New Eph Receptor Ligand

Abstract

A point mutation (P56S) in VAPB (VAMP-associated membrane protein B) is associated with certain forms of familial amyotrophic lateral sclerosis (ALS), which is characterized by progressive loss of motor neuron function. VAPB is a member of the VAP family of proteins, which are typically associated with the endoplasmic reticulum (ER) and thought to function in lipid metabolism. They have an N-terminal major sperm protein (MSP) domain, a coiled-coil motif, and a transmembrane domain. Tsuda et al . found with immunofluorescent analysis of either endogenous proteins or versions tagged on the N and C termini with different epitope tags that the Drosophila homolog of human VAPB, which they call dVAP, was localized both intracellularly and extracellularly in wing imaginal discs. Western blot analysis of fly extracts or human white blood cells suggested that a cleaved form of the protein was present in addition to the full-length protein, and detection of the short forms in human serum suggested that the cleavage product was secreted. Analysis of a version of dVAP in which the residue homologous to the one associated with ALS was mutated (P58S) showed that the protein was not present extracellularly but instead formed aggregates in the wing disc cells or neurons. These P58S aggregates also included wild-type dVAP and ER proteins and appeared to trigger the ER stress response in motor neurons. Antibodies against ubiquitin recognized the aggregated P58S protein on Western blots of immunoprecipitated P58S, and ubiquitin colocalized with the aggregates in Drosophila neurons. Although presynaptic overexpression of wild-type dVAP alters motor neuron morphology and electrophysiological properties and causes muscle defects, as well as impairs flight, overexpression of P58S did not cause such phenotypes. Furthermore, the P58S mutant rescued the lethality of null mutations in dVAP; thus, P58S retains some biological activity but not all properties of the wild-type protein. Because VAPs have a MSP domain and MSPs are ligands for Eph homologs in Caenorhabditis elegans , the authors performed genetic experiments that placed dVAP and Eph in common developmental pathways in flies and placed VPR-1, the C. elegans VAP homolog, and VAB-1, the C. elegans Eph receptor, in common pathways in worms. Fluorescein isothiocyanate (FITC)-conjugated versions of the MSP domain of human VAP, dVAP, or VPR-1 were incubated with isolated C. elegans gonads, and all three proteins bound in the same pattern as that of MSP-FITC. Additionally, the hVAP MSP domain competed with MSP-FITC, suggesting that they recognized the same binding sites. The MSP domain of hVAP also competed with EphrinB2 for binding to EphA4 in a dose-dependent manner, suggesting that VAPs may serve as Eph ligands in mammals as well. Ackerman and Cox provide insight into the importance of these results in understanding the mechanism underlying ALS. H. Tsuda, S. M. Han, Y. Yang, C. Tong, Y. Q. Lin, K. Mohan, C. Haueter, A. Zoghbi, Y. Harati, J. Kwan, M. A. Miller, H. J. Bellen, The amyotrophic lateral sclerosis 8 protein VAPB is cleaved, secreted, and acts as a ligand for Eph receptors. Cell 133 , 963-977 (2008). [PubMed]