The Interaction Between RTN3/HAP And Related Proteins And Their Intracellular Function

hap, a gene encoding an ASY(NogoB) interacting protein, was identified by cloning complementary DNA from human lung cell line (WI238) cDNA library using yeast two hybrid system. Sequence analysis showed that both HAP and ASY belong to RTN family. HAP is RTN3, while ASY is RTN4B/NogoB. Last ten years, our lab has focused on the structure, dimerization, apoptotic function of HAP and a lot of reports on HAP have been published. Based on previous results, we studied the interactions of RTN3/HAP and related proteins and their intracellular function. RTN3/HAP took part in many apoptosis pathways and was crucial to the cross-talking among those pathways. We also discussed the relation of RTN3 and CRELD1, and revealed the molecular mechanism of AVSD more or less.In a preliminary experiment, Dr Liu carried out yeast two-hybrid screen in the embryonic Drosophila melanogaster library, using hRTN3 as the bait to find its potential associated candidates. A protein identical to the truncated form (1-220aa) of dFADD (AF295103) was identified as hRTN3 associated partner. This interesting result prompted us to investigate whether RTN3 could interact with counterpart of dFADD in the mammalian cells. Firstly, to determine that speculation, Flag-tagged hFADD and HA-tagged RTN3 were co-expressed in human HeLa cells. The co-immunoprecipitation experiments indicated that over-expressed or endogenous FADD and RTN3 could indeed interact in the mammalian cells. We observed that the dominant negative form of FADD (DN-FADD) containing the death domain but with DED deletion could still interact with RTN3, indicating that the death domain in FADD may mediate the association with RTN3. To map the region in the RTN3 responsible for interaction with FADD, three truncated forms of RTN3 were constructed: del-N-RTN3 (49-236aa), del-Nogo66 and del-C-RTN3 (1-191aa). The results from co-immunoprecipitation assays indicated that there was detectable association between RTN3 containing C terminal and FADD, whereas del-C-RTN3 failed to interact with FADD. It has been well established that FADD plays a critical role in the membrane bound death-inducing signaling complexes. Caspase-8 is an essential apoptotic caspase that is activated through oligomerization via association with FADD. To examine if activation of caspase-8 is involved in the RTN3 induced apoptosis, we studied the activation level of caspase-8 in HeLa cells with RTN3 over-expressed. The results indicated that the cells exhibited strong apoptosis as expected and notably, considerable amounts of caspase-8 were processed into active forms in response to RTN3 over-expression. With a construct of dominant negative FADD, we further investigated whether RTN3 elicited activation of caspase-8 is FADD-dependent and found that DN-FADD completely abolished the caspase-8 processing. Moreover, we found DN-FADD could not absolutely inhibit RTN3 induced apoptosis, hinting that there may be other initiator caspases activation in this process. Bid processing and cytochrome c release play important roles in the cellular apoptosis. Notably, we found DN-FADD could inhibit Bid cleavage and cytochrome c release in this process, further indicating that these cellular affairs in response to RTN3 over-expression are caspase-8 dependent. The interaction of FADD-RTN3 partly influence tunicamycin-induced apoptosis and caspase8 activation. FADD is an effective adaptor that participate various apoptosis processes. Our results suggested that besides death domain mediated interactions between FADD and TRADD or other DD family members, the death domain in FADD is still functioning in a way of mediating interaction(s) with the non-DD containing protein(s).Furthermore, NogoB/ASY, a homologue of RTN3 and a potential RTN3 interacting protein, also associated with FADD and induced cytochrome c release in a FADD dependent manner.Bcl-2 is known as a critical inhibitor of apoptosis triggered by a broad range of stimuli, mainly acting on the mitochondria. In a preliminary experiment, we performed a screen for RTN3-associated proteins using the T7 select phage display system (Novagen). We found a member of the reticulon (RTN) family, RTN3, which did not belong to the Bcl-2 family and was predominantly localized on the endoplasmic reticulum (ER), can interact with Bcl-2 in vitro and in vivo. Our results identified that RTN3 bound Bcl-2 on the ER and reduced the anti-apoptotic activity of Bcl-2 and increasing amount of ectopic RTN3 raised the apoptosis ratio of HeLa cells. The more ectopic Bcl-2 decreased, the more of the cell apoptosis inducing by equal RTN3. In HeLa cells stably expressing Bcl-2, when the expression of RTN3 increased by TM stimuli or transiently transfection, Bcl-2 in the mitochondria increased and the cell apoptosis induced by TM or STS reduced. These results suggest that RTN family proteins could modulate the anti-apoptotic activity of Bcl-2 by binding with it.RTN3/HAP was generally regarded as a novel human apoptosis-inducing protein, and little is known about its function in physiology. Herein, we demonstrated that CRELD1, a cell adhesion molecular playing a critical role in Atrioventricular septal defects (AVSD), could interact with RTN3 in vitro. Moreover we reported that ectopic CRELD1 could interact with ectopic or endogenous RTN3. When RTN3 and CRELD1 were over-expressed in the same cell, the cell growth trend was higher than the normal control HeLa cell but lower than the cell transiently transfected with pcDNA3.1-Flag-CRELD1 alone. Their interaction increased the localization of RTN3 on the plasma membrane, influenced the cell proliferation and moderately reduced the apoptotic activity of RTN3. Furthermore, the tunicamycin inducing cell apoptosis was partly suppressed by their interaction. These results suggest that CRELD1 can modulate the apoptotic activity of RTN3 by binding with it and can change its localization from the ER to the plasma membrane.