Moretta, A., and L. Moretta. 1997. HLA class I specific inhibitory receptors. Curr. Opin. Immunol. 9: 694-701 [Medline]. |
11. | Biassoni, R., C. Cantoni, M. Falco, S. Verdiani, C. Bottino, M. Vitale, R. Conte, A. Poggi, A. Moretta, and L. Moretta. 1996. The human leukocyte antigen (HLA)-C-specific "activatory" or "inhibitory" natural killer cell receptors display highly homologous extracellular domains but differ in their transmembrane and intracytoplasmic portions. J. Exp. Med. 183: 645-650 [Abstract]. |
12. | Olcese, L., A. Cambiaggi, G. Semenzato, C. Bottino, A. Moretta, and E. Vivier. 1997. Human killer cell activatory receptors for MHC class I molecules are included in a multimeric complex expressed by natural killer cells. J. Immunol. 158: 5083-5086 [Abstract]. |
13. | Lanier, L.L., B.C. Corliss, J. Wu, C. Leong, and J.H. Phillips. 1998. Immunoreceptor DAP12 bearing a tyrosine-based activation motif is involved in activating NK cells. Nature. 391: 703-707 [Medline]. |
14. |
Hayami, K.,
D. Fukuta,
Y. Nishikawa,
Y. Yamashita,
M. Inui,
Y. Ohyama,
M. Hikita,
H. Ohmori, and
T. Takai.
1997.
Molecular cloning of a novel murine cell-surface glycoprotein homologous to killer cell inhibitory receptors.
J.
Biol. Chem.
272:
7320-7327
|
15. |
Kubagawa, H.,
P.D. Burrows, and
M.D. Cooper.
1997.
A
novel pair of immunoglobulin-like receptors expressed by B
cells and myeloid cells.
Proc. Natl. Acad. Sci. USA.
94:
5261-5266
|
16. |
Bléry, M.,
H. Kubagawa,
C.-C. Chen,
F. Vély,
M.D. Cooper, and
E. Vivier.
1998.
The paired Ig-like receptor PIR-B
is an inhibitory receptor that recruits the protein-tyrosine
phosphatase SHP-1.
Proc. Natl. Acad. Sci. USA.
95:
2446-2451
|
17. |
Maeda, A.,
M. Kurosaki,
M. Ono,
T. Takai, and
T. Kurosaki.
1998.
Requirement of tyrosine phosphatase SHP-1 and
SHP-2 for PIR-B-mediated inhibitory signal.
J. Exp. Med.
187:
1355-1360
|
18. |
Ra, C.,
M.-H. Jouvin, and
J.P. Kinet.
1989.
Complete structure of the mouse mast cell receptor for IgE (Fc![]() |
19. | Takata, M., H. Sabe, A. Hata, T. Inazu, Y. Homma, T. Nukada, H. Yamamura, and T. Kurosaki. 1994. Tyrosine kinases Lyn and Syk regulate B cell receptor-coupled Ca2+ mobilization through distinct pathways. EMBO (Eur. Mol. Biol. Organ.) J. 13: 1341-1349 [Abstract]. |
20. |
Sugawara, H.,
M. Kurosaki,
M. Takata, and
T. Kurosaki.
1997.
Genetic evidence for involvement of type 1, type 2 and
type 3 inositol 1,4,5-trisphosphate receptors in signal transduction through the B-cell antigen receptor.
EMBO (Eur.
Mol. Biol. Organ.) J.
16:
3078-3088
|
21. |
Ravetch, J.V., and
B. Perussia.
1989.
Alternative membrane
forms of Fc![]() |
22. |
Fleit, H.B.,
S.L. Wright, and
J.C. Unkeless.
1982.
Human
neutrophil Fc![]() |
23. | Kurosaki, T., I. Gander, and J.V. Ravetch. 1991. A subunit common to an IgG Fc receptor and the T-cell receptor mediates assembly through different interactions. Proc. Natl. Acad. Sci. USA. 88: 3837-3841 [Abstract]. |
24. | Grynkiewicz, G., M. Poenie, and R.Y. Tsien. 1985. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J. Biol. Chem. 260: 3440-3450 [Abstract]. |
25. | Ravetch, J.V., and J.P. Kinet. 1991. Fc receptors. Annu. Rev. Immunol. 9: 457-492 [Medline]. |
26. | Colonna, M.. 1998. Unmasking the killer's accomplice. Nature. 391: 642-643 [Medline]. |
27. |
Morton, H.C.,
I.E. van den Herik-Oudijk,
P. Vossebeld,
A. Snijders,
A.J. Verhoeven,
P.J.A. Capel, and
J.G.J. van de
Winkel.
1995.
Functional association between the human
myeloid immunoglobulin A Fc receptor (CD89) and FcR ![]() |