Principles for the post-GWAS functional characterisation of risk loci

Several challenges lie ahead in assigning functionality to susceptibility SNPs. For example, most effect sizes are small relative to effects seen in monogenic diseases, with per allele odds ratios usually ranging from 1.15 to 1.3. It is unclear whether current molecular biology methods have enough resolution to differentiate such small effects. Our objective here is therefore to provide a set of recommendations to optimize the allocation of effort and resources in order maximize the chances of elucidating the functional contribution of specific loci to the disease phenotype. It has been estimated that 88% of currently identified disease-associated SNP are intronic or intergenic. Thus, in this paper we will focus our attention on the analysis of non-coding variants and outline a hierarchical approach for post-GWAS functional studies

Signaling mechanisms of the semaphorin receptor plexin -B1.

Semaphorins are chemorepulsive molecules that signal via plexin-containing receptor complexes to effect changes in the cytoskeleton required for growth cone turning or collapse. Semaphorin 4D is one such member which directly interacts with plexin-B1. The biochemical signaling mechanisms downstream of this receptor have not been well characterized, however, they appear to involve regulation of members of the Rho family of small GTPases which is a method common to other guidance receptor signaling systems. We observed that the intracellular domain of plexin-B1 is able to directly interact with the Rae GTPase. Rho and Cde42 do not interact with plexin-B1 and the interaction is specific to plexin-B1 and not plexin-A or -C family members. The interaction is dependent on the effector domain of Rae and was found to be, enhanced upon Sema4D co-expression. We also observed that plexin-B1 can compete with PAK for Rae and inhibit Rae-induced PAK activation. This effectively sequesters Rae activity. This is consistent with events downstream of other repulsive guidance receptors where Rae and Cdc42 are inhibited and Rho is activated. We also observed that expression of active Rae can enhance the ability of plexin-B1 to interact with Sema4D. Active Rae is able to stimulate the number of receptors at the cell surface without affecting plexin-B1 expression. To further direct signaling events at the level of the plexin-B1 receptor, we purified a plexin-B1 interacting protein from mouse brain called LARG. LARG is a known exchange factor for Rho and our analysis show that the PDZ domain of LARG directly interacts with the C-terminal type I PDZ-binding site of plexin-B1. Furthermore, Sema-4D stimulated activation of Rho is dependent on the interaction of plexin-B1 with LARG. All together our observations provide molecular descriptions for the role of Rae and Rho GTPases downstream of the plexin-B1 receptor.Ph.D.BiochemistryBiological SciencesNeurosciencesPure SciencesUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/123490/2/3079543.pd

The Plexin-B1/Rac interaction inhibits PAK activation and enhances Sema4D ligand binding

The small GTPase Rac has been implicated in growth cone guidance mediated by semaphorins and their receptors. Here we demonstrate that plexin-B1, a receptor for Semaphorin4D (Sema4D), and p21-activated kinase (PAK) can compete for the interaction with active Rac and plexin-B1 can inhibit Rac-induced PAK activation. We have also demonstrated that expression of active Rac enhances the ability of plexin-B1 to interact with Sema4D. Active Rac stimulates the localization of plexin-B1 to the cell surface. The enhancement in Sema4D binding depends on the ability of Rac to bind plexin-B1. These observations support a model where signaling between Rac and plexin-B1 is bidirectional; Rac modulates plexin-B1 activity and plexin-B1 modulates Rac function