Here we show that blocking the adhesive function of αvβ3 integrin with soluble RGD ligands, such as osteopontin or cilengitide, promoted association of Rab-coupling protein (RCP) with α5β1 integrin and drove RCP-dependent recycling of α5β1 to the plasma membrane and its mobilization to dynamic ruffling protrusions at the cell front. These RCP-driven changes in α5β1 trafficking led to acquisition of rapid/random movement on two-dimensional substrates and to a marked increase in fibronectin-dependent migration of tumor cells into three-dimensional matrices. Recycling of α5β1 integrin did not affect its regulation or ability to form adhesive bonds with substrate fibronectin. Instead, α5β1 controlled the association of EGFR1 with RCP to promote the coordinate recycling of these two receptors. This modified signaling downstream of EGFR1 to increase its autophosphorylation and activation of the proinvasive kinase PKB/Akt. We conclude that RCP provides a scaffold that promotes the physical association and coordinate trafficking of α5β1 and EGFR1 and that this drives migration of tumor cells into three-dimensional matrices.

Here, we report a direct interaction between the beta1 integrin cytoplasmic tail and Rab25, a GTPase that has been linked to tumor aggressiveness and metastasis. Rab25 promotes a mode of migration on 3D matrices that is characterized by the extension of long pseudopodia, and the association of the GTPase with alpha5beta1 promotes localization of vesicles that deliver integrin to the plasma membrane at pseudopodial tips as well as the retention of a pool of cycling alpha5beta1 at the cell front. Furthermore, Rab25-driven tumor-cell invasion into a 3D extracellular matrix environment is strongly dependent on ligation of fibronectin by alpha5beta1 integrin and the capacity of Rab25 to interact with beta1 integrin. These data indicate that Rab25 contributes to tumor progression by directing the localization of integrin-recycling vesicles and thereby enhancing the ability of tumor cells to invade the extracellular matrix.

Rabaptin-5 functions as an effector for the small GTPase Rab5, a regulator of endocytosis and early endosome fusion. We have searched for structural determinants that confer functional specificity on Rabaptin-5. Here we report that native cytosolic Rabaptin-5 is present in a homodimeric state and dimerization depends upon the presence of its coiledcoil predicted sequences. A 73 residue C-terminal region of Rabaptin-5 is necessary and sufficient both for the interaction with Rab5 and for Rab5-dependent recruitment of the protein on early endosomes. Surprisingly, we uncovered the presence of an additional Rabbinding domain at the N-terminus of Rabaptin-5. This domain mediates the direct interaction with the GTPbound form of Rab4, a small GTPase that has been implicated in recycling from early endosomes to the cell surface. Based on these results, we propose that Rabaptin-5 functions as a molecular linker between two sequentially acting GTPases to coordinate endocytic and recycling traffic.

SummarySeveral protein families control intracellular transport processes in eukaryotic cells. Here, we show that the Rab11 GTPase effector protein Rab11-FIP3 (henceforth, FIP3) directly interacts with the dynein light intermediate chain 1 (DLIC-1, gene symbol DYNC1LI1) subunit of the cytoplasmic dynein 1 motor protein complex. We show that Rab11a, FIP3 and DLIC-1 form a ternary complex and that DLIC-1 colocalises with endogenous FIP3 and Rab11a in A431 cells. We demonstrate that association between FIP3 and DLIC-1 at the cell periphery precedes minus-end-directed microtubule-based transport, that FIP3 recruits DLIC-1 onto membranes, and that knockdown of DLIC-1 inhibits pericentrosomal accumulation of key endosomal-recycling compartment (ERC) proteins. In addition, we demonstrate that expression of a DLIC-1-binding truncation mutant of FIP3 disrupts the ability of ERC proteins to accumulate pericentrosomally. On the basis of these and other data, we propose that FIP3 links the Rab11 GTPase and cytoplasmic dynein to mediate transport of material from peripheral sorting endosomes to the centrally located ERC.

Rab4 and Rab11 are small GTPases belonging to the Ras superfamily. They both function as regulators along the receptor recycling pathway. We have identified a novel 80-kDa protein that interacts specifically with the GTP-bound conformation of Rab4, and subsequent work has shown that it also interacts strongly with Rab11. We name this protein Rab coupling protein (RCP). RCP is predominantly membrane-bound and is expressed in all cell lines and tissues tested. It colocalizes with early endosomal markers including Rab4 and Rab11 as well as with the transferrin receptor. Overexpression of the carboxyl-terminal region of RCP, which contains the Rab4-and Rab11-interacting domain, results in a dramatic tubulation of the transferrin compartment. Furthermore, expression of this mutant causes a significant reduction in endosomal recycling without affecting ligand uptake or degradation in quantitative assays. RCP is a homologue of Rip11 and therefore belongs to the recently described Rab11-FIP family.