Receptor palytoxin

Mechanism of Palytoxin Action on the Epidermal Growth Factor Receptor... 

Many environmental toxins interact with specific cellular receptors, including enzymes, ion channels and ion pumps, and thus provide natural tools for the study of cellular signalling pathways. Palytoxin, a compound isolated from the coelen-terate of genus Palythoa, is one such useful and intriguing compound. The structure of palytoxin was first determined in 1981 independently by Hirata (7) and Moore (2). As one of the most potent marine toxins known, palytoxin has been studied in a variety of systems ranging from erythrocytes to neurons. As a tumor promoter of the non 12-O-tetradecanoylphorbol-13-acetate (TPA) type, palytoxin can also be studied in the context of a growth control system. 

To ensure that the inhibition of EGF binding by palytoxin was not a consequence of cell toxicity, the effect of palytoxin on DNA synthesis in Swiss 3T3 cells was monitored. When cells were incubated in the presence of palytoxin, 10% fetal calf serum, and H-thymidine for 19.5 hr, no depression in the extent of H-thymidine incorporation into DNA was detected up to 3.7 pM palytoxin (Table I). Although 11 pM palytoxin was toxic when present for a prolonged period, under the conditions of the assays described above no toxicity was detected (Table I). When cells were incubated in the presence of palytoxin, 0.1% fetal calf serum, and H-thymidine, palytoxin did not stimulate significant incorporation of H-thymidine into DNA. Thus, although it can modulate the EGF receptor system under these conditions, palytoxin alone does not appear to be mitogenic for Swiss 3T3 cells. 

Inhibition of EGF binding by palytoxin could be due to a decrease in receptor affinity, as in the case of TPA-type tumor promoters, and/or a decrease in receptor number. In Swiss 3T3 cells there are two classes of EGF receptors. The dissociation constants for the two EGF receptor classes were determined to be approximately 2 X 10 M and 2 x 10" M, corresponding to approximately 1 x 10 and 1 X 10 receptor molecules per cell, respectively (33). Scatchard analysis revealed that treatment of Swiss 3T3 cells with palytoxin, like PDBu, caused an apparent loss in high-affinity binding (Figure 2). However, in contrast to PDBu, palytoxin also caused a significant (approximately 50%) loss of low affinity EGF binding. 

Na also appears to play a role in palytoxin action in some systems. To determine if there is a Na requirement for palytoxin action on the EGF receptor, Swiss 3T3 cells were assayed for palytoxin activity in Na containing medium versus Na deficient medium. When NaCl is replac by cholineCl, palytoxin can no longer inhibit EGF binding in Swiss 3T3 cells (Figure 5). By contrast, PDBu is equipotent in both Na containing and Na free media (data not shown). 

Because these results suggest that extracellular Na is required for inhibition of EGF binding by palytoxin in these cells, we determined if palytoxin caused Na influx in Swiss 3T3 cells. When Na influx was monitored at an early time point (7 min), it was found that palytoxin causes an influx of Na and that the rate of Na influx is dose dependent (Figure 6). In parallel with its effect on EGF binding, palytoxin at different doses increases intracellular Na to the same final level (42). Although Na influx occurs prior to the inhibition of EGF binding, these results and the apparent Na dependence of the palytoxin effect suggest a role for Na in the action of palytoxin on the EGF receptor. 

Figure 7. Comparison of effects of palytoxin and phorbol dibutyrate on EGF receptor binding.

In contrast to the TPA-type tumor promoters, palytoxin, thapsigargin, and okadaic acid are classified as non-TPA type tumor promoters, which do not bind to phorbol ester receptors, or activate protein kinase C in vitro (Table II) (6,25-27). In this chapter, thapsigargin is not discussed, because it is derived from terrestrial plants. 

A, aplysiatoxin, or palytoxin. Therefore, we proposed that okadaic acid binds to its own receptor and induces various biological effects, including tumor promoting activity, through this receptor (27). We are now investigating the nature of its receptor. 

Wattenberg, E.V, McNeil, P.L., Eujiki, H., and Rosner, M.R. 1989. Palytoxin down-modulates the epidermal growth factor receptor through a sodium-dependent pathway. J Biol Chem 264, 213-219. 

The Na-K ATPase is the known receptor to palytoxin and ostreocins. The interaction shows lower potency for ostreocins, the natnre and site of the binding of palytoxin is unknown, it acts as a functional antagonist of ouabain, and their binding sites on the receptor are probably different. ... 

Definitive evidence supporting the presence of a receptor would include classic binding studies, which due to scarcity of MTX are difficult to conduct. Nevertheless, if MTX acts like most known nonpeptide marine toxins, one would expect a receptor. For instance, toxins with structural similarities to MTX, like brevetoxins and palytoxin (PTX) have recognized receptors [46-49]. The case of PTX is of particular interest, since it shares some of its cellular effects with MTX, such as membrane depolarization, [Ca i increases and oncotic cell death [50]. 

Among the other possible cellular receptors tested, it was found that palytoxin caused K " efflux in yeast expressing a hybrid between the Na KVATPase and the H, K -ATPase, converting this enzyme into and open channel. Interestingly, subsequent studies have described palytoxin-stimu-lated cation fluxes that were blocked by vanadate but resistant to ouabain in rat colon, suggesting that the toxin was able to convert a vanadate-sensitive H K ATPase into an electrogenic cation transporter and consequently, that the pore-forming action of palytoxin was not restricted to Na, K -ATPase since it was also observed with the coloific H, KVATPase, which is related to the sodium pmnp. The toxin was also found to interfere with the sarcolemmal calcium pump in cardiac myocytes as a secondary effect. Further smdies are required to determine how palytoxin interferes with each of the P-type ATPase pmnps or if the effects of the toxin on these pumps are consequences of structural similarities between the enzymes. ... 

When we analyzed the different pathways that could potentially contribute to the palytoxin-induced calcium entry in neurons, we found that the effect of palytoxin was dependent on the presence of extracellular calcium and glutamate receptors activation. Furthermore, secondary activation of voltage-dependent sodium and calcium channels and the reversal of the NaVCa" exchanger also contributed to the rise in calcium caused by the toxin." ... 

Another common cellular target of palytoxin and TPA is the EGF receptor. It has been described that palytoxin inhibited the binding of EGF to low- and high-affinity receptors in a manner independent of protein kinase C. Moreover, picomolar concentrations of palytoxin inhibited EGF binding in the absence of cytosolic calcium increase. Further studies indicated that the palytoxin effects on EGF receptor were not due to common secondary effects of sodium influx, including membrane depolarization, changes in intracellular pH, or inhibition of protein synthesis. ... 

Wattenberg, E.V. et al., Palytoxin down-modnlates the epidermal growth factor receptor throngh a sodium-dependent pathway,/. Biol. Chem. 264, 213, 1989. 

Wattenberg, E.V. et al., Sodium as a mediator of non-phorbol tumor promoter action. Down-modulation of the epidermal growth factor receptor by palytoxin, J. Biol. Chem. 264, 14668, 1989. Kuroki, D.W. et al., Activation of stress-activator protein kinase/c-Jun N-terminal kinase by the non-TPA-type tumor promoter palytoxin. Cancer Res. 56, 637, 1996. 

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