Intracellular calcium levels are differentially regulated in T lymphocytes triggered by anti-CD2 and anti-CD3 monoclonal antibodies

Vol. 7. No, 3, pp. 287 293, 1995. Copyright © 1995 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0898~,568/95 $9.50 + 0.00

CellularSigmdling

Pergamon 0898-6568(94)00079-4

INTRACELLULAR REGULATED

CALCIUM

LEVELS

IN T LYMPHOCYTES ANTI-CD3

ARE DIFFERENTIALLY

TRIGGERED

MONOCLONAL

BY ANTI-CD2

AND

ANTIBODIES

F A B R I Z I O SPINOZZI,*I" E L I S A B E T T A A G E A , * O N E L I A BISTONI,* S I L V I A BELIA,+ A N N A M A R I A TRAVETTI,* R O B E R T O GERLI,* C H R I S T O P H E R M U S C A T * and A L B E R T O BERTOTTO§ Departments of *Internal Medicine, ~Cellular Biology and §Pediatrics, University of Perugia, 1-06100 Perugia, Italy

(Received 9 September 1994; and accepted 1 October 1994) Abstract--Antigen and/or mitogen-driven T-cell activation is mediated by a rise in intracellular free Ca 2+, as second messenger. A regulatory key role for this process is represented by membrane-associated [Ca2+/Mg2+] ATP-ase that is mainly devoted to extrusion of intracellular ion excess. In the present study we have investigated the kinetics of Ca 2+ fluxes in both resting and already activated (Jurkat T-cell line) T lymphocytes after CD3 and CD2 (T112 and T113) triggering and focused our attention on plasma membrane [CaZ+/Mg2+] ATP-ase activity. In both resting T cells and Jurkat cell line, the CD2 stimulation was able to determine a rise in intracellular free Ca 2+ higher than that observed after CD3 triggering. In addition, this calcium signal was independent of negative feedback control exerted by [Ca2+/Mg2÷] ATP-ase, as well as of IP3 generation. Thus the CD2 molecular system may, together with cell-adhesion properties, act as an amplifier of Ca 2+ signals that, if delivered in the context of other molecular systems, such as CD3 or MHC class II antigens, are essentially devoted to the polyclonal co-stimulatory recruitment of a larger cellular repertoire.

Keywords: Intracellular calcium CD3 and CD2 stimulation, T lymphocytes, Jurkat T cells, IP3 generation, Ca2+/Mg2+ ATP-ase.

.INTRODUCTION

senger. However, the CD3 and CD2 m A b s have been found to differ in their ability to transduce activating signals which lead to intracellular Ca 2+ m o b i l i z a t i o n . In particular, p h o s p h o r y l a t i o n o f tyrosine substrates, CD3-~ and -r I chains, phospholypase-C activation and inositol 1,4,5-triphosphate (IP3) production may take place in different ways depending on the type of the m A b used [8, 9]. The source of the increased intracellular Ca 2+ has been the subject of investigation for several years. Studies of permeabilized Jurkat cells e x p o s e d to p u r i f i e d IP 3 suggest that the initial transient Ca 2+ peak is due to Ip3-mediated release from intracellular stores [10]. This mechanism, however, is insufficient to account for the sustained elevation of intracellular Ca 2+ at approximately twice the level of a resting cell. A second mechanism for calcium accumulation is an altered

T lymphocytes can be activated by specific antigens [ 1] or by polyclonal T-cell activators, such as m A b s directed against structures closely related to the TcR [2]. Both the TcR complex and appropriate A P C are i n v o l v e d in the activation process induced by the mitogenic stimulus [3, 4]. The subsequent priming of T cells is mediated by a rise in cytoplasmic free calcium [5-7], as a second mes-

+ A u t h o r to w h o m c o r r e s p o n d e n c e s h o u l d be a d d r e s s e d at: lstituto di C l i n i c a M e d i c a 1, P o l i c l i n i c o M o n t e l u c e , 1-06122 P e r u g i a , Italy. Abbreviations: - - I P 3 inositol 1,4,5-triphosphate; - - T c R T cell receptor for antigen: APC -- antigen-presenting cell; mAb -- monoclonal antibody; PBMC -- peripheral blood m o n o n u c l e a r .cells; S R B C - - s h e e p r e d b l o o d cells.

287

288

F. SPINOZZI el al.

f l u x a c r o s s the p l a s m a m e m b r a n e . T h e s l o w e d extrusion o f cytosolic Ca 2+ by plasma m e m b r a n e [CaX+/Mg -'+] A T P - a s e a n d / o r the e n h a n c e d Ca 2+ i n f l u x a c r o s s the m e m b r a n e by an ion c h a n n e l may all be implicated [2, 6, 11]. The aim o f the present study was to investigate the kinetics o f Ca 2+ flux in freshly isolated normal human T l y m p h o c y t e s cultured in the presence o f anti-CD3 or appropriate pairs (T1 l. and T113) of a n t i - C D 2 m A b s . The kinetic pattern o f an already activated T cell was investigated by carrying out identical e x p e r i m e n t s on the Jurkat leukemic cell line.

14]. The cells were washed and resuspended in a standard medium containing 125 mM NaC1, 5 mM KCI, 1 mM MgSO4, 1 mM KH2PO4, 5.5 mM glucose, 1 mM CaCI2 and 20 mM HEPES pH 7.4. Three micromolar Fura-2/AM and 250 gm Sulphinpyrazone were then added and the cell suspension incubated at 37°C for 30 rain. The cells were washed, resuspended in the standard medium with 250 btM Sulphinpyrazone plus antiCD3 or anti-CD2 mAb and transferred into thermostatically controlled cuvettes equipped for magnetic stirring. Fluorescence measurements were made on a Perkin-Elmer LS-5B (excitation: 340 nm, emission: 485 nm) connected with a computering system. Final calibration was done with 0.3 M EGTA-2 M Tris pH 8.5, 0.05% Triton X-100, 4 mM CaCI~. Intracellular Ca ~+ concentration was calculated according to the general formula:

MATERIALS AND METHODS F - Fin,n

Cell preparation

PBMC from 10 healthy control subjects were isolated by F i c o l l - H y a q u e (Lymphoprep, Nycomed AS, Oslo, Norway) density gradient centrifugation, resuspended in RPMI-1640 supplemented with 10% FCS, 4 mM L-glutamine, 100 U/ml penicillin and 100 pg/ml streptomycin (complete medium) (Gibco, Grand Island, NY, U.S.A.) and separated into SRBC rosette-enriched (E +) and rosette-depleted ( E ) subsets, as previously described [12]. The E cell suspensions were then irradiated at 3000 rad and used as source of APC. E+ cell populations were passed through nylon wool columns and treated with the OKMI mAb (Ortho, Raritan, N J, U.S.A.) plus rabbit C (Cedarlane, Ornby, Ontario, Canada) to eliminate residual contaminating monocytes. On the basis of their reactivity with the anti-CD3 mAb OKT3 (Ortho) (> 98% by flow cytometric analysis, FACScan©, Becton Dickinson, Mountain View, CA, U.S.A.), these mononuclear cells were considered highly purified T-cell subsets. The Jurkat cell line was continuously grown in coinplete medium at 3 T C in 5% COe atmosphere for all experiments, and both purified normal T cells and Jurkat cells were stimulated with a n t i - C D 3 mAb (Tissue type culture, U.S.A., work dilution 0.1 ~g/ml, unless otherwise specified) or appropriate pairs of antiCD2 mAbs (anti-TI le and T117, a generous gift of Dr S. F. Schlossman, Dana Farber Cancer Center, Boston, MA, U.S.A., work dilution 1:400 of ascite fluids). Control experiments were also performed with mouse ascite fluid (from 1:100 to 1:400 working dilution). hm'acellular calcium levels

Concentration of intracellular Ca -'+ was measured in highly purified T-cell suspensions using Fura-2/AM (a calcium fluorescent ester chelator and indicator) [13,

[Ca~÷l~-

×

Kd.

where: K<~ = F u r a - 2 - C a e+ dissociation constant: F = intracellular indicator fluorescence; F,,,~,, = fluorescence measured after addition of 0.05% Triton X-100 and 0.3 M EGTA-2M Tris; F ..... = cell fluorescence alter CacL saturation. Plotted values are expressed as nmol of intracellular Ca :+. IP s determination

T cells (10 × 106 cells) were stimulated with antiCD3 (work dilution 0.1 lug/ml) or anti-CD2 (T11~ and TI 1> work dilution 1:400 and 1:800 of ascite fluids) mAb. After proteic sedimentation by microfuge ( 12,000 r.p,m, for 1 rain), the supernatants were extracted four times with H~O-saturated diethylether and, alter evaporation of residual ether (2 h in forced ventilation box), neutralized (pH 7-7.5) with I N NaOH. The samples were then applied to A m p r e p - S A X - m i n i c o l u m n s (Amersbam) and IP~ was eluted with 5 ml 0.17 M KHCO3. The quantitative determination of collected IP 3 was carried out according to Amersham's assay system and, after comparison with a standard curve, plotted as pmol/ml. [Ca-'+/Mf ÷] A TP-ase activiO, assay

The activity was measured on membranes obtained by sonication of the cells (10 pulses, l/s). The assay was carried out in 1 ml of incubation medium in the presence of 100 J,tg of membrane protein. The final salt concentration used in the medium was 100 btM CaCI,, 60 btM K-EGTA, 10 mM KCI, 5 mM MgCI~ 300 mM sucrose, 5 mM HEPES (final pH 7.4L 2.5 mM ATP. After 10 rain at 37°C, the reaction was stopped by addilion of I ml of 10% TCA and the precipitate removed

CD3- and CD2-regulated intracellular calcium levels 20

700

A

600

B

anti-CD3 -li-

G'

500

ra

~

G

400

o~

+ +

anti-CD2

C

o

Ee-,

289

0

Q. 10

300

200

/

o

100

/

2, j

5

o©oooG 0

O O

0

,

10

20

30

40

50

60

0

Time (seconds)

'

|

2o

,

|

|

|

4o

|

=

so

n

so

,

|

loo

,

12o

Time (minutes)

Fig. 1. Intracellular Ca 2+ levels (A) and IP~ generation (B) in normal highly purified T lymphocytes triggered by anti-CD3 (OKT3, 0.1 gg/ml) or appropriate pairs (T 11~_and TI 1~, final dilution 1:400) of anti-CD2 mAbs. To avoid false positive results due to the source of anti-CD2 mAb (ascite fluid), control experiments were also performed with mouse ascite fluid, that do not contained anti-human antibodies, at the same dilutions. In these conditions no significant ion fluctuations were recorded. by centrifugation at 5000 g for 10 min. The released orthophosphate was estimated on I ml of clear supernatant, as previously described [15]. The enzymatic activity was expressed as pmol/Pi released per mg of protein per rain. RESULTS The addition of anti-CD3 (OKT3) or anti-CD2 (TI 1~ and T I 1~) m A b s to freshly isolated normal T l y m p h o c y t e s r e s u l t e d in a rapid i n t r a c e l l u l a r

Ca 2+ mobilization. However, the elevation pattern was m o r e rapid and p r o n o u n c e d with a n t i - C D 2 (Fig. 1A). A better u n d e r s t a n d i n g of this p h e n o m e n o n was sought by testing to see whether there were any parallel m o d i f i c a t i o n s in IP 3 turnover. S u r p r i s i n g l y , the results r e v e a l e d that a n t i - C D 2 was u n a b l e to elicit like rises in IP3 g e n e r a t i o n (Fig. I B). The fact that c a l c i u m release from internal stores is a metabolic process presumed to be

SmM EQTA

120

120

•

$mM EGTA

.::i 110 100 100 90 80

i "

CD3 [Ix)

(2x)

"1

1

70 60

(3x)

L

...

..: ..... ',.'~ . . . . . •..

/

•

80

100

i•

CD2 [lxl

(2x)

1

l

.*

/

,,.. . ....o , ....,.... . • ,

"U.. "'41

'0

"o 40

I

I

I

200

300

400

Time (seconds)

i •

50

I

i

t

Q

.." :.,

"., •. . . .." .... ~i"

80

.:/''.,..

•

.

1

1;o

2o0

3~o

,~o

Time [seconds)

Fig. 2. Transmembrane Ca 2÷ fluxes were blocked by 5raM EGTA pre-treatment of normal T cells incubated with different anti-CD2 (from 1:400 to 1:200 final dilution) and anti-CD3 concentrations (OKT3 from 0.1 to 0.4 gg/ml).

290

F. S P I N O Z Z I

1500

CD2 (lx)

(2x)

l

1250

(Sx]

I

l

900 I- CD3 (lx}

(8x)

.t~ ...

1000

et al.

1"

i

(2x)

I 750 l"

...

/

[4x) .......

o ......

....6

5oo [

",.oy"

O"..... ........

..1~ 750

450 ]'-

, ...,..O'*" i

o

5OO

.......O.........0 . y ..0'

250 q ;'..,..,

° ....... , ........ ,";'"', ........ ,o........ ~,""","~ ....... , 0

I 200

0

400

....O ' " "

15o t

''~' .....

300

5 trim EGTA

600

! 1000

800

Time (seconds)

, o . . ,''''''O'''''"

~.

0

5 mM EGTA

'1"-'-'O"-',"O 0

100

200

. . . . . t ....... • . . . . . ql- ..... • 300

400

500

Time (seconds)

Fig. 3. Intracellular Ca 2+ levels in Jurkat T-cell line incubated with different concentrations of anti-CD2 (from 1:400 to 1:50) and anti-CD3 (from 0.1 to 0.4 gg/ml). The bottom of the graphic represent the same experiments in the presence of 5 mM EGTA. to avoid false positive results due to the source of anti-CD2 mAb (ascite fluid), control experiments were also performed with mouse ascite fluid, that do not contained anti-human antibodies, at the same dilutions. In these conditions no significant ion fluctuations were recorded. regulated by IP3 generation, mediated in turn by receptor-induced polyphosphoinositide hydrolysis [16], m a y partially account for the rapid rise in i n t r a c e l l u l a r Ca 2+ o b s e r v e d in f r e s h l y i s o l a t e d CD3-triggered, but not CD2-stimulated, T cells. However, 5 m M E G T A was effective in blocking transmembrane calcium influx in both CD3- and CD2-mediated T-cell activation (Fig. 2). This suggests that low conductance, cation non-selective calcium-permeable channels may be opened without the intervention of IP~ as second m e s s e n g e r [17] and that phospholipase C may have a differ-

~, r, -6

5

CD2 Q~

-.A

"O"

CD3

3

2

o

'5

lO

~'s

20

Minutes

Fig. 4. IP~ generation in Jurkat T-cell line following anti-CD3 (0. I mg/ml) or anti-CD2 (1:400) stimulation.

ent role in soluble anti-CD2-mediated signal transduction. Resting (GO phase of the cell cycle) T lymphocytes usually retain the option of returning to the cell cycle when activated by growth factors. Both G - p r o t e i n - l i n k e d receptors (i.e. bombesin, bradykinin) and t y r o s i n e - k i n a s e - l i n k e d receptors (i.e. p l a t e l e t - d e r i v e d g r o w t h f a c t o r , e p i t h e l i a l growth factor, insulin growth factor, and TcR) can stimulate proliferation. IP3 and diaglycerol are common to both p a t h w a y s , but in c e r t a i n cells an increase in phosphoinositide turnover is not itself a sufficient stimulus to induce m i t o g e n e s i s [18]. However, in Jurkat T cells, both anti-CD3 and antiCD2 e l i c i t e d similar rises in i n t r a c e l l u l a r Ca 2+, which were almost entirely sustained by an external flux from ion channels and/or pores (Fig, 3). In fact, the EGTA-mediated (5 raM) inhibition of external Ca 2+ fluxes not only reduced the basal intracellular ion level, but also blocked its rise after maximal mitogenic stimulation. These results are consistent with the finding that neither CD3- nor CD2-mediated stimulation is able to modify IP 3 generation (Fig. 4). The p h y s i o l o g i c a l role that the plasma membrane [Ca2+/Mg 2+] ATP-ase play in regulating the extrusion o f cytosolic free Ca 2+ had never been investigated in these experimental systems, where, in the absence o f external Ca 2+ sources (culture mediums Ca 2+ and Mg 2+ tree), this may be of func-

CD3- and CD2-regulated intracellular calcium levels

14

A

12 oD 10

E

.E

E o) :=L

8 6 4 2 0 basal

anti-CD3

14

B

12 o~ E

anti-CD2

10

6 4

bas~

anti-CD3

anti-CD2

Fig. 5. [Ca2+/Mg2+] ATP-ase activity on T lymphocyte (A) or Jurkat T-cell (B) membranes, incubated for 10 min with anti-CD3 (0.1 lag/ml) or anti-CD2 (1:400) mAbs. Values are expressed as mg inorganic phosphorus (Pi) generated in 1 rain per gg of protein membrane lysate.

tional relevance. In fact, [Ca2+/Mg 2+] ATP-ase activity fell in normal T cells after they were treated with anti-CD2, but rose when they were stimulated with anti-CD3 (Fig. 5A). Although [Ca2+/Mg 2+] ATP-ase activity was detectable in Jurkat T cells, it remains substantially unmodified after both anti-CD3 and anti-CD2 stimulation (Fig. 5B).

DISCUSSION Various ligands that bind the TcR complex induce phosphoinositide hydrolysis and Ca 2÷

291

mobilization. They include appropriately presented Ag, mAb anti-CD3 and mitogenic lectins. In addition, mAbs directed against other cell surface molecules, including CD2, may also initiate a rise in intracellular free calcium [2, 19, 20, 21]. IP 3, which is the recognized second messenger releasing Ca 2+ from intracellular stores, may induce a second independent effect by enhancing calcium influx through plasma membrane permeable channels [22]. Once initiated, the process leads to the activation of PKC and to a wide variety of cellular responses which culminate in IL-2 mRNA transcription and finally in cell proliferation and division [23]. If the common result of the interaction of a resting T lymphocyte with multiple activating molecules is focused on few second-messenger systems, it would be of interest to distinguish between single molecule-related activating signals. In addition, net changes in intracellular Ca 2+ can arise from release from intracellular stores, an increase in Ca 2+ uptake, decreased rate of efflux or a combination of these events. In particular, the rise in Ca 2+ common to both classical CD3-mediated and alternative CD2-mediated T-cell activation pathways, as well as the functional control that phosphoinositide turnover and [CA2+/Mg 2+] ATP-ase exerts in different experimental conditions, require more extensive investigation if one is to understand how T lymphocytes respond to a stimulatory signal mediated by multiple specialised molecules [24]. The dramatic rise in intracellular free Ca 2. documented'in highly purified resting T lymphocytes after anti-CD2 stimulation in the present experiments was not sustained by a parallel increase in IP3 generation. Similar results were also obtained in Jurkat T-cells. In fact, they showed elevated basal IP3 levels but no mitogen-induced modifications. It is worth noting that the calcium flux in the stimulated cells was three to fourfold higher than the resting range and twice that of the CD3stimulated control T lymphocytes. The drop in IP3 generation observed after CD2 triggering of normal T lymphocytes may be the consequence of an already described elevation in cAMP [25] which acts n e g a t i v e l y on D i a g l y c e r o l h y d r o l y s i s . Interestingly, this phenomenon may also occur, in

292

F. SPINOZZ1et al.

the absence of other stimuli, after interaction of CD2 with l y m p h o c y t e - f u n c t i o n - a s s o c i a t e d Ag-3 [26]. Ca 2+ uptake through a selective, conductive channel is sensitive to depolarization of the plasma membrane, as occurs after antigen or mitogen stimulation, whereas IP3-mediated release from intracellular stores was shown to be independent of changes in plasma membrane potentials in both B and T lymphocytes [27, 28]. For these reasons it was difficult to explain the rise in intracellular Ca 2+ that follows CD2 stimulation without measuring [Ca2+/Mg 2÷] ATP-ase function. The extrusion of intracellular free calcium is under the strict c o n t r o l o f this m e m b r a n e - a s s o c i a t e d e n e r g y dependent pump [23]. In effect, the modification in the functional activity of [Ca2+/Mg 2~] ATP-ase recorded after CD3 triggering, did not occur after CD2 stimulation. A similar phenomenon, d e s c r i b e d as c a p a c i t a t i v e c a l c i u m entry, m a y occur with substances known as inhibitors of the [Ca2+/Mg 2+] ATP-ase, such as thapsigargin, that cause depletion of intracellular Ca 2÷ pools without IP3 production and as result mimic the ability of surface membrane IP3-1inked agonists to activate Ca 2+ entry [29]. On the other hand, the distinctly different IP3 generation and [Ca2÷/Mg 2+] ATP-ase patterns seen in the Jurkat leukemic cell line may reflect the upregulated activation state of other substrates involved in intracellular Ca 2. turnover, such as t y r o s i n e k i n a s e (Ick or fvn) [21], The d y n a m i c s o f these different transducing mechan i s m s have b e e n c o m p a r e d in N I H 3T3 cells, w h i c h c a r r y both a G - p r o t e i n l i n k e d r e c e p t o r (bradykinin) and a tyrosine-kinase-linked receptor (platelet-derived-growth-factor). The IP3 formation induced by platelet-derived growth factor was much slower than that elicited by bradykinin and the resulting Ca 2+ r e s p o n s e had a much l o n g e r latency [30]. The adhesion functions now recognized in the CD2-1ymphocyte function associated Ag-3 [31] are certainly different and less complex than those that reside in the functional receptorial site constituted by the T c R / C D 3 m u l t i c h a i n system. It is therefore not surprising that the calcium signal delivered by the CD3 complex is carefully regulated by multiple internal negative and positive

feedback controls, such as the Ca 2+ levels per se, IP~ generation (influenced by both phospholipaseCf3 t and phospholipase-Cy0, [CaWMg 2+] ATP-ase activity and PKC-mediated phosphorylation of the CD3y subunit, which act as a feedback inhibitor of the CD3/TcR function [23]. The resulting intracellular Ca 2+ levels are in this way optimally modulated to induce transcription of m R N A for IL-2R and IL-2 secretion, as well as cell-cycle progression in the stimulated cells. The CD2 molecular system may, together with cell-adhesion properties, act as an a m p l i f i e r o f Ca 2+ signals that, if delivered in the context of other molecular systems, such as CD3 or MHC class II Ag, are essentially d e v o t e d to the p o l y c l o n a l c o - s t i m u l a t o r y recruitment of a larger cellular repertoire. REFERENCES 1. White J., Herman A., Pullen A. M., Kubo R., Kappler J. W. and Marrack P. (1989) Cell 56, 27-35. 2. Weiss A., lmboden J., Shoback D. and Stobo J. (1984) Proc. hath. Acad. Sci. U.S.A. 81, 4169-4173. 3. Ahmann G. B., Sachs D. M. and Hodes R. J. (-1978) Eur. J. Immunol. 8, 1981-1989, 4. F r e l i n g e r J. A. (1977) Eur. J. l m m u n o l . 7, 447-451. 5. Hesketh T. R., Smith G. A., Morre J. P., Taylor M. V. and Metcalfe J. C. (1983) J. biol. Chem. 258, 4876-4882. 6. Oettgen H. C., Terhorst C., Cantley L. C. and Rosoff P. M. (1985) Cell 40, 583-590. 7. Ledbetter J. A., June C. H,, Grosmaire L. S. and Rabinovitch P. S. (1987) Proc. hath. Acad. Sci. U.S.A, 84, 1384-1388. 8. Samelson L. E., Fletcher M. C., Ledbetter J. A. and June C. H. (1990) J. lmmunol. 145, 2448-2454. 9. Weissman A. M., Ross P., Luong E. T., GarciaMorales P., Jelachich M. L., Biddison W. E., Klausner R. D. and Samelson L. E, (1988) J. lmmunol. 141, 3532-3536. 10. Imboden J. B. and Stobo J. D. (1985) J. exp. Med. 161,446-456. 1 I. Gardner P., Alcover A., Kuno M., Moingeon P., Weyand C. M., Goronzy J. and Reinherz E. L. (1989) J. biol. Chem. 264, 1068-1076. 12. Spinozzi F., Bertotto A., Rondoni F., Gerli R., Scalise F. and Grignani F. (1991) hnmunology 73, 140-146.

13. Grynkiewicz G., Poenie M. and Tsien R. Y. (1985) J. biol. Chem. 260, 3440-3450.

CD3- and CD2-regulatedintracellularcalcium levels 14. Di Virgilio F., Steinberg T. H. and Silverstein (1990) Cell Calcium 11, 57-62. 15. Taussky H. H. and Shorr E. (1952) J. biol. Chem. 202, 675. 16. Nel A. E., Wooten M. W. and Galbraith R. M. (1987) Clin. I m m u n o l . I m m u n o p a t h o l . 44, 167-186. 17. Pecht I., Corcia A., Liuzzi M. P. T., Alcover A. and Reinherz E. L. (1987) EMBO J. 6, 1935. 18. Pouyssfgur J. and Seuwen K. (1992) A. Rev. Physiol. 54, 195. 19. Nisbet-Brown E., Cheung R. K. and Goldstein S. (1987) Nature 316, 545. 20. Weiss M. J., Daley F. F., Hodgdon J. C. and Reinherz E. L. (1984) Proc. natn. Acad. Sci. U.S.A. 81, 6836-6840. 21. Tsien R. Y., Pozzan T. and Rink T. J. (1982) Nature 295, 68-71. 22. Berridge M. J. (1993) Nature 361, 315-325. 23. Crabtree G. (1989) Science 243, 355.

293

24. Gardner P. (1989) Cell 59, 15-26. 25. Carrera A. C., Rincon, M., deLandazuri M. O. and Lopez-Botet, B. P. (1988) Eur. J. Immunol. 18, 961-964. 26. Miller G. T., Hochman P. S., Meier W., Tizard R., Bixler S. A., Rosa M. D. and Wallner B. P. (1993), J. exp. Med. 178, 211-222. 27. Gelfand E. W., MacDougall S. I., Cheung R. K. and Grinstein S. (1989) J. exp. Med. 170, 315. 28. Gelfand E. W., Cheung R. K. (1990) Eur. J. Immunol. 20, 1237-1241. 29. Putney J. W. and Bird G. S. J. (1993) Cell 75, 199-201. 30. Renard D. C., Bolton M. M., Rhee S. G., Mergolis B. L., Zilberstein A., Schlessinger J., Thomas A. P. (1992) Biochem. J. 281, 775-784. 31. Selvaragj J., Plunkett M. L., Dustin M., Sanders M. E., Shaw S. and Springer T. A. (1987) Nature 326, 400-403.