ABSTRACT
Rab GTPases are compartment-specific molecular switches that regulate intracellular vesicular transport in eukaryotes. GDP/GTP exchange factors (GEFs) control Rab activation, and current models propose that localised and regulated GEF activity is important in targeting Rabs to specific membranes. Here, we investigated the mechanism of GEF function using the Rab27a GEF, Rab3GEP (also known as MADD), in melanocytes as a model. We show that Rab3GEP-deficient melanocytes (melan-R3GKO) manifest partial disruption of melanosome dispersion, a read-out of Rab27a activation and targeting. Using rescue of melanosome dispersion in melan-R3GKO cells and effector pull-down approaches we show that the DENN domain of Rab3GEP (conserved among RabGEFs) is necessary, but insufficient, for its cellular function and GEF activity. Finally, using a mitochondrial re-targeting strategy, we show that Rab3GEP can target Rab27a to specific membranes in a GEF-dependent manner. We conclude that Rab3GEP facilitates the activation and targeting of Rab27a to specific membranes, but that it differs from other DENN-containing RabGEFs in requiring DENN and non-DENN elements for both of these activities and by lacking compartment-specific localisation.
Subject(s)
Biological Transport/physiology , Guanine Nucleotide Exchange Factors/metabolism , rab27 GTP-Binding Proteins/metabolism , Animals , Melanocytes/cytology , Melanocytes/metabolism , Melanosomes/metabolism , Mice , Multiple Acyl Coenzyme A Dehydrogenase Deficiency/metabolism , Primary Cell Culture , rab GTP-Binding Proteins/metabolism , rab3 GTP-Binding Proteins/metabolismABSTRACT
Chemotactic and angiogenic factors secreted within the tumor microenvironment eventually facilitate the metastatic dissemination of cancer cells. Calcium-sensing receptor (CaSR) activates secretory pathways in breast cancer cells via a mechanism driven by vesicular trafficking of this receptor. However, it remains to be elucidated how endosomal proteins in secretory vesicles are controlled by CaSR. In the present study, we demonstrate that CaSR promotes expression of Rab27B and activates this secretory small GTPase via PI3K, PKA, mTOR and MADD, a guanine nucleotide exchange factor, also known as DENN/Rab3GEP. Active Rab27B leads secretion of various cytokines and chemokines, including IL-6, IL-1ß, IL-8, IP-10 and RANTES. Expression of Rab27B is stimulated by CaSR in MDA-MB-231 and MCF-7 breast epithelial cancer cells, but not in non-cancerous MCF-10A cells. This regulatory mechanism also occurs in HeLa and PC3 cells. Our findings provide insightful information regarding how CaSR activates a Rab27B-dependent mechanism to control secretion of factors known to intervene in paracrine communication circuits within the tumor microenvironment.