Mutation at the Processing Site of Chicken Low Density Lipoprotein Receptor-related Protein Impairs Efficient Endoplasmic Reticulum Exit, but Proteolytic Cleavage Is Not Essential for Its Endocytic Functions

Abstract

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...

Mutant Lrp1 Knock-In Mice Generated by Recombinase-Mediated Cassette Exchange Reveal Differential Importance of the NPXY Motifs in the Intracellular Domain of LRP1 for Normal Fetal Development

“…Nonetheless, it cannot be excluded that the presumed residual processing by a different protease may contribute to the rescue of the phenotype. On the other hand, the results are in agreement with analysis in vitro, which showed no effect of mutation of the furin cleavage site on endocytic functions, although ER exit was somewhat retarded (14). Thus, the furin-dependent processing of LRP1 to form the characteristic heterodimer of the mature receptor (an event not unlike the Notch receptor) is apparently of minor importance for its function.…”

“…Initially synthesized as a single-chain 600-kDa precursor protein, LRP1 is proteolytically cleaved by a furinlike endoprotease into two subunits of 515 kDa and 85 kDa (10,11). The functional significance of this maturation step is not known; however, it does not seem to be essential for its endocytic functions (14). The very large, extracellular ␣-subunit contains the ligand-binding domains and remains attached to the membrane by a noncovalent association with the smaller ␤-subunit, containing an extracellular part, the membrane spanning domain and the cytoplasmic or intracellular domain (LRP1-ICD).…”

Congenital sucrase-isomaltase deficiency arising from cleavage and secretion of a mutant form of the enzyme

“…In most of these cases the protein is blocked intracellularly, often in the ER, and is ultimately degraded. Examples of this phenotype are the ∆F508 mutant of the cystic fibrosis transmembrane conductance regulator (30), several cases of the sodium-glucose transporter in glucose galactose malabsorption (31), and mutants of the LDL receptor in familial hypercholesterinemia (32). Phenotype I of SI in CSID also undergoes a transport block in the ER (4,5).…”

Nuclear Targeting by the Growth Factor Midkine

“…Since midkine is functional even at low pH, as assessed by using a system involving midkine-induced plasminogen activator activity (37), the function of midkine may remain intact in the endosomes. Pseudomonas exotoxin (PE) provides one model for the translocation of an exogenous protein into the cytosol via LRP-mediated internalization (16,36,52). After binding to LRP on the cell surface, PE is translocated as a cargo of endosomes to the endoplasmic reticulum, where furin cleaves it, producing an ADP-ribosylating fragment.…”