The Role of Protein Kinase C in T Lymphocyte Proliferation

Vol. 263, No. 33, Issue of November 25, pp. 17467-17470, 1988 Printed in U.S.A.

THEJOURNAL OF BIOLOGICAL CHEMISTRY Q 1988 by The American Society for Biochemistry and Molecular Biology, Inc.

The Role of Protein KinaseC in T Lymphocyte Proliferation EXISTENCE OF PROTEIN KINASE C-DEPENDENT AND -INDEPENDENT PATHWAYS* (Received for publication, January 11, 1988)

Juan Miguel Redondo$, Abelardo Lopez-Rivasg, Victoria Vilan, Edward J. Cragoe, Jr. 11, and Manuel Fresno$** From the SCentrode Biologia Molecular, Consejo Superior de Znvestigaciones Cientificas, Universidad Autonoma de Madrid, Canto Blanco, 28049 Madrid, the SZnstituto deZnvestigaciones Biomidicas, Consejo Superior deZnvestigaciones Cientificas, Madrid. the IlLaboratorio de ZnmunoDatolopia, - . Centro Ramon y Cajal, Madrid, and 112211 Oak Terrace Drive, Landsdale, Pennsylvania19446

The mouse cytotoxic T cell clone (CTLL-2) was able to grow in the presence of culture medium supplemented only with transferrin, 2-mercaptoethanol, and recombinant interleukin 2 (IL-2). This lymphokine stimulated the synthesis of DNA in these cells. Similarly, phorbol esters, which activate protein kinase C, induced DNA synthesis in this clone. Furthermore, this later proliferation was not blocked by anti-IL-2 receptor antibodies, which inhibited IL-%induced proliferation, suggesting that it was not indirectly due to the secretion of IL-2 by the cells. CTLL-2 cells pretreated with high doses of phorbol esters for 48 h down regulated protein kinase C and were depleted of this enzyme. This was shown by: 1) purification and in vitro assay of protein kinase C; 2) the lack of effect of phorbol esters in the stimulation of the Na+/H+antiporter which has been directly linked to the activation of protein kinase C. As expected, those protein kinase C-depleted cells no longer synthesized DNA and proliferated in response to phorbol esters. However, they proliferated identically to control cells in response to IL-2. Therefore, our results suggest two differentpathways for T cell proliferation, one which involves protein kinaseC and the other which does not.

Interleukin-2 (IL-2)' is a lymphokine which stimulates the differentiation and growth of T lymphocytes (1).Interaction of IL-2 with its specific receptor, which is expressed on activated T cells, induces the progression of the cells through the cell cycle (2), but the molecular bases of its signal transducing mechanism are stillcontroversial. Thus, contradictory data on the ability of IL-2 to stimulate phosphoinositide hydrolysis (3-5) and Ca2+ fluxes (6-8) have been reported. Furthermore, the data on the participation of a GTP binding protein (G protein) in the action of IL-2, whichcouldbe

* This work was supported by Grants 443/85 from the comisi6n Asesora de Investigacion Cientifica y Tecnica-Consejo Superior de Investigaciones Cientificas, Plan Concertado 41/82 from Antibibticos S. A., and from Fondo de Investigaciones Sanitarias de la Seguridad Social. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ** To whom correspondence should be addressed. The abbreviations used are: IL-2, interleukin-2; rIL-2, recombinant interleukin-2; DME, Dulbecco'smodified Eagle's medium; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; EGTA, [ethylenebis(oxyethylenenitrilo)]tetraaceticacid; PDBu, phorbol dibutyrate; protein kinase C, Ca2+/phospholipid-dependent enzyme; TPA, phorbol myristate acetate.

responsible for the activation of the phosphoinositide metabolism (9), have been challenged (10). Farrar and Anderson (11)have reported a translocation of protein kinase C by IL2, probably as a consequence of phosphoinositide hydrolysis (4).Since phorbol esters translocate protein kinase C in a similar manner, activate protein kinase C activity (12, 13), and mimic some of the IL-2 actions, such as the induction of some genes (14)or T cell proliferation (15), the same authors have proposed the activation of protein kinase C by IL-2 as a crucial step in the signal transduction elicited by IL-2 (11). Here, we present evidence that activation of protein kinase C is not an obligatory step inthe signal transduction elicited by IL-2. In addition, our results indicate the existence of two signal transducing pathways in T cell proliferation, one which involves protein kinase C and theother which does not. EXPERIMENTALPROCEDURES

Measurement ofthe InternalpH-MouseCTLL-2 cloned cells were continuously grown in Dulbecco's modified Eagle's medium (DME) supplemented with 5% fetal cal serum, 5 X M 2-mercaptoethanol, and 20 units/ml rIL-2 (16). CTLL-2 cells (106/ml) grown as above were treated or not with 500 ng/ml PDBu 24 h after the addition of IL-2 and cultured for 48 h at 37 'C. Then, they were harvested and washed thoroughly with DME, 10% fetal calf serum and resuspended a t 5 X lo' cells/ml in the same medium. The cells were loaded with the fluorescent pH-sensitive acetoxymethyl ester of 2',7'-bis-carboxyethyl-5,6-carboxyfluorescein(2 pg/ml) for 30 min at 37 "C. Then the cells were washed and resuspended in an electrolite solution containing 140 mM NaCl, 5 mM KCl, 0.9 mM M&~z,1.8 mM CaC12, 25 mM glucose, amino acid mixture at the same concentrations as that in DME, buffered with 16 mM Hepes and 6 mM Tris-HC1 to a pH of7.2 at 2 X lo6 cells/ml. The internalpH was determined fluorimetrically at 500 nm wavelength for excitation and 525 nm for emission at 37 "C using a Perkin-Elmer LS spectrofluorimeter and calibrated with nigericin and KC1 as described (17). Purification and in Vitro Assayof Protein KinaseC-CTLL-2 cells (10') from each group (control or PDBu-pretreated) prepared as above were collected and washed 3 times. The sediment of the cells was lysed with 20 mM Hepes-HC1buffer, pH 7.5, containing 2mM EDTA, 10 mM EGTA, 0.3 M saccharose, 0.5% (v/v) Triton X-100, 0.5 mM phenylmethylsulfonyl fluoride, 2 mM dithiothreitol. The suspension was homogenizedby 20 strokes in a homogenizer and thencentrifuged at 100,000 X g for 30 min at 4 "C. The supernatantwas collected, and the protein kinase C was partially purified by chromatography on DEAE-cellulose (0.5 X 10 cm) equilibrated with 20 mM Tris-HC1, pH 7.5, 1 mM EDTA, 1 mM EGTA, and 25% glycerol. The column was washed extensively with this buffer, and the enzyme was eluted with the same buffer but containing 0.1 M NaCl. 200-pl fractions were collected, and 20 pl of each was assayed for protein kinase activity which was determined by measuring the incorporation of 32P from [Y-~'P]ATP into histone HI. The standard reaction contained, in 150 pl, 20 mM Hepes, pH 7.5, 10 mMMgC12, 1 mg/ml histone H1, 20 pCi/ ml [-y-32P]ATP,20 p~ ATP with or without protein kinase C cofactors 5 mM CaClZ,100 pg/ml phosphatidylserine, and 10 pg/ml 1,2-diolein.

17467

and T Cell Proliferation

C Protein Kinase

17468

Reaction was started by the addition of column fractions. After 15 min of incubation at 30 “C, 35 pl of the mixture was filtered through phosphocellulose filters. The amount of protein in the samples was determined by the Bradford method (18). Incorporation of fH/Thymidine-Mouse CTLL-2 control cells or cells treated for 48 h with PDBu 500 ng/ml as described above were washed twice with DME and resuspended at lo6 cells/ml in DME, 5 X lo-’ M 2-mercaptoethanol, and 5 pg/ml human transferrin. Aliquots of 100 pl were placed in multiwell plates, and different concentrations of rIL-2 or PDBu were added to thewells and incubated at 37 “C in the presence or absence of anti-IL-2 receptor antibodies (PC 61) (19). 20 h later 1pCi/well [‘Hlthymidine was added, and 4 h laterthe cells were harvested, filtered through glass fiber, and the radioactivity incorporated was estimated in ascintillation spectrometer. RESULTS

The mouse CTLL-2 T lymphocyte cloned cells can grow in culture medium only supplemented with transferrin, 2-mercaptoethanol, andrIL-2 (16). In normal culture conditions 72 h after the last addition of IL-2 these cells were mostly in a “quiescent” state (90-98% in the Go/G1 phase of the cell cycle), and they did not incorporate [3H]thymidine into DNA (data not shown). When they were restimulated with fresh rIL-2 they progressed through the cell cycle, and 16 h later they started to incorporate [3H]thymidine into DNA. This incorporation was dependent on thedose of IL-2 used (Table I). The same cells also responded to a different mitogenic stimulus provided by a phorbol ester, such as PDBu, a known activator of protein kinase C (12). The optimal PDBu dose was 10-20 ng/ml, although the cells never synthesized DNA in response to phorbol esters as well as to IL-2 (Table I). Although phorbol esters have been shown to induce IL-2 secretion by some T lymphocytes (20), the PDBu-induced proliferation of CTLL-2 cells was not due to the indirect secretion of IL-2 by the cells, because it was not blocked by antibodies to the IL-2 receptor which inhibited the IL-2dependent proliferation (Table I). In several cell types, including thymocytes, treatment with high concentrations of phorbol esters for long incubation periods leads to the disappearance of the protein kinase C activity, and this has allowed the study of the importance of protein kinase C o n the action of hormones and growth factors (21, 22). In CTL clones, this treatment could not be done with PDBu alone since the incubation of CTLL-2 cells in the absence of IL-2 greatly reduced cell viability (not shown). Therefore, CTLL-2 cells were treated for 48 h in the presence of 500 ngfml PDBu together with IL-2 to maintain the same viability as the control cells. To determine the activity of protein kinase C in these cells, we studied the effect of IL-2

and PDBu on the Na+/H+ antiporter whose activity is enhanced through activation of protein kinase C (23, 24). In control CTLL-2 cells, rIL-2 even at very high concentrations (5,000 units/ml) was unable to increase the internal pH, suggesting that in these cells both the protein kinase C and the antiporter were not activated by IL-2. However, PDBu elevated the internal pH by 0.13& 0.02 units after alag period of 2 min. This cytoplasmic alkalinization was prevented by the previous addition and reversed by the posterior addition of 5-(N-ethyl-N-isopropyl)amiloride, a known antiporter inhibitor (25) (Fig. 1). The 5-(N-ethyl-N-isopropyl)amiloride was synthesized specifically for this study by the previously published method (26). By contrast, PDBu was unable to induce any increase in theinternalpHin CTLL-2 cells

PUB

Amiloride

FIG.1. Activity of theNa+/H+antiport in CTLL-2 cells. Control cells were stimulated with rIL-2 (5000 units/ml) (upper) or PDBu (PDB) (100 ng/ml) (upper and middle). PDBu-pretreated cells as described under “Experimental Procedures” were stimulated with PDBu (100 ng/ml) (lower). Where indicated 5-(N-ethyl-N-isopropyl)amiloride, a potent inhibitor of the Na+/H+ antiport (26) was added a t 10 p~ (middle) or 50 p~ (lower).

TABLE I DNA synthesis induced by ZL-2 or by PDBu; effectof anti-ZL-2 receptor antibodies CTLL-2 cells were obtained as described under “Experimental Procedures,” placed in multiwell plates, and affinity purified anti-IL2 receptor antibody PC 61 (10 pg/ml) was added before the appropriate stimulus. Proliferation was assayed by [3H]thymidineincorporation. Results shown are the mean f the standard deviation of triplicate culture. ~~

~~

[3H]Thymidine incorporated (cpm)

Stimulus

IL-2

PDBu

unitslrnl

nglrnl

2.5 5 5 20 28.266 20 2.5

Control

+Anti-IL-2 receptor

1,446 f 624 19,396 f 1,019 33,172 f 543 5,884 f 515 8,832 f 365 f 2.264

1,720 f 212 4,274 200 9,610 f 454 5,779 f 324 9,344 f 302 16,710 k 454

*

Ok

08

12

1.6

ELUTION VOLUME lmll

2 CONTROL

PRETREATED

FIG.2. Partial purification of protein kinase from control or PDBu-treatedCTLL-2 cells. A, protein kinase C elution profile of control cells (0)or PDBu-pretreated cells (0).B, requirements of protein kinase C activity from control or PDBu-pretreated CTLL-2 cells. The fractions with activity of A were pooled and assayed for protein kinase C activity. Complete reaction mixture (dotted bars), the same but with PDBu instead of 1,2-diolein (block bars), without Ca2+(white bars), orwithout phosphatidylserine (lined bars).

Protein Kinase

C and T Cell Proliferation

17469

This was further confirmed in proliferation assays measuring the mitotic index over a period of 72 h. In agreement with the results of DNA synthesis, CTLL-2 cells proliferated in response to phorbol esters and IL-2(Fig. 4A).By contrast, protein kinase C-depleted cells proliferated identically to control cells in response to IL-2 but not in response to PDBu. However, some mitogenic response to PDBu was observed at later times. This most likely reflects the time needed for protein kinase C to reexpress to sufficient levels to be stimulated by mitogenic concentrations of PDBu. Furthermore, protein kinase C-depleted CTLL-2 cells cultured in the presence of high concentrations of PDBu to keep the enzyme down-regulated for the entire 72-h period also responded to IL-2 (Fig. 4B).

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DISCUSSION

Recently, there has been a growing interest in the molecular 10 100 ' PDBu (ng/ml) mechanism of signal transduction elicited by IL-2. However, I I I the mechanism by which IL-2 exerts itspleiotropic effect has 10 20 not yet been defined. T cell clones which respond to IL-2, as rlL-2 (U/ml) CTLL-2, represent a good model system to analyze the interFIG. 3. DNA synthesis by CTLL-2 cells. Control (0,W) or protein kinase C-depleted cells pretreated with PDBu for 48 h as action of IL-2 with a homogeneous, nontransformed populathere described under "Experimental Procedures" (0,0 ) were stimulated tion of cells. Since IL-2 is basically a growth factor (l), with different concentrations of rIL-2 (0,0),or PDBu(W, 0).Results have been some attempts to involve it in the same signal are the mean of duplicate cultures. TdR,thymidine. tranducing pathways described for other growth factors (12, 28). Thus, it has been suggested that after receptor IL-2 interaction, an endogenous phospholipase C is activated, probably coupled to a G protein (9) which hydrolyzes phosphatidylinositol (4), to generate two types of second messengers. One is inositol phosphates responsible for Ca2+mobilization and the other is diacylglycerol, whichactivated protein kinase C (12, 28). However, the overall model has been questioned. The failure of IL-2 to mobilize intracellular Ca2+ (6, 7)' or generate inositol phosphates in lympocytes shown by otherauthors (3, 5)' raises the question of whether this mechanism is necessary for IL-2 activity (10). Based on a number of indirect evidences, Farrar et al. (14) have suggested a central role for protein kinase C in their model of IL-2 response: (i) the effect of phorbol esters and IL-2 in T lymT I M E (hrsl T I C (hrrl phocyte proliferation (14, 15); (ii)IL-2 activation of the Na'/ FIG. 4. Proliferation of CTLL-2 cells. A , control CTLL-2 cells H' antiporter whose activation is linked to phosphorylation cultured with IL-2 for 3 days to deprive IL-2 by consumption were washed and reestimulated with fresh IL-2, 20 units/ml (O), PDBu, by protein kinase C (17); (iii) redistributionof protein kinase 10 ng/ml (A)or 20 ng/ml (O),or with IL-2 plus PDBu, 10 ng/ml (A) C from the cytosol to themembrane (11). By contrast, there are other evidences that point out the and IL-2 plus PDBu, 20 ng/ml (a); unstimulated controlcells (W). B, protein kinase C-depleted CTLL-2 cells cultured with IL-2 and PDBu, existence of two different pathways for phorbol esters and IL500 pg/ml, for 3 days were washed and reestimulated with IL-2, 20 2. Since TPA induces proliferation in some T cell clones and units/ml, in the absence (0)or in the presence (0)of PDBu, 500 ng/ not in others and IL-2 induces it inall of them (15), thiscould ml, or with PDBu, 20 ng/ml alone (0). also be taken as aproof that IL-2 and TPA may be acting by different mechanisms. TPA alone stimulates the incorporapreviously pretreated with high doses of phorbol esters (Fig. tion of thymidine by CTLL-2 cells, but thisproliferation does 1). not seem to occur via an autocrine mechanism mediated by To corroborate the lack of protein kinase C activity in the induction of IL-2 synthesis, since it was not inhibited by phorbol ester-treated cells, the enzyme was extracted from a monoclonal antibody against the IL-2 receptor (19) which both types of cells, with Triton X-100, which is known to blocks IL-2-induced proliferation (Table I). This indicates solubilize most of the protein kinase C (11,27), and activity its that in these cells the activation of protein kinase C is measured in vitro. As shown in Fig. 2, a substantial amount sufficient to trigger proliferation. of kinase activity was detected in total extracts of control Phorbol esters also mimic IL-2 for the induction of some CTLL-2 cells. This phosphorylation showed the characteris- genes such as y-interferon and IL-2 receptor (14). However, tics of protein kinase C activity since it required the three there is some evidence that TPA and IL-2 induced different cofactors, Ca2+,phosphatidyl serine, and diacylglycerol, which sets of genes (29, 30), suggesting that IL-2 may transduce could be substituted by phorbol esters (Fig. 2B). The protein signals in addition to those associated with protein kinase C kinase C activity of cells treated with high doses of phorbol activity. esters for 48 h was less than 3% of that of the control cells. The Na'/H' antiporter is activated after the binding of As expected, these proteinkinase C-depleted T cells no longer many growth factors probably through activation of the prosynthesized DNA in response to mitogenic doses of PDBu. However, they incorporated [3H]thymidine identically to con* J. M. Redondo, A. Lopez-Rivas, and M. Fresno, unpublished trol cells in response to IL-2 (Fig. 3). results.

17470

Protein Kinase

C and T Cell Proliferation

tein kinase C (31),suggesting that theresultant intracellular alkalinization is important in the initiation of growth. Mills et al. (17) have described a stimulation of the antiportby IL2, but this required the use of extremely high concentrations of IL-2 (17). Furthermore,concentrations of IL-2 several times higher than thephysiological concentration needed for proliferation were uneffective (17). By contrast, IL-2 was unable to induce activation of the antiport inour clone even at 5000 units/ml. The reason for this discrepancy is unknown. Moreover, in Mills experiments, although the addition of IL2 to T cell clones may increase the internal pH, theproliferation occurs in presence of amiloride concentrations which blocked this increase (17), suggesting that neither cellular alkalinization nor the activation of Na+/H+ antiport is essential for IL-2-dependent proliferation, which is in agreement with our results. Altogether, these results indirectly indicate that IL-2 does not activate protein kinase C. Recently, it has been shown that the protein kinase C inhibitor H-7 inhibits IL-%induced proliferation (32). However, this compound is by no means a specific inhibitor of protein kinase C since it also inhibits other enzymes. Furthermore, the compound was present duringthe entire proliferation assay and affected cell viability. Translocation of protein kinase C from the cytosol to the membrane has been described and taken as a proof of the involvement of protein kinase Cinthe IL-2transducing pathway (17). However, the same group has reported that in 12-day T cell blasts whose growth is supposedly dependent on IL-2 the lymphokine did not induce translocation of protein kinase C (33). Our data on the inability of IL-2 to raise the internal pH through the antiporter, which is a direct in vivo measurement of protein kinase C activation (17), suggest that if translocation is taking place it should be nonfunctional. It was recently reported that prolonged incubation with high concentrations of phorbol esters causes a decrease in protein kinase C activity (21, 22). This method was widely used to study the role of protein kinase C in growth factor activity (27, 34). We describe here for the first time the method successfully applied to cytotoxic T cell clones. After this treatment we barely detected protein kinase C in CTLL2 cells. Our results directly show that these cells which have been depleted of protein kinase C activity still respond with the same dose response to IL-2 in DNA synthesis and proliferation. Since protein kinase C took at least 24 h to reexpress to a level enough to promote proliferation by phorbol esters, and IL-2 was able to support cell division for 5 days in the continuous presence of high concentrations of PDBu, it is very unlikely that IL-2-induced proliferation in protein kinase C-depleted cells is due to a resurface of this enzyme. In summary, our results allow us to discard the protein kinase C in the signal transducing mechanism of IL-2. In support of our hypothesis, it was recently shown that IL-2 and PDBu do not induce the phosphorylation of the same substrates in T cells (35). Since phorbol esters also induce the proliferation of CTLL-2 and enhance the activity of protein kinase C, it is likely that different but perhaps convergent pathways of T cell proliferation exist: the conventional pathway which does not require protein kinase C and an IL-2independent one which can be mediated by protein kinase C activation. Further experiments are required to characterize

where these two pathways converge. Acknowledgments-We thank Benilde Jimenez for helping us with the protein kinase C assay and Dr. J. Avila for the critical reading of this manuscript. REFERENCES 1. Smith, K. A. (1984) Annu. Rev. Immunol. 2 , 319-333 2. Cantrell, D. A. & Smith, K. A. (1984) Science 224,1312-1316 3. Kozumbo, W. J., Harris, D. T., Gromowski, S., Cerottini, J. C. & Cerutti, P. A. (1987) J. Immunol. 138,606-612 4. Bonvini, F., Ruscetti, F. W., Ponzoni, M., Hoffman, T. & Farrar, W. L. (1987) J. Biol. Chem. 262,4160-4164 5. Mills, G. B., Stewart, D. J., Mellors, A. & Gelfand, E. W. (1986) J. Zmmunol. 136,3019-3024 6. Mills, G. B., Cheung, R. K., Grinstein, S. & Gelfand, E. W. (1985) J. Immunol. 134,2431-2435 7. Harris, D. T., Kozumbo, N. J., Cerutti, P. & Cerottini, J. C. (1987) J. Immunol. 138,600-605 8. Rossio, J. L., Farrar, W. L. & Ruscetti, F. W. (1986) in Leucocytes and Host Defense (Oppenheim, J. J., and Jacobs, D. M., eds) pp. 131-136, Alan R. Liss, Inc., New York 9. Evans, S. W., Beckner, S. K. & Farrar, W. L. (1987) Nature 3 2 6 , 166-168 10. Bourne, H. R. (1987) Nature 326,833-834 11. Farrar, W. L. & Anderson, W. B. (1985) Nature 3 1 6 , 233-235 12. Nishizuka, Y. (1984) Nature 308,693-698 13. Kraft, A. S. & Anderson, W. B. (1983) Nuture 301,621-623 14. Benjamin, W. R., Steeg, P. S. & Farrar, J. J. (1982) Proc. Nutl. Acad. Sci. U. S. A. 79,5379-5383 15. Kim, D. K., Otten, G., Moldwin, R. L., Dunn, D. E., Nau, G. J. & Fitch, F. W. (1986) J. Immunol. 137,2755-2760 16. Redondo, J. M., L6pez Rivas, A. & Fresno, M. (1986) FEBS Lett. 206,199-202 17. Mills, G.B., Cragoe, E. J., Jr., Gelfand, E. W. & Grinstein, S. (1985) J. B i d . Chem. 2 6 0 , 12500-12507 18. Bradford, M. M. (1976) Anal. Biochem. 7 2 , 248-254 19. Lowenthal, J. W., Corthisy, P., Toregic, C., Lees, R., MacDonald, H. R. & Nabholz, M. (1985) J. Immunol. 136,3988-3994 20. Farrar, J. J., Fuller-Farrar, J., Hilfikes, M.L., Stadler, B.M., Farrar, W. C. & Simon, P. L. (1980) J. Immunol. 1 2 6 , 25552558 21. Grinstein, S., Mack, E. & Mills, G. B. (1986) Biochem. Biophys. Res. Commun. 134,8-13 22. Rodriguez-Pefia, A. & Rozengurt, E. (1984) Biochem. Biophys. Res. Commun. 120,1053-1059 23. Rosoff, P. M., Stein, L. F. & Cantley, L. C. (1984) J. Biol. Chem. 269,7056-7060 24. Hesketh, T. R., Moore, J. A., Morris, D., Taylor, M. W., Rogers, J., Smith, G. A. & Metcalfe, J. C. (1985) Nature 313,481-484 25. Besterman, J. M., May, W. S., Jr., Levine, H., 111, Cragoe, E. J., Jr. & Cuatrecasas, P. (1985) J. Biol. Chem. 260,1155-1159 26. Cragoe, E. J., Jr., Woltersdorf, 0. W., Jr., Bicking, J. B., Kwang, S. F. & Jones, J. H. (1967) J. Med. Chem. 10,66-75 27. McArdle, C.A., Huckle, W. R. & Conn, P. M. (1987) J. Biol. Chem. 262,5028-5035 28. Berridge, M. J. (1984) Biochem. J. 220,345-360 29. Heckford, S. E., Gelmann, E. P., Agnor, C. L.,Jacobson, s.,Zinn, S. & Matis, L. A. (1986) J. Immunol. 137,3652-3663 30. Albert, F., Hua, C., Truneh, A., Pierres, M. & Schmitt-Verhulst, A. (1985) J. Immurwl. 134, 3649-3655 31. Macara, I. G. (1985) Am. J. Physiol. 2 4 8 , C3-Cll 32. Clark, R. B., Love, J. T., Jr., Sgrai, D., Lingenheld, F. G. & Sha’afi, R. I. (1987) Biochem. Biophys. Res. Commun. 1 4 6 , 666-672 33. Farrar, W. L. & Ruscetti, F. W. (1986) J. Zmmunol. 136,12661273 34. Frick, K. K., Womer, R. B. & Scher, C. B. (1988) J. Biol. Chem. 263,2948-2952 35. Friedrich, B. & Gullberg, M. (1988) Eur. J. Immunol. 1 8 , 489492