W. Scott Argraves

LDL receptor-related protein, a multifunctional ApoE receptor, binds secreted β-amyloid precursor protein and mediates its degradation

The secreted form of beta-amyloid precursor protein (APP) containing the Kunitz proteinase inhibitor (KPI) domain, also called protease nexin II, is internalized and degraded by cells. We show that the low density lipoprotein (LDL) receptor-related protein (LRP) is responsible for the endocytosis of secreted APP. APPs770 degradation is inhibited by an LRP antagonist called the receptor-associated protein (RAP) and by LRP antibodies and is greatly diminished in fibroblasts genetically deficient in LRP. APPs695, which lacks the KPI domain, is a poor LRP ligand. Since LRP also binds apolipoprotein E (apoE)-enriched lipoproteins and inheritance of the epsilon 4 allele of the apoE gene is a risk factor for Alzheimers disease (AD), these data link in a single metabolic pathway two molecules strongly implicated in the pathophysiology of AD.

Fibulin, a novel protein that interacts with the fibronectin receptor β subunit cytoplasmic domain

A 100 kd protein was isolated from tissue and cell extracts by affinity chromatography on a synthetic peptide representing the cytoplasmic domain of the fibronectin receptor beta subunit. The 100 kd protein also bound to native fibronectin receptor, and this binding could be reversed with EDTA. Calcium may be the divalent cation required for the binding since the 100 kd protein was found to bind 45Ca2+. The N-terminal amino acid sequence of the 100 kd protein was not similar to any sequence in a protein data base. Immunofluorescent staining of cells cultured on fibronectin showed the 100 kd protein coinciding with the fibronectin receptor beta subunit in sites of substrate contact. Therefore this protein, which we term fibulin, interacts with the fibronectin receptor in vitro and associates with the receptor in vivo. Fibulin is a potential mediator of interactions between adhesion receptors and the cytoskeleton.

Fibulin is localized at sites of epithelial-mesenchymal transitions in the early avian embryo☆

Fibulin is a 100-kDa calcium-binding, extracellular matrix (ECM), and plasma glycoprotein (Argraves et al., Cell 58, pp. 623-629, 1989; Argraves et al., J. Cell Biol. 111, 3155-3164). Immunoprecipitation analysis showed that antibodies against human fibulin react with an avian isoform (M(r) 100,000). The spatial and temporal distribution of fibulin was examined in the early avian embryo using immunofluorescence microscopy. In stage 15-22 quail embryos fibulin is a constituent of most basement membranes. Areas undergoing epithelial-mesenchymal transitions such as the endocardial cushions, developing myotomes, and neural crest display especially prominent immunostaining. In the early heart fibulin expression was most pronounced in the cardiac jelly at sites where endocardial cushion cells begin the migrations that lead to the formation of valvular and septal primordia. Laser scanning confocal microscopy showed extensive extracellular accumulations of fibulin on the surface of endocardial mesenchyme cells that were motile at the time of fixation (stage 19). These data suggest that enhanced deposition of fibulin at sites of epithelial-mesenchymal transitions may influence cell behavior.

Human fibulin-1D: molecular cloning, expression and similarity with S1-5 protein, a new member of the fibulin gene family.

Fibulin-1 is an extracellular matrix (ECM) component of basement membranes and connective tissue elastic fibers, and a blood protein. Multiple forms of fibulin-1 that differ in their C-terminal regions are produced through the process of alternative splicing of their precursor RNA. Two transcripts of 2.4 and 2.7 kb are the predominant fibulin-1 mRNAs expressed in human tissues and cultured cells. While the 2.4 kb transcript had been shown to encode fibulin-1C, the 2.7 kb transcript did not correspond to any of the previously identified human fibulin-1 variants. Herein, we report on the isolation and sequencing of cDNA corresponding to the 2.7 kb fibulin-1 transcript which encodes a novel, alternatively spliced form of human fibulin-1 that we term the D form. The deduced amino acid sequence of the D form is identical in its first 566 residues to the three known fibulin-1 variants (fibulin-1A-C); however, it has a unique 137 amino acid-C-terminal segment encoded by the alternatively spliced portion of its transcript. RNA hybridization analysis showed that the fibulin-1D transcript is coordinately expressed with that of fibulin-1C both in tissues and in cultured cells. Using antibodies specific to the unique C-terminal segment of fibulin-1D and -1C, both proteins were found to be expressed in human placenta. Recombinant fibulin-1D generated in transfected mammalian cells displayed similar ligand-binding properties as placenta-derived fibulin-1 and recombinant fibulin-1C, and it was capable of incorporating into cultured cell ECM in the absence of other fibulin-1 forms. A comparative sequence analysis revealed that the unique C-terminal region of fibulin-1D is similar to the C-terminal regions of fibulin-1C and fibulin-2. Furthermore, the C-terminal regions of fibulin-1C, -1D and -2 are similar to the C-terminal region of a recently described protein termed S1-5. In addition to this C-terminal similarity, S1-5 also contains repeated EGF-like modules and a conserved N-terminal element, thereby leading to the conclusion that S1-5 is a third member of the fibulin gene family.

The α2-Macroglobulin Receptor/Low Density Lipoprotein Receptor-Related Protein and the Receptor-Associated Protein

a*-Macroglobulin (azM) is a proteinase inhibitor that inhibits all four classes of proteinase. The mechanism of inhibition is unique; aZM is cleaved by the proteinase in a region termed the “bait region” and then undergoes conformational changes that activate internal thiol ester bonds, providing sites for covalent attachment of proteinases and other molecules. These conformational changes also expose receptorbinding domains on the a*M-proteinase complex that mediate its interaction with a specific cell surface receptor that is responsible for removing these complexes from the circulation. The receptor responsible for a*M-proteinase clearance was isolated by ligand affinity chromatography from human placenta tissue’,* and from liver.’ Sequencing studies revealed that the a2M receptor is identical to low density lipoprotein (LDL) receptor-related protein (LRP)!.’ The purified receptor contains a 5 15kDa heavy chain and an 85-kDa light chain that are noncovalently associated. A 39-kDa polypeptide copurifies with the receptor’,* and has been termed the receptorassociated protein (RAP).6 In this report, we will review the structure of LRP, the numerous ligands that are now known to bind to LRP, and the potential function of LRP in their metabolism, as well as review the structure of the receptor-associated protein and its potential function in vivo.

An Overview of the Structure and Function of Glycoprotein 330, a Receptor Related to the α2-Macroglobulin Receptora

Glycoprotein 330 (gp330) is a member of an emerging family of receptors that include the low density lipoprotein receptor (LDLR), the very low density lipoprotein receptor (VLDLR), the az-macroglobulin receptorLDLR-related protein (LRP), an LRP-like protein from C. elegans, and several avian ovarian receptors.z4 The following is a brief overview of our knowledge of the structure and function of gp330. The available evidence indicates that gp330, like LRP, is a multifunctional receptor potentially involved in lipoprotein metabolism, as well as protease regulation.

Role of the Ldl Receptor-Related Protein in Proteinase and Lipoprotein Catabolism

Proteinases play an important role in biological processes and consequently their activity is carefully regulated. This often occurs by reaction of the proteolytic enzyme with specific inhibitors. Removal of the inhibited proteinase is then accomplished by its interaction with cell surface receptors which mediate its internalization and subsequent degradation. The LDL receptor-related protein/α2M receptor (LRP) is large cell surface receptor that mediates the removal of proteinases and proteinase-inhibitor complexes1–5. In addition, this receptor plays an important role in the hepatic clearance of certain apolipoprotein E- and lipoprotein lipase-enriched lipoproteins6–8. Thus, LRP serves a unique role in biology by virtue of its capacity to mediate the cellular uptake of both proteinases and lipoproteins.