ABSTRACT. RME-8 is a DnaJ-domain-containing protein that was first identified in Caenorhabditis elegans as being required for uptake of yolk proteins. RME-8 has also been identified in other species, including flies and mammals, and the phenotypes of their RME-8 mutants suggest the importance of this protein in endocytosis. In the present study, we cloned human RME-8 (hRME-8) and characterized its biochemical properties and functions in endocytic pathways. hRME-8 was found to be a peripheral protein that was tightly associated with the membrane via its N-terminal region. It partially colocalized with several early endosomal markers, but not with late endosomal markers, consistent with observations by immunoelectron microscopy. When cells were transfected with a panel of dominant-active Rab proteins, hRME-8 was confined to large vacuoles induced by expression of Rab5aQ79L, but not by Rab7Q67L. Expression of C-terminally-truncated hRME-8 mutants led to the formation of large puncta and vacuoles, and compromised endocytic pathways through early endosomes, i.e., recycling of transferrin and degradation of epidermal growth factor. Taken together, these results indicate that hRME is primarily involved in membrane trafficking through early endosomes, but not through degradative organelles, such as multivesicular bodies and late endosomes.

Cell adhesion to the extracellular matrix inhibits apoptosis, but the molecular mechanisms underlying the signals transduced by different matrix components are not well understood. Here, we examined integrin-mediated antiapoptotic signals from laminin-10/11 in comparison with those from fibronectin, the best characterized extracellular adhesive ligand. We found that the activation of protein kinase B/Akt in cells adhering to laminin-10/11 can rescue cell apoptosis induced by serum removal. Consistent with this, wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase, or ectopic expression of a dominant-negative mutant of Akt selectively accelerated cell death upon serum removal. In contrast to laminin-10/11, fibronectin rescued cells from serum depletion-induced apoptosis mainly through the extracellular signal-regulated kinase pathway. Cell survival on fibronectin but not laminin was significantly reduced by treatment with PD98059, a specific inhibitor of mitogen-or extracellular signal-regulated kinase kinase-1 (MEK1) and by expression of a dominant-negative mutant of MEK1. Laminin-10/11 was more potent than fibronectin in preventing apoptosis induced by serum depletion. These results, taken together, demonstrate laminin-10/11 potency as a survival factor and demonstrate that different extracellular matrix components can transduce distinct survival signals through preferential activation of subsets of multiple integrin-mediated signaling pathways.Cell adhesion to the ECM 1 generates intracellular signals that modulate cell proliferation, survival, and differentiation (1, 2). Normal epithelial cells deprived of matrix attachment undergo programmed cell death, a form of apoptosis termed anoikis (3). Malignant transformation by oncogenic Ras can prevent this process of apoptosis after denial of ECM attachment (3) or withdrawal of survival factors (4).One approach to study the effects of ECM signals independently of signals from other extracellular sources has been to deprive cells of serum and then to analyze the effects of specific ECM ligands on cellular functions such as adhesion, migration, and survival. Using this approach, many cell biologic functions of ECM signals have been elucidated (2). Most studies of integrin-mediated signaling events have been performed on cells adhering to FN through the ␣ 5 ␤ 1 integrin, which seems to be involved in regulating apoptosis triggered by serum deprivation in many cell types (5, 6). This integrin has also been reported to protect neuronal cells against apoptosis triggered by ␤-amyloid peptide (7). In breast epithelial cells, the ␣ 6 ␤ 1 integrin receptor for laminin-1 has been shown to cooperate with insulin-signaling pathways to protect cells from apoptosis (8). In endothelial cells, functional inhibition of the ␣ v ␤ 3 integrin can lead to programmed cell death (9). Thus, several distinct integrins have been implicated in protection against apoptosis in different cell types. However, the signaling events transduced by the ␣ 3 ␤ 1 integrin, the major recepto...

CD151 (PETA-3 ⁄ SFA-1) is a member of the tetraspanin family of proteins, possessing four membranespanning domains [1]. CD151 was initially identified in platelets [2] and T-cell leukemic cells [3] and has been found to be expressed in a wide variety of cells, including epithelial, endothelial, muscle, Schwann and dendritic cells. Studies using antibodies and small interfering RNA (siRNA) against CD151 have revealed that treatments with these antibodies and siRNA attenuate cell adhesion to and migration on substrates, and disturb epithelial cell-cell adhesion and polarization in cultured cells [4][5][6][7][8][9][10], indicating that CD151 is involved in the regulation of cell-cell and cell-substratum adhesions. Several studies performed in vivo highlight the physiological importance of CD151: in humans, a nonsense mutation in CD151 causes diseases, including end-stage hereditary nephropathy, pretibial epidermolysis bullosa and sensorineural deafness [11]. CD151-null mice show defects in platelet aggregation, keratinocyte migration, T-cell proliferation and pathological angiogenesis, although these mice are viable and fertile [12][13][14][15]. Recently, Sachs et al. [16] reported that CD151 knockout mice show strain-dependent severe renal defects caused by abnormalities of the glomerular basement membrane, loss of podocyte foot processes, glomerulosclerosis and cystic tubular dilation.CD151 interacts with various membrane proteins, including the laminin-binding integrins a3b1, a6b1, a6b4 and a7b1, and other tetraspanin family members, The tetraspanin CD151 forms a stable complex with integrin a3b1, a widely expressed laminin receptor, and is implicated in the regulation of integrin a3b1-mediated cellular responses, including cell attachment, spreading and migration. However, the molecular mechanism by which CD151 regulates integrin a3b1 functions remains unclear. To address this issue, we knocked down CD151 expression in A549 human lung adenocarcinoma cells by RNA interference. When plated on laminin-511 (laminin-10), the CD151-knocked-down cells showed aberrant membrane protrusions and exhibited reductions in the tyrosine phosphorylation of focal adhesion kinase, Src, p130Cas and paxillin. The formation of membrane protrusions was attenuated when the cells were either plated on surfaces coated with higher concentrations of laminin-511 or treated with the integrin b1-activating mAb TS2 ⁄ 16; however, neither treatment could rescue the reduced tyrosine phosphorylation. These results indicate that CD151 knockdown weakens the integrin a3b1-mediated adhesion to laminin-511 and thereby provokes an aberrant morphology, but this reduced adhesive activity is not involved in the decline of signaling events in CD151-knocked-down cells. Thus, our results suggest that CD151 regulates integrin a3b1 functions in two independent aspects: potentiation of integrin a3b1-mediated cell adhesion and promotion of integrin a3b1-stimulated signaling events involving tyrosine phosphorylation.Abbreviations EGFP, enhanced green fluorescent...