A microassay for the determination of binding parameters of estrogen and androgen receptors employing affinity immobilization on Cibacron blue 3GA-Sepharose 6B

The problems of currently available ligand-binding assays for sex-steroid receptor proteins include the relatively large mass of tissue required, the interference by sex hormone-binding globulin (SHBG), and use in the androgen receptor (AR) assay of the unstable synthetic ligand methyltrienolone. To overcome these difficulties the stabilizing effect of the dye Cibacron blue 3GA on AR and estrogen receptor (ER) proteins, and its ability to bind to these proteins, was utilized in developing an assay system for each receptor that could be applied to small samples. Use of the affinity gel Cibacron blue 3GA-Sepharose 6B (Blue gel) for the immobilization of AR, ER, and the steroid ligands bound to these receptors in the standard two-tier column assay system enabled the use of a 1:100 (original tissue weight:volume) concentration, making possible full (5-7 point) Scatchard analysis on tissue specimens of a mass as low as 15-20 mg. Significant stabilization of AR and ER was observed and association constants for these receptors were of a similar order of magnitude to those obtained either by Sephadex LH-20 gel filtration or the dextran-coated charcoal adsorption technique. Inactivation by dilution was shown to be largely prevented based on results obtained with cytosol concentrations from 1:5 to 1:100 (original tissue weight:volume). Because Blue gel does not bind SHBG, the natural steroid 5 alpha-androstan-17 beta-ol-3-one (DHT) may be employed as a ligand in the AR assay.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evidence for a two-step mechanism involved in assembly of functional signal recognition particle receptor

The signal recognition particle (SRP) and SRP receptor act sequentially to target nascent secretory proteins to the membrane of the ER. The SRP receptor consists of two subunits, SR alpha and SR beta, both tightly associated with the ER membrane. To examine the biogenesis of the SRP receptor we have developed a cell-free assay system that reconstitutes SR alpha membrane assembly and permits both anchoring and functional properties to be assayed independently. Our experiments reveal a mechanism involving at least two distinct steps, targeting to the ER and anchoring of the targeted molecule on the cytoplasmic face of the membrane. Both steps can be reconstituted in vitro to restore translocation activity to ER microsomes inactivated by alkylation with N-ethyl-maleimide. The characteristics elucidated for this pathway distinguish it from SRP-dependent targeting of secretory proteins, SRP-independent ER translocation of proteins such as prepromellitin, and direct insertion mechanisms of the type exemplified by cytochrome b5.     

Binding of ribosomes to the rough endoplasmic reticulum mediated by the Sec61p-complex

The cotranslational translocation of proteins across the ER membrane involves the tight binding of translating ribosomes to the membrane, presumably to ribosome receptors. The identity of the latter has been controversial. One putative receptor candidate is Sec61 alpha, a multi- spanning membrane protein that is associated with two additional membrane proteins (Sec61 beta and gamma) to form the Sec61p-complex. Other receptors of 34 and 180 kD have also been proposed on the basis of their ability to bind at low salt concentration ribosomes lacking nascent chains. We now show that the Sec61p-complex has also binding activity but that, at low salt conditions, it accounts for only one third of the total binding sites in proteoliposomes reconstituted from a detergent extract of ER membranes. Under these conditions, the assay has also limited specificity with respect to ribosomes. However, if the ribosome-binding assay is performed at physiological salt concentration, most of the unspecific binding is lost; the Sec61p- complex then accounts for the majority of specific ribosome-binding sites in reconstituted ER membranes. To study the membrane interaction of ribosomes participating in protein translocation, native rough microsomes were treated with proteases. The amount of membrane-bound ribosomes is only slightly reduced by protease treatment, consistent with the protease-resistance of Sec61 alpha which is shielded by these ribosomes. In contrast, p34 and p180 can be readily degraded, indicating that they are not essential for the membrane anchoring of ribosomes in protease-treated microsomes. These data provide further evidence that the Sec61p-complex is responsible for the membrane- anchoring of ribosomes during translocation and make it unlikely that p34 or p180 are essential for this process.     

Estrogen receptor variants and mutations

There is a large and increasing body of experimental and clinical data supporting the existence of variant estrogen receptor (ER) proteins in both normal and neoplastic estrogen target tissues, including human breast. Therefore, future examination of ER signal transduction and/or measurement of ER protein must take into account variant ER expression. The functions of variant ER proteins, either physiological or pathological, remain unclear, although a role(s) for some ER variants in breast tumorigenesis and breast cancer progression would be consistent with the accumulated data. Possible tissue specific expression leads to the speculation that ER variants may have a role in tissue specific estrogen action. The following review focuses on the current knowledge available in the scientific literature with respect to the type and characteristics of estrogen receptor variants and mutations that have been identified to occur naturally in tissues and cell lines.     

Identification of ERGIC-53 as an intracellular transport receptor of alpha1-antitrypsin

Secretory proteins are exported from the endoplasmic reticulum (ER) by bulk flow and/or receptor-mediated transport. Our understanding of this process is limited because of the low number of identified transport receptors and cognate cargo proteins. In mammalian cells, the lectin ER Golgi intermediate compartment 53-kD protein (ERGIC-53) represents the best characterized cargo receptor. It assists ER export of a subset of glycoproteins including coagulation factors V and VIII and cathepsin C and Z. Here, we report a novel screening strategy to identify protein interactions in the lumen of the secretory pathway using a yellow fluorescent protein-based protein fragment complementation assay. By screening a human liver complementary DNA library, we identify alpha1-antitrypsin (alpha1-AT) as previously unrecognized cargo of ERGIC-53 and show that cargo capture is carbohydrate- and conformation-dependent. ERGIC-53 knockdown and knockout cells display a specific secretion defect of alpha1-AT that is corrected by reintroducing ERGIC-53. The results reveal ERGIC-53 to be an intracellular transport receptor of alpha1-AT and provide direct evidence for active receptor-mediated ER export of a soluble secretory protein in higher eukaryotes.     

Endoplasmic reticulum quality control of unassembled iron transporter depends on Rer1p-mediated retrieval from the golgi

Endoplasmic reticulum (ER) quality control is a conserved process by which misfolded or unassembled proteins are selectively retained in the endoplasmic reticulum (ER). Failure in oligomerization of multisubunit membrane proteins is one of the events that triggers ER quality control. The transmembrane domains (TMDs) of unassembled subunits are determinants of ER retention in many cases, although the mechanism of the TMD-mediated sorting of unassembled subunits remains elusive. We studied a yeast iron transporter complex on the cell surface as a new model system for ER quality control. When Fet3p, a transmembrane subunit, is not assembled with the other membrane subunit, Ftr1p, unassembled Fet3p is exclusively localized to the ER at steady state. The TMD of Fet3p contains a determinant for this process. However, pulse-chase analysis and in vitro budding assays indicate that unassembled Fet3p rapidly escapes from the ER. Furthermore, Rer1p, a retrieval receptor for ER-resident membrane proteins in the Golgi, is responsible for the TMD-dependent ER retrieval of unassembled Fet3p. These findings provide clear evidence that the ER quality control of unassembled membrane proteins can be achieved by retrieval from the Golgi and that Rer1p serves as a specific sorting receptor in this process.     

Efficient and selective photoaffinity labeling of the estrogen receptor using two nonsteroidal ligands that embody aryl azide or tetrafluoroaryl azide photoreactive functions

3-(4-Azido-2,3,5,6-tetrafluorobenzoyl)-6-hydroxy-2-(4- hydroxyphenyl)benzo[b]thiophene 1 (tetrafluoroaryl azide, TFAA) and its protio analogue 3-(4-azidobenzoyl)-6- hydroxy-2-(4-hydroxyphenyl)benzo[b]thiophene 2 (protioaryl azide, PAA), photoaffinity labeling (PAL) reagents for the estrogen receptor (ER), have been prepared in high specific activity tritium-labeled form (19 Ci/mmol) and shown to undergo selective and efficient photocovalent attachment to ER from rat uterus. Both azides 1 and 2 demonstrate high binding affinity for ER as determined by both a competitive binding assay (relative binding affinities: estradiol = 100; TFAA = 9.3; PAA = 66) and a direct binding assay (Kd: estradiol = 0.24 nM; TFAA = 2.64 nM; PAA = 0.37 nM). When unlabeled TFAA and PAA are irradiated at greater than 315 nm, they demonstrate site-specific photoinactivation of ER that reaches 43% and 55%, respectively, by 30 min. Specific photocovalent attachment to ER can be effected by irradiation of the tritium-labeled azides; the covalent attachment efficiency is good (1 = 20-30%, 2 = ca. 25%) and the selectivity of ER labeling is high. Characterization of the photolabeled proteins by SDS-polyacrylamide gel electrophoresis shows specific labeling of a major component at Mr 60,000 and a minor species at Mr 46,000, the same two species that are labeled by [3H]tamoxifen aziridine, a well-characterized affinity label for ER. The ER-specific antibodies H222Sp gamma and D547Sp gamma show a clean precipitation of only these two species. In the MCF-7 human breast cancer cell line, PAA is a full estrogen agonist in terms of stimulation of cell proliferation and induction of progesterone receptor. These two azides provide the first system in which the photocovalent attachment efficiency of an aryl azide can be compared to its tetrafluorosubstituted aryl azide analogue in a complex biological receptor system. Azides 1 and 2 are the most efficient and selective PAL reagents prepared to date for ER, and they should be useful in further studies of the hormone-binding domain of this protein.     

Estrogen receptor β and its domains interact with casein kinase 2, phosphokinase C, and N-myristoylation sites of mitochondrial and nuclear proteins in mouse brain

The localization of estrogen receptor (ER)β in mitochondria suggests ERβ-dependent regulation of genes, which is poorly understood. Here, we analyzed the ERβ interacting mitochondrial as well as nuclear proteins in mouse brain using pull-down assay and matrix-assisted laser desorption ionization mass spectroscopy (MALDI-MS). In the case of mitochondria, ERβ interacted with six proteins of 35-152 kDa, its transactivation domain (TAD) interacted with four proteins of 37-172 kDa, and ligand binding domain (LBD) interacted with six proteins of 37-161 kDa. On the other hand, in nuclei, ERβ interacted with seven proteins of 30-203 kDa, TAD with ten proteins of 31-160 kDa, and LBD with fourteen proteins of 42-179 kDa. For further identification, these proteins were cleaved by trypsin into peptides and analyzed by MALDI-MS using mascot search engine, immunoprecipitation, immunoblotting, and far-Western blotting. To find the consensus binding motifs in interacting proteins, their unique tryptic peptides were analyzed by the motif scan software. All the interacting proteins were found to contain casein kinase (CK) 2, phosphokinase (PK)C phosphorylation, and N-myristoylation sites. These were further confirmed by peptide pull-down assays using specific mutations in the interacting sites. Thus, the present findings provide evidence for the interaction of ERβ with specific mitochondrial and nuclear proteins through consensus CK2, PKC phosphorylation, and N-myristoylation sites, and may represent an essential step toward designing selective ER modulators for regulating estrogen-mediated signaling.     

Conformational and electrostatic properties of unoccupied and liganded estrogen receptors determined by aqueous two-phase partitioning

The technique of aqueous two-phase partitioning (ATPP) has been used to characterize conformational and electrostatic properties of unoccupied and liganded rat uterine estrogen receptors. The adaptation of the hydroxylapatite receptor assay with ATPP systems has permitted estrogen receptor (ER) partition coefficients to be accurately determined, even when the partitioning process results in significant loss of ER binding capacity. The pH and salt dependences of estrogen receptor partition coefficients indicate that the theory governing partitioning behavior can be accurately applied to partitioning data obtained with crude cytosols. This technique has revealed a ligand-induced change in the properties of the unoccupied receptor that precedes the process of heat-induced transformation in vitro. The difference in partitioning behavior between unoccupied and nontransformed estrogen receptor is observed in all combinations of buffers and salts tested and is of equal magnitude as the difference between partition coefficients of nontransformed and transformed ER. The partition coefficients of both unoccupied and nontransformed ER are constant over the ER concentration range in which binding cooperativity has been previously demonstrated. The combined effects of salt and pH on ER partition coefficients indicate a pI of approximately 5.5 for both unoccupied and nontransformed estrogen receptors. However, the partition coefficients at the pI differ. It is concluded that estradiol binding to its unoccupied receptor results in a change in surface properties of the ER monomer that is independent of receptor transformation and makes the receptor less hydrophobic.     

Quantitation of the immunocytochemical assay for estrogen receptor protein (ER-ICA) in human breast cancer by television imaging

A Quantimet 720D Image Analysis System has been programmed for light microscopic evaluation of the nuclear estrogen receptor distribution in frozen sections of human breast cancer stained by the peroxidase-antiperoxidase method using monoclonal antibodies to estrogen receptor protein (ER). This method provides precise criteria for distinguishing ER-positive and -negative cells and a sensitive and reproducible means for densitometric quantification of the staining patterns. Although imaging sequence and graphic analysis are automated by computer programs, light pen interaction provides supervision of feature selection. Imaging of the immunocytochemical assay (ER-ICA) in 50 patients revealed marked heterogeneity of nuclear estrogen receptor concentration varying over a nearly 100 fold concentration range. Various ER concentration patterns were evident: (I) distributions with a single peak (CV = 5%) present at various concentration levels; (II) bimodal distributions, revealing co-existent ER-positive and ER-negative subpopulations; (III) multimodal distributions with a number of resolvable concentration peaks; and (IV) highly skewed distributions with or without discernible peaks, frequently extending over the entire concentration range. Statistical methods of de-convolution were applied to determine the frequency and ER concentration characteristics of component subpopulations in the mosaic cases and for resolving the proportion of ER-positive and -negative cells. An approach for evaluating nuclear ER content in conjunction with ER concentration patterns in individual patients revealed whether spread in the ER concentration distribution resulted from differences in nuclear ER content or from variability in nuclear volume distribution.     

Detection of the level of estrogen receptor and functional variants in human breast cancers by novel assays

The level of estrogen receptor (ER) is a key determinant for the management of ER-positive [ER(+)] breast cancer patients. Growth of many human breast cancers is regulated by estrogen (E2) and progesterone (Pr). Generally, the ER in ER(+) breast cancer patients is targeted for therapy with antihormones. However 40% of ER(+) patients do not respond to antihormone therapy. Thus, the identification of antihormone resistant ER(+) breast cancers is essential for therapeutic predictions. Although 3H-E2 binding and immunodetection can identify ER, these procedures do not assess the functional state of the receptor molecule. In this study we describe a novel and rapid assay for the detection of ER and its functional state on the basis of the downstream interaction with its response element (ERE) based on the preferential binding of DNA-protein complex (ERE-ER) to a nitrocellulose membrane (NMBA). This method permits measurement of both the total and the functional fraction of ER. The ER status was examined in breast cancer cell lines and in breast cancer biopsy specimens by (i) 3H-E2 binding assay, (ii) immunodetection assays and (iii) by its interaction with 32P-ERE. The sensitive NMBA assay was validated with well-characterized ER(+) breast cancer cell lines and also identified functional variants of ER among breast tumor biopsy specimens.     

A redox-sensitive luciferase assay for determining the localization and topology of endoplasmic reticulum proteins

Correct localization and transmembrane topology are crucial for the proteins residing and functioning in the endoplasmic reticulum (ER). We have developed a rapid and convenient assay, based on the redox-sensitive luciferase from Gaussia princeps (Gluc) and green fluorescence protein (GFP), to determine the localization or topology of ER proteins. Using the tandem Gluc-GFP reporter fused to different positions of a target protein, we successfully characterized the topologies of two ER transmembrane proteins Herp and HRD1 that are involved in the ER quality control system. This assay method may also be applicable to the proteins in secretory pathway, plasma membrane, and other compartments of cells.     

WRB and CAML Are Necessary and Sufficient to Mediate Tail-Anchored Protein Targeting to the ER Membrane

Tail-Anchored (TA) proteins are inserted into the endoplasmic reticulum (ER) membrane of yeast cells via the posttranslational Guided Entry of Tail-Anchored protein (GET) pathway. The key component of this targeting machinery is the ATPase Get3 that docks to the ER membrane by interacting with a receptor complex formed by the proteins Get1 and Get2. A conserved pathway is present in higher eukaryotes and is mediated by TRC40, homolog of Get3, and the recently identified membrane receptors WRB and CAML. Here, we used yeast lacking the GET1 and GET2 genes and substituted them with WRB and CAML. This rescued the growth phenotypes of the GET receptor mutant. We demonstrate that WRB and CAML efficiently recruit Get3 to the ER membrane and promote the targeting of the TA proteins in vivo. Our results show that the membrane spanning segments of CAML are essential to create a functional receptor with WRB and to ensure TA protein membrane insertion. Finally, we determined the binding parameters of TRC40 to the WRB/CAML receptor. We conclude that together, WRB and CAML are not only necessary but also sufficient to create a functional membrane receptor complex for TRC40. The yeast complementation assay can be used to further dissect the structure-function relationship of the WRB/CAML heteromultimer in the absence of endogenous receptor proteins.     

人乳腺癌cAMP结合蛋白

建立了测定人乳腺癌胞浆ｃＡＭＰ结合蛋白（ｃＡＭＰｂ．ｐ．）方法。综合研究了其温度、保温时间、配体浓度、稳定性等条件。ｃＡＭＰｂ．ｐ．的Ｋ_Ｄ值为２．９０×１０~（－８）ｍｏｌ／Ｌ．并测定了６０例雌激素受体（ＥＲ）Ｆｕ性乳腺癌标本的ｃＡＭＰｂ．ｐ．含量。此组病人术后均接受系统的内分泌治疗,ＥＲ／ｃＡＭＰｂｐ,比值范围为７．７～３６２×１０~（－３）,ＥＲ／ｃＡＭＰｂ．ｐ．比值≥４０×１０~（－３）的五年生存率明显高于比值＜４０×１０~（－３）组,（ｐ＜０．００５）．表明测定ＥＲ／ｃＡＭＰｂ．ｐ．比值对预测患者内分泌治疗疗效,优于单独测定ＥＲ．     

An in vitro assay for the selective endoplasmic reticulum associated degradation of an unglycosylated secreted protein

The endoplasmic reticulum (ER) represents the first compartment into which nascent secreted proteins traffic, and not coincidentally the ER lumen houses a high concentration of factors that facilitate protein folding, such as molecular chaperones. To off-set the potentially lethal consequences of mis-folded secreted protein accumulation, aberrant proteins may be selected for degradation via a process known as ER associated degradation (ERAD). After their selection ERAD substrates are retro-translocated back to the cytoplasm and then degraded by the 26S proteasome. Key features of the selection, retro-translocation, and degradation steps that constitute the ERAD pathway were elucidated through the development of an in vitro ERAD assay. In this assay the fates of two yeast proteins can be distinguished after their translocation, or import into ER-derived microsomes. Whereas a wild type, glycosylated protein ("Gp(alpha)F") is stable, a non-glycosylated version of the same protein ("p(alpha)F") is rapidly degraded when microsomes containing radiolabeled forms of these substrates are incubated in cytosol and ATP. The purpose of this chapter is first to discuss the experimental findings from the use of the in vitro assay, and then to describe the assay in detail. Finally, future potential uses of the in vitro system are illustrated.     

The mouse FKBP23 binds to BiP in ER and the binding of C-terminal domain is interrelated with Ca2+ concentration

FK506 binding protein 23 from mouse (mFKBP23) is a peptidyl-prolyl cis-trans isomerase (PPIase) from the endoplasmic reticulum (ER), which consists of an N-terminal PPIase domain and a C-terminal domain with Ca(2+) binding sites. The assay of adsorption from ER extract with glutathione S-transferase-mFKBP23 attached to glutathione-Sepharose 4B shows that mFKBP23 binds to mouse immunoglobulin binding protein (mBiP). The same assay with the recombinant proteins of the N- and C-termini of mFKBP23 shows that the binding of the C-terminus is Ca(2+)-dependent and the switch point is between 2 and 3 mM. By high concentration of Ca(2+) this binding cannot be detected. Furthermore, the Ca(2+)-regulated binding of mFKBP23 and mBiP in ER can be detected by means of co-immunoprecipitation.