The regulation of low density lipoprotein receptor-related protein (LRP) activity by insulin was studied using 3T3-L1 adipocytes. The LRP mRNA and protein expression were independent of differentiation state of the cells and of insulin treatment. In differentiated cells, insulin treatment acutely stimulated the cell surface presentation of LRP (approximately 2-fold) as evidenced by methylamine-activated alpha(2)-macroglobulin binding and by biotinylation of cell surface LRP. The increased cell surface presentation was accompanied by a 39% decrease in LRP level in the low density microsomes. The magnitude of insulin-stimulated cell surface presentation of LRP was similar to that of transferrin receptor but was much less than that of GLUT4. Both the increases in LRP and GLUT4 cell surface presentation upon insulin treatment were abolished by inhibition of phosphatidylinositide 3-kinase. The increased cell surface presentation of LRP was associated with proportionally increased endocytic activity, and the internalization rate constant (K(e)) was not decreased by insulin treatment. Thus, insulin treatment most likely stimulates recycling of LRP from an endosomal pool to the plasma membrane, which is regulated in a phosphatidylinositide 3-kinase-dependent manner in 3T3-L1 adipocytes.

The small GTP-binding protein Rab4 has been involved in the recycling of ␣v␤3 integrins in response to platelet-derived growth factor (PDGF) stimulation suggesting a role for Rab4 in cell adhesion and migration. In this study, we explored the role of Rabip4 and Rabip4, two Rab4 effector proteins, in migration of NIH 3T3 fibroblasts. In these cells, Rabip4 and Rabip4, collectively named Rabip4s, were partially co-localized with the early endosomal marker EEA1. PDGF treatment re-distributed endogenous Rabip4s toward the cell periphery where they colocalized with F-actin. In cells expressing green fluorescent protein (GFP)-Rabip4 or GFP-Rabip4, constitutive appearance of GFP-Rabip4s at the cell periphery was accompanied by local increase in cortical F-actin in membrane ruffles at the leading edge. The expression of GFP-Rabip4 induced an increased migration compared with control cells expressing GFP alone, even in the absence of PDGF stimulation. On the contrary, in cells expressing a mutated form of Rabip4s unable to interact with Rab4, lack of typical leading edge was observed. Furthermore, PDGF treatment did not stimulate the migration of these cells. Furthermore, down-regulation of the expression of Rabip4s inhibited PDGF-stimulated cell migration. Endogenous Rabip4s were localized with ␣v integrins at the leading edge following PDGF treatment, whereas in cells expressing GFP-Rabip4s, ␣v integrins, together with GFP-Rabip4s, were constitutively localized at the leading edge. In contrast, reduction in Rabip4s expression levels using small interfering RNA was associated with impaired PDGF-induced translocation of ␣v integrins toward the leading edge. Taken together, our data provide evidence that Rabip4s, possibly via their interaction with Rab4, regulate integrin trafficking and are involved in the migration of NIH 3T3 fibroblasts.Cell migration is a fundamental process that is required during embryonic morphogenesis, tissue repair, and regeneration, as well as, in progression of certain diseases including cancer, mental retardation, atherosclerosis, and arthritis (1, 2). Cell migration involves the formation of extended protrusions in the direction of migration, a process that is driven by actin polymerization (3), and subsequent stabilization of the leading edge protrusions via integrins adhering to the extracellular matrix (2, 4). Integrins are heterodimeric receptor molecules that participate in an endo-exocytic cycle in which they are continually internalized from the plasma membrane, delivered to the endosomes, and recycled to the plasma membrane for reutilization (5-7). Recent studies have shown that, in fibroblasts, platelet-derived growth factor (PDGF) 4 -stimulated recycling of ␣v␤3 integrins from the early endosomes is dependent on Rab4a, suggesting a role for Rab4a in the mechanisms regulating integrin trafficking during cell adhesion (7,8).Rab4a is a small GTPase implicated in endocytosis in multiple cell types (9, 10). We identified Rabip4, a ubiquitous 69-kDa protein, as an effector of Rab4a on the...

We determined the role of N-linked glycosylation of apolipoprotein B (apoB) in the assembly and secretion of lipoproteins using transfected rat hepatoma McA-RH7777 cells expressing human apoB-17, apoB-37, and apoB-50, three apoB variants with different ability to recruit neutral lipids. Substituting Asn residue with Gln at the single glycosylation site within apoB-17 (N 158 ) decreased its secretion efficiency to a level equivalent to that of wild-type apoB-17 treated with tunicamycin, but had little effect on its synthesis or intracellular distribution. When selective N-to-Q substitution was introduced at one or more of the five N-linked glycosylation sites within apoB-37 (N 158 , N 956 , N 1341 , N 1350 , and N 1496 ), secretion efficiency of apoB-37 from transiently transfected cells was variably affected. When all five N-linked glycosylation sites were mutated within apoB-37, the secretion efficiency and association with lipoproteins were decreased by Ͼ 50% as compared with wild-type apoB-37. Similarly, mutant apoB-50 with all of its N-linked glycosylation sites mutagenized showed decreased secretion efficiency and decreased lipoprotein association in both d Ͻ 1.02 and d Ͼ 1.02 g/ml fractions. The inability of mutant apoB-37 and apoB-50 to associate with very low-density lipoproteins was attributable to impaired assembly and was not due to the limitation of lipid availability. The decreased secretion of mutant apoB-17 and apoB-37 was not accompanied by accumulation within the cells, suggesting that the proportion of mutant apoB not secreted was rapidly degraded. However unlike apoB-17 or apoB-37, accumulation of mutant apoB-50 was observed within the endoplasmic reticulum and Golgi compartments. These data imply that the N-glycans at the amino terminus of apoB play an important role in the assembly and secretion of lipoproteins containing the carboxyl terminally truncated apoB. The asparagine (N)-linked oligosaccharides of proteins are an important component of the quality control mechanisms of eucaryotic cells. Multiple roles have been assigned to N-linked oligosaccharides, including folding of nascent polypeptides, protection from proteolytic degradation, intracellular trafficking, secretion, cell surface expression, maintenance of protein conformation, and enzymatic activity (1-3). Human apolipoprotein (apo) B-100, a major structural protein of VLDL synthesized in the liver, is a 4,536 amino acid glycoprotein. There are 20 potential N-linked glycosylation sites within apoB-100, of which 16 Asn residues are conjugated with oligosaccharides on plasma LDL. Each mole of apoB-100 contains 5-6 mol of high-mannose type, and 8-10 mol of complex type oligosaccharides (4). As a member of the vitellogenin family of lipid transport and storage proteins (5-7), apoB-100 possesses numerous amphipathic ␣ -helices and ␤ -strands that constitute the major structural framework for the assembly and integrity of triglyceride-rich lipoproteins (8, 9). The precise amino acid sequences within apoB-100 that are involved ...

The low density lipoprotein receptor-related protein (LRP) is synthesized as a proreceptor that undergoes post-translational proteolytic processing, yielding a noncovalently associated ␣␤ dimer as the mature LRP. We tested the role of processing by creating a mutant in which the P1 residue (Arg 3942 ) of the consensus site for furin cleavage (Arg-Asn-Arg-Arg 3942 2) was replaced with Ser in chicken LRP. Transfection of the mutant LRP (designated LRP-RS) into a Chinese hamster ovary cell line lacking endogenous LRP resulted in expression of the unprocessed full-length proreceptor. Comparison of cell lines stably expressing either the wild-type LRP (LRP-wt) or the unprocessed LRP-RS showed that at comparable expression levels, both receptors restored the sensitivity of cellular protein synthesis to Pseudomonas exotoxin A (IC 50 ‫؍‬ 25 ng/ml). Subcellular fractionation and neuraminidase treatment showed that both LRP forms were transported to the plasma membrane. In addition, LRP-RS exhibited kinetics of binding, endocytosis, and degradation of methylamine-activated ␣ 2 -macroglobulin that were identical to those of LRP-wt. The internalization rate constant was similar for LRP-wt (K e ‫؍‬ 0.259 min ؊1 ) and mutant LRP-RS (K e ‫؍‬ 0.252 min ؊1 ), suggesting that it takes about 4 min for the entire surface LRP pool to be internalized. Sorting of LRP from the endosomal compartment to lysosomes or recycling to the plasma membrane were also unaltered in mutant LRP-RS. Pulse-chase analysis showed that the lack of processing of LRP had no effect on the stability of its post-endoplasmic reticulum form or on the rate of its intracellular transit from the endoplasmic reticulum to the Golgi apparatus. However, the exit of mutant LRP from the endoplasmic reticulum was retarded by the Arg 3942 -to-Ser substitution, as evidenced by prolonged retention within the endoplasmic reticulum (t1 ⁄2 ‫؍‬ 4 h for LRP-wt and t1 ⁄2 > 13 h for LRP-RS).The low density lipoprotein (LDL) 1 receptor-related protein (LRP), a member of the LDL receptor gene family, adopts the topology of a type I integral membrane protein (1). A major structural feature of this family of receptor proteins is the arrangement of clusters of ligand-binding repeats (designated class A repeats) interspersed between clusters of epidermal growth factor precursor repeats within the extracellular domains (2). Variations in the makeup of the modular extracellular domains appear to dictate the ligand binding characteristics of the various family members, which in the case of LRP shows multifunctionality for a wide range of proteins, including ␣ 2 -macroglobulin (␣ 2 M) and Pseudomonas exotoxin A (PEA). However, all of the members of this receptor family share a capacity to bind a 39-kDa receptor-associated protein, or RAP (3). Furthermore, the cytoplasmic domain of LRP contains two copies of the NPXY motif that is important for the clathrincoated pit-mediated internalization of the LDL receptor (4).LRP is translated as a single polypeptide (600 kDa) consisting of 4525 amino a...