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Transglutaminase and receptor signaling: Concepts and realities
TRANSGL~TAMINASE AND R~C~~T~R SIGNALINGS CONCEPTS AND REALITIES L. FBsUs, J. HARSFALVI, A. HORVATH and M. SANDOR* Department of Clinical Chemistry. University School of Medicine, I&4012, Debrecen, Hungary and *Department of Immunology, EGtvijs L&and University, H-2131, Gild, Hungary
INTRODUCTION Sear&kg to find a function far the ubiquitous transglutaminases (Folk, 1980) has led to the intriguing proposition that it might have a significant role in the biochemical pathway of receptor signaling. The first studies carried out in Pastan’s laboratory have shown that primary amines, lysyl and glutaminyl peptides, which share the property of being competitive inhibitors of the cellular transglutaminase~ can inhibit ligand internalization through coated pits and vesicles (Davies er al., 1980). Since then the same approach has been applied to the receptors of a diverse group of molecules, including aZmacroglobulin, catecholamines, tr~iodothyron~ne, low-density lipoproteins, epiderma1 growth factor, insulin, luteinizing hormone and immune complexes, yielding similar results [for references see F&is (1984)].
incorporatian can be detected at 1OOpM concn, whereas hydrolysis could proceed in the 10pl4 range with a high reaction velocity. (c) It has been shown to be Iocalized in all cell compartments, including the nucleus and the cell surface membrane. (d) Transglutaminase has a hydrophobic portion, enabling it to interact with the hydrocarbon chain of phospho~ipids~ becoming a membrane-localized enzyme (F&is et al., 1983). It is inactive in the membrane of a resting cell but can be activated by several means which perturb the membrane structure, like phospholipase and protease action, lipid phase separation, membrane fusion etc. (Fbsiis, 1984). It has been also claimed that the membrane-bound responding form has distinct characteristics compared to the well-characterized “liver enzyme”, and its presence or activation is always associated with the stimulating/proliferating state of the cell (Chang and Chung, 1982).
TRANSGLUTA~INASE ACTION AND ITS REGULATION IN THE CELLULAR ~VIRON~~NT~EN~RAL OYERVIEW
Cellular transglutaminase is an excellent candidate for playing a regulatory rote in almost any significant cellular phenomena, including receptor signaling, for several reasons: (a) The results of any of its actions [namely an acyt transfer reaction between peptide-bound glutamine residues and a series of primary amines, including the c-amino group of lysine residues in appropriate peptides when the result is the formation of an ~“(y-glutaminyl)lysine cross-link between two proteins, hydrotysis at the carboxamide group of pcpt~de-buund glutamine, and hydrolysis and amidotysis of certain aliphatic amines and active esters] can be exampies of posttranslational modifications utilizing naturally occurring substances like polyamines, histamine etc. and resulting in “aberrant” proteins with altered charges (Folk, 1980). (b) It is a Ca2’ -regulated enzyme, one of the Ca2+-binding proteins of the cell. Though the purified enzyme requires a high concn of Ca*’ for optimal activity, the catalytic reactions, especially the hydrolysis, may proceed at much lawer Ca*+ concns as shown in Fig. 1. In extracts of lymphocytes amine
POSSIBLE WAYS OF TRANSGLUTA~NASE DURING RECEPTOR SIGNALlNG
ACTtON
Despite all of the aforementioned encouraging findings and several excellent hypotheses coupling the enzyme to aging, immune protection, cell cycle regulation etc. the role of transglutaminase in the eel is still an enigma. The reason is mainly the lack of sufficient indisputable evidence to support the ideas. Its suggested role in receptor signaling is not an exception. The correlation between the extent of inhibition of the enzyme by competitive inhibitors ranging in potency over three orders of magnitude and the inhibition of receptor clustering is striking (Davies et al., 1980). Whenever it was measured the enzyme activity was higher in cells stimulated through receptors than in the nonst~mulated counterparts. The CaZ+ requirement of the enzyme is in accord with the observation that Ca?+ influx into the cells is one of the first consequences of receptor triggering. In fact, the addition of the Ca2+ ionophore A23187 to the cells can induce an increased transglutaminase activity (of course, an increased cellular transgtutaminase activity always results in cell lysates; a 1161
L. FBsiis et al,
1162
0
01
hydrolysts
10
05 Caz+(mmol/l)
Fig. 1. Effect of various CaZf concns on the velocity of transglu~minase-catalyzed [3H]putrescine inco~oration into /3-casein (transfer reaction) and the release of NH, from fi-casein (hydrolysis). Aliquots of cell extracts prepared by sonication from human peripheral blood lymphocytes were used as source of enzyme activity. Values shown represent the means of triplicate determinations.
continuous transglutaminase activity in intact cells has not been proven yet) like several of the numerous responses listed as consequences of receptor triggering. However, when one tries to specify the action of transglutaminase in these circumstances the mostly indirect evidence is not too convincing (F&is, 1982). Transglutaminase may cross-link receptors or covalently attach ligand-receptor complexes to each other or to other membrane proteins
The direct catalytic action of transglutaminase on receptor proteins seems to be the most obvious possibility, especially in the light of results demonstrating that cellular (Szabo T., personal communication) as well as thrombin-activated plasma transglutaminase (Bruhn and Zurborn, 1982) can directly trigger cell proliferation, that is they can act as mitogens themselves. The cell surface proteins modified by the enzymes have not been identified yet, though by using radioactive amine substrates several cell surface proteins can be labeled by means of transglutaminases (F&is and Laki, 1976). As far as receptors or receptor-ligand complexes are concerned no transglutaminase-catalyzed covalent ligand-re~ptor complex formation has been observed so far. Only purified Fc receptors were studied directly and found to be polymerized by the enzyme (F&is et al., 1982). The cross-linked Fc receptor was functionally different compared to the monomeric one, suggesting that transglutaminase may have a role in controlling Fe receptor heterogeneity as well as internalization through these receptors. However, the demonstration of cross-linked Fc receptors in an intact cell could not be achieved, though it has been clearly shown that primary amines were potent inhibitors of Fc receptor mediated phagocytosis by macrophages (F&is et al., 1981) as well as the
triggering of murine B-lymphocytes to form clones of antibody-producing plasma cells (julian et al., 1983). In the latter study the primary amines appeared to inhibit an early, T-cell-inde~ndent event in the B-cell activation pathway. Considering other membrane or submembranous proteins, µglobulin, the common component of HLA and some tumor antigens, was demonstrated to be cross-linked by transglutaminase in solution as well as in the membrane of proliferating cells (F&s& ef al., 1981). The intracellular region of HLA-A and -B antigens contains two glutamine residues which are used as amine acceptors by transglutaminase (Pober and Strominger, 1981). Some components of the cytoskeletal structure, like actin, filamine (Cohen et al., 1981) vinculin etc., as well as fibronectin (Birckbichler and Patterson, 1978), have also been shown to contain transglutaminase-sensitive glutamine residues. Whether these findings have something to do with receptor signaling remains to be substantiated. It may modifr ligartd-receptor complexes or other membrane protrim to make them find their way to coated pits This assumption is closely related to the previous one and almost all the findings and arguments listed there are also applicable here. The transglutaminaseinhibiting agents usually do not affect binding but prevent internalization. which may be explained as the enzyme being prevented from modifying receptors, receptorrligand complexes or membrane proteins, causing irreversible clustering or internalization. Again, no such modification is known and the inhibitory primary amines may exert their action by other means as well, like perturbation of membrane structure, lysosomotropic activity (see later), noncovalent binding to receptors or regulatory proteins [like dansylcadaverine to calmodulin (Cornwell et al., 198311etc. Some of the side effects might not exist in isolated membranes, in which Dickson (1981) could show that transglutaminase inhibitors blocked the change of low-affinity receptors to high-affinity ones after the binding of ligands. Transglutaminase may participate in the biochemical mechanism of receptor recycling
In some of the studies when the effect of primary amines on the binding and clustering of receptors was excluded a decrease in the number of available receptors could be found (Leuven, 1980) suggesting an interference with receptor recycling. The lysosomotropic effect of the primary amines in increasing intracellular (lysosomal) pH, thereby preventing ligand-receptor dissociation, can explain the inhibition of recycling. However, lysosomotropic chloroquine, secondary as well as tertiary amines, or other derivatives (like properly dimethylated dansylcadaverine and histamine), which are not transglutaminase substrates, could not inhibit receptor signaling in several cases (Davies, 1980; Leuven,
1163
Transglutaminase and receptor signaling
1
I!fl 0
2
a, TIME (MXRS)
72
Fig. 2. Transglutaminase activity in the nucleus of human peripheral blood lymphocytes various time intervals following phytohaemagglutinin (PHA) stimulation as compared to nonstimulated ones. Activity measurement: incorporation of [3H]putrescine into dimethylated casein. For details see F&is (1984).
1980; Julian et al., 1983). On the other hand, it was also concluded that transglutaminase may not be involved in the mechanism of growth-factormediated receptor. internalization or mitogenesis because well-known inhibitors of the enzyme had little or no effect (King, et al., 1981). The data are very controversial in this respect, which may be partially explained by differences in the systems used or that the results reflect the existence of several parallel mechanisms. Transglutaminase-formed protein-protein or proteinprimary amine conjugates may serve as signal structures
Since clustering and endocytosis of receptors are not necessarily followed by mitogen response, the role of transglutaminase in the events leading to mitogenic response might be more specific and important in the signaling process than simply being essential in irreversible receptor binding and uptake of the ligand-receptor complex. In most of the studies on this mitogen-induced lymphocytes were used, in which transglutaminase activity is abruptly increased following mitogen addition (Novogrodsky et al., 1978) just as well as the level of polyamines. It was demonstrated by Folk et al. (1980) that polyamines are natural substrates of transglutaminase. When normal human blood lymphocytes were treated with mitogens to induce blastogenesis in the presence of labeled putrescine, part of the label was incorporated into proteins. Labeled y-glutamyl-putrescine, -spermidine and -spermin were identified in the cellular protein fraction. Though the proteins have not yet been identified it could be postulated that covalent protein-polyamine conjugates serve as signaling structures for mitogenesis for that this kind of posttranslational covalent modification is one way of regulating enzymes important in this respect. The first possible example of such an enzyme modification has
been recently demonstrated in the case of ornithine decarboxylase (Russel and Manen, 1982) into which could be incorporated by transputrescine glutaminase, making it inactive, but meanwhile being capable of stimulating RNA polymerase I activity. In our studies an increased transglutaminase activity could be observed in the nucleus during the first 24 hr after stimulation of lymphocytes (Fig. 2) by phytohaemagglutinin. Similar results could be obtained using several other kinds of mitogen (data not shown). Not surprisingly, dansylcadaverine, the most potent inhibitor of transglutaminase, could inhibit blastogenesis (Fig. 3). Meanwhile the formation of several dansylcadaverine-labeled nuclear proteins was also observed. The nature of these proteins is currently being investigated. The concn of these amine-labeled proteins formed either using naturally occurring amines at labeling concns or others at inhibitory concns is very low for conventional isolation procedures, to say nothing of subsequent methods to prove that the amine was incorporated into y -glutamyl residues by transglutaminase (in none of the aforementioned cases including the receptor as well as other protein studies has been this criterion been met so far). Since the discovery of the catabolizing enzyme y-glutamylamine cyclotransferase (Fink et al., 1980) one should consider the possibilty that protein “aberrations” resulting from transglutaminase action may signal preferentially rapid proteolytic degradation of these proteins (and substrated for the catabolyzing enzyme which is capable of using only those polypeptides in which the proteolysis at the glutamine side of either the cross-link or the amine derivative is complete) are rapidly formed making it exceptionally difficult to catch these transient derivatives. Naturally, the same is true as far as the postulated modifications of receptors or membrane proteins are concerned.
001 0025 0 0 DANSYLCADAVERINE (mmol/
01
I)
Fig. 3. Inhibition of PHA-induced stimulation of human peripheral blood lymphocytes (2 x 105ml) by dansylcadaverine. Radioactivity measurements were carried out 72 hr following PHA addition. For details see F&is (1984).
L. FBsiis et al.
1164
fortunately, that has not turned out to be the case, at least so far. Although the transglutaminaseinhibitory primary amines have become widely used methodological tools in receptor studies, their mechanism of action is not fully understood. Due mainly to methodological difficulties direct and final evidence of transglutaminase-catalyzed modification of receptors, cellular proteins or other has not been found. However, the number of encouraging results is high enough to stimulate further efforts to find new methodological approaches utilizing ail the available data and to consider the possibility of the involvement of transglutaminase in receptor signaling as an open question. Acknowledgement-rhis
Fig. 4. Transglutaminase activities measured in cell extracts of monocytes and lymphocytes prepared from patients with systemic lupus erythematosus (SLE) or Hodgkin’s disease and healthy individuals. Method: incorporation of [jH]putrescine into dimethylated casein. For details see F&is (1984).
PHAGOCYTOSIS
AND MACROPHAGE
ACTIVATION
An interesting aspect of the involvement of transglutaminase in cell stimulation processes is the case of macrophages. We have previously shown that agents which inhibit transglutaminase activity in rat macrophages block Fc receptor mediated phagocytosis (F&is et al., 1981). The endocytosis of immune complexes via the Fc receptor produces an activation of transglutaminase and the stimulation of amine incorporation into membrane proteins of intact cells (these proteins have not been identified either). Similar results were obtained later using guinea pig (Leu et al., 1982) as well as murine (Schroff et al., 1981) macrophages. The inflammatory peritoneal macrophages have higher levels of transglutaminase activity than resident nonactivated macrophages. According to a recent report the synthesis of transglutaminase is greatly accelerated by certain types of macrophage stimulation (Mortaugh et al., 1983). -hese studies seem to have a clinical relevance as well. We have found that in human monocytes an increased transglutaminase activity can be measured not only following in eitro stimulation but also in cells isolated from patients with SLE and Hodgkin’s disease when other signs of in vivo monocyte activation are also apparent (Fig. 4). The transglutaminase activity of lymphocytes was not changed. CONCLUDING
REMARKS
Based on the first reports in 1980 which hay suggested a strong involvement of transglutaminase in receptor signaling one expected that a crucial and specific biochemical step in the biochemical cascade of cell triggering might be soon revealed. Un-
the National Foundation MD.
work was partially supported by for Cancer Research, Bethesda,
REFERENCES
Birckbichler P. J. and Patterson M. K., Jr (1978) Cellular trans~lutaminase, growth and transfo~ation. Ann. N. I: Acad- Sci. 312, 354-365. Bruhn H. D. and Zurborn K. H. 119821 Factor XIII and fibronectin as regulators of cell &oli&ration. In Factor XIII and Fibronectin (Edited by Enbring R. and Klingemann H. G.), pp. 229-236. Die Gedizinische, Marburg. Chane, S. K. and Chung S. I. (1982) Characterization of act&ated trassglutamiiase in stimilated cell. Fedn Proc. 41, 641.
Cohen I., Glaser T., Veis A. and Bruner-Lorand J. (1981) Ca’+-dependent cross-linking processes in human platelets. Biochim. biophvs. Acta 676, 137-147. _ Cornwell M. M.. Jul&o R. L. and Davies P. J. (1983) Inhibition of the adhesion of Chinese hamster ovar; cells by the naphthylsulfonamides dansylcadaverine and N{6-aminohexyi)-S-chloro-l-naphtylenesulfonamide (W7). Bioch~~~.biophys. Acta 762, 414419.
Davies P. .I. A., Davies D. R., Levitzki A., Maxfield F. R., Milhaud P.. Willincrham M. C. and Pastan I. H. (19801 Transglutaminase is essential in receptor-mediated kndo: cytosis of cqmacroglobulin and polypeptide hormones. Nature, Land. 283, 162-167. Dickson R. B. (1981) Inhibition by bacitracin of high affinity binding of ‘%Y~M to plasma membranes. FEBS Lett. 126, 265-268.
F&is L. (1982) Transglutaminase activation: significance with respect to immunologic phenomena. Sure. Immun. Res. 1, 291-304. F&is L. (1984) Transglutaminase and immune phenomena. Compeid. Immun. 5.
F&is L.. Erdei A.. SBndor M. and Gereelv J. 09821 The: influence of tissuk transglutaminase on”&; function ‘of Fc receptors. Motet. Immun. 19, 39-43. F&.is L., Falus A., Erdei A. and Laki K. (1981a) Human µglobulin is a substrate of tissue transglutaminase: polymerization in solution and on the cell surface. J. Cell Biol. 89, 706-710. F&s L., Horvath A. and HBrsfalvi J. (1983) Interaction between tissue transglutaminase and phospholipid vesicles. FEBS Lat. 155, 1-5. F&is L. and Laki K. (1976) On coupling of bovine fibrinogen to the surface of malignant murine plasma cells by means of transglutaminase. Biochem. biophys. Res. Commun. 72, 131-137.
F&is L., SAndor M., Horvath L. I., Bagyinka Cs., Erdei A. and Gergely J. (19816) Immune-complex-induced transglutaminase activation: its role in the Fe receptor-
Transglutaminase
and receptor
mediated transmembrane effect on peritoneal macrophages. Molec. Immun. 18, 633-638. Fink M. L., Chung S. I. and Folk J. E. (1980) y-Glutamylamine cyclotransferase: specificity toward c-(7-glutamyl)-t.-lysine and related compounds. Proc. natn. Acad. Sci. U.S.A. 71, 4564-4568. Folk J. E. (1980) Transglutaminases. A. Reo. Biochem. 49, 517-531. Folk J. E., Park M. H., Chung S. I., Schrode J., Lester E. P. and Cooper H. L. (1980) Polyamines as physiological substrates for transglutaminases. J. biol. Chem. 255, 3695-3699. Julian C., Spech N. A. and Pierce S. K. (1983) Primary amines inhibit the triggering of B-lymphocytes to antibody synthesis. J. Immun. 130, 91-96. King A. C., Hernaez-Davis L. and Cuatrecasas P. (1981) Lysosomotropic amines inhibit mitogenesis induced by growth factors. Proc. natn. Acad. Sci. U.S.A. 78,717-721. Leu R. W., Herriott M. J., Moore P. E., Orr G. R. and
signaling
1165
Birckbichler P. J. (1982) Enhanced transglutaminase activity associated with macrophage activation. Possible role in Fc mediated phagocytosis. Exp/ Cell Res. 141, 191-199. Leuven F. V. (1980) Primary amines inhibit recycling of a,M receptors in fibroblasts. Cell 20, 3743. Mortaugh M. P., Mehta K., Johnson J., Myers M., Juliano R. L. and Davies P. J. (1983) Induction of tissue transglutaminase in mouse peritoneal macrophages. J. biol. Chem. 258, 1107411081. Novogrodsky A., Quittner S., Rubin A. L. and Stenczel K. H. (1978) Transglutaminase activity in human lymphocytes Proc. natn. Acad. Sci. U.S.A. 75, 1157-I 161. Pober J. S. and Strominger J. L. (1981) Transglutaminase modifies the carboxy-terminal intracellular region of HLA-A and -B antigens. Nature, Lond. 289, 819-821. Russel D. H. and Manen C. A. (1982) Posttranslationally modified ornithine decarboxylase may regulate RNA polymerase I activity. Biochem. Pharmac. 31, 3373-3378.
INTRODUCTION Sear&kg to find a function far the ubiquitous transglutaminases (Folk, 1980) has led to the intriguing proposition that it might have a significant role in the biochemical pathway of receptor signaling. The first studies carried out in Pastan’s laboratory have shown that primary amines, lysyl and glutaminyl peptides, which share the property of being competitive inhibitors of the cellular transglutaminase~ can inhibit ligand internalization through coated pits and vesicles (Davies er al., 1980). Since then the same approach has been applied to the receptors of a diverse group of molecules, including aZmacroglobulin, catecholamines, tr~iodothyron~ne, low-density lipoproteins, epiderma1 growth factor, insulin, luteinizing hormone and immune complexes, yielding similar results [for references see F&is (1984)].
incorporatian can be detected at 1OOpM concn, whereas hydrolysis could proceed in the 10pl4 range with a high reaction velocity. (c) It has been shown to be Iocalized in all cell compartments, including the nucleus and the cell surface membrane. (d) Transglutaminase has a hydrophobic portion, enabling it to interact with the hydrocarbon chain of phospho~ipids~ becoming a membrane-localized enzyme (F&is et al., 1983). It is inactive in the membrane of a resting cell but can be activated by several means which perturb the membrane structure, like phospholipase and protease action, lipid phase separation, membrane fusion etc. (Fbsiis, 1984). It has been also claimed that the membrane-bound responding form has distinct characteristics compared to the well-characterized “liver enzyme”, and its presence or activation is always associated with the stimulating/proliferating state of the cell (Chang and Chung, 1982).
TRANSGLUTA~INASE ACTION AND ITS REGULATION IN THE CELLULAR ~VIRON~~NT~EN~RAL OYERVIEW
Cellular transglutaminase is an excellent candidate for playing a regulatory rote in almost any significant cellular phenomena, including receptor signaling, for several reasons: (a) The results of any of its actions [namely an acyt transfer reaction between peptide-bound glutamine residues and a series of primary amines, including the c-amino group of lysine residues in appropriate peptides when the result is the formation of an ~“(y-glutaminyl)lysine cross-link between two proteins, hydrotysis at the carboxamide group of pcpt~de-buund glutamine, and hydrolysis and amidotysis of certain aliphatic amines and active esters] can be exampies of posttranslational modifications utilizing naturally occurring substances like polyamines, histamine etc. and resulting in “aberrant” proteins with altered charges (Folk, 1980). (b) It is a Ca2’ -regulated enzyme, one of the Ca2+-binding proteins of the cell. Though the purified enzyme requires a high concn of Ca*’ for optimal activity, the catalytic reactions, especially the hydrolysis, may proceed at much lawer Ca*+ concns as shown in Fig. 1. In extracts of lymphocytes amine
POSSIBLE WAYS OF TRANSGLUTA~NASE DURING RECEPTOR SIGNALlNG
ACTtON
Despite all of the aforementioned encouraging findings and several excellent hypotheses coupling the enzyme to aging, immune protection, cell cycle regulation etc. the role of transglutaminase in the eel is still an enigma. The reason is mainly the lack of sufficient indisputable evidence to support the ideas. Its suggested role in receptor signaling is not an exception. The correlation between the extent of inhibition of the enzyme by competitive inhibitors ranging in potency over three orders of magnitude and the inhibition of receptor clustering is striking (Davies et al., 1980). Whenever it was measured the enzyme activity was higher in cells stimulated through receptors than in the nonst~mulated counterparts. The CaZ+ requirement of the enzyme is in accord with the observation that Ca?+ influx into the cells is one of the first consequences of receptor triggering. In fact, the addition of the Ca2+ ionophore A23187 to the cells can induce an increased transglutaminase activity (of course, an increased cellular transgtutaminase activity always results in cell lysates; a 1161
L. FBsiis et al,
1162
0
01
hydrolysts
10
05 Caz+(mmol/l)
Fig. 1. Effect of various CaZf concns on the velocity of transglu~minase-catalyzed [3H]putrescine inco~oration into /3-casein (transfer reaction) and the release of NH, from fi-casein (hydrolysis). Aliquots of cell extracts prepared by sonication from human peripheral blood lymphocytes were used as source of enzyme activity. Values shown represent the means of triplicate determinations.
continuous transglutaminase activity in intact cells has not been proven yet) like several of the numerous responses listed as consequences of receptor triggering. However, when one tries to specify the action of transglutaminase in these circumstances the mostly indirect evidence is not too convincing (F&is, 1982). Transglutaminase may cross-link receptors or covalently attach ligand-receptor complexes to each other or to other membrane proteins
The direct catalytic action of transglutaminase on receptor proteins seems to be the most obvious possibility, especially in the light of results demonstrating that cellular (Szabo T., personal communication) as well as thrombin-activated plasma transglutaminase (Bruhn and Zurborn, 1982) can directly trigger cell proliferation, that is they can act as mitogens themselves. The cell surface proteins modified by the enzymes have not been identified yet, though by using radioactive amine substrates several cell surface proteins can be labeled by means of transglutaminases (F&is and Laki, 1976). As far as receptors or receptor-ligand complexes are concerned no transglutaminase-catalyzed covalent ligand-re~ptor complex formation has been observed so far. Only purified Fc receptors were studied directly and found to be polymerized by the enzyme (F&is et al., 1982). The cross-linked Fc receptor was functionally different compared to the monomeric one, suggesting that transglutaminase may have a role in controlling Fe receptor heterogeneity as well as internalization through these receptors. However, the demonstration of cross-linked Fc receptors in an intact cell could not be achieved, though it has been clearly shown that primary amines were potent inhibitors of Fc receptor mediated phagocytosis by macrophages (F&is et al., 1981) as well as the
triggering of murine B-lymphocytes to form clones of antibody-producing plasma cells (julian et al., 1983). In the latter study the primary amines appeared to inhibit an early, T-cell-inde~ndent event in the B-cell activation pathway. Considering other membrane or submembranous proteins, µglobulin, the common component of HLA and some tumor antigens, was demonstrated to be cross-linked by transglutaminase in solution as well as in the membrane of proliferating cells (F&s& ef al., 1981). The intracellular region of HLA-A and -B antigens contains two glutamine residues which are used as amine acceptors by transglutaminase (Pober and Strominger, 1981). Some components of the cytoskeletal structure, like actin, filamine (Cohen et al., 1981) vinculin etc., as well as fibronectin (Birckbichler and Patterson, 1978), have also been shown to contain transglutaminase-sensitive glutamine residues. Whether these findings have something to do with receptor signaling remains to be substantiated. It may modifr ligartd-receptor complexes or other membrane protrim to make them find their way to coated pits This assumption is closely related to the previous one and almost all the findings and arguments listed there are also applicable here. The transglutaminaseinhibiting agents usually do not affect binding but prevent internalization. which may be explained as the enzyme being prevented from modifying receptors, receptorrligand complexes or membrane proteins, causing irreversible clustering or internalization. Again, no such modification is known and the inhibitory primary amines may exert their action by other means as well, like perturbation of membrane structure, lysosomotropic activity (see later), noncovalent binding to receptors or regulatory proteins [like dansylcadaverine to calmodulin (Cornwell et al., 198311etc. Some of the side effects might not exist in isolated membranes, in which Dickson (1981) could show that transglutaminase inhibitors blocked the change of low-affinity receptors to high-affinity ones after the binding of ligands. Transglutaminase may participate in the biochemical mechanism of receptor recycling
In some of the studies when the effect of primary amines on the binding and clustering of receptors was excluded a decrease in the number of available receptors could be found (Leuven, 1980) suggesting an interference with receptor recycling. The lysosomotropic effect of the primary amines in increasing intracellular (lysosomal) pH, thereby preventing ligand-receptor dissociation, can explain the inhibition of recycling. However, lysosomotropic chloroquine, secondary as well as tertiary amines, or other derivatives (like properly dimethylated dansylcadaverine and histamine), which are not transglutaminase substrates, could not inhibit receptor signaling in several cases (Davies, 1980; Leuven,
1163
Transglutaminase and receptor signaling
1
I!fl 0
2
a, TIME (MXRS)
72
Fig. 2. Transglutaminase activity in the nucleus of human peripheral blood lymphocytes various time intervals following phytohaemagglutinin (PHA) stimulation as compared to nonstimulated ones. Activity measurement: incorporation of [3H]putrescine into dimethylated casein. For details see F&is (1984).
1980; Julian et al., 1983). On the other hand, it was also concluded that transglutaminase may not be involved in the mechanism of growth-factormediated receptor. internalization or mitogenesis because well-known inhibitors of the enzyme had little or no effect (King, et al., 1981). The data are very controversial in this respect, which may be partially explained by differences in the systems used or that the results reflect the existence of several parallel mechanisms. Transglutaminase-formed protein-protein or proteinprimary amine conjugates may serve as signal structures
Since clustering and endocytosis of receptors are not necessarily followed by mitogen response, the role of transglutaminase in the events leading to mitogenic response might be more specific and important in the signaling process than simply being essential in irreversible receptor binding and uptake of the ligand-receptor complex. In most of the studies on this mitogen-induced lymphocytes were used, in which transglutaminase activity is abruptly increased following mitogen addition (Novogrodsky et al., 1978) just as well as the level of polyamines. It was demonstrated by Folk et al. (1980) that polyamines are natural substrates of transglutaminase. When normal human blood lymphocytes were treated with mitogens to induce blastogenesis in the presence of labeled putrescine, part of the label was incorporated into proteins. Labeled y-glutamyl-putrescine, -spermidine and -spermin were identified in the cellular protein fraction. Though the proteins have not yet been identified it could be postulated that covalent protein-polyamine conjugates serve as signaling structures for mitogenesis for that this kind of posttranslational covalent modification is one way of regulating enzymes important in this respect. The first possible example of such an enzyme modification has
been recently demonstrated in the case of ornithine decarboxylase (Russel and Manen, 1982) into which could be incorporated by transputrescine glutaminase, making it inactive, but meanwhile being capable of stimulating RNA polymerase I activity. In our studies an increased transglutaminase activity could be observed in the nucleus during the first 24 hr after stimulation of lymphocytes (Fig. 2) by phytohaemagglutinin. Similar results could be obtained using several other kinds of mitogen (data not shown). Not surprisingly, dansylcadaverine, the most potent inhibitor of transglutaminase, could inhibit blastogenesis (Fig. 3). Meanwhile the formation of several dansylcadaverine-labeled nuclear proteins was also observed. The nature of these proteins is currently being investigated. The concn of these amine-labeled proteins formed either using naturally occurring amines at labeling concns or others at inhibitory concns is very low for conventional isolation procedures, to say nothing of subsequent methods to prove that the amine was incorporated into y -glutamyl residues by transglutaminase (in none of the aforementioned cases including the receptor as well as other protein studies has been this criterion been met so far). Since the discovery of the catabolizing enzyme y-glutamylamine cyclotransferase (Fink et al., 1980) one should consider the possibilty that protein “aberrations” resulting from transglutaminase action may signal preferentially rapid proteolytic degradation of these proteins (and substrated for the catabolyzing enzyme which is capable of using only those polypeptides in which the proteolysis at the glutamine side of either the cross-link or the amine derivative is complete) are rapidly formed making it exceptionally difficult to catch these transient derivatives. Naturally, the same is true as far as the postulated modifications of receptors or membrane proteins are concerned.
001 0025 0 0 DANSYLCADAVERINE (mmol/
01
I)
Fig. 3. Inhibition of PHA-induced stimulation of human peripheral blood lymphocytes (2 x 105ml) by dansylcadaverine. Radioactivity measurements were carried out 72 hr following PHA addition. For details see F&is (1984).
L. FBsiis et al.
1164
fortunately, that has not turned out to be the case, at least so far. Although the transglutaminaseinhibitory primary amines have become widely used methodological tools in receptor studies, their mechanism of action is not fully understood. Due mainly to methodological difficulties direct and final evidence of transglutaminase-catalyzed modification of receptors, cellular proteins or other has not been found. However, the number of encouraging results is high enough to stimulate further efforts to find new methodological approaches utilizing ail the available data and to consider the possibility of the involvement of transglutaminase in receptor signaling as an open question. Acknowledgement-rhis
Fig. 4. Transglutaminase activities measured in cell extracts of monocytes and lymphocytes prepared from patients with systemic lupus erythematosus (SLE) or Hodgkin’s disease and healthy individuals. Method: incorporation of [jH]putrescine into dimethylated casein. For details see F&is (1984).
PHAGOCYTOSIS
AND MACROPHAGE
ACTIVATION
An interesting aspect of the involvement of transglutaminase in cell stimulation processes is the case of macrophages. We have previously shown that agents which inhibit transglutaminase activity in rat macrophages block Fc receptor mediated phagocytosis (F&is et al., 1981). The endocytosis of immune complexes via the Fc receptor produces an activation of transglutaminase and the stimulation of amine incorporation into membrane proteins of intact cells (these proteins have not been identified either). Similar results were obtained later using guinea pig (Leu et al., 1982) as well as murine (Schroff et al., 1981) macrophages. The inflammatory peritoneal macrophages have higher levels of transglutaminase activity than resident nonactivated macrophages. According to a recent report the synthesis of transglutaminase is greatly accelerated by certain types of macrophage stimulation (Mortaugh et al., 1983). -hese studies seem to have a clinical relevance as well. We have found that in human monocytes an increased transglutaminase activity can be measured not only following in eitro stimulation but also in cells isolated from patients with SLE and Hodgkin’s disease when other signs of in vivo monocyte activation are also apparent (Fig. 4). The transglutaminase activity of lymphocytes was not changed. CONCLUDING
REMARKS
Based on the first reports in 1980 which hay suggested a strong involvement of transglutaminase in receptor signaling one expected that a crucial and specific biochemical step in the biochemical cascade of cell triggering might be soon revealed. Un-
the National Foundation MD.
work was partially supported by for Cancer Research, Bethesda,
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