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.     

The LOX-1 scavenger receptor cytoplasmic domain contains a transplantable endocytic motif

Oxidized low-density lipoprotein particles is a pro-atherogenic factor implicated in atherosclerotic plaque formation. The LOX-1 scavenger receptor binds OxLDL and is linked to atherosclerotic plaque initiation and progression. We tested the hypothesis that the LOX-1 cytoplasmic domain contains a transplantable signal for membrane protein endocytosis. Structural modeling of the LOX-1 cytoplasmic domain reveals that a tripeptide motif (DDL) implicated in LOX-1 endocytosis is part of a curved β-pleated sheet structure. The two aspartic acid residues within this structural model are highly solvent-accessible enabling recognition by cytosolic factor(s). A triple alanine substitution of the DDL motif within the LOX-1 scavenger receptor substantially reduced endocytosis of OxLDL. Transplantation of the LOX-1 cytoplasmic domain into a transferrin receptor reporter molecule conferred efficient endocytosis on this hybrid protein. Mutation of the DDL motif within the hybrid LOX-1-TfR protein also substantially reduced receptor-mediated endocytosis. Thus a transplantable endocytic motif within the LOX-1 cytoplasmic domain is needed to ensure efficient internalization of pro-atherogenic OxLDL particles.     

The role of scavenger receptor class A in the adhesion of cells is dependent on cell type and cellular activation state.

Scavenger receptor class A (SR-A) facilitates the development of atherosclerosis, which might be due to its role in the uptake of modified low-density lipoproteins. However, the receptor is also suggested to be important for cell adhesion, thereby potentially influencing the residence time of cells in vivo. Using SR-A-deficient mice, we investigated the role of SR-A in the adhesion of peritoneal macrophages (PM) and tissue macrophages (Kupffer cells). In resident PM no effect of the absence or presence of SR-A on cell adhesion was observed, either in the presence or in the absence of serum. However, in thioglycollate-induced PM, SR-A is important for adhesion both in the presence and in the absence of serum and more than 85% of the divalent-cation-independent adhesion in the presence of serum is mediated by SR-A. In unactivated Kupffer cells, like in resident PM, adhesion is not influenced by the absence or presence of SR-A. In vivo administration of phorbol 12-myristate 13-acetate leads to the activation of Kupffer cells, and it appears that under these conditions SR-A does contribute to adhesion, since both in the absence and in the presence of serum SR-A is responsible for about 35% of cell adhesion. It is concluded that SR-A is important for the divalent-cation-independent adhesion of activated PM and Kupffer cells, suggesting that SR-A may influence the residence time of cells at sites of cellular activation, e.g., in atherosclerotic plaques and during liver infection.     

The agonist-binding domain of the calcium-sensing receptor is located at the amino-terminal domain.

The calcium-sensing receptor (CaR) is a G-protein-coupled receptor that displays 19-25% sequence identity to the gamma-aminobutyric acid type B (GABAB) and metabotropic glutamate (mGlu) receptors. All three groups of receptors have a large amino-terminal domain (ATD), which for the mGlu receptors has been shown to bind the endogenous agonist. To investigate whether the agonist-binding domain of the CaR also is located in the ATD, we constructed a chimeric receptor named Ca/1a consisting of the ATD of CaR and the seven transmembrane region and C terminus of mGlu1a. The Ca/1a receptor stimulated inositol phosphate production when exposed to the cationic agonists Ca2+, Mg2+, and Ba2+ in transiently transfected tsA cells (a transformed HEK 293 cell line). The pharmacological profile of Ca/1a (EC50 values of 3.3, 2.6, and 3.9 mM for these cations, respectively) was very similar to that of the wild-type CaR (EC50 values of 3.2, 4.7, and 4.1 mM, respectively). For the mGlu1a receptor, it has been shown that Ser-165 and Thr-188, which are located in the ATD, are involved in the agonist binding. An alignment of CaR with the mGlu receptors showed that these two amino acid residues have been conserved in CaR as Ser-147 and Ser-170, respectively. Each of these residues was mutated to alanines and tested pharmacologically using the endogenous agonist Ca2+. CaR-S147A showed an impaired function as compared with wild-type CaR both with respect to potency of Ca2+ (4-fold increase in EC50) and maximal response (79% of wild-type response). CaR-S170A showed no significant response to Ca2+ even at 50 mM concentration. In contrast, each of the two adjacent mutations, S169A and S171A, resulted in pharmacological profiles almost identical to that of the wild-type receptor. These data demonstrate that Ser-170 and to some extent Ser-147 are involved in the Ca2+ activation of the CaR, and taken together, our results reveal a close resemblance of the activation mechanism between the CaR and the mGlu receptors.     

Secreted forms of the amyloid-beta precursor protein are ligands for the class A scavenger receptor

Upon activation, platelets secrete a 120-kDa protein that competes for the binding and internalization of acetyl low density lipoproteins (AcLDL) by macrophages. From the amino-terminal amino acid sequence, amino acid composition, and immunoblot analysis, we identified the active factor in platelet secretion products as sAPP, an alpha-secretase cleavage product of the beta-amyloid precursor protein (APP), that contains a Kunitz-type protease inhibitor (KPI) domain. We showed that both sAPP751 (also called Nexin II) and sAPP695, which does not contain a KPI domain, are ligands for the class A scavenger receptor (SR-A). Chinese hamster ovary cells stably transfected to express the SR-A bound and internalized 4-fold more human platelet-derived sAPP than control cells. The binding and internalization of sAPP were inhibited by the SR-A antagonist fucoidin. In addition, sAPP competed as effectively as fucoidin for SR-A-mediated cell association and degradation of (125)I-AcLDL. To determine if the KPI domain is required for the binding of sAPP to the SR-A, APP751 and APP695 were expressed in Chinese hamster ovary cells, and sAPP751 and sAPP695 purified from the medium were tested for their binding to the SR-A. sAPP751 and sAPP695 were equally effective in competing for the cell association of (125)I-AcLDL by SR-A-expressing cells, demonstrating that the KPI domain is not essential for binding. We also found that sAPP751 is present in extracts of atherosclerotic lesions and that sAPP competes for the SR-A-mediated cell association of oxidized low density lipoprotein. Deletion mutagenesis indicated that a negatively charged region of APP (residues 191-264) contributes to binding to the SR-A. These results suggest that the SR-A contributes to the clearance of sAPP and that sAPP competes for the cell association of other SR-A ligands.     

Human transferrin receptor internalization is partially dependent upon an aromatic amino acid on the cytoplasmic domain.

The objective of this work is to identify the elements of the human transferrin receptor that are involved in receptor internalization, intracellular sorting, and recycling. We have found that an aromatic side chain at position 20 on the cytoplasmic portion of the human transferrin receptor is required for efficient internalization. The wild-type human transferrin receptor has a tyrosine at this position. Replacement of the Tyr-20 with an aromatic amino acid does not alter the rate constant of internalization, whereas substitution with the nonaromatic amino acids serine, leucine, or cysteine reduces the internalization rate constant approximately three-fold. These results are consistent with similar studies of other receptor systems that have also documented the requirement for a tyrosine in rapid internalization. The amino terminus of the transferrin receptor is cytoplasmic, with the tyrosine 41 amino acids from the membrane. These two features distinguish the transferrin receptor from the other membrane proteins for which the role of tyrosine in internalization has been examined, because these proteins have the opposite polarity with respect to the membrane and because the tyrosines are located closer to the membrane (within 25 amino acids). The externalization rate for the recycling of the transferrin receptor is not altered by any of these substitutions, demonstrating that the aromatic amino acid internalization signal is not required for the efficient exocytosis of internalized receptor.     

The physiological scavenger receptor function of hepatic sinusoidal endothelial and Kupffer cells is independent of scavenger receptor class A type I and II

N-cadherin, a cell adhesion molecule normally found in neural cell tissue, has been found recently to be expressed on the surface of malignant T-cells. The function of N-cadherin on these cells remains unclear. Heterotypic assays between Molt-3 T lymphoblastic leukemia cells and Caco-2 epithelial monolayers were examined under different conditions to assess the functional role of N-cadherin. The results indicate that adherence of Molt-3 cells to Caco-2 monolayers was reduced significantly following pretreatment of Molt-3 cells with 100 M of an N-cadherin-derived antagonist decapeptide. In contrast, pretreatment of Molt-3 cells with an anti-N-cadherin antibody raised against the first 20 amino acids of N-cadherin sequence led to a surprisingly marked enhancement of Molt-3 cell adherence to Caco-2 monolayers. In addition, the presence of anti-N-cadherin antibody neutralized the inhibitory effect of anti-ICAM-1 on Molt-3 adhesion to Caco-2 monolayers. This novel finding demonstrates that external stimulus through the N-cadherin amino terminus can modulate adhesion of malignant T-cells to epithelia and may promote their ability to invade or metastasize to inflammatory sites.     

Uptake and fate of class B scavenger receptor ligands in HepG2 cells.

Class B scavenger receptors (SR-Bs) interact with native, acetylated and oxidized low-density lipoprotein (LDL, AcLDL and OxLDL), high-density lipoprotein (HDL3) and maleylated BSA (M-BSA). The aim of this study was to analyze the catabolism of CD36- and LIMPII-analogous-1 (CLA-1), the human orthologue for the scavenger receptor class B type I (SR-BI), and CD36 ligands in HepG2 (human hepatoma) cells. Saturation binding experiments revealed moderate-affinity binding sites for all the SR-B ligands tested with dissociation constants ranging from 20 to 30 microg.mL-1. Competition binding studies at 4 degrees C showed that HDL and modified and native LDL share common binding site(s), as OxLDL competed for the binding of 125I-LDL and 125I-HDL3 and vice versa, and that only M-BSA and LDL may have distinct binding sites. Degradation/association ratios for SR-B ligands show that LDL is very efficiently degraded, while M-BSA and HDL3 are poorly degraded. The modified LDL degradation/association ratio is equivalent to 60% of the LDL degradation ratio, but is three times higher than that of HDL3. All lipoproteins were good cholesteryl ester (CE) donors to HepG2 cells, as a 3.6-4.7-fold CE-selective uptake ([3H]CE association/125I-protein association) was measured. M-BSA efficiently competed for the CE-selective uptake of LDL-, OxLDL-, AcLDL- and HDL3-CE. All other lipoproteins tested were also good competitors with some minor variations. Hydrolysis of [3H]CE-lipoproteins in the presence of chloroquine demonstrated that modified and native LDL-CE were mainly hydrolyzed in lysosomes, whereas HDL3-CE was hydrolyzed in both lysosomal and extralysosomal compartments. Inhibition of the selective uptake of CE from HDL and native modified LDL by SR-B ligands clearly suggests that CLA-1 and/or CD36 are involved at least partially in this process in HepG2 cells.     

The cytoplasmic domain of the T cell receptor zeta chain is sufficient to couple to receptor-associated signal transduction pathways.

The function of the T cell antigen receptor (TCR) invariant chains, CD3 gamma, delta, epsilon, and zeta, is poorly understood. Evidence suggests that CD3 couples receptor ligand binding to intracellular signaling events. To examine the role of the CD3 zeta chain in TCR-mediated signal transduction, a chimeric protein linking the extracellular and transmembrane domains of CD8 to the cytoplasmic domain of the zeta chain was constructed. The CD8/zeta chimera is expressed independently of the TCR and is capable of transducing signals that, by criteria of early and late activation, are indistinguishable from those generated by the intact TCR. These data indicate that CD8/zeta can activate the appropriate signal transduction pathways in the absence of CD3 gamma, delta, and epsilon, and suggest that the role of CD3 zeta is to couple the TCR to intracellular signal transduction mechanisms.     

Oxidative stress impairs endocytosis of the scavenger receptor class A

We report the characterization of a cell system employing Chinese hamster ovary (CHO) cells and CHO cells transfected with the scavenger receptor class A (CHO-SRA) using extracellularly produced reactive oxygen species (ROS) in order to study the endocytic function of the scavenger receptor. The oxidative environment was produced using tert-butyl hydroperoxide (TBH) and characterized by flow cytometry and cell viability. Once an adequate oxidative environment was established, binding and internalization studies of radiolabeled acetylated LDL particles (125I-labeled Ac-LDL) with CHO-SRA cells were carried out. RT-PCR analysis using total RNAs from CHO-SRA cells revealed that oxidative stress does not alter the expression of the scavenger receptor. However, internalization of 125I-labeled Ac-LDL through this receptor carried out by these cells was completely abolished under extracellularly oxidative conditions. Together, these results support the idea that an oxidative stress produced extracellularly, inhibiting the endocytosis of the scavenger receptor, could help to understand and explain the mechanisms by which several physiologically important ligands are accumulated in the extracellular space with its consequent cell damage.     

Activation of signaling pathways by putative scavenger receptor class A (SR-A) ligands requires CD14 but not SR-A

Macrophage scavenger class A type I and type II receptors (SR-A) are trimeric, integral membrane glycoproteins that bind an unusually broad array of macromolecular ligands. These ligands include modified proteins and lipoproteins, nucleic acids, and a variety of plant and microbial cell wall constituents, such as fucoidan and lipoteichoic acid. Early studies of SR-A functions indicated that the receptors bound, internalized, and degraded their ligands without provoking any macrophage activating signaling events. More recent studies have provided evidence that several SR-A ligands can activate macrophage gene expression via utilization of a receptor-linked, PI3-kinase pathway. To investigate the role of SR-A in engaging signal transduction events, we employed macrophages taken from mice lacking these receptors. Using either fucoidan or lipoteichoic acid, we confirm that both ligands stimulate tyrosine phosphorylation of PI3-kinase and production of modest levels of the cytokine, TNFalpha. However, macrophages taken from SR-A null mice did not differ from wild type macrophages in these responses, indicating that these signaling events arise independently of SR-A activity. Employing mice lacking CD14, a GPI anchored receptor that binds bacterial lipopolysaccharide and signals via activation of Toll-like receptors, we show that the fucoidan and lipoteichoic acid responses are largely abrogated when CD14 is absent. These data do not provide support for direct SR-A involvement in signal transduction events and suggest that the early characterization of these receptors as initiators of a non-phlogistic, pathogen clearance pathway was correct.     

Separation of lipid transport functions by mutations in the extracellular domain of scavenger receptor class B,type I

Scavenger receptor class B, type I (SR-BI) shows a variety of effects on cellular cholesterol metabolism, including increased selective uptake of high density lipoprotein (HDL) cholesteryl ester, stimulation of free cholesterol (FC) efflux from cells to HDL and phospholipid vesicles, and changes in the distribution of plasma membrane FC as evidenced by increased susceptibility to exogenous cholesterol oxidase. Previous studies showed that these multiple effects require the extracellular domain of SR-BI, but not the transmembrane and cytoplasmic domains. To test whether 1) the extracellular domain of SR-BI mediates multiple activities by virtue of discrete functional subdomains, or 2) the multiple activities are, in fact, secondary to and driven by changes in cholesterol flux, the extracellular domain of SR-BI was subjected to insertional mutagenesis by strategically placing an epitope tag into nine sites. These experiments identified four classes of mutants with disruptions at different levels of function. Class 4 mutants showed a clear separation of function between HDL binding, HDL cholesteryl ester uptake, and HDL-dependent FC efflux on one hand and FC efflux to small unilamellar vesicles and an increased cholesterol oxidase-sensitive pool of membrane FC on the other. Selective disruption of the latter two functions provides evidence for multiple functional subdomains in the extracellular receptor domain. Furthermore, these findings uncover a difference in the SR-BI-mediated efflux pathways for FC transfer to HDL acceptors versus phospholipid vesicles. The loss of the cholesterol oxidase-sensitive FC pool and FC efflux to small unilamellar vesicle acceptors in Class 4 mutants suggests that these activities may be mechanistically related.     

The two-pore domain K+ channel, TRESK, is activated by the cytoplasmic calcium signal through calcineurin

Agonist-induced cytoplasmic calcium signals often have profound effects on the membrane potential during cellular activation. In the present study, we report that cytoplasmic calcium elevation can regulate the membrane potential by a novel mechanism. TRESK, a recently described member of the two-pore domain potassium (2PK(+)) channel family, was activated 5-15-fold after stimulation of various Ca(2+)-mobilizing receptors in Xenopus oocytes. Extracellular application of ionomycin, as well as the microinjection of inositol 1,4,5-trisphosphate or calcium, also evoked TRESK activation, whereas microinjection of EGTA or pretreatment of the oocytes with thapsigargin prevented the receptor-mediated effect. These data indicate that TRESK is activated by increased cytoplasmic calcium concentration. However, application of Ca(2+) to inside-out membrane patches failed to influence TRESK single channel activity, suggesting that cytoplasmic factors are also required for the regulation. Cyclosporin A and FK506, specific inhibitors of the calcium/calmodulin-dependent protein phosphatase (calcineurin), completely eliminated TRESK activation. Coexpression of a constitutively active form of calcineurin with TRESK increased the basal background K(+) current and attenuated the response of the channel to the calcium signal, indicating that TRESK was activated by the permanent calcineurin activity. Serine 276 was identified as the major functional target of calcineurin in TRESK by alanine-scanning mutagenesis. This is the first example of calcineurin being involved in the regulation of a two-pore domain K(+) channel, and thus, TRESK channels may regulate the excitability of neurons and other cell types in response to Ca(2+)-mobilizing hormones and neurotransmitters in a manner that is sensitive to immunosuppressive drugs.     

An autonomous signal for basolateral sorting in the cytoplasmic domain of the polymeric immunoglobulin receptor

The polymeric immunoglobulin receptor is normally delivered from the Golgi to the basolateral surface of epithelial cells and then transports polymeric IgA and IgM to the apical surface. We now report that a 14 residue segment of the 103 amino acid cytoplasmic domain, proximal to the plasma membrane, directs the receptor to the basolateral surface. A mutant receptor lacking these 14 amino acids is sorted directly to the apical surface from the Golgi. Furthermore, this sequence is sufficient to redirect an apical membrane protein, placental alkaline phosphatase, to the basolateral plasma membrane. We conclude that this sequence contains an autonomous signal, which specifies sorting from the Golgi to the basolateral surface, a process previously postulated to occur by default.     

Apoptotic thymocyte clearance in scavenger receptor class A-deficient mice is apparently normal

Studies of apoptotic cell uptake by phagocytes in vitro have implicated a number of different receptors capable of mediating ingestion. However, there is currently little evidence for involvement of any of these candidate receptors in vivo. Previously, we have shown by the use of a blocking mAb against the class A scavenger receptor (SR-A) and thymic macrophages prepared from SR-A null mice, that this receptor is responsible for approximately 50% of the uptake of apoptotic thymocytes in vitro. In this study we have investigated the frequency of dying cells in the thymus of mice lacking SR-A. Our inability to demonstrate increased frequencies of nonphagocytosed Annexin V+, TUNEL+, or propidium iodide+ apoptotic thymocytes suggests there is no deficiency in apoptotic thymocyte clearance in these mice. Even when the rate of thymocyte apoptosis was increased by exposure of receptor-deficient mice to gamma irradiation, we did not detect a difference in the numbers of dying cells compared with similarly treated wild-type animals. This provides the first direct evidence of redundancy in apoptotic cell clearance mechanisms in vivo.     

The effect of class a scavenger receptor deficiency in bone

Class A scavenger receptor (SR-A) is predominantly expressed by macrophages, and because osteoclasts are of monocyte/macrophage lineage, SR-A is of potential interest in osteoclast biology. In addition to modified low density lipoprotein uptake, SR-A is also important in cell attachment and signaling. In this study we evaluated the effect of SR-A deletion on bone. Knock-out animals have 40% greater body weight than wild type. Body composition analyses demonstrated that total lean and fat body mass were greater in knock-out animals, but there was no significant difference in percent fat and lean body mass. Bone mineral density and content were significantly greater in knock-out compared with wild type animals. Micro-computed tomography analyses confirmed that total volume, bone volume as well as trabecular number, thickness, and connectivity were significantly greater in knock-out mice. As expected, trabecular separation was greater in wild type mice. The phenotype appears to be explained by 60% fewer osteoclasts in females and 35% fewer in males compared to wild type mice with a paradoxical increase in nuclei/osteoclast in knock-out animals. Furthermore, there were no differences in adipocyte number and osteoblast number or activity. The addition of the soluble extracellular domain of SR-A to RAW264.7 cells stimulated a concentration-dependent increase in osteoclast differentiation that was receptor activator of nuclear factor-kappaB ligand (RANKL)-dependent. Soluble SR-A had no effect on cell proliferation in the presence of RANKL but stimulated a 40% increase in numbers in the absence of RANKL. We conclude that SR-A plays a role in normal osteoclast differentiation, suggesting a novel role for this receptor in bone biology.     

The human scavenger receptor class B type I is a novel candidate receptor for the hepatitis C virus

We discovered that the hepatitis C virus (HCV) envelope glycoprotein E2 binds to human hepatoma cell lines independently of the previously proposed HCV receptor CD81. Comparative binding studies using recombinant E2 from the most prevalent 1a and 1b genotypes revealed that E2 recognition by hepatoma cells is independent from the viral isolate, while E2-CD81 interaction is isolate specific. Binding of soluble E2 to human hepatoma cells was impaired by deletion of the hypervariable region 1 (HVR1), but the wild-type phenotype was recovered by introducing a compensatory mutation reported previously to rescue infectivity of an HVR1-deleted HCV infectious clone. We have identified the receptor responsible for E2 binding to human hepatic cells as the human scavenger receptor class B type I (SR-BI). E2-SR-BI interaction is very selective since neither mouse SR-BI nor the closely related human scavenger receptor CD36, were able to bind E2. Finally, E2 recognition by SR-BI was competed out in an isolate-specific manner both on the hepatoma cell line and on the human SR-BI-transfected cell line by an anti-HVR1 monoclonal antibody.     

Functional diversification between two related Plasmodium falciparum merozoite invasion ligands is determined by changes in the cytoplasmic domain

The pathogenesis of Plasmodium falciparum depends on efficient invasion into host erythrocytes. Parasite ligands encoded by multi‐gene families interact with erythrocyte receptors. P. falciparum reticulocyte binding protein homologues (PfRhs) are expressed at the apical surface of invasive merozoites and have divergent ectodomains that are postulated to bind different erythrocyte receptors. Variant expression of these paralogues results in the use of alternative invasion pathways. Two PfRh proteins, PfRh2a and PfRh2b, are identical for 2700 N‐terminal amino acids and differ only in a C‐terminal 500 amino acid region, which includes a unique ectodomain, transmembrane domain and cytoplasmic domain. Despite their similarity, PfRh2b is required for a well‐defined invasion pathway while PfRh2a is not required or sufficient for this pathway. Mapping the genomic region encoding these proteins revealed a recombinogenic locus with PfRh2a and PfRh2b in a head‐to‐head orientation. We have generated viable PfRh2a/2b chimeric parasites to identify the regions required for alternative invasion pathway utilization. We find that the differential ability to use these pathways is conferred by the cytoplasmic domains of PfRh2a and PfRh2b, not the ectodomain or transmembrane regions. Our results highlight the importance of the cytoplasmic domain for functional diversification of a major adhesive ligand family in malaria parasites.