On the biophysics of transmembrane signalling

Molerular Immunology, Vol. 25, No. Printed in Great Bntain.

ON THE

I I, pp. 1075-1080,

BIOPHYSICS

0161.5890/88 $3.00 + 0.00 Pergamon Press plc

1988

OF TRANSMEMBRANE

L. TR~N, A. ASZAL&, M. BALAZS, S. A. MULHERN, Biomedical Cyclotron Laboratory, Department of Biophysics, H-4012 Hungary and Food and Drug Administration, (First received 4 January

J. SZ~LL~SI

SIGNALLING and S. DAMJANOVICH

Medical University School of Debrecen, Washington, D.C. 20204, U.S.A. 1988)

Abstract-Transmembrane signalling involves a number of physical translocations, changes in proximity of membrane elements like receptor subunits, or sequestration of proteins from the membrane. The monitoring of such changes with flow cytometric energy transfer revealed a new putative subunit of the IL-2 receptor and a possible intermolecular interaction between HLA class I and class II antigens. Lateral diffusion of the components of the multi-subunit IL-2 receptor was also followed. Changes in the intracellular pH were considered as a measure of efficient signal transfer in a number of cases. An overview and critical comparison of data is presented in the paper.

Transmembrane

signalling

and

its

regulation

is a

believed that a single general mechanism cannot account for the function of different systems (Cuatrecasas, 1986). In many cases, the plasma membrane receptors play the role of the primary target and their interaction with hormones, drugs, growth factors, neurotransmitters or even light represents the initial elementary step in the sequence of events during this process. The receptors themselves are subject to various forms of regulation as reflected by changes in their biological activity and subcellular distribution. As a result of the receptor-ligand interaction, the receptors may undergo a covalent modification, frequently a phosphorylation (Sibley and Lefkowitz, 1985; Benovic et al., 1986; Strasser et al., 1986b). Phosphorylation of one or several serine, threonine or tyrosine residues can be followed by the receptor’s sequestration or internalization (Beguinot et al., 1985), or translocation of an enzyme from the cytosol into the plasma membrane (Kuhn, 1978; Strasser er al., 1986a). Some of the membrane receptors display phosphokinase activity and intramolecular autophosphorylation occurs (Czech, 1985; Kahn, 1985; Sefton and Hunter, 1984: Hunter and Cooper, 1985; Pike and Krebs, 1986). Other receptors mediate endocytosis, giving rise to the internalization of many types of macromolecules and their intracellular transport. Changes in macromolecular associations frequently occur, following receptor-ligand binding. For example, binding of epidermal growth factor (Schlessinger, 1979), nerve growth factor (Levi et al., 1980), CQ macroglobulin (Maxfield et al., 1979), and insulin (Schlessinger et al., 1978) to their corre-

complex

phenomenon.

This work was carried

It is widely

out as part of the research program sponsored by the Hungarian Academy of Sciences (Grant No. OTKA-I 12).

sponding receptors results in a redistribution of the latter followed by a series of prompt biological responses. A representative field, where biophysical approach may help the understanding of transmembrane signalling, is in the hormone-receptor interaction. As an example, investigations on the structure of interleukin-2 (IL-2) are discussed here. IL-2 is produced by stimulated T-lymphocytes. This T-cell growth factor permits the induction of T-cell immune response having bound to IL-2 receptor expressed on the surface of the appropriate T-cells (Robb et al., 1981; Uchiyama et al., 1981; Waldmann, 1986; for review see Greene and Leonard, 1986). Membrane receptors for IL-2 have been detected in low and high affinity forms, characterized with a dissociation constant of l&30 @l and 5-15 pM, respectively (Robb et al., 1984). While there are cells with both low and high affinity IL-2 receptors, several types of cells like Eb, a murine T-cell lymphoma line (Diamantstein et al., 1985), express exclusively low affinity binding sites. Both classes of receptors share the same 55 kDa glycoprotein called Tat or p-chain (Uchiyama et al., 1981; Waldmann, 1986) characterized by MAbs in rat, mouse and human (Osawa and Diamantstein, 1983, 1984; Leonard et al., 1982; Malek et al., 1983). Immunoprecipitation experiments revealed a 75 kDa protein (P-chain) which binds IL-2 with an intermediate affinity (Kuo et al., 1986; Sharon et al., Tsudo et al., 1986; Robb et al., 1987). It was also proposed that the high affinity IL-2 receptor is a complex of the 55 and 75 kDa peptides (Tsudo et al., 1987). Based on the results of cross-linking and immunoprecipitation investigations by Herrmann and Diamantstein (1987), the existance is suggested of a 105-l 15 kDa complex specifically associated with high affinity IL-2 receptors in mice containing a third molecule. a putative y-chain, in addition to the a and/or p-chain.

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Based on the subunit structure and the different affinities (at least two) of the IL-2 receptors, it is assumed that the structure and affinity may be more intricately related than it was thought. Furthermore, it is assumed that in the process of signal transduction via IL-2 receptor-ligand interaction, membrane protein associations may occur. To verify these assumptions, the authors decided to carry out proximity measurements on distinct human T-cell activation antigens. Although there were several similar suggestions that the differing subunit structure of the IL-2 receptor may manifest in different affinity (Sharon et ai., 1986; Tsudo PI (11.. 1986, 1987; Smith, 1987). it has to be noted that according to alternative interpretations, different classes of IL-2 receptors might be coded by different genes (Greene and Leonard, 1986) or might be products of different processing (Kondo et cti., 1986). The HUT 102B2 cell line derived from a human T-cell lymphoma was chosen as the object of the proximity measurements because these cells expressed an extraordinarily high amount of IL-2 rcceptors and our flow cytometric energy transfer (FCET) method (Tron of 01.. 1984) was used. FCET allows the determination of absolute fluorescence resonance energy transfer effcicncy between donorand acceptor-ladled specific sites of the cytoplasmic membrane on a cell-by-cell basis. This is a measure of the separation distance of these sites. Ligdnds used for labeling the Tat antigen and T27 peptide, a 95 kDa membrane protein co-precipitating with the 55 kDa Tat, were the following fluorescein (F) and rhodamine (R) conjugated MAbs: anti-Tat (Uchiyama ef al., 1981) and 0KT27 (Sziillosi rf ul., 1987b). The F-anti-Tat served as donor and the R-OKT27 as acceptor in accord with the higher abundance of the T27 antigens, compared to the number of Tat antigens expressed by the HUT 10282 cells. This is explained by the fact that antigens of higher abundance are preferably used as acceptors in energy transfer measurements (Tron t’t a(.. 1984; Sziillijsi et al., 1984, 1987a; Damjanovich c’t al., 1983) because of higher energy transfer efficiency relative to the inverse case. The measured high efficiencyof energy transfer between F-anti-Tat and R-OKT27 antibodies (7%) in nine independent experiments provided strong evidence for these antibodies binding to loci very close to each other (Szolliisi et al.. 1987b). The number of Tat and T27 antigens per cell are sufficiently high to allow FCET measurements with easily measurable transfer efficiency, but not too high to cause trivia1 energy transfer in case of random relative distribution. This was proved by the fact that no energy transfer was found between F-anti-Tat and R-antitransferrin receptor. Efficient energy transfer was measured between donor and acceptor conjugated 0KT27 antibodies. which showed that the majority of the T27 antigens are in an aggregated form. Thcsc aggregates contain both the Tat and T27 antigens

with more than a single copy of the latter on average. FCET measurements also made it possible to calculate the number of F-anti-Tat and R-0KT27 binding sites per cell on a cell-by-cell basis and correlate the energy transfer efficiency with the donor to acceptor ratio. Our measurements resulted in a fairly broad distribution of this ratio, thus arguing against a strict stoichiometry of the involved antigens in the complexes as they are expressed on the cell surface. In addition to the cells with approximately the same amount of donor and acceptor binding sites. there is a large number of cells with si~ni~cantly higher abundance of T27 antigens. These facts can be interpreted such that either a certain fraction of the latter is not associated with the Tat antigen, or the stoichiometry of these antigens in the appropriate macromolecular complex is not strictly defined. This is consistent with the experimental finding that certain IL-2 binding cells expressing the Tat antigen do not bind the OKT27 antibody. The issue of the molecular association between the Tat and T27 antigens was further investigated with diffusion measurements. Lateral diffusion constants (D) for the Tat and T27 antigens on HUT 102B2 cells were measured by fluorescence recovery after photobleaching (FRAP) using F-anti-Tat and F-OKT27 as labeled ligands (Edidin er al., 1988). For the TX peptide, D values around IO ‘“cm’jsec were routinely obtained, a value typical for the diffusion rate of a large number of intramembrane proteins. While D values were not changed, the percentage recovery which is regarded as a measure of the mobile fraction of the labeled Tat antigen was significantly lower when cells were incubated with unlabeled 0KT27 antibodies prior to the FRAP measurement. At the same time OKT27 pretreatment resulted in an increased number of HUT 102B2 cells with no Auorescence recovery at all in the F-anti-Tat labeled sample, The diffusion constant on these cells is lower than 10. “cm’isec based on the time scale of the experiments. These results show direct or indirect interaction between the Tat and T27 antigens. The very high frequency of cells with no recovery at all in the F-OKT27 stained sample contrasts with the almost ten-fold lower value in the F-anti-Tat sample. This remarkable difference argues against a stable complex between Tat and T27 under the conditions of the FRAP measurement. However, the changing fraction of cells with no recovery at all and the changing extent of recovery in the F-anti-Tat sample as a result of 0KT27 pretr~tment, as well as the significant energy transfer between F-anti-Tat and R-0KT27 bound to HUT l02B2 cells, give strong evidence for the existence of interaction between these antigens. Results of the FCET and FRAP experiments suggest that the proximity relations (or complex formation pattern) of these antigens depend on their liganded state. For the interpretation of the FCET and FRAP

On the biophysics of transmembrane data, a model with the following features is proposed: (I) in addition to the hindered lateral diffusion of both the Tat and T27 antigens, the latter are restricted in long range diffusion; (2) T27 and Tat antigens are forming complexes with a stoichiometry of 1 : 1 or 2: 1 for the former, and I : 1 for the latter; (3) these complexes possess high values of dissociation-association rate constants compared to the time window of the FRAP measurements; (4) the Kd of this complex depends on the liganded state of the antigens. Apart from the results of the FCET and FRAP measurements, this model can explain the inconsistence of the Tat-T27 co-precipitation results (Sz(illiisi et al., 1987b). It is known that there are Tat negative cells expressing T27 antigens and OKT27 negative IL-2 binding cells. Thus the recent finding on associated Tat and T27 antigens on the surface of HUT 102B2 cells cannot be general. Nevertheless, the complexes with their varying association-dissociation state as a function of the ligand binding may have a role in signal transmission. Details of this role have to be elicited by further biochemical, biophysical and immunological experiments. In the case of IL-2 binding 0KT27 negative cells some other specific membrane proteins can substitute for the T27 antigens. Antigens coded by the human major histocompatibility complex, HLA, play an essential role in numerous processes of immunological relevance among which many belong to the par excellence transmembrane signalling processes. They are involved in different cell-cell interactions controlling cellular recognition, T- and B-cell differentiation and in the humoral and cell mediated host versus graft response. The class I HLA-A, -B and -C, the classic transplantation antigens, contain a 44 kDa heavy chain and the &-microglobulin (Klein and Figueroa, 1986), a II.5 kDa light chain which is non-covalently associated with the former. These molecules control the killing of virus infected cells by T-killer cells and are responsible for graft rejection. The class II antigens also contain an a or heavy (34 kDa) and a nonconvalently linked b or light chain (29 kDa). The HLA-D region is comprised of three subregions HLA-DP, HLA-DQ and HLA-DR (Giles and Capra, 1985). These molecules are involved in the activation of T-helper cells and will assist virus specific B-cells carrying the same class II molecules. They also play an active role in other communication between lymphoid cells. Several recent investigations supplied experimental evidence for interactions between MHC class I and class II antigens. In capping experiments using pairs of MAbs with various specificities to class I and class II antigens, uni- and bi-directional co-capping was frequently observed (Neppert and Mueller-Eckhardt, 1984. 1985). It has been reported that human Ia-like inhibited secondary lymphodeterminants proliferative responses (Pawelec et al., 1982). Sterkers

signalling

1077

et al. (1983) observed that antibodies against class I molecules can inhibit class II restricted immune reactions. It has been shown also that antigen presentation by macrophages can be inhibited by antibodies with class I specificity (McCalmon et al., 1975; Bright and Munro, 1981). Anichini et al. (1985) reported that MHC restricted (through class I antigens) cytotoxicity can be abolished by class II specific MAbs. In our experiments carried out on a human Blymphoblastoid cell line, PGF (Brodsky, 1984), we were looking for direct evidence for the possible intermolecular interactions between HLA class I and class II antigens. Donor and acceptor conjugated MAbs were used to label these antigens as it is required by the FCET method (Trbn et al., 1984; SzSlliisi et al., 1984). For labeling the class I antigens the &-microglobulin specific L368 antibody and the monomorphic W6/32 antibody with HLA-A, -B and -C specificity were selected (Brodsky, 1984). This way, both L368 and W6/32 MAbs were suitable for labeling all class I antigens. Class II antigens were labeled with fluorescein or rhodamine conjugated polymorphic Genox 3.53 and anti-LeulO antibodies. While the latter binds to all (al/l, and al&) DQ molecules, the Genox 3.53 recognizes only a subpopulation (a, b, ). Energy transfer between F-L368 and R-L368 bound to PGF cells was not found. Thus it can be argued that there is a disperse distribution of the MHC class I antigens on these cells. If these molecules are members of any macromolecular associations, they exist in a single copy with each complex. Contrary to this finding, a relatively high efficiency (8%) of energy transfer was observed when DQ specific LeulO antibodies conjugated either with fluorescein or rhodamine were mixed and this mixture was used for labeling the cells (Tr6n et al., 1987). As the number of LeulO antibody binding sites on a DQ molecule is not known, the close proximity of the epitopes LeulO is specific either for a dimeric form of DQ antigens or more than a single copy is present per antigen. Relative distribution of class I and II antigens is non-random. This conclusion was drawn from the results of energy transfer efficiency measurements on PGF cells labeled with donor and acceptor conjugated antibodies, specific to class I and II antigens. The 6% average energy transfer efficiency was calculated from the results of four sets of measurements using four different combinations of the class I and II specific ligands (F-L368 + R-LeulO; F-W6/32 + R-LeulO; F-LeulO + R-L368 and FGenox 3.53 + R-L368). Based on the results, the authors suggest the following model of the human MHC antigens. Class I and II molecules form complexes on the cell surface. These complexes contain a single HLA-A, -B or -C molecule and, depending on the number of L368 binding sites per DQ molecule, one or two molecules.

1078

L. TR~N et

In summary, the FCET measurements provided strong evidence for the physical association between HLA class I and If antigens existing prior to capping or co-capping. The close proximity of these antigens supplied the structural background for most of the interactions between these antigens as listed earlier. The measure of the efficient signal transfer is a function, at least in some cases, of the intracellular pH (pH,). It is known that the pH within a given cell may influence virtually all forms of biochemical activity. Substantial changes in the functionaf state of a cell therefore can be accompanied by an altered value of the pH,. It has been shown that a number of mitogenic stimuli cause @ cytoplasmic afkalinization (Reuss et ul., 1986). In particular Moolenaar et al. (1983) observed that growth stimulation of quiescent human fibroblasts by epidermic growth factor or serum factors induced a rapid and persistant elevation of pH,. A combination of platelet derived growth factor, vasopressin and insulin increased similarly the intracellular pH of Swiss 3T3 cells (Schufdiner and Rozengurt, 1982). It was also shown that Concanavalin A (Hesketh er uf., 1985; Gerson and Kiefer, 1982, 1983), LPS (Gerson and Kiefer, 1982, 19X3), phorbol ester ~Moolenaar rt cd., 1984; Rosoff and Cantley, 1985: Hesketh el ul., 198S), or Caionophore A 23187 (Hesketh CI a/.. 1985) activation of different cells was accompanied with an alkalinization phase. Cyclosporin A (CsA), a potent immunoregulator. became a widely used agent in solid organ transplantation (Cohen et al., 1984) for preventing allograft rejection without suppressing humoral immunity. The cellular and molecular mechanism of its selective effect on cytotoxic lymphocytes is not understood. As there is a growing body of evidence that cell proliferation is paralleled by a cytoplasmic pH elevation. the pH, is cell cycle related during cxponential growth (Musgrove et ~1.. 19871 and cell cycling can be arrested in G 1 at an acidic pH (Taylor and Hodson, 1984). The authors were interested in the study of the effect of CsA treatment on the intracellular pH of lymphocytes. A fluorescent pH indicator 2’-7’-bis-carboxyethyl-5and-6-carboxy-fluoresccin (BCECF) and flow cytometry were used in intracellular pH measurements. This method allowed the measurement of pH, distribution with excellent statistics on a ccll-bycell basis, making it possible to detect eventual subpopulations in the response, in contrast to other techniques using microelectrodes or F’” NMR measurements (Deutsch et al., 1982). Increasing concn of CsA (0.0.5-l @g/ml) up to the highest pharmacological dose did not cause any change in the intracellular pH of mouse spleen and human peripheral blood lymphocytes within 30 min. It should be mentioned that CsA does change the transmcmbrane ion fluxes within half a minute (Damjanovich CT/nl., 1986, 1987; Matyus et al.. 1986). One can then conclude that CsA affects ion fluxes without per-

al.

turbation of processes which play an active role in pH, homeostasis. This might mean, among other things, that CsA does not interfere with the Nat/H’ exchange which was shown to be involved in intracellular pH regulation of different cells (Grinstein et ok, 198.5a. b). However, at higher CsA cone (3 pg/ml), a transient decrease in the intracellular pH of human peripheral blood lymphocytes was observed and at a concn of IOpg/ml. CsA causes persistent acidification. As no subpopulation with different (pH,) response to the CsA administration at pharmacological doses was observed, it seems unlikely that GA inhibits cellular immune response, blocking the characteristic pH changes required for proliferation. The homogeneous pH, response of the lymphocytes was similar to the homogeneous change in the transmembrane potentials caused by CsA. One can argue that the specific effect of the drug on the signal transmission of a defined lymphocyte subpopulation is not based on primary effects such as change in the transmembrane ion fluxes, membrane potential or intracellular pH, but is decided in a later step in the chain ofevcnts showing a subset-specific dependence on the parameters of the previous primary or early effects. Using three ditrerent systems, WC demonstrated that biophysical investigations, like fluorescence resonance energy transfer, lateral diffusion of membrane proteins, membrane potential and intracellular pli measurements, can provide remarkable contribution to the understanding of the signal transduction. The clarification of the entire mechanism of any defined form of transmembrane signalling process, however, is a very complex task requiring a multitude of tcchniqucs. In the collection of the different approaches. the biophysicaf approach is only one but a very substantial component.

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