Osteoclastic bone resorption is a highly dynamic process that requires the tight ordering of intracellular trafficking events in order to maintain the structural and functional polarization of the ruffled border and basolateral domains. Rab3 proteins are a subfamily of GTPases, known to mediate membrane transport in eukaryotic cells and play a role in exocytosis. Our recent data indicates that Rab3D modulates a post-TGN trafficking step that is required for osteoclastic bone resorption (1). Here, to identify down-stream regulatory molecules of Rab3D, we have performed a yeast two-hybrid screen. Amongst several candidate Rab3D-interacting proteins identified, Rab3D was found to associate with calmodulin, an established regulator of osteoclastic bone resorption. As an initial effort to better define the interaction between Rab3D and calmodulin, we generated several mutants of Rab3D which interfere with the GDP/GTP nucleotide exchange (Rab3DQ81L, Rab3DN135I) and/or membrane attachment of Rab3D (Rab3D-CXC). By in vivo bioluminescence resonance energy transfer (BRET) assay, Calmodulin was found to associate equivalently with wild type Rab3D as well as Rab3DN135I and Rab-3DCXC variants. Over expression of constitutively active Rab3D (Rab3DQ81L) enhanced this interaction suggesting that the active form of Rab3D (i.e. GTP-bound) might recruit additional effector molecules which further potentiate it’s binding to calmodulin. In an attempt to address the impact of calmodulin activity on Rab3D-calmodulin interaction and osteoclastic bone resorption, we performed complementary BRET and in vitro bone resorption assays in the presence of the calmodulin inhibitor, calmidazolium chloride. Interestingly, we show that suppression of calmodulin activity via calmidazolium chloride impairs the association of Rab3D with calmodulin, an affect that correlates with a disruption in osteoclastic bone resorption. We propose that the recruitment of calmodulin by Rab3D might be an important requirement for osteoclast-mediated bone resorption.
