Development of a specific radioimmunoassay for the placental folate receptor and related high-affinity folate binding proteins in human tissues

High-affinity membrane-associated and soluble folate binding proteins (FBPs) from human placenta, milk, and KB cells appear to share antigenic determinants [A. C. Antony et al. (1981) J. Biol. Chem. 256, 9684-9692 and (1985) 260, 14911-14917]. Iodination of a highly purified preparation of placental folate receptor (PFR) by various techniques resulted in significant denaturation of the PFR as evidenced by additional peaks of radioactivity on Sephacryl S-200 gel filtration in 1% Triton X-100. These denatured species had similar molecular weights on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as radioiodinated and native PFR, and were also recognized, albeit with less efficiency, by specific rabbit antiserum raised against purified PFR. Since these denatured species failed to bind folate, they were specifically excluded from 125I-PFR by their inability to bind pteroylglutamate-Sepharose. This ws accomplished in a single step by iodination of PFR bound to the affinity column and elution of 125I-PFR under identical conditions that the native PFR was purified. The purified 125I-PFR comigrated with unlabeled PFR on SDS-PAGE and its elution profile on Sephacryl S-200 gel filtration was identical to radioligand bound PFR. The resulting radioimmunoassay standard curve using this affinity chromatography purified 125I-PFR, unlabeled PFR, and anti-human PFR serum had a range for measurement between 5 and 500 ng of PFR and was not affected by the concentration of folate in the sample. The practical utility of this radioimmunoassay for measuring cross-reacting material to the PFR was validated by its ability to quantitate the 40,000 and 160,000 Mr FBPs which are the two major forms of high-affinity FBPs in human tissues.     

Membrane topology and insertion of membrane proteins: search for topogenic signals.

Integral membrane proteins are found in all cellular membranes and carry out many of the functions that are essential to life. The membrane-embedded domains of integral membrane proteins are structurally quite simple, allowing the use of various prediction methods and biochemical methods to obtain structural information about membrane proteins. A critical step in the biosynthetic pathway leading to the folded protein in the membrane is its insertion into the lipid bilayer. Understanding of the fundamentals of the insertion and folding processes will significantly improve the methods used to predict the three-dimensional membrane protein structure from the amino acid sequence. In the first part of this review, biochemical approaches to elucidate membrane protein topology are reviewed and evaluated, and in the second part, the use of similar techniques to study membrane protein insertion is discussed. The latter studies search for signals in the polypeptide chain that direct the insertion process. Knowledge of the topogenic signals in the nascent chain of a membrane protein is essential for the evaluation of membrane topology studies.     

Sensitivity to Hsp90-targeting drugs can arise with mutation to the Hsp90 chaperone, cochaperones and plasma membrane ATP binding cassette transporters of yeast.

The Hsp90 molecular chaperone catalyses the final activation step of many of the most important regulatory proteins of eukaryotic cells. The antibiotics geldanamycin and radicicol act as highly selective inhibitors of in vivo Hsp90 function through their ability to bind within the ADP/ATP binding pocket of the chaperone. Drugs based on these compounds are now being developed as anticancer agents, their administration having the potential to inactivate simultaneously several of the targets critical for counteracting multistep carcinogenesis. This investigation used yeast to show that cells can be rendered hypersensitive to Hsp90 inhibitors by mutation to Hsp90 itself (within the Hsp82 isoform of yeast Hsp90, the point mutations T101I and A587T); with certain cochaperone defects and through the loss of specific plasma membrane ATP binding cassette transporters (Pdr5p, and to a lesser extent, Snq2p). The T101I hsp82 and A587T hsp82 mutations do not cause higher drug affinity for purified Hsp90 but may render the in vivo chaperone cycle more sensitive to drug inhibition. It is shown that these mutations render at least one Hsp90-dependent process (deactivation of heat-induced heat shock factor activity) more sensitive to drug inhibition in vivo.     

Localization of basic residues required for receptor binding to the single alpha-helix of the receptor binding domain of human alpha2-macroglobulin.

To better understand the structural basis for the binding of proteinase-transformed human alpha2-macroglobulin (alpha2M) to its receptor, we have used three-dimensional multinuclear NMR spectroscopy to determine the secondary structure of the receptor binding domain (RBD) of human alpha2M. Assignment of the backbone NMR resonances of RBD was made using 13C/15-N and 15N-enriched RBD expressed in Escherichia coli. The secondary structure of RBD was determined using 1H and 13C chemical shift indices and inter- and intrachain nuclear Overhauser enhancements. The secondary structure consists of eight strands in beta-conformation and one alpha-helix, which together comprise 44% of the protein. The beta-strands form three regions of antiparallel beta-sheet. The two lysines previously identified as being critical for receptor binding are located in (Lys1374), and immediately adjacent to (Lys1370) the alpha-helix, which also contains an (Arg1378). Secondary structure predictions of other alpha-macroglobulins show the conservation of this alpha-helix and suggest an important role for this helix and for basic residues within it for receptor binding.     

Ca binding to the human red cell membrane: characterization of membrane preparations and binding sites.

Inside out and right side out vesicles were used to study the sidedness of Ca binding to the human red cell membrane. It was shown that these vesicles exhibited only a limited permeability to Ca, enabling the independent characterization of Ca binding to the extracellular and cytoplasmic membrane surfaces...     

Mapping the human membrane proteome: a majority of the human membrane proteins can be classified according to function and evolutionary origin

Background  

Membrane proteins form key nodes in mediating the cell's interaction with the surroundings, which is one of the main reasons why the majority of drug targets are membrane proteins.     

Structure and evolution of insulins: implications for receptor binding.

Insulin is a member of a family of hormones, growth factors and neuropeptides which are found in both vertebrates and invertebrates. A common 'insulin fold' is probably adopted by all family members. Although the specificities of receptor binding are different, there is a possibility of co-evolution of polypeptides and their receptors.     

Sequence requirements for membrane assembly of polytopic membrane proteins: molecular dissection of the membrane insertion process and topogenesis of the human MDR3 P-glycoprotein.

The biogenesis of membrane proteins with a single transmembrane (TM) segment is well understood. However, understanding the biogenesis and membrane assembly of membrane proteins with multiple TM segments is still incomplete because of the complexity and diversity of polytopic membrane proteins. In an attempt to investigate further the biogenesis of polytopic membrane proteins, I used the human MDR3 P-glycoprotein (Pgp) as a model polytopic membrane protein and expressed it in a coupled cell-free translation/translocation system. I showed that the topogenesis of the C-terminal half MDR3 Pgp molecule is different from that of the N-terminal half. This observation is similar to that of the human MDR1 Pgp. The membrane insertion properties of the TM1 and TM2 in the N-terminal half molecule are different. The proper membrane anchorage of both TM1 and TM2 of the MDR3 Pgp is affected by their C-terminal amino acid sequences, whereas only the membrane insertion of the TM1 is dependent on the N-terminal amino acid sequences. The efficient membrane insertion of TM3 and TM5 of MDR3 Pgp, on the other hand, requires the presence of the putative TM4 and TM6, respectively. The TM8 in the C-terminal half does not contain an efficient stop-transfer activity. These observations suggest that the membrane insertion of putative TM segments in the human MDR3 Pgp does not simply follow the prevailing sequential event of the membrane insertion by signal-anchor and stop-transfer sequences. These results, together with my previous findings, suggest that different isoforms of Pgp can be used in comparison as a model system to understand the molecular mechanism of topogenesis of polytopic membrane proteins.     

Partial primary structure of the 48- and 90-kilodalton core proteins of cell surface-associated heparan sulfate proteoglycans of lung fibroblasts. Prediction of an integral membrane domain and evidence for multiple distinct core proteins at the cell surface of human lung fibroblasts

Heparitinase treatment of cell surface-associated heparan sulfate proteoglycans (HSPG) of human lung fibroblasts reveals core proteins with apparent Mr values of 125,000, 90,000, 64,000, 48,000 and 35,000 (Lories, V., De Boeck, H., David, G., Cassiman, J.-J., and Van den Berghe, H. (1987) J. Biol. Chem. 262, 854-859). The 90- and 48-kDa core proteins share the epitope of the monoclonal antibody 6G12 which was used to screen a human lung fibroblast expression cDNA library. Rescreening of the libraries yielded clone 48K5 with an insert of 3439 base pairs. Polyclonal antibodies were raised in rabbits against a fragment of the protein encoded by the 48K5 cDNA different from the part carrying the 6G12 epitope. These antibodies specifically recognize the 90- and 48-kDa core proteins on Western blots of total cellular extracts of human lung fibroblast HSPG. The specific reactivity of the polyclonal antiserum confirms the identity of the 48K5 clone and further distinguishes the 48- and the 90-kDa core proteins, which do share the 6G12-defined epitope and at least one additional antigenic determinant with the 48K5 cDNA-encoded protein, from the 125-, 64-, and 35-kDa core proteins of cell surface HSPG of human lung fibroblasts which do not react with either antibody preparation. The protein encoded by the 48K5 clone contains a stop-transfer sequence indicative of an integral membrane protein and three potential glycosaminoglycan attachment sites. The 48K5 clone detects two major poly(A)+ RNA species in human lung fibroblasts presumably generated by the use of alternative polyadenylation signals. The 48K5 gene was mapped to chromosome 8q23 by in situ hybridization and hybridization to DNA of somatic cell hybrids.     

Mutational analysis of the human KDEL receptor: distinct structural requirements for Golgi retention, ligand binding and retrograde transport.

The KDEL receptor is a seven-transmembrane-domain protein that is responsible for the retrieval of endoplasmic reticulum (ER) proteins from the Golgi complex. It is a temporary resident of the Golgi apparatus: upon binding a KDEL-containing ligand, it moves to the ER, where the ligand is released. We have expressed mutant forms of the human receptor in COS cells and examined their intracellular locations and ligand-binding capacities. We show that ligand binding is dependent on charged residues within the transmembrane domains. Surprisingly, retrograde transport of occupied receptor is unaffected by most mutations in the cytoplasmic loops, but is critically dependent upon an aspartic acid residue in the seventh transmembrane domain. Retention in the Golgi apparatus requires neither ligand binding nor this aspartate residue, and thus is independent of receptor recycling. We suggest that movement of the receptor is controlled by conformational changes and intermolecular interactions within the membrane bilayer.     

Binding of selected iodothyronine analogues to receptor sites of isolated rat hepatic nuclei. High correlation between structural requirements for nuclear binding and biological activity.

The limited capacity, high affinity binding of 35 iodothyronine analogues by rat liver nuclei has been examined in an in vitro system. The in vitro nuclear binding of all the analogues tested was highly correlated with their published thyromimetic potencies in the intact animals. Binding and biological activity are greater for 3'-mono-than 3',5'-di-substituted iodothyronines. A 4'-hydroxyl group is essential, but the 3' substituent can be several halogen or non-halogen groups for which the distal conformation is preferred. The ether linkage can be replaced equally well by a methylene or sulfur group. The presence of both 3 and 5 groups which are limited to halogens or small alkyl groups are necessary for the maintenance of significant activity. Halogen-free iodothyronines have very low, but significant activity both in vitro and in vivo. The data provide information on the structural requirements for thyroid hormone action and further support the physiological relevance of the nuclear sites.     

Fusion properties of cells infected with human parainfluenza virus type 3: receptor requirements for viral spread and virus-mediated membrane fusion.

Cells can be persistently infected with human parainfluenza virus type 3 (HPF3) by using a high multiplicity of infection (MOI) (> or = 5 PFU per cell). The persistently infected cells exhibit no cytopathic effects and do not fuse with each other, yet they readily fuse with uninfected cells. We have previously shown that the failure of the persistently infected cells to fuse with each other is due to the lack of a receptor on these cells for the viral hemagglutinin-neuraminidase glycoprotein, and we have established that both fusion and hemagglutinin-neuraminidase proteins are needed for cell fusion mediated by HPF3. We then postulated that the generation of persistent infection and the failure of cells infected with HPF3 at high MOI to form syncytia are both due to the action of viral neuraminidase in the high-MOI inoculum. In this report, we describe experiments to test this hypothesis and further investigate the receptor requirements for HPF3 infection and cell fusion. A normally cytopathic low-MOI HPF3 infection can be converted into a noncytopathic infection by the addition of exogenous neuraminidase, either in the form of a purified enzyme or as UV-inactivated HPF3 virions. Evidence is presented that the receptor requirements for an HPF3 virus particle to infect a cell are different from those for fusion between cells. By treating infected cells in culture with various doses of neuraminidase, we demonstrate that virus spreads from cell to cell in the complete absence of cell-cell fusion. We compare the outcome of HPF3 infection in the presence of excess neuraminidase with that of another paramyxovirus (simian virus 5) and provide evidence that these two viruses differ in their receptor requirements for mediating fusion.     

Donor-acceptor tetrahydrochrysenes, inherently fluorescent, high-affinity ligands for the estrogen receptor: binding and fluorescence characteristics and fluorometric assay of receptor.

We have examined the binding behavior and fluorescence characteristics of a series of novel ligands for the estrogen receptor (ER). These ligands are derivatives of 5,6,11,12-tetrahydrochrysene (THC), a structure that embodies a stilbene chromophore, found in many nonsteroidal estrogens, within a rigid tetracyclic system where it cannot easily be distorted from planarity, thus providing the conjugation and rigidity required for efficient fluorescence. Additional steric bulk, as trans-disposed ethyl substituents at the internal C-5 and C-11 positions, is required for the highest relative binding affinity (RBA), and the trans-5,11-diethyl-2,8-dihydroxy-THC derivative binds to ER with an affinity greater than that of estradiol. The replacement of one of the phenolic hydroxyl groups of this THC derivative with an electron-withdrawing group (COMe, COOMe, CONH2, CN, or NO2) yields unsymmetrical THCs with binding affinities 15-40% that of estradiol (E2). The fluorescence emission shifts from about 380 nm for the dihydroxy THC to 475-688 nm for the donor-acceptor THCs. The emission of these donor-acceptor THCs is highly solvatochromic and shifts to longer wavelengths as the solvent polarity increases. In ethanol, the fluorescence quantum yield of the first four of these compounds is high (phi f = 0.43-0.69), but the fifth compound, the nitro-THC, is almost nonemissive in protic solvents. When they are incubated with protein solutions containing ER (approximately 10(-9) M), the emission from the donor-acceptor THCs bound specifically to ER is in the 500-570-nm range, whereas fluorescence from non-receptor-bound fluorophores is in the 425-460-nm range. Thus, fluorescence from these probes bound specifically to ER could be measured under equilibrium conditions as well as after the removal of free and non-receptor-bound material by treatment with charcoal-dextran. This is one of the first demonstrations of ligands whose fluorescence is distinctly different when free, when bound to ER, or when bound to non-receptor proteins. It is also the first demonstration of ER assay by fluorescence under equilibrium conditions.     

Binding of insulin to solubilized insulin receptor from human placenta. Evidence for a single class of noninteracting binding sites

Binding of 125I-insulin to insulin receptor was studied at equilibrium using plasma membrane protein, from human placenta, solubilized in Triton X-100. Measured at 1 nM 125I-insulin, the amount of radioligand bound specifically was dependent upon pH, the optimal association at 4 degrees C occurring in the pH range 8.2-8.7. The Adair equation was employed for analysis of binding isotherms generated at several pH values for concentrations of radioligand varying from 0.01-8.0 nM. Association constants derived from these analyses showed the same pH dependence described above and were found to be independent of receptor concentration. Hill plots derived from these titrations consistently yielded Hill coefficients of 1 and Scatchard plots gave virtually straight lines. Close correspondence was found between theoretical analyses and observations under all experimental conditions and graphical methods employed. These results are consistent with a single class of noninteracting 125I-insulin binding sites on the solubilized insulin receptor.     

A high-affinity receptor for urokinase plasminogen activator on human keratinocytes: characterization and potential modulation during migration.

Low passage cultures of normal human keratinocytes produce several components of the plasminogen activator/plasmin proteolytic cascade, including urokinase plasminogen activator (uPA), tissue plasminogen activator (tPA), and two specific inhibitors. Studies here presented demonstrate that these cells also contain a high-affinity (Kd = 3 x 10(-10) M) plasma membrane-binding site for uPA. High molecular weight uPA, either as the single-chain precursor or two-chain activated form, bound to the receptor; however, low molecular weight (33 kD) uPA, tPA, or epidermal growth factor did not compete for binding, demonstrating specificity. Acid treatment, which removed endogenous uPA from the receptor, was required to detect maximal binding (45,000 sites per cell). To investigate the possibility that the uPA receptor on keratinocytes may be involved in epithelial migration during wound repair, cultures were wounded and allowed to migrate into the wounded site. Binding sites for uPA were localized by autoradiographic analysis of 125I-uPA binding as well as by immunocytochemical studies using anti-uPA IgG. With both techniques uPA binding sites were detected selectively on the plasma membrane of cells at the leading edge of the migrating epithelial sheet. This localization pattern suggests that uPA receptor expression on keratinocytes may be coupled to cell migration during cutaneous wounding.     

Pharmacological and signaling analysis of human chemokine receptor CCR-7 stably expressed in HEK-293 cells: high-affinity binding of recombinant ligands MIP-3beta and SLC stimulates multiple signaling cascades.

The chemokine receptor CCR-7 is expressed in T, NK, and dendritic cells in a time-ordered and stimulus-dependent manner. Thorough analyses of the pharmacological profiles of the recombinant ligands for CCR-7, MIP-3beta/ELC/CK-beta 11, and SLC/Exodus-2/TCA4/6C-kine, using CCR-7-expressing HEK-293E transfectants determine that ligands both bind with a K(d) in the 100 pM range-10- to 100-fold greater affinities than published K(d) values. High-affinity binding of each ligand is associated with rapid mobilization of intracellular calcium and cell migration as predicted for chemokine GPCRs, and in keeping with more recent evidence, robust activation of mitogen-activated protein kinase (MAPK).     

iLoc-Hum: using the accumulation-label scale to predict subcellular locations of human proteins with both single and multiple sites

Although numerous efforts have been made for predicting the subcellular locations of proteins based on their sequence information, it still remains as a challenging problem, particularly when query proteins may have the multiplex character, i.e., they simultaneously exist, or move between, two or more different subcellular location sites. Most of the existing methods were established on the assumption: a protein has one, and only one, subcellular location. Actually, recent evidence has indicated an increasing number of human proteins having multiple subcellular locations. This kind of multiplex proteins should not be ignored because they may bear some special biological functions worthy of our attention. Based on the accumulation-label scale, a new predictor, called iLoc-Hum, was developed for identifying the subcellular localization of human proteins with both single and multiple location sites. As a demonstration, the jackknife cross-validation was performed with iLoc-Hum on a benchmark dataset of human proteins that covers the following 14 location sites: centrosome, cytoplasm, cytoskeleton, endoplasmic reticulum, endosome, extracellular, Golgi apparatus, lysosome, microsome, mitochondrion, nucleus, peroxisome, plasma membrane, and synapse, where some proteins belong to two, three or four locations but none has 25% or higher pairwise sequence identity to any other in the same subset. For such a complicated and stringent system, the overall success rate achieved by iLoc-Hum was 76%, which is remarkably higher than that by any of the existing predictors that also have the capacity to deal with this kind of system. Further comparisons were also made via two independent datasets; all indicated that the success rates by iLoc-Hum were even more significantly higher than its counterparts. As a user-friendly web-server, iLoc-Hum is freely accessible to the public at or . For the convenience of most experimental scientists, a step-by-step guide is provided on how to use the web-server to get the desired results by choosing either a straightforward submission or a batch submission, without the need to follow the complicated mathematical equations involved.     

SecA protein needs both acidic phospholipids and SecY/E protein for functional high-affinity binding to the Escherichia coli plasma membrane.

Translocation of preproteins across the Escherichia coli inner membrane requires acidic phospholipids. We have studied the translocation of the precursor protein proOmpA across inverted inner membrane vesicles prepared from cells depleted of phosphatidylglycerol and cardiolipin. These membranes support neither translocation nor the translocation ATPase activity of the SecA subunit of preprotein translocase. We now report that inner membrane vesicles which are depleted of acidic phospholipids are unable to bind SecA protein with high affinity. These membranes can be restored to translocation competence by fusion with liposomes containing phosphatidylglycerol, suggesting that the defect in SecA binding is a direct effect of phospholipid depletion rather than a general derangement of inner membrane structure. Reconstitution of SecY/E, the membrane-embedded domain of translocase, into proteoliposomes containing predominantly a single synthetic acidic lipid, dioleoylphosphatidylglycerol, allows efficient catalysis of preprotein translocation.     

Ca(2+)-dependent translocation of cytosolic proteins to isolated granule subpopulations and plasma membrane from human neutrophils.

In order to identify cytosolic proteins involved in control of granule exocytosis in human neutrophils, subcellular fractions enriched in each of the 3 major granule subsets were incubated with cytosol from neutrophils in the presence or absence of Ca2+. After washing, proteins were eluted from the organelles by EGTA. Annexins I, II, IV and VI were found to bind to all organelles studied. In addition, a 28-kDa protein was found to bind exclusively to plasma membranes and secretory vesicles, the most readily exocytosed organelle of neutrophils. Ca(2+)-dependent association of cytosolic proteins to different granule subsets may control differential exocytosis of granules.