REGULATION OF EPIDERMAL GROWTH-FACTOR RECEPTOR ACTIVITY BY CROTOXIN, A SNAKE-VENOM PHOSPHOLIPASE A(2) TOXIN - A NOVEL GROWTH-INHIBITORY MECHANISM

Crotoxin (CT), a phospholipase A(2) (PLA(2)) derived from the venom of Crotalus durissus terrificus, is a heterodimeric protein composed of subunit B with enzymatic activity and a binding regulatory subunit (A) without enzyme activity. Although the PLA(2) activity of CT may be im portant in its anti-proliferative activity, its cytostatic mechanism i s unknown. In this study, we examined the cytostatic effect of PLA(2)- associated CT activity on squamous carcinoma cells expressing distinct levels of epidermal growth factor receptor (EGFr). CT was most effect ive in suppressing growth on cells expressing high intrinsic levels of EGFr. Cardiotoxin, another membrane active toxin with no intrinsic PL A(2) activity, had no differential anti proliferative activity on cell s expressing high EGFr levels, suggesting a correlation between EGFr e xpression and CT-directed antiproliferative activity. Both chemically modified CT (MCT) devoid of PLA(2) activity and covalently cross linke d CT (CCT), which is functionally unable to utilize cellular membranes as PLA(2) substrate, were also without growth inhibitory activity. No evidence for direct binding of CT to EGFr was found, although pretrea tment with EGF was able to partially suppress the anti-proliferative a ctivity of CT. Tyrosine phosphorylation of EGFr, however, was stimulat ed by CT in intact A431 cells. Tyrosine phosphorylation of EGFr was co ncentration-dependently stimulated (3- to 8-fold) in cellular membrane s of A431 cells treated in vitro with CT but not with anti-proliferati vely inactive MCT or CCT. The data provide evidence for transmembrane receptors involved in growth signaling (namely EGFr) as cellular targe ts and potential effectors of PLA(2)-mediated anti-proliferative activ ity of snake venom.