The RHD loop region has been detected both on the surface of cells and intracellularly, and it has been suggested that the RHD hydrophobic regions might either span the ER membrane or plasma membrane completely or might double back on themselves to form a hairpin (Figure 2b). Antibodies against the amino-terminal domain of RTN4 bind to the surface of chick oligodendrocytes in live spinal cord explants 8 and cultured oligodendrocytes interact specifically with both amino-terminal domain-specific antibodies and antibodies directed against the RTN4 66-amino-acid loop (66-loop) 16. These findings suggest that the amino terminus and the 66-loop project into extracellular space, and therefore that the first RHD hydrophobic region must double back on itself in the membrane. However, other data suggest that the amino-terminal domain is intracellular. Antibodies against the 66-loop region of RTN4 detect small amounts of this epitope on the surface of live COS-7 cells, but antibodies against c-Myc tags fused to either the amino or the carboxy terminus do not bind to live cells 8.

More recent data from non-neuronal cells in which RTN4 is overexpressed strongly support a third model, in which most of both the amino-terminal domain and the 66-loop are cytoplasmic. In COS cells treated with maleimide polyethylene glycol, cysteines in the amino-terminal domain and the loop regions of ER-associated RTN4 were found to be modified by the reagent after detergent disruption of the plasma membrane but not the ER membrane 6. Cysteines in the carboxy-terminal region were only partially modified. All these results suggest that mammalian reticulons might have different topologies in the ER and plasma membranes; such multiple conformations may enable them to carry out multiple roles in the cell. Another protein with multiple membrane topologies is the mammalian prion protein (PrP); overexpression of a certain transmembrane form of the prion protein, ^CtmPrP, causes neurodegenerative disease distinct from that caused by the natural pathogenic prion form PrP^Sc 1920. Another possibility is that reticulons assume different topologies in different cell types: the reticulon amino-terminal region has been detected only in the cytoplasm in COS-7 cells, but has been found on both the surface and in the cytoplasm of oligodendrocytes. Again, this may reflect the diverse roles of reticulon proteins in different cell types.

The solution structure of the RHD loop of RTN4, known as Nogo66, has recently been probed by circular dichroism (CD) and nuclear magnetic resonance (NMR). Nogo66 is soluble in pure water and consists of three alpha helices, two short flanking one long, spanning residues 6-15, 21-40 and 45-53, followed by the unstructured residues 55-60 2122. The Nogo66 loop is involved in several RTN4-specific signaling cascades, including interaction with the Nogo receptor (NogoR) to inhibit neurite outgrowth 23, and with the cell adhesion molecule contactin-associated protein (Caspr) 24 to mediate the localization of potassium channels at axonal paranodes. The human RTN1 and RTN3 66-loops share 71% and 63% identity with the RTN4 loop; mouse RTN1 and RTN3 identity with human RTN4 is 67% and 59%. Despite this high degree of identity, the RTN1 66-loop does not bind to NogoR, and the function of the 66-loops in RTN1 and RTN3 is unknown in both mammals and lower organisms.

As mentioned above, the amino-terminal regions of different reticulons are highly divergent in sequence. The amino-terminal domains of the human RTN4 isoforms appear to be highly unstructured, even under physiological conditions. In silico analysis and measurements by CD and NMR of the human isoforms RTN4A and RTN4B reveal a high degree of disorganization, with only short alpha helices and beta sheets that exist transiently 25. Recent studies have shown that intrinsically unstructured proteins (IUPs) are more likely to form multiprotein complexes than are proteins with stable tertiary structure 26, are better able to 'moonlight' - carry out alternative functions 25 - and may fold upon binding to their partners 27. It has been shown that up to 33% of eukaryotic proteins contain long disordered regions, compared with 2% of archeal proteins 25. The characterization of RTN4 as an intrinsically unstructured/disordered protein may explain its involvement in many physiological processes, as explained below.

There is growing evidence that reticulons are involved in bending and shaping the ER membrane, in trafficking of material from the ER to the Golgi apparatus, and in apoptosis (Figure 3). Antibody-mediated inhibition of RTN4A in mammalian cells prevents GTP-induced formation of elongated membrane tubules in vitro 6, and knocking out both the C. elegans RTNL RET-1 and its associated protein YOP-1 interferes with ER formation during mitosis in the worm 33. RTN4A also localizes to subdomains of the Xenopus nuclear envelope, and its inhibition by anti-RTN4A antibodies limits nuclear envelope assembly 35. C. elegans RET-1 also interacts with the protein RME-1, a regulator of endocytic recycling 363738. In a yeast two-hybrid screen, the mammalian RTN1 isoforms RTN1A and RTN1B were found to interact with a component of the mammalian endocytosis adaptor complex AP-2. RTN1C, in contrast, may be involved in exocytosis. It associates with calreticulin-negative regions of the ER and co-immunoprecipitates with the SNARE proteins syntaxin 1, syntaxin 7, syntaxin 13 and VAMP2 39. Overexpression of a fragment of RTN1C increases the rate of exocytosis in PC12 cells. RTN1, RTN2, and RTN4 were all identified in a yeast two-hybrid screen using the vesicle fusion protein chaperone β-SNAP as bait, although these results were not confirmed by co-immunoprecipitation 40. In summary, reticulons interact with proteins involved in vesicular formation and fusion such as SNAREs and SNAPs. Reticulons also appear to play a role in ER morphogenesis; nevertheless, cells lacking reticulon expression do not have major defects in ER, endosomal or microsomal structure.

Reticulons are also involved in intracellular trafficking - a close cousin of vesicle formation and recycling. Overexpression of RTN3 in HeLa cells prevents retrograde transport of proteins from the Golgi complex to the ER 41. In yeast, RTNL1B forms complexes with Yip3p, the yeast ortholog of the mammalian Rab-GDI displacement factor (GDF). Small GTPases of the Rab family facilitate vesicle trafficking between organelles, and are regulated by GDFs 42. In C. elegans, inhibition of RET-1 and YOP-1 disrupted nuclear envelope assembly, and of 29 Rabs screened, depletion of Rab5 mimicked this phenotype closely 33. In a screen in human cells for GTPase-activating proteins (GAPs), which inhibit Rab function, the protein TBC1D20 was found to be a GAP for Rab1 and Rab2, and in the same study, interaction between RTN1C and TBC1D20 was identified in a yeast two-hybrid screen, a further argument for a role for reticulons as regulators of Rab-regulated intracellular trafficking 43.

In mammalian cells, reticulons may also play a role in apoptosis. Both RTN1C and what is now known to be RTN4A were identified in a screen for interactors with Bcl-X_L, a powerful inhibitor of apoptosis 44. RTN1C was found to inhibit Bcl-X_L, and RTN4A was found to inhibit both Bcl-X_L and another apoptosis inhibitor, Bcl-2, demonstrating a pro-apoptotic role for reticulons. More recently, RTN1C was shown to modulate apoptosis by upregulating the sensitivity of the ER to stressors in neuroblastoma cells 45. Several labs have shown that RTN3 also enhances apoptosis via interaction with Bcl-2 464748. Although these and other data indicate that reticulons may have a role in tumor suppression via upregulation of apoptosis, this topic is not without controversy 49.

The longest isoform of RTN4, RTN4A, has been extensively characterized in the mammalian CNS (recently reviewed by Liu et al. 50). It had long been known that in contrast to the myelin of the peripheral nervous system, myelin from the CNS appeared to prevent neuronal regeneration after injury 51. In 1988, by size fractionation of rat CNS myelin, Caroni and Schwab discovered a 250 kDa inhibitor of neurite outgrowth 52. This protein was later identified as a novel reticulon (RTN4A) and also named Nogo-A after its inhibitory effect on neuronal regeneration 81314. As the protein is generally called by this name in neuronal regeneration studies, we shall use that name in the following discussion. GrandPré et al. 8 showed that the extracellular/ER luminal portion of Nogo-A, the 66-loop termed Nogo66, is a potent inhibitor of neurite outgrowth. The receptor for Nogo66 was subsequently identified and termed NogoR 23. Inhibition of NogoR using the antagonist peptide NEP1-40 releases myelin-mediated inhibition of neurite outgrowth in culture, and both the acute intrathecal delivery and delayed systemic delivery of NEP1-40 promotes axonal regeneration of corticospinal tract fibers after dorsal hemisection in rats 18535455. Work on the amino-terminal domain of Nogo-A demonstrated its capacity to induce growth-cone collapse independent of NogoR via a region now called Δ20 1623, whereas another amino-terminal region, termed Nogo-A-24, is known to enhance the binding affinity of Nogo66 for NogoR when fused to Nogo66 15. Interestingly, the RHD region common to all isoforms of Nogo (RTN4) is alone sufficient to delay nerve regeneration after sciatic nerve crush 56.

Numerous in vivo studies in animals have found that either genetic ablation or pharmacological inhibition of the Nogo-A-NogoR interaction promotes axon growth and behavioral recovery after spinal cord injury 17185354575859606162, and significant improvement of recovery after similar prevention of Nogo-A action is also seen after stroke injury 636465. The field is not free from controversy, however 6667. The genetic background can alter the effects of Nogo inhibition 68, and studies of spinal cord injury in Nogo-knockout animals generated in different laboratories have yielded variable results 697071. The weight of evidence for a role for Nogo-A as an inhibitor of neurite outgrowth and a limitor of axon growth in spinal cord injury, however, make it a prime target for therapeutic intervention. Indeed, clinical trials of anti-Nogo antibodies are already under way.

Although the mechanism of action of reticulon in the ER remains to be elucidated, the mechanism by which Nogo-A inhibits neurite outgrowth and axon regeneration in the CNS is well characterized (Figure 4, Table 1). The receptor - NogoR - for the Nogo66 region was identified in 2001 21, but this receptor lacks a transmembrane and signaling domain and so must interact with a co-receptor or other signal transducer. Several candidates for this role have been discovered: the neurotrophin receptor p75, the transmembrane protein LINGO-1 and the orphan tumor necrosis factor family member TAJ/TROY have all been shown to bind NogoR and participate in the inhibition of neurite outgrowth in vitro 72737475767778 (see Figure 4, Table 1). The epidermal growth factor receptor (EGFR) may indirectly be a signal transducer for NogoR - the kinase activity of EGFR has been shown to be required for the inhibitory action of Nogo66 in culture - but EGFR does not directly bind NogoR 78. A crystal structure of the ligand-binding domain of the NogoR receptor has been determined 7980.

NogoR and all its putative co-receptors rely on the small GTPase RhoA for their downstream effects. Upon RhoA activation as a result of NogoR signaling, Rho-activated kinase (ROCK) stimulates actinomyosin activity, causing growth-cone collapse 5081. Blocking Rho activity either pharmacologically or with dominant-negative RhoA releases Nogo66-mediated neurite outgrowth inhibition in vitro 82838485.