Research ArticleSlow endocytosis of the LDL receptor-related protein 1B: Implications for a novel cytoplasmic tail conformation
Introduction
The LDL receptor-related protein 1 (LRP1) is an endocytic receptor of the low-density lipoprotein receptor (LDLR) family. LRP1 is a 600-kDa, type I, single-pass transmembrane protein that is cleaved by the endopeptidase furin in the trans-Golgi network to generate a non-covalently associated heterodimer of 515 and 85 kDa [1], [2]. The 515 kDa subunit consists of four extracellular cysteine-rich ligand binding domains that are interspersed with EGF and YWTD repeats. The 85 kDa subunit contains the transmembrane domain and 100 amino acid-cytoplasmic tail. Through its ligand binding domains, LRP1 binds and facilitates the internalization of numerous structurally and functionally distinct ligands including proteases, protease inhibitors, growth factors, lipoproteins, and viral and bacterial proteins [3]. LRP1 undergoes constitutive and rapid endocytosis with a t1/2 of ∼ 0.5 min, consistent with its predominant distribution in clathrin coated structures and endosomes [4]. Following endocytosis, LRP1 is recycled back to the cell surface while internalized ligands are primarily trafficked to the lysosome for degradation [4]. By modulating the metabolism of its ligands, LRP1 plays roles in coagulation, lipid homeostasis, wound healing, and long-term potentiation of memory, as well as in the pathogenesis of Alzheimer's disease [3]. The cytoplasmic tail of LRP1 contains several potential endocytosis motifs, including two NPXY motifs (X = any amino acid), one YXXØ motif (Ø = a bulky hydrophobic residue), and two dileucine motifs. The dominant endocytosis motif within the cytoplasmic tail of LRP1 is the tyrosine-based YATL sequence located 63–66 amino acids from the transmembrane domain, although the distal dileucine motif also contributes to rapid endocytosis [5]. Both YXXØ and dileucine motifs are known to interact with the AP-2 adaptor complex, which links receptor tails to the structural coat protein clathrin. Nevertheless, it has recently been shown that multiple and redundant factors are required for the endocytosis of LRP1, suggesting that LRP1 endocytosis is somewhat unique [6].
LRP1B, a close homologue of LRP1, was initially discovered as a putative tumor suppressor in non-small cell lung carcinoma, and inactivating mutations have since been identified in multiple types of cancer [7], [8], [9], [10], [11]. LRP1 and LRP1B have very similar domain structures and are 59% identical at the amino acid level. The two features that distinguish LRP1B from LRP1 are the presence of an extra ligand binding repeat in the fourth ligand binding domain and a unique insertion of 33 amino acids within the cytoplasmic tail. Although the biological functions of LRP1B have not yet been defined, LRP1B has been shown to bind several of LRP1's ligands, and therefore may also play roles in a wide variety of cellular processes [12]. Despite the many similarities between LRP1 and LRP1B, the endocytosis rate of an LRP1B minireceptor is much slower (t1/2 ∼ 8 min) than that of a similar LRP1 minireceptor (t1/2 ∼ 0.5 min) [12]. This difference in endocytic rate could have important implications for the biological and tumor suppressor functions of LRP1B, as LRP1 and LRP1B may compete for binding to ligands while exerting opposite effects on their clearance from the cell surface [13]. Since the cytoplasmic tail of LRP1B contains the same potential endocytosis motifs as LRP1, we sought to uncover the mechanism underlying the distinct rates of endocytosis between LRP1 and LRP1B. We hypothesized that sequences within the LRP1B tail inhibit interactions between the endocytosis motifs in the tail and the endocytic machinery. We were specifically interested in the 33 amino acid insertion, which resides between potential endocytosis motifs and may have cytoplasmic binding partners that would sterically prevent endocytic adaptors from binding the adjacent motifs. In this study, we constructed a series of chimeric and mutant minireceptors in order to determine which sequences are critical for the slower endocytic rate of LRP1B.
Section snippets
Materials
Human recombinant RAP was expressed as a glutathione S-transferase fusion protein and isolated as described previously [14]. Mouse anti-HA antibody was from Covance (HA.11). Carrier-free Na125I was purchased from PerkinElmer Life Sciences. Proteins were iodinated using the IODO-GEN method as described previously [5]. Mirus Trans-IT CHO transfection kit was from Mirus. Restriction enzymes were from New England Biolabs. QuikChange mutagenesis kit and Pfu ultra DNA polymerase were from Stratagene.
LRP1B undergoes slow endocytosis and localizes to the cell surface
The cytoplasmic tail of LRP1 contains 5 potential endocytosis motifs: two NPXY, one YXXØ, and two dileucine motifs, and the rapid endocytosis of LRP1 is primarily dependent on the YXXØ and the dileucine motif distal to the plasma membrane [5]. Although all of these motifs are present in the LRP1B tail within the same local context as the LRP1 tail, the endocytosis rate of LRP1B is more than 15-fold slower than that of LRP1 (Fig. 2A) [12]. In order to study the functional differences between
Discussion
The cytoplasmic domains of LRP1 and LRP1B are highly homologous, yet the endocytosis rate of LRP1B is markedly slower. The present study aimed to identify sequences within the LRP1B cytoplasmic tail that are responsible for this difference in endocytosis rate. Our data suggest that one factor influencing this difference could be the utilization of different clathrin adaptors for internalization. Whereas LRP1 uses the YXXØ and distal LL motifs for rapid endocytosis, LRP1B can use either of its
Acknowledgments
We thank Dr. Michelle Schlief and Dr. Stuart Kornfeld for their critical reading of the manuscript. We are also grateful to Dr. Maria Isabel Yuseff and members of the Bu lab for helpful discussion of this work. This work was supported by National Institutes of Health Grant R01 CA100520 and by a grant from the Alzheimer's Association (to G.B.). G.B. is an Established Investigator of the American Heart association. Y.L. is partially supported by a grant from the American Heart Association
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