Differential effects of estradiol and estradiol-BSA conjugates.

The steroid 17beta-estradiol (E2) acts to modulate transcription through classical nuclear estrogen receptors (ER-alpha and ER-beta). However, E2 also induces a number of rapid responses (<10 min) within cells, including cells devoid of classical ERs, consistent with the presence of a membrane receptor for E2. Membrane impermeable steroids, typically bovine serum albumin (BSA) conjugates, are commonly used to characterize these non-genomic actions of E2 to exclude the involvement of nuclear ERs. Here we report that E2-BSA conjugate preparations, but not unconjugated E2, activate extracellular signal-regulated protein kinases (ERK1 and ERK2) in the SK-N-SH neuroblastoma cell line, raising concerns regarding the use of these reagents as E2 mimics. Freshly prepared solutions of E2-BSA were found to contain free immunoassayable E2 (iE2), which could be removed by filtration. E2-BSA solutions devoid of free iE2 failed to compete for binding of 125I16alpha-iodo-E2 to ER-alpha or ER-beta. Furthermore, in contrast to E2, E2-BSA conjugates did not bind to ER-alpha or ER-beta as assessed by electrophoretic mobility shift analyses. Protein analysis demonstrated that certain E2-BSA preparations were of very high molecular weight, suggesting extreme protein cross-linking. These findings suggest that E2-BSA does not mimic E2 and is not an appropriate ligand for investigating estrogen receptors. This underscores the need to design stable, cell impermeable analogs of estrogen for the characterization of membrane estrogen receptors.

Pharmacological characterization of soluble human FSH receptor extracellular domain: facilitated secretion by coexpression with FSH.

Follicle-stimulating hormone (FSH) is a member of the glycoprotein hormone family that regulates gametogenesis and steroidogenesis. Glycoprotein hormones signal through a unique class of G-protein-coupled receptors (GPCRs) that have a long extracellular domain (ECD), which is the primary site for hormone binding. The hFSHR-ECD was expressed in insect cells as a C-terminal, epitope-tagged protein resulting in production of soluble active receptor in the intracellular compartment and in the secreted culture medium. Coexpression of hFSHR-ECD with FSHbeta or FSHalpha/beta increased the secretion of the truncated receptor. Pharmacological studies to assess ligand-receptor interactions without the transmembrane domains showed higher affinity values (K(D)S) for [125I]hFSH using mammalian-expressed full-length receptor, secreted hFSHR-ECD, or secreted hFSHR-ECD coexpressed with FSHbeta, whereas the K(D) value for hFSHR-ECD coexpressed with FSHalpha/beta subunits showed lower affinity. Competition of other glycoprotein hormones for secreted hFSHR-ECD coexpressed with FSHbeta or mammalian full-length hFSHR resulted in similar binding profiles, indicating analogous pharmacology. Finally, we have demonstrated that a small molecule, suramin, which has been reported to interact with the mammalian full-length FSHR, competes for the binding of [125I]hFSH by interacting directly at the hFSHR-ECD.

Expression of estrogen receptor-beta protein in rodent ovary.

Estrogen is an essential hormone for the LH surge and ovulation. The primary source of estrogen is from ovarian granulosa cells and in rats, estrogen, in turn, increases granulosa cell number and enhances FSH-stimulated gene expression in these cells. Thus, rat granulosa cells both respond to and synthesize estrogen. To further elucidate the mechanisms mediating the actions of estrogen in granulosa cells, we have identified and characterized the estrogen receptor-beta (ER-beta) subtype in rodent granulosa cells. ER-beta protein was localized to the nuclei of rat granulosa cells in preantral and antral follicles by immunocytochemistry, coincident with the location of ER-beta messenger RNA (mRNA). Immunoprecipitation and Western blot analysis using ER-beta specific antisera demonstrated a protein of approximately 60 kDa in granulosa cells prepared from PMSG-primed immature mice and estrogen-treated immature rats. Extracts from granulosa cells specifically bound an estrogen response element and the complex was recognized by antisera to ER-beta. A synthetic steroid estrogen radioligand, [125I]-17alpha-iodovinyl-11beta-methoxyestradiol ([125I]-VME2), bound to cytosolic granulosa cell preparations with high affinity (estimated K(D) value of 401 +/- 83 pM, and Bmax value of 102 +/- 9 fmol/mg protein). ER-beta protein levels rapidly declined following hCG treatment consistent with the reported decrease in binding activity and ER-beta mRNA levels by high levels of gonadotropins. Overall, we have demonstrated that 1) ER-beta protein is the dominant estrogen receptor subtype present in rodent granulosa cells, 2) this receptor is functional, and 3) it is regulated by ovulatory doses of gonadotropins. Thus, ER-beta is likely to be a mediator of estrogen action in rodent granulosa cells during follicular development.

A novel human estrogen receptor beta: identification and functional analysis of additional N-terminal amino acids.

A novel human estrogen receptor beta (hERbeta) was cloned from human testis mRNA, ovary and thymus cDNA utilizing PCR and 5' RACE methods. The 5' end of hERbeta contained an additional open reading frame, in-frame and upstream of the published clones. hERbeta encodes a protein of 530 amino acids with an approximate molecular weight of 63 kDa and is larger than the previously reported rat, mouse and human protein. To determine the functional role of additional N-terminal amino acids, we compared the transcription functions of receptor lacking (hERbetaT) and receptor containing (hERbetaL) this N-terminal extension. hERbetaL is more active than hERbetaT in transactivating ERE-based reporter genes. hERbetaL, but not hERbetaT, attenuated cytokine mediated NFkappaB activation. Taken together, the additional N-terminal amino acids appear to play a role in modulating estrogen responsive gene expression in vitro.

Induction of promiscuous G protein coupling of the follicle-stimulating hormone (FSH) receptor: a novel mechanism for transducing pleiotropic actions of FSH isoforms.

Under physiological conditions, FSH is secreted into the circulation as a complex mixture of several isoforms that vary in the degree of glycosylation. Although it is well established that the glycosylation of FSH is important for the serum half-life of the hormone and coupling of the receptor to adenylate cyclase, little is known concerning how physiologically occurring glycosylation patterns of this hormone affect receptor signaling. In this study, we have examined the biological activity of deglycosylated human FSH (DeGly-phFSH), recombinant mammalian-expressed hFSH (CHO-hFSH), and insect cell-expressed hFSH (BV-hFSH, alternatively glycosylated) as compared with that of purified human pituitary FSH (phFSH) using a Chinese hamster ovarian cell line stably expressing the hFSH receptor (3D2 cells). Differentially glycosylated forms of FSH did not bind to the FSH receptor in the same manner as phFSH. Although all hormones showed similar potency in competing for [125I]phFSH binding to the hFSH receptor, competition curves for deglycosylated and insect cell-produced FSH were steeper. Similarly, glycosylation of FSH had a profound effect on bioactivity of the hormone. Purified hFSH produced a sigmoidal dose-dependent stimulation in cAMP production, whereas DeGly-phFSH and BV-hFSH induced biphasic (bell-shaped) dose-response curves. BV-hFSH also elicited biphasic effects on steroidogenesis in primary cultures of rat granulosa cells. The cellular response to BV-hFSH was dependent on the degree of receptor-transducer activation. BV-hFSH bioactivity was strictly inhibitory when combined with the ED80 of phFSH. Lower concentrations of phFSH resulted in a gradual shift from inhibition to a biphasic activity in the presence of the ED20 of phFSH. Biphasic responses to BV-hFSH were attributed to activation of different G protein subtypes, since treatment of 3D2 cells with cholera toxin or pertussis toxin differentially blocked the two phases of BV-hFSH bioactivity. These data suggest that alternative glycosylation of FSH leads to a functionally altered form of the hormone. Functionally different hormones appear to convey distinct signals that are transduced by the receptor-transduction system as either stimulatory or inhibitory intracellular events via promiscuous, glycosylation-dependent G protein coupling. Promiscuity in signaling of the FSH receptor, in turn, may represent a potentially novel mechanism for FSH action, whereby the gonad may respond in diverse ways to complex hormonal signals such as those presented by circulating FSH isoforms.

Variant cDNA sequences of human ATP:citrate lyase: cloning, expression, and purification from baculovirus-infected insect cells.

ATP:citrate lyase (ACL) is a major generator of cytosolic acetyl-coenzymeA, which is required for both fatty acid and cholesterol biosynthesis. The human ACL (hACL) cDNA was cloned by RT-PCR, and our results indicate the existence of previously unknown sequence variations in hACL. Expression of the hACL cDNA in Spodoptera frugiperda 9 insect cells resulted in the production of high levels of soluble, active enzyme. The recombinant protein (re-hACL) was purified to homogeneity from the soluble lysate of infected cells and was observed to exist as a tetramer by gel filtration chromatography. Kinetic analyses indicated that the re-hACL and rat ACL have very similar enzymological properties. The facile preparation of milligram quantities of purified, active re-hACL affords the opportunity to characterize the enzyme for structure-based design of hypolipidemic drugs, and to further examine the functional significance of the sequence variations.

Production, purification and characterization of non-myristylated human T-cell protein tyrosine kinase in a baculovirus expression system.

A non-myristylated form (LCK M) of the human T-lymphocyte-specific protein tyrosine kinase (LCK) was produced at high levels in a baculovirus expression system (BVES) using two strategies. First, LCK M was produced by direct expression of a Gly2 --> Ala mutant of LCK. Second, LCK was produced as a glutathione S-transferase (GST) fusion, and LCK M was derived from the fusion protein by cleavage with thrombin. Both recombinant proteins (re-proteins) were produced at 5% of the total protein of infected Spodoptera frugiperda (Sf9) cells and were purified to >95% homogeneity. The enzymatic properties of the re-proteins and their inhibition by protein kinase inhibitors were comparable to the native enzyme (LCK N) derived from Jurkat cells and wild-type LCK derived from the BVES. The high production levels will facilitate the recovery of large quantities of re-protein for use in biochemical and biophysical studies.

p56lck interactions accelerate autophosphorylation and enhance activity toward exogenous substrates.

p56lck, which is a lymphoid-restricted tyrosine kinase, plays a significant role both in activation of mature T lymphocytes and in T cell development. Recombinant human p56lck was expressed in a baculovirus system and purified to > 95% purity. Purified recombinant p56lck was found to have enzymatic characteristics indistinguishable from those of the native enzyme expressed in the Jurkat human T lymphocytic cell line. The comparisons of recombinant and native p56lck were performed using an assay in which the enzyme was immobilized with antibodies onto the wells of a microtiter plate, enabling in situ purification of the native enzyme. Further characterization of the purified recombinant p56lck revealed significantly different enzyme concentration curves for peptide phosphorylation by immobilized and soluble p56lck. In particular, there was very low activity at low concentrations of enzyme assayed in solution. The activity at low enzyme concentrations could be substantially increased by preincubation of high concentrations of enzyme with ATP prior to dilution for the peptide phosphorylation reaction. We examined the relationship between autophosphorylation and activation for peptide phosphorylation. As for peptide phosphorylation, nonlinear enzyme concentration curves were also observed for autophosphorylation in solution, with very low levels of autophosphorylation at low enzyme concentrations. There was a correlation between the enzyme concentration dependency for autophosphorylation and for preincubation with ATP for maximal enhancement of subsequent peptide phosphorylation. The results suggest that autophosphorylation activates p56lck for phosphorylation of exogenous substrates and that high enzyme concentrations accelerate intermolecular autophosphorylation and enzyme activation.

High level expression and purification of the enzymatically active cytoplasmic region of human CD45 phosphatase from yeast.

The cytoplasmic region of human CD45 corresponding to residues 584-1281 was inserted downstream of the alcohol dehydrogenase promoter and transfected into a haploid strain of yeast. Expression of recombinant CD45 in yeast reached as high as 5% of the soluble protein. Following removal of cellular debris by centrifugation and an ammonium sulfate precipitation step, the enzyme was purified using phenyl-Sepharose chromatography, preparative gel filtration, Mono Q anion exchange chromatography and a final analytical gel filtration step. Enzymatically active material with a purity of > or = 98% was obtained with a yield approaching 50%. The final product gave a Km of 5.5 mM and a Vmax of 87.5 U/mg with p-nitrophenylphosphate and a Km and Vmax of 0.167 mM and 185 U/mg, respectively, with a phosphotyrosine peptide. The native enzyme purified from Jurkat cells showed comparable Kms with both substrates to the recombinant enzyme but displayed substantially lower Vmax values for both substrates.

Overexpression, site-directed mutagenesis, and mechanism of Escherichia coli acid phosphatase.

Site-directed mutagenesis was used to examine the catalytic importance of 2 histidine and 4 arginine residues in Escherichia coli periplasmic acid phosphatase (EcAP). The residues that were selected as targets for mutagenesis were those that were also conserved in a number of high molecular weight acid phosphatases from eukaryotic organisms, including human prostatic and lysosomal acid phosphatases. Both wild type EcAP and mutant proteins were overproduced in E. coli using an expression system based on the T7 RNA polymerase promoter, and the proteins were purified to homogeneity. Examination of the purified mutant proteins by circular dichroism and proton NMR spectroscopy revealed no significant conformational changes. The replacement of Arg16 and His17 residues that were localized in a conserved N-terminal RHGXRXP motif resulted in the complete elimination of EcAP enzymatic activity. Critical roles for Arg20, Arg92, and His303 were also established because the corresponding mutant proteins exhibited residual activities that were not higher than 0.4% of that of wild type enzyme. In contrast, the replacement of Arg63 did not cause a significant alteration of the kinetic parameters. The results are in agreement with a previously postulated distant relationship between acid phosphatases, phosphoglycerate mutases, and fructose-2,6-bisphosphatase. These and earlier results are also consistent with the conclusion that 2 histidine residues participate in the catalytic mechanism of acid phosphatases, with His17 playing the role of a nucleophilic acceptor of the phospho group, whereas His303 may act as a proton donor to the alcohol or phenol.

Cloning, expression, and catalytic mechanism of the low molecular weight phosphotyrosyl protein phosphatase from bovine heart.

The first representative of a group of mammalian, low molecular weight phosphotyrosyl protein phosphatases was cloned, sequenced and expressed in Escherichia coli. Using a 61-mer oligonucleotide probe based on the amino acid sequence of the purified enzyme, several overlapping cDNA clones were isolated from a bovine heart cDNA library. A full-length clone was obtained consisting of a 27-bp 5' noncoding region, an open reading frame encoding the expected 157 amino acid protein, and an extensive 3' nontranslated sequence. The identification of the clone as full length was consistent with results obtained in mRNA blotting experiments using poly(A)+ mRNA from bovine heart. The coding sequence was placed downstream of a bacteriophage T7 promoter, and protein was expressed in E. coli. The expressed enzyme was soluble, and catalytically active and was readily isolated and purified. The recombinant protein had the expected Mr of 18,000 (estimated by SDS-PAGE), and it showed cross-reactivity with antisera that had been raised against both the bovine heart and the human placenta enzymes. The amino acid sequence of the N-terminal region of the expressed protein showed that methionine had been removed, resulting in a sequence identical to that of the enzyme isolated from the bovine tissue, with the exception that the N-terminal alanine of the protein from tissue is acetylated. A kinetically competent phosphoenzyme intermediate was trapped from a phosphatase-catalyzed reaction. Using 31P NMR, the covalent intermediate was identified as a cysteinyl phosphate. By analogy with the nomenclature used for serine esterases, these enzymes may be called cysteine phosphatases.

Covalent structure, disulfide bonding, and identification of reactive surface and active site residues of human prostatic acid phosphatase.

The pairing of the half-cysteine residues of human prostatic acid phosphatase was established by proteolytic digestion and analysis of the resulting peptide mixtures by fast atom bombardment mass spectrometry (FAB-MS). An independently derived, full length cDNA clone was used as the basis for the interpretation of the FAB-MS data. The sequence of the native protein is that predicted from the present cDNA sequence, except for the carboxyl-terminal end and some possible post-translational deamidations. Isolated human prostatic acid phosphatase was found to have multiple carboxyl-terminal ends, terminating in Thr, Glu, and Asp, corresponding to residues 349-351 of the 354-residue protein that is predicted from the cDNA sequence after removal of a leader peptide. The protein contains no free sulfhydryl groups. The identical monomer chains of the dimeric native enzyme are found to contain three disulfide bonds, specifically Cys-129 to Cys-340, Cys-183 to Cys-281, and Cys-315 to Cys-319. In view of the conserved positions of cysteines in the homologous human and rat liver lysosomal acid phosphatases, an identical disulfide bonding pattern may be predicted for those proteins. The location of a potential antigenic site was established by selective labeling of proximate tyrosine residues predicted to be on the surface. A conserved RHGXRXP sequence is present in the prostatic, lysosomal, Escherichia coli, and yeast acid phosphatases and is predicted to be of mechanistic significance. In addition, residue Arg-54 is shown to be an active site residue by reaction of the enzyme with phenylglyoxal. Interestingly, this residue is present in a sequence RXRY (R,H) that is also present in lysosomal phosphatase and in recently described protein tyrosine phosphatases.

A novel xylB-based positive selection vector.

Expression of a plasmid-borne Escherichia coli xylulokinase gene (xylB) under the control of the lac promoter yields constitutively high levels of xylulokinase activity. When a plasmid containing this lac-xylB fusion (pLEK100) is transformed into a xylB- mutant the Xyl+ phenotype is restored on xylose-containing media. When the same transformants are plated on xylitol medium, growth inhibition is observed. Positive selection is achieved by cloning DNA into the unique restriction sites of pLEK100, to disrupt xylB expression, transforming E. coli, and then plating transformants on xylitol medium. With this protocol only transformants with insert containing plasmids will be obtained. This results in a considerable reduction in the time and effort needed to construct genomic libraries or perform routine DNA cloning experiments. Three unique sites are available which are suitable for positive selection of DNA fragments, via the disruption of translation (BglII) or transcription (HindIII, SalI, and BglII) of the xylB gene.

Cloning of the Pachysolen tannophilus Xylulokinase Gene by Complementation in Escherichia coli.

The gene coding for xylulokinase has been isolated from the yeast Pachysolen tannophilus by complementation of Escherichia coli xylulokinase (xylB) mutants. Through subcloning, the gene has been localized at one end of a 3.2-kilobase EcoRI-PstI fragment. Expression of the cloned gene was insensitive to glucose inhibition. Furthermore, the cloned gene did not cross-hybridize with E. coli and Saccharomyces cerevisiae xylulokinase genes.

Positive selection vectors based on xylose utilization suppression.

High levels of xylose isomerase activity in wild-type Escherichia coli strains results in a Xyl- phenotype. This phenomenon was exploited for the development of a versatile positive selection system. The xylA promoter was deleted with the exonuclease BAL 31 and the resulting structural gene was inserted into the SmaI site of pUC9, yielding the prototype vector, pLX100. In this construct xylA expression is placed under the transcriptional control of the lac promoter. Transformation of any wild-type E. coli strain with pLX100 results in high levels of xylose isomerase and a Xyl- phenotype. Decreasing the activity below a critical level (approx. 100 u) restores the Xyl+ phenotype. pLX100 contains contiguous restriction sites for HindIII, PstI, BamHI and XhoI, suitable for positive selection cloning experiments. E. coli transformants containing pLX100 cannot grow in minimal medium with xylose unless a DNA fragment is inserted into any one of the unique restriction sites. This makes the plasmid an ideal positive-selection cloning vector.

Cloning and characterization of the xyl genes from Escherichia coli.

Specific xylose utilization mutants of Escherichia coli were isolated that had altered xylose isomerase ( xylA ), xylulokinase ( xylB ), and regulatory ( xylR ) or transport ( xylT ) activities. We screened the Clarke and Carbon E. coli gene bank and one clone, pLC10 -15, was found to complement the xyl mutants we had characterized. Subcloning and DNA restriction mapping allowed us to locate the xylA and xylB genes on a 1.6 kbp Bg/II fragment and a 2.6 kbp HindIII-Sa/I fragment, respectively. The identification and mapping of xyl gene promoters suggest that the xylA and xylB genes are organized as an operon having a single xylose inducible promoter preceding the xylA gene.