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.

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.

The endosomal recycling pathway lies at the heart of the membrane trafficking machinery in the cell. It plays a central role in determining the composition of the plasma membrane and is thus critical for normal cellular homeostasis. However, defective endosomal recycling has been linked to a wide range of diseases, including cancer and some of the most common neurological disorders. It is also frequently subverted by many diverse human pathogens in order to successfully infect cells. Despite its importance, endosomal recycling remains relatively understudied in comparison to the endocytic and secretory transport pathways. A greater understanding of the molecular mechanisms that support transport through the endosomal recycling pathway will provide deeper insights into the pathophysiology of disease and will likely identify new approaches for their detection and treatment. This review will provide an overview of the normal physiological role of the endosomal recycling pathway, describe the consequences when it malfunctions, and discuss potential strategies for modulating its activity.

Inflammation induces dramatic changes in the biosynthetic profile of the liver, leading to increased serum concentrations of positive acute phase (AP) proteins and decreased concentrations of negative AP proteins. Serum amyloid A (SAA) and the pentraxins C-reactive protein (CRP) and serum amyloid P component (SAP) are major AP proteins: their serum levels can rise by 1000-fold, indicating that they play a critical role in defense and/or the restoration of homeostasis. We have cloned SAA and a SAP-like pentraxin from salmonid fish species. The salmonid SAA shares approximately 70% amino acid identity with mammalian AP SAA. When salmonids are challenged with an AP stimulus, i.e., Aeromonas salmonicida, SAA responds dramatically as a major AP reactant. The salmonid pentraxin shows approximately 40% amino acid identity to both mammalian SAP and CRP. Evolutionary analysis suggests the presence of only a single such protein in teleosts and lower animal species. Surprisingly, the salmonid pentraxin behaves as a negative AP reactant, reminiscent of the SAP-like Syrian hamster female protein, in that hepatic mRNA concentrations decline to 50% of prestimulus levels. This study reinforces the hypothesis that SAA induction is an essential and universal feature of the vertebrate AP response and that it represents part of an ancient host defense system. Conversely, the species-dependent heterogeneity of pentraxin expression during the vertebrate AP response supports the possibility that its most important ancestral (and perhaps present) function is not related to its AP behavior.

Myosin Va is a widely expressed actin-based motor protein that binds members of the Rab GTPase family (3A, 8A, 10, 11A, 27A) and is implicated in many intracellular trafficking processes. To our knowledge, myosin Va has not been tested in a systematic screen for interactions with the entire Rab GTPase family. To that end, we report a yeast two-hybrid screen of all human Rabs for myosin Va-binding ability and reveal 10 novel interactions (3B, 3C, 3D, 6A, 6A′, 6B, 11B, 14, 25, 39B), which include interactions with three new Rab subfamilies (Rab6, Rab14, Rab39B). Of interest, myosin Va interacts with only a subset of the Rabs associated with the endocytic recycling and post-Golgi secretory systems. We demonstrate that myosin Va has three distinct Rab-binding domains on disparate regions of the motor (central stalk, an alternatively spliced exon, and the globular tail). Although the total pool of myosin Va is shared by several Rabs, Rab10 and Rab11 appear to be the major determinants of its recruitment to intracellular membranes. We also present evidence that myosin Va is necessary for maintaining a peripheral distribution of Rab11- and Rab14-positive endosomes.

Rab11-FIP2 is a recently described member of the Rip11/Rab11-FIP/Rab coupling protein family of Rab11 interacting proteins. Rab11-FIP2 interacts with both Rab11 and myosin Vb and co-localizes with Rab11 in both HeLa and Madin-Darby canine kidney cells (Hales, C. M., Griner, R., Hobdy-Henderson, K. C., Dorn, M. C., Hardy, D., Kumar, R., Navarre, J., Chan, E. K., Lapierre, L. A., and Goldenring, J. R. (2001)J. Biol. Chem. 276, 39067–390751). Here, we characterized the specificity of the interaction between Rab11-FIP2 and Rab11 and report that it does not interact with Rab4, Rab3, Rab5, Rab6, or Rab7. We demonstrate that the COOH-terminal region of Rab11-FIP2, which contains the Rab11 binding domain (RBD), is necessary and sufficient for its early endosomal membrane association. In contrast, the amino-terminal region, which contains a phospholipid binding C2-domain, by itself was insufficient for membrane binding. Expression of a deletion mutant of Rab11-FIP2, containing the RBD, caused tubulation of a transferrin receptor-positive early endosomal compartment in HeLa cells. Endogenous Rab11 was also associated with this compartment. This phenotype cannot be reversed by excess wild-type Rab11, or dominant-positive Rab11 (Rab11Q70L), suggesting that Rab11-FIP2 functions downstream of Rab11 in endosomal trafficking.