Localization of Low Density Lipoprotein Receptor-related Protein 1 to Caveolae in 3T3-L1 Adipocytes in Response to Insulin Treatment

Abstract

The insulin-induced translocation of low density lipoprotein receptor-related protein 1 (LRP1) from intracellular membranes to the cell surface in 3T3-L1 adipocytes was differentiation-dependent and did not occur in 3T3-L1 fibroblasts. Prompted by findings that the plasma membrane of 3T3-L1 adipocytes was rich in caveolae, we determined whether LRP1 became caveolae-associated upon insulin stimulation. The caveolae domain was isolated by the well characterized detergent solubilization and sucrose density ultracentrifugation methodology. Under basal conditions, only a trace amount of LRP1 was caveolae-associated despite the markedly elevated caveolin-1 and caveolae after adipocytic cell differentiation. Upon insulin treatment, the amount of LRP1 associated with caveolae was increased by 4-fold within 10 min, which was blocked completely by pretreatment with wortmannin prior to insulin. The caveolar localization of LRP1 in adipocytes was specific to insulin; treatment with platelet-derived growth factor-bb isoform did not promote but rather decreased caveolar localization of LRP1 below basal levels. The insulin-induced caveolar localization of LRP1 was also observed in 3T3-L1 fibroblasts where translocation of LRP1 from intracellular membranes to the cell surface was absent, suggesting that association of LRP1 with caveolae was achieved, at least in part, through lateral transmigration along the plane of plasma membranes. Immunocytochemistry studies revealed partial co-localization of LRP1 (either endogenous LRP1 or an epitopetagged minireceptor) with caveolin-1 in cells treated with insulin, which was confirmed by co-immunoprecipitation of LRP1 with caveolin-1 in cells treated with insulin but not platelet-derived growth factor-bb. These results suggest that the localization of LRP1 to caveolae responds selectively to extracellular signals.The low density lipoprotein receptor-related protein 1 (LRP1) 1 is a large type I membrane protein (4525 amino acids) and belongs to the low density lipoprotein receptor (LDLR) gene family (1, 2). Mammalian LRP1 is abundantly expressed in hepatocytes, neurons, smooth muscle cells, fibroblasts, and adipocytes. LRP1 is cleaved post-translationally by the endopeptidase furin in the distal Golgi, and the resulting extracellular ␣-chain (515 kDa) and transmembrane ␤-chain (85 kDa) form a non-covalent heterodimer (3). The ␣-chain is composed of three major structural modules, namely class A ligand-binding (complement-type) repeats, the epidermal growth factor (EGF) precursor-type repeats, and YWTD ␤-propellers. The ligand-binding repeats are dispersed throughout the LRP1 extracellular domain in four clusters denoted I to IV, of which clusters II and IV possess the ability to bind a variety of structurally unrelated ligands (4, 5). The cytoplasmic tail of the LRP1 ␤-chain contains motifs resembling those required for receptor-mediated endocytosis through clathrin-coated pits (e.g. NPXY and YXXL) that are found in other LDLR family members (6, 7). In addition, the cytoplasmic tail of th...

Pro-cathepsin D interacts with the extracellular domain of the β chain of LRP1 and promotes LRP1-dependent fibroblast outgrowth

“…Our results reveal that pro-cath-D colocalized with LRP1 at the cell surface in lipid rafts. At the cell surface, LRP1 has been shown to be localized in clathrin-coated pits and in lipid rafts (Boucher et al, 2002;Wu and Gonias, 2005;Zhang et al, 2004). Here, we show that LRP1 overexpression directs pro-cath-D to the lipid rafts.…”

“…Interestingly, some of the cell surface LRP1 is located in lipid rafts (Wu and Gonias, 2005;Zhang et al, 2004). Therefore, we next investigated whether pro-cath-D and LRP1 might colocalize at the cell surface in these micro-domains.…”

Dissection of the insulin signaling pathway via quantitative phosphoproteomics

“…The low density lipoprotein receptor-related proteins (LRPs) are another important protein family involved in formation of lipid rafts (45). Besides LRP-1, we also detected LRP-6, which was not known to be involved in insulin signaling.…”

Cathepsin D is partly endocytosed by the LRP1 receptor and inhibits LRP1-regulated intramembrane proteolysis

“…There was also a decrease in pro-cath-D endocytosis in LRP1-silenced fibroblasts ( Figure 1A, panel d) when uptake was measured over 18 h ( Figure 1A, panel c, compare lanes 3 and 4, and panel e for quantification). We then investigated pro-cath-D internalization by LRP1 using MEF that lacked M6P receptors ( Figure 1B Pro-cath-D and ectopic cath-D do not modulate LRP1b-chain tyrosine phosphorylation in fibroblasts The LRP1 at the plasma membrane is located in clathrin-coated pits and lipid rafts (Boucher et al, 2002;Zhang et al, 2004;Wu and Gonias, 2005), and it has been suggested that there are LRP1-induced signal transduction pathways triggered by tyrosine phosphorylation or RIP in lipid rafts (Boucher et al, 2002; von Cathepsin D, endocytosis and LRP1 RIP D Derocq et al Arnim et al, 2005;Wu and Gonias, 2005). We observed that LRP1b overproduction directs pro-cath-D to the lipid rafts (Beaujouin et al, 2010), suggesting that cath-D modulates the tyrosine phosphorylation of LRP1, as shown for the PDGF-BB (Boucher et al, 2002;Loukinova et al, 2002;Boucher and Gotthardt, 2004;Newton et al, 2005) and CTGF (Yang et al, 2004) growth factors, the tPA serine protease (Hu et al, 2006) and in fibroblasts transformed with v-Src (Barnes et al, 2001(Barnes et al, , 2003.…”