Identification of a Major Cyclic AMP-Dependent Protein Kinase A Phosphorylation Site within the Cytoplasmic Tail of the Low-Density Lipoprotein Receptor-Related Protein: Implication for Receptor-Mediated Endocytosis

Abstract

The low-density lipoprotein (LDL) receptor-related protein (LRP) is a multiligand endocytic receptor that belongs to the LDL receptor family. Recently, studies have revealed new roles of LDL receptor family members as transducers of extracellular signals. Our previous studies have demonstrated LRP phosphorylation within its cytoplasmic tail, but the nature of LRP phosphorylation and its potential function was unknown. In the present study using both in vivo and in vitro analysis, we found that LRP phosphorylation is mediated by the cAMP-dependent protein kinase A (PKA). Using site-directed mutagenesis and LRP minireceptor constructs, we further identified the predominant LRP phosphorylation site at serine 76 of its cytoplasmic tail. Finally, we demonstrated that mutations of serine 76, which abolish LRP phosphorylation by PKA, result in a decrease in the initial endocytosis rate of LRP and a lower efficiency in delivery of ligand for degradation. Thus, the role of PKA phosphorylation of LRP in receptor-mediated endocytosis may provide a mechanism by which the endocytic function of LRP can be regulated by external signals.The low-density lipoprotein (LDL) receptor-related protein (LRP) is a member of the LDL receptor (LDLR) gene family, which also include in mammals: LDLR itself, the very-lowdensity lipoprotein receptor (VLDLR), megalin-LRP-2, apolipoprotein E receptor-2 (apoER2)-LR8B, and LR11-sorLA-1 (30,44,54,55). LRP is synthesized as a 600-kDa single-chain precursor, which undergoes posttranslational proteolytic processing within the trans-Golgi compartment by the endopeptidase furin (21,53). This posttranslational processing results in a formation of mature LRP as a noncovalently associated heterodimer, consisting of the extracellular 515-kDa chain and the transmembrane 85-kDa chain (21). The 515-kDa subunit contains all the putative ligand-binding domains including 31 copies of complement-type ligand-binding repeats arranged in four clusters of 2, 8, 10, and 11. In addition, there are 22 copies of the cysteine-rich epidermal growth factor (EGF)-type repeat flanking the ligand-binding clusters (30,44). The multiple domain structure of LRP provides potential binding sites for many structurally and functionally diverse ligands including apolipoprotein E-lipoproteins, ␣ 2 -macroglobulin, plasminogen activators, and ␤-amyloid precursor protein (7,30,44). Ligand interactions with LRP can be antagonized by a 39-kDa receptor-associated protein (RAP), a unique LRP ligand frequently used as a tool in the study of ligand-receptor interaction. RAP also functions intracellularly as a molecular chaperone for LRP and facilitates LRP folding and trafficking within the secretory pathway (7). Increasing evidence has shown that LRP plays important roles in lipoprotein remnant catabolism (52), protease regulation (49), cell migration (50, 51), neuronal process outgrowth (23), and the pathogenesis of Alzheimer's disease (29,48).Despite extensive studies on the extracellular domains of the LDLR members in ligand binding, inform...

The Putative Tumor Suppressor LRP1B, a Novel Member of the Low Density Lipoprotein (LDL) Receptor Family, Exhibits Both Overlapping and Distinct Properties with the LDL Receptor-related Protein

“…An HA epitope was inserted near the amino terminus to facilitate immunological detection of the minireceptor protein in transfected cells, as has been done with the LRP minireceptors. Thus, the structure of this minireceptor is very similar to that of LRP domain IV minireceptor (mLRP4), which undergoes similar trafficking with the full-length LRP (29,32,33). The subcloned DNA sequence was confirmed by DNA sequencing and in vitro translation before it was used for cell transfection.…”

“…As seen in Fig. 4, C and D, the K d of ϳ3.3 nM is similar to that seen for mLRP4 (33). Thus, we next examined the internalization/endocytic rates for 125 I-RAP in mLRP1B4-and mLRP4-expressing cells.…”

Low Density Lipoprotein (LDL) Receptor-related Protein 1B Impairs Urokinase Receptor Regeneration on the Cell Surface and Inhibits Cell Migration

“…The reaction was quenched with 20 mM Tris, pH 7.5, containing 150 mM NaCl for 10 min at 4°C, and cell lysates were prepared in 10 mM CHAPS (Sigma). Immunoprecipitation was carried out with anti-HA antibody, as described before (19), and immunoprecipitated soluble uPAR was analyzed by SDS-PAGE and autoradiography.…”

“…This LRP1B minireceptor is analogous to the LRP minireceptor mLRP4, which we developed previously to study the trafficking and function of the receptor. We have previously shown (13,19,20) that both the rate of endocytosis and the intracellular trafficking of mLRP4 are identical to those of the full-length receptor. mLRP1B4 includes both of the sequences that are not present within LRP (in domain IV and in the cytoplasmic tail) and therefore allows us to identify the functional differences between these two receptors.…”

cAMP‐dependent protein kinase A and the dynamics of epithelial cell surface domains: Moving membranes to keep in shape

“…In some instances, PKA directly phosphorylates the 'cargo' protein. Indeed, the low density lipoprotein-related protein LRP is phosphorylated by PKA at Ser76, and mutations of Ser76 result in a decrease in the initial endocytosis rate of LRP and a lower efficiency in delivery of ligand for degradation (85) . While PKA activity may promote the endocytosis of LRP, the agonist-stimulated endocytosis of glutamate receptors is inhibited by PKA activation, which may reduce the interaction of glutamate receptors with G-coupled receptor kinase 2 and arrestins (87) .…”