SREC-II,a new member of the scavenger receptor type F family,trans-interacts with SREC-I through its extracellular domain

The scavenger receptor expressed by endothelial cells (SREC) with an extremely large cytoplasmic domain, was originally identified in a human endothelial cell line. In this study, we have cloned a second isoform named SREC-II and shown that there is a heterophilic interaction between SREC-I and -II at their extracellular domains. The cDNA for murine SREC-II encodes an 834-amino acid protein with 35% homology to SREC-I. Similar to SREC-I, SREC-II contains multiple epidermal growth factor-like repeats in its extracellular domain. However, in contrast to SREC-I, SREC-II had little activity to internalize modified low density lipoproteins (LDL). A Northern blot analysis revealed a tissue expression pattern of SREC-II similar to that of SREC-I with predominant expression in human heart, lung, ovary, and placenta. Mouse fibroblast L cells with no tendency to associate showed noticeable aggregation when SREC-I was overexpressed in these cells, whereas overexpression of SREC-II caused only slight aggregation. Remarkably, intense aggregation was observed when SREC-I-expressing cells were mixed with those expressing SREC-II. Deletion of almost all of the cytoplasmic receptor domain had no effect on the receptor expression and cell aggregation, indicating that solely the extracellular domain is involved in cell aggregation. The association of SREC-I and -II was effectively suppressed by the presence of scavenger receptor ligands such as acetylated LDL and oxidized LDL. These findings suggest that SREC-I and -II show weak cell-cell interaction by their extracellular domains (termed homophilic trans-interaction) but display strong heterophilic trans-interaction through the extracellular epidermal growth factor-like repeat domains.     

SREC-I, a type F scavenger receptor, is an endocytic receptor for calreticulin

Calreticulin and gp96 (GRP94) traffic associated peptides into the major histocompatibility complex class-I cross-presentation pathway of antigen-presenting cells (APCs). Efficient accession of the cross-presentation pathway requires APC receptor-mediated endocytosis of the chaperone/peptide complexes. Previously, scavenger receptor class-A (SRA) was shown to play a substantial role in trafficking gp96 and calreticulin into macrophages, accounting for half of total receptor-mediated uptake. However, the scavenger receptor ligand fucoidin competed the chaperone uptake beyond that accounted for by SRA, indicating that another scavenger receptor(s) may also contribute. Consistent with this hypothesis, we showed that the residual calreticulin uptake into SRA(-/-) macrophages is competed by the scavenger receptor ligand acetylated low density lipoprotein (LDL). We now report that an additional scavenger receptor, SREC-I (scavenger receptor expressed by endothelial cell-I), mediates the endocytosis of calreticulin and gp96. Ectopic expression of SREC-I in Chinese hamster ovary cells yielded chaperone recognition and uptake, and these processes were competed by the inhibitory ligands fucoidin and acetylated (Ac)LDL. Although AcLDL competes for the chaperone interactions with SRA and SREC, we showed that not all of the scavenger receptors, which bind AcLDL, bind calreticulin or gp96. The overexpression of SREC-I in macrophages increased chaperone endocytosis, indicating that SREC-I functions in APCs and that the cytosolic components necessary for the endocytosis of SREC-I and its cargo are present and not limiting in APCs. These data identify a novel class of ligands for SREC-I and provide insight into the mechanisms by which APCs and potentially endothelial cells traffic chaperone/antigen complexes.     

Molecular cloning and functional characterization of a human scavenger receptor with C-type lectin (SRCL), a novel member of a scavenger receptor family

Using a human placenta cDNA library, we cloned a novel member belonging to the scavenger receptor family. Complementary DNA of this clone encodes a type II transmembranous glycoprotein containing a collagen-like domain, which are typical structural characteristics of scavenger receptor class A. This protein also contains a C-type lectin/carbohydrate recognition domain (C-type CRD) located at the C-terminus. We designated this as Scavenger Receptor with C-type Lectin (SRCL) type I. We also isolated human SRCL type II, which lacks the C-type CRD. Northern blot analysis revealed that hSRCL type I and type II mRNAs are abundantly expressed in adult human tissues. When hSRCL type I and type II were expressed in CHO-K1 cells, they were localized in the plasma membrane forming clusters on the surface. Ligand-binding studies of CHO-K1 cells expressing hSRCL type I and type II demonstrated their specific binding capacity in Escherichia coli and Staphylococcus aureus. These results indicate that hSRCL is a novel bacteria-binding receptor containing a C-type CRD and this receptor may play an important role in host defense.     

Molecular cloning of a mouse scavenger receptor with C-type lectin (SRCL)(1), a novel member of the scavenger receptor family.

The cDNA clone encoding a mouse scavenger receptor with C-type lectin (SRCL), a novel member of the scavenger receptor family, has been isolated from a mouse embryonic cDNA library. The predicted cDNA sequence contains a 2226 bp open reading frame encoding a coiled-coil, collagen-like, C-type lectin/carbohydrate recognition domain with an overall sequence identity of 92% to human SRCL. In contrast to human, mouse SRCL mRNA was expressed ubiquitously in various adult tissues including the liver and spleen, in which human SRCL mRNA was under detection limits. Mouse SRCL mRNA was expressed in the macrophage cell line J774A.1 cells at a high level and in the embryo as early as E9.     

Cadherin-11 interacts with the FGF receptor and induces neurite outgrowth through associated downstream signalling

Cadherin-11 is a cell–cell adhesion molecule whose expression is often correlated with cellular migratory phenomena. We recently demonstrated that cadherin-11 activation by immobilized cad11–Fc (cadherin-11 ectodomain fused to Fc fragment) promotes axonal extension of spinal cord explants. Here, we show that this induced neurite outgrowth is dependent on the FGF receptor (FGFR) activity. Downstream, DAG lipase/CAM kinase and PI3 kinase pathways are required, but not the MAP kinase signalling. We also demonstrate that a tagged form of FGFR1 co-immunoprecipitates with β-catenin containing cadherin-11 immunocomplexes. FGFR1 and β-catenin show colocalization and enhanced association during cadherin-11 engagement, suggesting that FGFR1 interaction with cadherin-11 adhesion complexes is reinforced during cell contact formation. In vitro pull-down experiments using recombinant ectodomains suggest that cadherin-11/FGFR interact directly through their extracellular domains. Altogether, we propose that cadherin-11 recruits the FGFR upon adhesive engagement at nascent contacts, triggering the activation of downstream pathways involved in growth cone progression.     

Molecular cloning of human macrophage capping protein cDNA. A unique member of the gelsolin/villin family expressed primarily in macrophages.

Macrophage capping protein (MCP) is a Ca(2+)-sensitive protein which reversibly blocks the barbed ends of actin filaments but does not sever preformed actin filaments. The human cDNA for MCP has been cloned and sequenced. The derived amino acid sequence predicts a polypeptide of 38.4 kDa. Human MCP expressed in Escherichia coli using a pET12a vector was functionally identical to the native protein purified from rabbit alveolar macrophages with respect to Ca2+ sensitivity and ability to block monomer exchange at the barbed end of actin filaments. Sequence comparison with other actin-binding protein sequences indicates that MCP is a member of the gelsolin/villin family of barbed end blocking proteins. Unlike gelsolin, this protein has a limited tissue distribution being detected primarily in macrophages where it was abundant, representing 0.9-1% of the total cytoplasmic protein. Northern blot analysis of U937 and HL60 cells differentiated to macrophage-like cells demonstrated that MCP message increases to 2.6 and greater than 7 times initial levels, respectively. Human MCP displays a 93% amino acid sequence identity with two recently described mouse proteins, gCap39 and Mbh1. Its abundance in macrophages and the corresponding increases in mRNA levels upon promyelocyte and monocyte development into macrophages indicate that MCP may play an important role in macrophage function.     

Cd36, a member of the class b scavenger receptor family, as a receptor for advanced glycation end products

Interaction of advanced glycation end products (AGE) with AGE receptors induces several cellular phenomena potentially relating to diabetic complications. Five AGE receptors identified so far are RAGE (receptor for AGE), galectin-3, 80K-H, OST-48, and SRA (macrophage scavenger receptor class A types I and II). Since SRA is known to belong to the class A scavenger receptor family, and the scavenger receptor collectively represents a family of multiligand lipoprotein receptors, it is possible that CD36, although belonging to the class B scavenger receptor family, can recognize AGE proteins as ligands. This was tested at the cellular level in this study using Chinese hamster ovary (CHO) cells overexpressing human CD36 (CD36-CHO cells). Cellular expression of CD36 was confirmed by immunoblotting and immunofluorescent microscopy using anti-CD36 antibody. Upon incubation at 37 degrees C, (125)I-AGE-bovine serum albumin (AGE-BSA) and (125)I-oxidized low density lipoprotein (LDL), an authentic ligand for CD36, were endocytosed in a dose-dependent fashion and underwent lysosomal degradation by CD36-CHO cells, but not wild-type CHO cells. In binding experiments at 4 degrees C, (125)I-AGE-BSA exhibited specific and saturable binding to CD36-CHO cells (K(d) = 5.6 microg/ml). The endocytic uptake of (125)I-AGE-BSA by these cells was inhibited by 50% by oxidized LDL and by 60% by FA6-152, an anti-CD36 antibody inhibiting cellular binding of oxidized LDL. Our results indicate that CD36 expressed by these cells mediates the endocytic uptake and subsequent intracellular degradation of AGE proteins. Since CD36 is one of the major oxidized LDL receptors and is up-regulated in macrophage- and smooth muscle cell-derived foam cells in human atherosclerotic lesions, these results suggest that, like oxidized LDL, AGE proteins generated in situ are recognized by CD36, which might contribute to the pathogenesis of diabetic macrovascular complications.     

Shedding of CD163, a novel regulatory mechanism for a member of the scavenger receptor cysteine-rich family

The glucocorticoid-inducible transmembrane protein CD163 is a member of the scavenger receptor cysteine-rich (SRCR) family which is expressed exclusively on human monocytes and macrophages. The expression of the protein is significantly downregulated in response to phorbol 12-myristate 13-acetate (PMA) by a yet unknown mechanism. We now demonstrate that PMA induces shedding of a soluble form of CD163 rather than internalization, revealing a novel regulatory mechanism for a member of the SRCR family. Bisindolylmaleimide I was shown to inhibit phorbol ester-induced shedding, thus implying an involvement of protein kinase C (PKC). Furthermore, cleavage could be prevented by protease inhibitors. Therefore, we suggest that PMA-induced activation of PKC leads to protease-mediated shedding of CD163. These results indicate a specific release mechanism of soluble CD163 by human monocytes which could play an important role in modulating inflammatory processes.     

Calsarcin-3, a novel skeletal muscle-specific member of the calsarcin family, interacts with multiple Z-disc proteins

The Z-disc is a highly specialized multiprotein complex of striated muscles that serves as the interface of the sarcomere and the cytoskeleton. In addition to its role in muscle contraction, its juxtaposition to the plasma membrane suggests additional functions of the Z-disc in sensing and transmitting external and internal signals. Recently, we described two novel striated muscle-specific proteins, calsarcin-1 and calsarcin-2, that bind alpha-actinin on the Z-disc and serve as intracellular binding proteins for calcineurin, a calcium/calmodulin-dependent phosphatase shown to be integral in cardiac hypertrophy as well as skeletal muscle differentiation and fiber-type specification. Here, we describe an additional member of the calsarcin family, calsarcin-3, which is expressed specifically in skeletal muscle and is enriched in fast-twitch muscle fibers. Like calsarcin-1 and calsarcin-2, calsarcin-3 interacts with calcineurin, and the Z-disc proteins alpha-actinin, gamma-filamin, and telethonin. In addition, we show that calsarcins interact with the PDZ-LIM domain protein ZASP/Cypher/Oracle, which also localizes to the Z-disc. Calsarcins represent a novel family of sarcomeric proteins that serve as focal points for the interactions of an array of proteins involved in Z-disc structure and signal transduction in striated muscle.     

The microtubule-binding protein Hook3 interacts with a cytoplasmic domain of scavenger receptor A

The class A scavenger receptor (SR-A) is a multifunctional transmembrane glycoprotein that is implicated in atherogenesis, innate immunity, and cell adhesion. Despite extensive structure-function studies of the receptor, intracellular molecules that directly interact with SR-A and regulate the receptor trafficking have not been determined. In the current study, we have identified a microtubule-binding protein, Hook3, as a novel interacting partner of SR-A. The association between a rat Hook3 isoform and SR-A was suggested by yeast two-hybrid screening and mass spectrometry analysis of SR-A-cytoplasmic domain-bound proteins in rat alveolar macrophages. The binding of overexpressed and endogenous human Hook3 to SR-A was demonstrated by pull-down assay and co-immunoprecipitations. Furthermore, endogenous murine SR-A and HK3 co-sedimented from cell lysates isolated from Raw264.7 murine macrophage cells. The interaction of Hook3 with SR-A was significantly stimulated after SR-A had recognized the extracellular ligand. Studies using truncations demonstrated that the positively charged C-terminal Val614-Ala717 region of human Hook3 was required for the interaction with the negatively charged residues, Glu12, Asp13, and Asp15 in the human SR-A cytoplasmic domain. By transfecting small interfering RNA targeting Hook3, total and surface expression, receptor-mediated ligand uptake and protein stability of SR-A were significantly promoted, whereas the protein synthesis and maturation were not altered. We propose for the first time that Hook3 may participate in the turnover of the endocytosed scavenger receptor.     

Isolation, characterization, and cDNA cloning of chicken turpentine-induced protein, a new member of the scavenger receptor cysteine-rich (SRCR) family of proteins

Acute-phase serum proteins were induced by administrating a chicken with turpentine oil. One of these proteins was a new protein that appeared in front of albumin in polyacrylamide disc gel electrophoresis using a 4.5-16% gel. To purify this protein, turpentine-administrated chicken serum was fractionated by ammonium sulfate precipitation at 50% saturation, and the supernatant fraction was chromatographed on a DEAE-Toyopearl 650S column. The purified protein is a mannose-glycoprotein, and its N-terminal sequence, determined by the Edoman method, is not homologous from that of other reported acute-phase proteins. An analysis of physiological function with two different test systems, chemiluminescence measurement and electron spin resonance spectroscopy, showed that the purified protein has antioxidant activity and inhibits superoxide (O(2)) mediated by activation of the receptor. In support of these results, the complete amino acid sequence of 18-B is homologous to the scavenger receptor cysteine-rich (SRCR) family of proteins that participate in the regulation of leukocyte function. 18-B is composed of four SRCR domains, which is different from the previously characterized SRCR family of proteins such as Spalpha, CD6, and CD163. These findings indicate that turpentine-induced 18-B, a new member of scavenger receptor cysteine-rich family, may be implicated in regulation of cell function in a manner of inhibition of the overproduction of the reactive oxygen species.     

Solution structures of the C-terminal headpiece subdomains of human villin and advillin, evaluation of headpiece F-actin-binding requirements

Headpiece (HP) is a 76-residue F-actin-binding module at the C terminus of many cytoskeletal proteins. Its 35-residue C-terminal subdomain is one of the smallest known motifs capable of autonomously adopting a stable, folded structure in the absence of any disulfide bridges, metal ligands, or unnatural amino acids. We report the three-dimensional solution structures of the C-terminal headpiece subdomains of human villin (HVcHP) and human advillin (HAcHP), determined by two-dimensional 1H-NMR. They represent the second and third structures of such C-terminal headpiece subdomains to be elucidated so far. A comparison with the structure of the chicken villin C-terminal subdomain reveals a high structural conservation. Both C-terminal subdomains bind specifically to F-actin. Mutagenesis is used to demonstrate the involvement of Trp 64 in the F-actin-binding surface. The latter residue is part of a conserved structural feature, in which the surface-exposed indole ring is stacked on the proline and lysine side chain embedded in a PXWK sequence motif. On the basis of the structural and mutational data concerning Trp 64 reported here, the results of a cysteine-scanning mutagenesis study of full headpiece, and a phage display mutational study of the 69-74 fragment, we propose a modification of the model, elaborated by Vardar and coworkers, for the binding of headpiece to F-actin.     

Rdj2, a J protein family member, interacts with cellular prion PrP(C)

PrP(C) is a glycosylphosphatidylinositol (GPI) anchored glycoprotein of unknown function. Misfolding of normal cellular PrP(C) to the pathogenic PrP(Sc) is the hallmark of prion diseases (transmissible spongiform encephalopathies). Prion diseases are characterized by extensive neurodegeneration and early death. Understanding how PrP(C) maintains its correct conformation is a major endeavor of current inquiry. Here we demonstrate a novel interaction between PrP(C) and the J protein family member, Rdj2 (DjA2; Dj3, Dnj3, Cpr3, and Hirip4). The importance of the J protein family in the cellular folding machinery has been recognized for many years. The PrP(C)/Rdj2 association was direct and concentration-dependent. Other J proteins such as CSPalpha and auxilin did not associate with PrP(C) in the absence of ATP, demonstrating the specificity of the PrP(C)/J protein interaction. These findings suggest that the J protein family serves as a 'folding catalyst' for PrP(C) and implicates Rdj2 as a factor in the protection against prion diseases.     

Cloning and expression of the rat prolactin receptor, a member of the growth hormone/prolactin receptor gene family

The primary structure of the rat liver prolactin receptor has been deduced from a single complementary DNA clone. The sequence begins with a putative 19 amino acid signal peptide followed by the 291 amino acid receptor that includes a single 24 amino acid transmembrane segment. In spite of the fact that the prolactin receptor has a much shorter cytoplasmic region than the growth hormone receptor, there is strong localized sequence identity between these two receptors in both the extracellular and cytoplasmic domains, suggesting that the two receptors originated from a common ancestor.     

Human nectin3/PRR3: a novel member of the PVR/PRR/nectin family that interacts with afadin

We have isolated nectin3/PRR3, the fourth human member of the nectin/PRR family, also described as the alpha herpes virus receptor family. Nectin/PRR members are adhesion molecules expressed at intercellular junctions. Nectin3/PRR3 is a transmembrane protein, whose extracellular region contains three Ig-like domains (V, C and C) and shares approximately 30% identity with the other members. It is mainly expressed in testis and placental tissues. SDS-PAGE analyses demonstrate that nectin3/PRR3 has a molecular weight of 83kDa. Nectin1/PRR1L and nectin2/PRR2S and L were found to be specifically expressed at the intercellular junctions. This localization is in part due to the interaction of the C-terminal part of these receptors (ended by the consensus sequence A/EXYV) and the PDZ domain of afadin. In this report we demonstrate that the nectin3/PRR3 receptor carries the A/EXYV consensus sequence and interacts in vivo with both long and short isoforms of afadin. These results suggest that the human nectin3/PRR3 is a new afadin-associated molecule.