Research Article IL4/PGE 2 induction of an enlarged early endosomal compartment in mouse macrophages is Rab5-dependent Marisa J. Wainszelbaum 1 , Brandon M. Proctor 1 , Suzanne E. Pontow, Philip D. Stahl ⁎ , M. Alejandro Barbieri 2 Department of Cell Biology and Physiology, Washington University School of Medicine, 660 S. Euclid, Campus Box 8228, Saint Louis, MO 63110, USA ARTICLE INFORMATION ABSTRACT Article Chronology: Received 9 February 2006 Revised version received 18 March 2006 Accepted 21 March 2006 Available online 25 April 2006 The endosomal compartment and the plasma membrane form a complex partnership that controls signal transduction and trafficking of different molecules. The specificity and functionality of the early endocytic pathway are regulated by a growing number of Rab GTPases, particularly Rab5. In this study, we demonstrate that IL4 (a Th-2 cytokine) and prostaglandin E 2 (PGE 2 ) synergistically induce Rab5 and several Rab effector proteins, including Rin1 and EEA1, and promote the formation of an enlarged early endocytic (EEE) compartment. Endosome enlargement is linked to a substantial induction of the mannose receptor (MR), a well-characterized macrophage endocytic receptor. Both MR levels and MR- mediated endocytosis are enhanced approximately 7-fold. Fluid-phase endocytosis is also elevated in treated cells. Light microscopy and fractionation studies reveal that MR colocalizes predominantly with Rab5a and partially with Rab11, an endosomal recycling pathway marker. Using retroviral expression of Rab5a:S34N, a dominant negative mutant, and siRNA Rab5a silencing, we demonstrate that Rab5a is essential for the large endosome phenotype and for localization of MR in these structures. We speculate that the EEE is maintained by activated Rab5, and that the EEE phenotype is part of some macrophage developmental program such as cell fusion, a characteristic of IL4-stimulated cells. © 2006 Elsevier Inc. All rights reserved. Keywords: Endocytosis Small GTPases Endosomes Introduction The endosomal apparatus is a collection of vesicles and tubules that mediate and orchestrate transport of internalized fluid, receptor-associated ligands and membrane proteins to various intracellular destinations including the lysosomal compartment, trans-Golgi network, endoplasmic reticulum and plasma membrane [1]. In polarized cells, the endosomal apparatus plays a role in transcellular transport and in professional phagocytes, endosomes are the source of mem- brane as well as an entry point for access to lysosomes [2]. Rab GTPases regulate vesicular transport in endocytosis and exocytosis, and they have been associated with the control of vesicle docking and fusion [3,4]. Particularly, Rab5 plays a central role in both cargo receptor internalization and in receptor tyrosine kinase internalization [5,6]. Little is known about the dynamic relationships among the tubules and vesicles that make up the early endosomal compartment or EXPERIMENTAL CELL RESEARCH 312 (2006) 2238 – 2251 ⁎ Corresponding author. Fax: +1 314 361 1490. E-mail address: pstahl@cellbiology.wustl.edu (P.D. Stahl). 1 Both authors contributed equally to this work. 2 Present address: Department of Biological Sciences, Florida International University, Miami, FL 33199, USA. 0014-4827/$ – see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2006.03.025 available at www.sciencedirect.com www.elsevier.com/locate/yexcr