Unlike TNF-R1, internalisation kinase inhibitor Brefeldin A is not required for TRAIL-induced apoptosis, as the assembly of the TRAIL DISC already occurs at the cell membrane (Kohlhaas et al, 2007). However, internalisation of the ligated receptor has been suggested to play a role in other TRAIL-mediated signalling events such as activation of JNK or NF-��B (Varfolomeev et al, 2005). In our hands, internalisation of TRAIL receptors ligated by agonistic DR4 and DR5 antibodies or by rhTRAIL revealed no significant differences; in all cases, the ligated receptor complex was rapidly internalised. This indicates that the opposing apoptosis-modulatory effect of JNK activation induced by TRAIL or selective DR4/DR5 activation was not due to receptor internalisation but possibly different isoforms of JNK activated by the individual receptors.

Ten isoforms of JNK are known to exist as a result of alternative splicing of the three genes, jnk1, jnk2 and jnk3 (Gupta et al, 1996). Little is known about the role of these isoforms in apoptosis. Overexpression of JNK1��1 increases resistance to vesicular stomatitis virus-induced cell death in 3T3 fibroblasts, whereas overexpression of JNK1��1 and JNK1��1 potentiates cisplatin- and doxorubicin-induced cell death in sarcoma cell lines (Han et al, 2002; Koyama et al, 2006). Previous studies have demonstrated a role for either JNK1 or JNK2 in TNF��-induced apoptosis (Dietrich et al, 2004; Liu et al, 2004). Our results show that the chief JNK isotype activated by DR4 and DR5 is JNK1.

Furthermore, whereas TRAIL-mediated receptor activation led to activation of both the long and short isoforms of JNK1, selective ligation of DR4 or DR5 with cross-linked agonistic antibodies predominantly activated the short JNK1 isoforms (JNK1��1 and/or ��1) and the difference in cell death seen following JNK inhibition may be related to the different JNK1 isoforms, with the short isoforms GSK-3 of JNK1 (JNK1��1 and JNK1��1) transmitting an antiapoptotic signal and the long isoforms of JNK1 (JNK1��2 or JNK1��2) mediating a proapoptotic signal. As a reason why activation of individual TRAIL receptors by agonistic antibodies activates different JNK isoforms from rhTRAIL, it is likely that the agonistic DR4- and DR5-specific antibodies trigger different receptor clustering, or intracellular conformational changes from those induced by rhTRAIL. It is also possible that on rhTRAIL treatment, higher-order heteromeric receptor complexes (receptosomes) including both DR4 and DR5 are formed where the interaction between the death domains of the various receptor trimers allows for the recruitment of more and/or different adaptor proteins.