Endocrine Abstracts

Several Rab proteins control secretory granule trafficking and, hence, hormone release, by regulating the activity of different components of the secretory granule transport machinery. Particularly, Rab18 inhibits secretory granule movement, which leads to reduced neuropeptide and hormone secretion in PC12 pheochromocytoma cells and ACTH-secreting ATt20 cells, respectively, but how this GTPase accomplishes its role and the identity of the proteins that regulate Rab18 activity (effectors) remain unknown. In this work, we searched for the Rab18 effectors that ultimately determine its participation in neuroendocrine secretion. Time-lapse video-microscopy revealed that a functional microtubule-based cytoskeleton network is necessary for Rab18 to anchor to the secretory granules and to inhibit their movement. Furthermore, yeast two-hybrid experiments allowed us to identify a plethora of putative Rab18 interacting proteins, among which it is worth to highlight a subunit of the microtubule-associated molecular motor kinesin-1 for its well-known role in anterograde secretory granule transport. We confirmed Rab18/kinesin-1 interaction by Fluorescence Resonance Energy Trasnfer (FRET), which also showed that such an interaction only occurs with the active, GTP-bound conformation of Rab18. Furthermore, FRET also revealed that huntingtin (HTT), a protein that modulates intracellular membrane trafficking by recruiting the molecular motors dynein or kinesin to the vesicle surface, associates with the inactive, GDP-bound form of Rab18. On the other hand, HTT overexpression increased Rab18 association to the surface of secretory granules in PC12 cells, which suggests that HTT could act as a guanine exchange factor (GEF) for this Rab GTPase. Altogether, our results suggest that Rab18 reduces neuroendocrine secretion by interacting and regulating the activity of various components of the microtubule-based transport apparatus.