Mary W. McCaffrey

Rab-coupling protein coordinates recycling of α5β1 integrin and EGFR1 to promote cell migration in 3D microenvironments

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.

The mechanism of internalization of glycosylphosphatidylinositol-anchored prion protein.

The mode of internalization of glycosylphosphatidylinositol‐anchored proteins, lacking any cytoplasmic domain by which to engage adaptors to recruit them into coated pits, is problematical; that of prion protein in particular is of interest since its cellular trafficking appears to play an essential role in its pathogenic conversion. Here we demonstrate, in primary cultured neurons and the N2a neural cell line, that prion protein is rapidly and constitutively endocytosed. While still on the cell surface, prion protein leaves lipid ‘raft’ domains to enter non‐raft membrane, from which it enters coated pits. The N‐terminal domain (residues 23–107) of prion protein is sufficient to direct internalization, an activity dependent upon its initial basic residues (NH2‐KKRPKP). The effect of this changing membrane environment upon the susceptibility of prion protein to pathogenic conversion is discussed.

Distinct Rab-binding domains mediate the interaction of Rabaptin-5 with GTP-bound rab4 and rab5

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 coiled‐coil 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 Rab‐binding domain at the N‐terminus of Rabaptin‐5. This domain mediates the direct interaction with the GTP‐bound 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.

Rab coupling protein (RCP), a novel Rab4 and Rab11 effector protein.

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.

Rab4 affects both recycling and degradative endosomal trafficking

The small GTPases Rab4, Rab5 and Rab7 are endosomal proteins which play important roles in the regulation of various stages of endosomal trafficking. Rab4 and Rab5 have both been localized to early endosomes and have been shown to control recycling and endosomal fusion, respectively. Rab7, a marker of the late endosomal compartment, is involved in the regulation of the late endocytic pathway. Here, we compare the role of Rab4, Rab5 and Rab7 in early and late endosomal trafficking in HeLa cells monitoring ligand uptake, recycling and degradation. Expression of the Rab4 dominant negative mutant (Rab4AS22N) leads to a significant reduction in both recycling and degradation while, as expected, Rab7 mutants exclusively affect epidermal growth factor (EGF) and low density lipoprotein degradation. As also expected, expression of the dominant negative Rab5 mutant perturbs internalization kinetics and affects both recycling and degradation. Expression of Rab4WT and dominant positive mutant (Rab4AQ67L) changes dramatically the morphology of the transferrin compartment leading to the formation of membrane tubules. These transferrin positive tubules display swellings (varicosities) some of which are positive for early endosomal antigen‐1 and contain EGF. We propose that the Rab4GTPase is important for the function of the early sorting endosomal compartment, affecting trafficking along both recycling and degradative pathways.

Small GTP-binding proteins and their role in transport.

The Sec4/Ypt1/rab family of small GTP-binding proteins, a branch of the p21ras superfamily, is now recognizedas playing a central role in the regulation of intracellular transport in eukaryotic cells.

Rab11-FIP3 links the Rab11 GTPase and cytoplasmic dynein to mediate transport to the endosomal-recycling compartment.

Several 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.

The dynamic Rab11-FIPs.

The Rab11-FIPs (Rab11-family interacting proteins; also known as FIPs) constitute an evolutionarily conserved protein family that act as effector molecules for multiple Rab and Arf (ADP-ribosylation factor) GTPases. They were initially characterized by their ability to bind Rab11 subfamily members via a highly-conserved C-terminal RBD (Rab11-binding domain). Resolution of the crystal structure of Rab11 in complex with FIPs revealed that the RBD mediates homodimerization of the FIP molecules, creating two symmetrical interfaces for Rab11 binding and leading to the formation of a heterotetrameric complex between two FIP and two Rab11 molecules. The FIP proteins are encoded by five genes and alternative splicing has been reported. Based on primary structure, the FIPs were subcategorized into two classes: class I [Rip11, FIP2 and RCP (Rab-coupling protein)] and class II (FIP3 and FIP4). Recent studies have identified the FIPs as key players in the regulation of multiple distinct membrane trafficking events. In this mini-review, we summarize the Rab11-FIP field and discuss, at molecular and cellular levels, the recent findings on FIP function.

Rab GTPases and microtubule motors

Rab proteins are a family of small GTPases which, since their initial identification in the late 1980s, have emerged as master regulators of all stages of intracellular trafficking processes in eukaryotic cells. Rabs cycle between distinct conformations that are dependent on their guanine-nucleotide-bound status. When active (GTP-bound), Rabs are distributed to the cytosolic face of specific membranous compartments where they recruit downstream effector proteins. Rab-effector complexes then execute precise intracellular trafficking steps, which, in many cases, include vesicle motility. Microtubule-based kinesin and cytoplasmic dynein motor complexes are prominent among the classes of known Rab effector proteins. Additionally, many Rabs associate with microtubule-based motors via effectors that act as adaptor molecules that can simultaneously associate with the GTP-bound Rab and specific motor complexes. Thus, through association with motor complexes, Rab proteins can allow for membrane association and directional movement of various vesicular cargos along the microtubule cytoskeleton. In this mini-review, we highlight the expanding repertoire of Rab/microtubule motor protein interactions, and, in doing so, present an outline of the multiplicity of transport processes which result from such interactions.

Rab11 proteins in health and disease

Comprising over 60 members, Rab proteins constitute the largest branch of the Ras superfamily of low-molecular-mass G-proteins. This protein family have been primarily implicated in various aspects of intracellular membrane trafficking processes. On the basis of distinct subfamily-specific sequence motifs, many Rabs have been grouped into subfamilies. The Rab11 GTPase subfamily comprises three members: Rab11a, Rab11b and Rab25/Rab11c, which, between them, have been demonstrated to bind more than 30 proteins. In the present paper, we review the function of the Rab11 subfamily. We describe their localization and primary functional roles within the cell and their implication, to date, in disease processes. We also summarize the protein machinery currently known to regulate or mediate their functions and the cargo molecules which they have been shown to transport.