Megalin (gp330) is an endocytic receptor for thyroglobulin on cultured fisher rat thyroid cells

We recently reported that megalin (gp330), an endocytic receptor found on the apical surface of thyroid cells, binds thyroglobulin (Tg) with high affinity in solid phase assays. Megalin-bound Tg was releasable by heparin. Here we show that Fisher rat thyroid (FRTL-5) cells, a differentiated rat thyroid cell line, can bind and endocytose Tg via megalin. We first demonstrated that FRTL-5 cells express megalin in a thyroid-stimulating hormone-dependent manner. Evidence of Tg binding to megalin on FRTL-5 cells and on an immortalized rat renal proximal tubule cell line (IRPT cells), was obtained by incubating the cells with 125I-Tg, followed by chemical cross-linking and immunoprecipitation of 125I-Tg with antibodies against megalin. To investigate cell binding further, we developed an assay in which cells were incubated with unlabeled Tg at 4 degrees C, followed by incubation with heparin, which released almost all of the cell-bound Tg into the medium. In solid phase experiments designed to illuminate the mechanism of heparin release, we demonstrated that Tg is a heparin-binding protein, as are several megalin ligands. The amount of Tg released by heparin from FRTL-5 and IRPT cells, measured by enzyme-linked immunosorbent assay (ELISA), was markedly reduced by two megalin competitors, receptor-associated protein (RAP) and 1H2 (monoclonal antibody against megalin), indicating that much of the Tg released by heparin had been bound to megalin ( approximately 60-80%). The amount inhibited by RAP was considered to represent specific binding to megalin, which was saturable and of high affinity (Kd approximately 11.2 nM). Tg endocytosis by FRTL-5 and IRPT cells was demonstrated in experiments in which cells were incubated with unlabeled Tg at 37 degrees C, followed by heparin to remove cell-bound Tg. The amount of Tg internalized (measured by ELISA in the cell lysates) was reduced by RAP and 1H2, indicating that Tg endocytosis is partially mediated by megalin.     

Apolipoprotein B is a calcium binding protein

Human hepatocarcinoma Hep G2 cells were grown in culture medium containing [45Ca2+]. The secreted lipoproteins of d less than 1.063 g/ml and d 1.063-1.21 g/ml were isolated from the culture media and analyzed by 3.3% and 7% SDS-polyacrylamide gel electrophoresis. Radioactivity profiles of [45Ca] from the gels showed that the peak of radioactivity corresponded to the apolipoprotein B band. The molar ratio of the incorporated [45Ca2+] and apolipoprotein B was close to unity. No radioactivity was found associated with any other secreted apolipoproteins. To confirm these findings, apolipoprotein B-containing lipoproteins were precipitated with anti-apolipoprotein B and high density lipoproteins were precipitated with anti-apolipoprotein A-I. Only the former precipitate was radioactive. These results suggest that apolipoprotein B is a calcium binding protein.     

The endocytic adaptor protein Disabled-2 is required for cellular uptake of fibrinogen

Endocytosis is pivotal for uptake of fibrinogen from plasma into megakaryocytes and platelet α-granules. Due to the complex adaptor and cargo contents in endocytic vehicles, the underlying mechanism of fibrinogen uptake is not yet completely elucidated. In this study, we investigated whether the endocytic adaptor protein Disabled-2 (DAB2) mediates fibrinogen uptake in an adaptor-specific manner. By employing primary megakaryocytes and megakaryocytic differentiating human leukemic K562 cells as the study models, we found that fibrinogen uptake is associated with the expression of integrin αIIbβ3 and DAB2 and is mediated through clathrin-dependent manner. Accordingly, constitutive and inducible knockdown of DAB2 by small interfering RNA reduced fibrinogen uptake for 53.2 ± 9.8% and 59.0 ± 10.7%, respectively. Culturing the cells in hypertonic solution or in the presence of clathrin inhibitor chlorpromazine abrogated clathrin-dependent endocytosis and diminished the uptake of fibrinogen. Consistent with these findings, 72.2 ± 0.2% of cellular DAB2 was colocalized with clathrin, whereas 56.4±4.1% and 54.6 ± 2.0% of the internalized fibrinogen were colocalized with clathrin and DAB2, respectively. To delineate whether DAB2 mediates fibrinogen uptake in an adaptor-specific manner, K562 stable cell lines with knockdown of the adaptor protein-2 (AP-2) or double knockdown of AP-2/DAB2 were established. The AP-2 knockdown cells elicited normal fibrinogen uptake activity but the uptake of collagen was diminished. In addition, collagen uptake was further reduced in DAB2/AP-2 knockdown cells. These findings thereby define an adaptor-specific mechanism in the control of fibrinogen uptake and implicate that DAB2 is the key adaptor in the clathrin-associated endocytic complexes to mediate fibrinogen internalization.     

Calcyclin is a calcium and zinc binding protein

Calcyclin, a cell cycle regulated protein, was recently purified from Ehrlich ascites tumour (EAT) cells and shown to be a calcium binding protein. Here we show that calcyclin monomer and dimer also bind zinc ions. Zinc binding sites seem to be different from calcium binding sites since: preincubation with Ca2+ lacks effect on the binding of Zn2+, and Ca2+ (but not Zn2+) increases tyrosine fluorescence intensity. Binding of Zn2+ reduces the extent of the conformational changes induced by Ca2+, and seems to affect Ca2(+)-binding. The data suggest that Ca2+ and Zn2+ might trigger the biological activity of calcyclin.     

Renal calcium binding protein in the diabetic and vitamin D-depleted rat.

A calcium binding protein has been purified 220 fold from rat kidney. The molecular weight of this protein (26 000-28 000) is more than double that of the duodenal calcium binding protein of the rat. In response to the stimuli of both streptozotocin diabetes and depletion and repletion with vitamin D, changes in the renal protein are minimal. This contrasts markedly with responses of the duodenal protein to the same stimuli: (a) there was marked depression of duodenal calcium binding protein by vitamin D depletion and diabetes; (b) duodenal calcium binding protein was restored by vitamin D treatment of depleted rats. The renal protein appears to be identical with a previously described 28 000 molecular weight protein from the kidney purified by a different technique (Hermsdorf, C.L. and Bronner, F. (1975) Biochim. Biophys. Acta 379, 553-561). In contrast to findings of the current study, previous investigators were unable to isolate the protein from vitamin D-deficient rats and postulated vitamin D dependence. The protein activator of cyclic AMP phosphodiesterase is a calcium binding protein found in many tissues including kidney. Based on lack of response to stimuli we used and similarity in method of isolation and properties, our renal calcium binding protein may be this protein activator.     

Human complement protein C9 is a calcium binding protein. Structural and functional implications

Human complement protein C9 is shown to be a metalloprotein that binds 1 mol of Ca2+/mol of C9 with a dissociation constant of 3 micron as measured by equilibrium dialysis. Incubation with EDTA removes the bound calcium, resulting in a apoprotein with decreased thermal stability. This loss in stability leads to aggregation and, therefore, to loss of hemolytic activity upon heating to a few degrees above the physiological temperature. Heat-induced aggregation of apoC9 can be prevented by salts that stabilize proteins according to the Hofmeister series of lyotropic ions, suggesting that the ion in native C9 may ligand with more than one structural element or domain of the protein. Ligand blotting indicates that the calcium binding site is located in the amino-terminal half of the protein. Removal of calcium by inclusion of EDTA in assay mixtures has no effect on the hemolytic activity of C9, and its capacity to bind to C8 in solution, or to small unilamellar lipid vesicles at temperatures at or below the physiological range. Although we do not know yet the precise structural and functional role of the bound calcium, it is clear that it provides thermal stability to C9 and it may have a function in regulation of membrane insertion.     

Vps9p CUE domain ubiquitin binding is required for efficient endocytic protein traffic

Rab5 GTPases are key regulators of protein trafficking through the early stages of the endocytic pathway. The yeast Rab5 ortholog Vps21p is activated by its guanine nucleotide exchange factor Vps9p. Here we show that Vps9p binds ubiquitin and that the CUE domain is necessary and sufficient for this interaction. Vps9p ubiquitin binding is required for efficient endocytosis of Ste3p but not for the delivery of the biosynthetic cargo carboxypeptidase Y to the vacuole. In addition, Vps9p is itself monoubiquitylated. Ubiquitylation is dependent on a functional CUE domain and Rsp5p, an E3 ligase that participates in cell surface receptor endocytosis. These findings define a new ubiquitin binding domain and implicate ubiquitin as a modulator of Vps9p function in the endocytic pathway.     

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.     

The bovine cardiac receptor for calcium channel blockers is a 195-kDa protein

The cardiac receptor for calcium channel blockers was purified from bovine microsomal membranes which contained 235 +/- 33 fmol nimodipine-binding sites/mg protein (mean +/- SEM of nine preparations). To identify the receptor during the purification 20% of its binding sites were prelabeled with (+)[3H]PN200-110. The receptor was solubilized with 0.6% digitonin and was purified to a specific density of 157 pmol/mg using a combination of ion-exchange, wheat-germ-agglutinin-Sepharose chromatography and sucrose density gradient centrifugation. In the last sucrose gradient bound (+)[3H]PN200-110 comigrated with a 195-kDa protein. ( +/-)[3H]Azidopine and [3H]ludopamil, the photoaffinity ligands for the dihydropyridine and phenylalkylamine-binding site of the calcium channel, were incorporated specifically into the 195-kDa protein. These data indicate that the bovine cardiac receptor for calcium channel blockers is a 195-kDa protein. Its molecular mass suggests that the bovine cardiac receptor differs considerably from the rabbit skeletal muscle receptor protein for calcium channel blockers.     

The calcium binding protein EhCaBP6 is a microtubular‐end binding protein in Entamoeba histolytica

The genome of Entamoeba histolytica encodes several calcium binding proteins and those characterized thus far have been shown to participate predominantly in phagocytosis and endocytosis. Our study showed that EhCaBP6 has two EF‐hand domains EFI and EFIII; it can bind Ca²⁺ in vitro and undergoes conformational transition on binding Ca²⁺ suggesting that it can serve as a calcium signal sensor. EhCaBP6 is localized in the nucleus, cytoplasm and plasma membrane and is sensitive to heat stress. Unlike other Ca²⁺ binding proteins that have been studied in E. histolytica, EhCaBP6 is found at microtubule ends and at the intercellular bridge with the microtubules during cytokinesis. Furthermore, increased expression of EhCaBP6 was correlated with a significant increase in the number of microtubular structures suggesting that this protein may regulate chromosome segregation and cytokinesis in E. histolytica.     

The endocytic protein intersectin is a major binding partner for the Ras exchange factor mSos1 in rat brain

We recently identified intersectin, a protein containing two EH and five SH3 domains, as a component of the endocytic machinery. The N-terminal SH3 domain (SH3A), unlike other SH3 domains from intersectin or various endocytic proteins, specifically inhibits intermediate events leading to the formation of clathrin-coated pits. We have now identified a brain-enriched, 170 kDa protein (p170) that interacts specifically with SH3A. Screening of combinatorial peptides reveals the optimal ligand for SH3A as Pp(V/I)PPR, and the 170 kDa mammalian son-of-sevenless (mSos1) protein, a guanine-nucleotide exchange factor for Ras, con- tains two copies of the matching sequence, PPVPPR. Immunodepletion studies confirm that p170 is mSos1. Intersectin and mSos1 are co-enriched in nerve terminals and are co-immunoprecipitated from brain extracts. SH3A competes with the SH3 domains of Grb2 in binding to mSos1, and the intersectin-mSos1 complex can be separated from Grb2 by sucrose gradient centrifugation. Overexpression of the SH3 domains of intersectin blocks epidermal growth factor-mediated Ras activation. These results suggest that intersectin functions in cell signaling in addition to its role in endocytosis and may link these cellular processes.     

Renal calcium binding protein in the diabetic and vitamin D-depleted rat

A calcium binding protein has been purified 220 fold from rat kidney. The molecular weight of this protein (26 000–28 000) is more than double that of the duodenal calcium binding protein of the rat. In response to the stimuli of both streptozotocin diabetes and depletion and repletion with vitamin D, changes in the renal protein are minimal. This contrasts markedly with responses of the duodenal protein to the same stimuli: (a) there was marked depression of duodenal calcium binding protein by vitamin D depletion and diabetes; (b) duodenal calcium binding protein was restored by vitamin D treatment of depleted rats. The renal protein appears to be identical with a previously described 28 000 molecular weight protein from the kidney purified by a different technique (Hermsdorf, C.L. and Bronner, F. (1975) Biochim. Biophys. Acta 379, 553–561). In contrast to findings of the current study, previous investigators were unable to isolate the protein from vitamin D-deficient rats and postulated vitamin D dependence. The protein activator of cyclic AMP phosphodiesterase is a calcium binding protein found in many tissues including kidney. Based on lack of response to stimuli we used and similarity in method of isolation and properties, our renal calcium binding protein may be this protein activator.     

钙结合蛋白calreticulin

钙结合蛋白ｃａｌｒｅｔｉｃｕｌｉｎ由Ｎ、Ｐ和Ｃ三个区域的氨在酸序列组成,具有很主的钙结合容量,主要存在于内质网上;其蛋白和基因在生物进化过程中具有极高的保守性,提示它在许多细胞功能的调节中发挥重要作用;它是一种独特的内质网膜分子伴侣,参与胞内钙信号、细胞粘际及基因表达的调控。     

The human fibroblast receptor for gp86 of human cytomegalovirus is a phosphorylated glycoprotein.

A human embryonic lung (HEL) cell receptor for gp86 of human cytomegalovirus that functions in virus-cell fusion was further characterized. Anti-idiotype antibodies that mimic gp86 were used to immunoprecipitate the 92.5-kDa fibroblast membrane receptor for gp86, which was preincubated with various endoglycosidases. The receptor, which has a pI ranging from 5.3 to 5.6, appears to be a glycoprotein with primarily N-linked sugar residues, some of which have high concentrations of mannose and some of which are complex oligosaccharides. Western blots (immunoblots) of electrophoretically transferred receptor incubated with various biotinylated lectins confirmed the presence of sugar moieties, including N-acetylglucosamine, glucose or mannose, and galactose, but not fucose or N-acetylgalactosamine. This gp86 receptor from uninfected HEL cells also incorporated radiolabeled phosphate from orthophosphoric acid, indicating that it is a constitutively phosphorylated receptor.     

Transferrin-binding protein complex is the receptor for transferrin uptake in Trypanosoma brucei

In Trypanosoma brucei, the products of two genes, ESAG 6 and ESAG 7, located upstream of the variant surface glycoprotein gene in a polycistronic expression site form a glycosylphosphatidylinositol- anchored transferrin-binding protein (TFBP) complex. It is shown by gel filtration and membrane-binding experiments that the TFBP complex is heterodimeric and binds one molecule of transferrin with high affinity (2,300 binding sites per cell; KD = 2.1 nM for the dominant expression site from T. brucei strain 427 and KD = 131 nM for ES1.3A of the EATRO 1125 stock). The ternary transferrin-TFBP complexes with iron-loaded or iron-free ligand are stable between pH 5 and 8. Cellular transferrin uptake can be inhibited by 90% with Fab fragments from anti-TFBP antibodies. After uptake, the TFBP complex and its ligand are routed to lysosomes where transferrin is proteolytically degraded. While the degradation products are released from the cells, iron remains cell associated and the TFBP complex is probably recycled to the membrane of the flagellar pocket, the only site for exo- and endocytosis in this organism. It is concluded that the TFBP complex serves as the receptor for the uptake of transferrin in T. brucei by a mechanism distinct from that in mammalian cells.     

Rab15 effector protein: a novel protein for receptor recycling from the endocytic recycling compartment

Sorting endosomes and the endocytic recycling compartment are critical intracellular stores for the rapid recycling of internalized membrane receptors to the cell surface in multiple cell types. However, the molecular mechanisms distinguishing fast receptor recycling from sorting endosomes and slow receptor recycling from the endocytic recycling compartment remain poorly understood. We previously reported that Rab15 differentially regulates transferrin receptor trafficking through sorting endosomes and the endocytic recycling compartment, suggesting a role for distinct Rab15-effector interactions at these endocytic compartments. In this study, we identified the novel protein Rab15 effector protein (REP15) as a binding partner for Rab15-GTP. REP15 is compartment specific, colocalizing with Rab15 and Rab11 on the endocytic recycling compartment but not with Rab15, Rab4, or early endosome antigen 1 on sorting endosomes. REP15 interacts directly with Rab15-GTP but not with Rab5 or Rab11. Consistent with its localization, REP15 overexpression and small interfering RNA-mediated depletion inhibited transferrin receptor recycling from the endocytic recycling compartment, without affecting receptor entry into or recycling from sorting endosomes. Our data identify REP15 as a compartment-specific protein for receptor recycling from the endocytic recycling compartment, highlighting that the rapid and slow modes of transferrin receptor recycling are mechanistically distinct pathways.     

SLIRP, a small SRA binding protein, is a nuclear receptor corepressor

Steroid receptor RNA activator (SRA), the only known RNA coactivator, augments transactivation by nuclear receptors (NRs). We identified SLIRP (SRA stem-loop interacting RNA binding protein) binding to a functional substructure of SRA, STR7. SLIRP is expressed in normal and tumor tissues, contains an RNA recognition motif (RRM), represses NR transactivation in a SRA- and RRM-dependent manner, augments the effect of Tamoxifen, and modulates association of SRC-1 with SRA. SHARP, a RRM-containing corepressor, also binds STR7, augmenting repression with SLIRP. SLIRP colocalizes with SKIP (Chr14q24.3), another NR coregulator, and reduces SKIP-potentiated NR signaling. SLIRP is recruited to endogenous promoters (pS2 and metallothionein), the latter in a SRA-dependent manner, while NCoR promoter recruitment is dependent on SLIRP. The majority of the endogenous SLIRP resides in the mitochondria. Our data demonstrate that SLIRP modulates NR transactivation, suggest it may regulate mitochondrial function, and provide mechanistic insight into interactions between SRA, SLIRP, SRC-1, and NCoR.     

C-terminal arginine cluster is essential for receptor binding of norovirus capsid protein

Noroviruses are the major viral pathogens of epidemic acute gastroenteritis affecting people worldwide. They have been found to recognize human histo-blood group antigens as receptors. The P domain of norovirus capsid protein was found to be responsible for binding to viral receptors, and the recombinant P protein forms P dimers and P particles in vitro. In this study, we demonstrate that a highly conserved arginine (R) cluster at the C terminus of the P domain is critical for receptor binding and P particle formation of the P proteins. Deletions of the R cluster abolished these functions. Replacement of the R cluster with histidines (another positively charged amino acid) resulted in low efficiency of receptor binding and P particle formation, while replacement with alanines led to loss of both functions completely. The R cluster also contains a highly conserved trypsin digestion site. A treatment of capsid protein or P domain mutants from both genogroup I (Norwalk virus) and genogroup II (VA387) noroviruses with trypsin resulted in a removal of the R cluster and the S domain, leaving a P polypeptide of 31.3 kDa (Norwalk virus) or 34.3 kDa (VA387), similar to the soluble P protein found in vivo. Our findings imply that the proteolytic process could be a necessary step for norovirus replication in the host.