Detecting Endogenous Rab8 Activation.

ABSTRACT

The family of Rab GTPases switch between GDP- and GTP-bound forms to interact with effectors and accessory proteins for the regulation of trafficking and signaling pathways in cells. The activation and recruitment of a specific Rab by stimulants or physiological changes can be detected and assessed by measuring the relative amount of the Rab in its active, "GTP-bound" state versus the inactive "GDP-bound" state. While GTP loading can be measured in vitro, current methods to detect the activation state of endogenous Rabs within a cellular context are limited. Here, we developed two molecular probes, based on domains of known Rab effectors, which can be used to pull down endogenous GTP-bound Rab8 from cell extracts as a measure of Rab8 activation. As a test system, we use the lipopolysaccharide (LPS) induced activation of Rab8 in mouse macrophages. The molecular probes compared for capture of GTP-bound Rab8 are derived from two Rab8 effectors, OCRL and PI3Kγ, with the former assessed as being more efficient. We describe how the OCRL-RBD probe is used to assess activation of Rab8 in cell extracts with a method that should be applicable to assessing GTP-bound Rab8 in other cell and tissue extracts.