Analysis of glucocorticoid receptor subspecies binding to DNA-cellulose and isolated nuclei

Conversion of the glucocorticoid receptor into a DNA-binding protein results in the generation of several distinct receptor subspecies (peaks A-E) which can be resolved by anion exchange chromatography. In vitro, the fraction of the receptor population (approx. 40%) which gains a capacity to bind DNA-cellulose is preferentially transformed into the peak A species by a process that was enhanced by the presence of KCl. At 0.4 M KCl, virtually all of the DNA-binding receptor was in the peak A form. Isolated nuclei also exhibit a receptor binding profile similar to that observed with DNA-cellulose.     

Characteristics of the nuclear translocation of progesterone receptor in fetal guinea pig uterus "in vivo", "in vitro" and in organ culture

The translocation of progesterone receptor from the cytosol into the nucleus was studied under "in vivo" and "in vitro" conditions in the uteri of guinea pig fetuses exposed to progesterone or a synthetic progestin, R5020. Progesterone treatment of estrogen-primed fetuses leads to a rapid (before 1h) transfer of cytosol progesterone receptor into the nucleus which is, however, short-lived (less than 3h). A rapid decrease in the retention of the estrogen receptor in the nucleus also occurs. In the "in vitro" incubations of whole fetal uteri, translocation of progesterone receptor is temperature-dependent and specific for progesterone and R5020; estradiol and cortisol have no effect. Putative progesterone receptors can also be induced in explants of fetal guinea pig uteri in organ culture which translocate from the cytosol into the nucleus under the same "in vitro" conditions as in whole uteri. Fetal uterine progesterone receptor, either stimulated "in vivo" by estrogen-priming or induced in organ culture, translocates from the cytosol into the nucleus and this process seems to be accompanied by a decrease in retention of the estrogen receptor in the nucleus which appears to be the mechanism by which progesterone antagonises estrogen action in fetal guinea pig uterus.     

Transformed mouse glucocorticoid receptor: generation and interconversion of the 3.8S, monomeric and 5.2S, oligomeric species

Recent studies have implicated subunit dissociation as a possible mechanism of glucocorticoid receptor transformation [Vedeckis, W.V. (1983) Biochemistry 22, 1983-1989; Raaka, B.M., & Samuels, H.H. (1983) J. Biol. Chem. 258, 417-425]. While it is becoming increasingly evident that the untransformed (non-nuclear-binding and non-DNA-binding) glucocorticoid receptor from mouse AtT-20 cells is a 9.1S oligomeric species (Mr 290 000-360 000), two transformed species have been described for this receptor. One of these has a sedimentation coefficient of 5.2 S (on molybdate-containing gradients), while the smallest nonproteolyzed, monomeric subunit is 3.8 S. The present study was undertaken to determine which is the most common form generated both in vitro and in vivo and the structural relationship between these two forms. A wide variety of in vitro transformation protocols all yielded the 5.2S form when analyzed on molybdate-containing sucrose gradients by using a vertical tube rotor. Kinetic studies showed that the appearance of the 5.2S form coincided precisely with the appearance of transformed receptor, as defined by DEAE-cellulose elution. Furthermore, when the 3.8S and 5.2S peaks were collected from sucrose gradients directly, they were transformed receptors as defined by both DEAE-cellulose and DNA-cellulose chromatography, while the 9.1S sucrose gradient peak was untransformed when the same criteria were used. The 3.8S monomer, when isolated from high-salt sucrose gradients and then desalted, reverted to the 5.2S form (molybdate-containing gradients) or a 6.6S form (low-salt, molybdate-free gradients).(ABSTRACT TRUNCATED AT 250 WORDS)     

A possible role for dephosphorylation in glucocorticoid receptor transformation

Addition of bovine intestinal alkaline phosphatase to mouse AtT-20 cell cytosol increases the rate of glucocorticoid receptor transformation, as evidenced by a change in sedimentation rate from 9.1S to 5.2S. Acid phosphatases are completely ineffective in this regard. Alkaline phosphatase-promoted receptor transformation is both time- and dose-dependent. A variety of phosphatase inhibitors are effective in inhibiting this process, the most potent being transition metal oxyanions such as molybdate, tungstate, and arsenate. The ability of the various inhibitors to suppress alkaline phosphatase-promoted receptor transformation does not correspond well with their potencies for inhibiting para-nitrophenyl phosphate hydrolysis. However, a better correspondence between the inhibition of endogenous receptor transformation and total cytosolic phosphatase activity is observed, and both sodium fluoride and glucose-1-phosphate inhibit endogenous receptor transformation. The protease inhibitors phenyl-methylsulfonyl fluoride and antipain have no effect on receptor transformation. Surprisingly, leupeptin is effective in inhibiting alkaline phosphatase-promoted receptor transformation. Although this raises the possibility of a contaminating protease activity in the alkaline phosphatase enzyme preparation, treatment of covalently affinity-labeled receptor with the enzyme shows no proteolysis of the receptor or any other non-specifically labeled cytosolic protein. Thus, it is possible that a novel action of leupeptin, unrelated to its protease-inhibitory activity, may be involved in the suppression of receptor transformation. The studies presented here suggest that dephosphorylation of some component in cytosol is involved in the destabilization of receptor subunit interactions, resulting in glucocorticoid receptor transformation.     

Quantification of estrogen- and progesterone receptors from cryostat sectioned human tumor tissue: comparison of ligand binding and immunocytochemical assays

We have recently described an assay procedure to measure estrogen and progesterone receptors in extracts from frozen sections by a ligand binding assay. With this methodological approach it is now possible to perform comparative experiments not only to DCC/Scatchard analyses from different tissue blocks, but also to immunocytochemical determinations in identical tissue blocks: (1) When receptor quantities measured by the two biochemical methods were compared, a high correlation of estrogen receptor content was found between determinations in supernatants from frozen sections and DCC/Scatchard analyses. A slightly poorer correlation in the comparison of the ligand binding assays was obtained for the progesterone receptor. (2) The percentage of tumor cells stainable by immunocytochemistry for estrogen and progesterone receptors could hardly be correlated to the receptor concentrations (fmol/mg) measured quantitatively by the two ligand binding assays. (3) As the final result tumor specimen could be grouped into classes of receptor status according to the presence or absence of a nuclear stain in immunocytochemical assays or according to receptor concentrations above or below distinct threshold which were fixed at 20 fmol/mg for Scatchard analyses of both receptor species, 20 fmol/mg for estrogen- and 40 fmol/mg for progestin receptors for the assay with sections. In this diagnostical consideration the concordance of both biochemical methods to the immunocytochemical assessment was high for estrogen and less pronounced for progesterone receptors. (4) In some breast cancer specimen analyzed biochemically an unspecific progestin binding component could be detected superimposed on the progesterone receptor peak after isoelectric focusing.     

Estimation of both the potency and efficacy of alpha7 nAChR agonists from single-concentration responses

The assessment of functional properties is a crucial step in the screening of potential new drug candidates. The development of moderate to high throughput electrophysiological recording systems such as OpusXpress (Molecular Devices) has facilitated the process of testing new drugs to a large degree. However, while the simple screening of multiple drugs at a single concentration identifies "hits" and "misses", the generation of full concentration-response studies is still a bottleneck in drug development. The alpha7 nicotinic acetylcholine receptor displays a unique concentration dependence of response kinetics which permits estimates of EC50 and Imax values for experimental drugs to be generated from single-concentration responses. This method is based on the analysis of 13 different concentration-response studies utilizing either human or rat alpha7 nAChR. Each experimental response was first normalized to an ACh control, and then a transformation of the pooled data was generated which, based on the relationship between the net charge and peak current to their respective EC50 values defined the "functional concentration" (the test concentration relative to the EC50 for the given agonist). At low functional concentrations, net charge is large relative to peak current amplitude and at higher functional concentration this relationship reverses. For any single-concentration response, the ratio of net charge to peak current can be used to estimate functional concentration. Efficacy can then be estimated by comparing the observed (net charge) response to the expected value for a full agonist at the estimated functional concentration. This extended analysis, combined with automated recording methods, should greatly increase the efficiency with which promising new drug candidates can be characterized.     

Characterization of steroid hormone receptors with ion-exchange fast protein liquid chromatography

Androgen, estrogen and progesterone receptors have been characterized with anion exchange Fast Protein Liquid Chromatography (FPLC) on a Mono Q column (Pharmacia). In the presence of sodium molybdate androgen receptors in cytosols from rat prostate, rat epididymis and calf uterus eluted as a single sharp peak at 0.32 M NaCl with recoveries of approx 90%. The molybdate-stabilized form of the androgen receptor from rat prostate was purified about 75-fold. The receptor containing FPLC-peak fractions sedimented in high salt (0.4 M KCl) linear sucrose gradients at 3.6 S (prostate) and at 4.6 S (epididymis and calf uterus) respectively. Multiple forms of the androgen receptor were present in cytosols from rat prostate prepared in the absence of sodium molybdate, probably due to proteolytic breakdown of the native form. Calf uterine estradiol and progesterone receptors prepared in the presence of sodium molybdate (20 mM) eluted from the Mono Q column at 0.32 M NaCl. The molybdate-stabilized forms of the oestradiol and progesterone receptors were purified approx 70-fold and 30-fold respectively. In the absence of molybdate the estradiol receptor dissociated into two major forms eluting at 0.23 M NaCl and 0.37 M NaCl. After heat induced transformation (30 min at 25 degrees C) of the estradiol receptor one major peak was eluted at 0.42 M NaCl, indicating a change in the surface charge of the estradiol receptor as a result of the 4 S to 5 S transformation. It is concluded that the FPLC anion exchange system is a powerful, fast tool for characterization and partial purification of steroid receptors. In addition this technique could be applied as a rapid procedure for the quantitative estimation of steroid receptors in small biological samples.     

Selective amplification of endothelin-stimulated inositol 1,4,5-trisphosphate and calcium signaling by v-src transformation of rat-1 fibroblasts.

The effects of the expression of the protein tyrosine kinase pp60v-src on endothelin- and thrombin-stimulated inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) production and calcium responses were investigated in Rat-1 fibroblasts. The ability of endothelin-1 to induce the accumulation of these second messengers was dramatically amplified by v-src transformation, with 6- and 3-fold enhancements of the peak Ins(1,4,5)P3 and peak calcium responses, respectively. In contrast, thrombin-dependent responses were slightly reduced following v-src transformation, demonstrating that the augmentation of endothelin-stimulated signal transduction is a selective effect. The magnitude of the stimulated accumulation of Ins(1,4,5)P3 presumably depends upon both the functional activation of phospholipase C to produce Ins(1,4,5)P3, and the activity of the enzymes that metabolize Ins(1,4,5)P3. Although the metabolism of Ins(1,4,5)P3 was strikingly altered by expression of pp60v-src, with a bias towards the production of higher inositol polyphosphates that is consistent with an activated Ins(1,4,5)P3 3-kinase, this change could not account for the marked increase in endothelin-stimulated signaling induced by v-src transformation. This suggests that an effect of pp60v-src is expressed at the level of the plasma membrane, through an interaction with one or more components in the receptor/guanine nucleotide binding protein (G protein)/phospholipase C system that transduces the endothelin signal into Ins(1,4,5)P3 production. Preparation of membranes from normal and v-src-transformed cells showed that, while there was no change in the number of high-affinity endothelin binding sites, the release of Ins(1,4,5)P3 in response to guanine nucleotides and endothelin-1 was significantly increased following v-src transformation. In contrast, the Ins(1,4,5)P3 responses to thrombin and high Ca2+ concentrations were unaffected by transformation. Thus the selective interactions within the G protein system that couples the endothelin receptor to phospholipase C are potential sites at which the v-src transformation process may act to amplify endothelin-dependent Ins(1,4,5)P3 production.     

Characterization of the nontransformed and transformed androgen receptor and heat shock protein 90 with high-performance hydrophobic-interaction chromatography

The hydrophobicity of the nontransformed and transformed androgen receptor from rat submandibular gland and heat shock protein 90 (hsp90) from rat submandibular gland and liver was characterized by using high-performance hydrophobic-interaction chromatography on TSK gel Ether-5PW. In the absence of molybdate, cytosol [3H]R1881-androgen receptor complexes were mainly eluted in the 1.3 M region (Peak 1) with a small peak in the 0.8 M region (Peak 2) of a descending salt gradient (2 to 0 M) of ammonium sulfate. In the presence of molybdate, Peak 2 was predominant. When labeled-cytosol was applied after being heated at 25 degrees C for 30 min, a third peak (Peak 3) at around 0.64 M ammonium sulfate was newly observed. Peaks 2 and 3 were observed, while Peak 1 completely disappeared with the labeled-cytosol precipitated at 40% saturated ammonium sulfate. The Stokes radius of Peak 1 was 7 nm, and of Peak 2 was 8 nm. Both peaks were retained poorly by DNA-cellulose but bound rather well to DEAE-cellulose. These results suggest that these two peaks represent the nontransformed receptor, indicating that there are isoforms of the nontransformed androgen receptor which are distinguished by their hydrophobic properties and Stokes radii. Peak 3 had a Stokes radius of 5 nm and preferentially bound to DNA-cellulose, suggesting that this peak corresponds to the transformed receptor. These results indicated that the transformation of the androgen receptor accompanies the enrichment of the hydrophobicity of the receptor molecule. Hsp90 purified from rat livers and hsp90 in the cytosol both from livers and submandibular glands were eluted from Ether-5PW at 0.8 M ammonium sulfate, at almost the same position as Peak 2. This finding suggests that the enrichment of hydrophobicity on transformation is due to dissociation of hsp90 from the nontransformed androgen receptor.     

Role of thyroglobulin endocytic pathways in the control of thyroid hormone release

Thyroglobulin (Tg), the thyroid hormoneprecursor, is synthesized by thyrocytes and secreted into the colloid.Hormone release requires uptake of Tg by thyrocytes and degradation inlysosomes. This process must be precisely regulated. Tg uptake occursmainly by micropinocytosis, which can result from both fluid-phasepinocytosis and receptor-mediated endocytosis. Because Tg is highlyconcentrated in the colloid, fluid-phase pinocytosis or low-affinityreceptors should provide sufficient Tg uptake for hormone release;high-affinity receptors may serve to target Tg away from lysosomes,through recycling into the colloid or by transcytosis into thebloodstream. Several apical receptors have been suggested toplay roles in Tg uptake and intracellular trafficking. A thyroidasialoglycoprotein receptor may internalize and recycle immature formsof Tg back to the colloid, a function also attributed to an as yetunidentified N-acetylglucosamine receptor. Megalin mediatesTg uptake by thyrocytes, especially under intense thyroid-stimulatinghormone stimulation, resulting in transcytosis of Tg from the colloidto the bloodstream, a function that prevents excessive hormone release.

     

Chromatographic separation of nontransformed and transformed glucocorticoid-receptor complexes from rat liver cytosol. Use of tungstate in the purification, inactivation, and resolution of receptor forms

Aliquots of rat liver cytosol glucocorticoid-receptor complexes (GRc) were transformed by an incubation with 8-10 mM ATP at 0 degrees C and were compared with those transformed by an exposure to 23 degrees C. The extent of receptor transformation was measured by chromatography of the samples over columns of DEAE-Sephacel. The ATP-transformed complexes, like those which were heat-transformed, exhibited lower affinity for the positively charged ion-exchange resin and were eluted with 0.12 M KCl (peak-I): the nontransformed complexes appeared to possess higher affinity and required 0.21 M KCl (peak II) for their elution. As expected, the receptor in the peak-I exhibited the DNA-cellulose binding capacity and sedimented as 4S in sucrose gradients. Peak II contained an 8-9S glucocorticoid receptor (GR) form that showed reduced affinity for DNA-cellulose. Presence of sodium tungstate (5 mM) prevented both heat and ATP transformation of the GRc resulting in the elution of the complexes in the region of nontransformed receptors. When parallel experiments were performed, binding of the cytosol GRc to rat liver nuclei or DNA-cellulose was seen to increase 10-15 fold upon transformation by heat or ATP: tungstate treatment blocked this process completely. The transformed and nontransformed GRc were also differentially fractionated by (NH4)2SO4: tungstate-treated (nontransformed) receptor required higher salt concentration and was precipitated at 55% saturation. In addition, the GRc could be extracted from DNA-cellulose by an incubation of the affinity resin with sodium tungstate resulting in approximately 500-fold purification of the receptor with a 30% yield. These studies show that the nontransformed, and the heat-, salt-, and ATP-transformed GRc from the rat liver cytosol can be separated chromatographically, and that the use of tungstate facilitates the resolution of these different receptor forms. In addition, extraction of the receptor from DNA-cellulose by tungstate provides another new and efficient method of partial receptor purification.     

Solubilization and affinity purification of the Y2 receptor for neuropeptide Y and peptide YY from rabbit kidney.

Neuropeptide Y (NPY) is an important neuropeptide in both central and peripheral neurones whereas peptide YY (PYY) is a gut hormone present in endocrine cells in the lower bowel. Both peptides interact with multiple binding sites that have been further classified into Y1 and Y2 receptors. We have solubilized native Y2 receptors both from basolateral membranes of proximal convoluted tubules from rabbit kidney and from rat hippocampal membranes. Solubilization of functional Y2 receptors was obtained with both 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and digitonin and resulted in each case in a single class of high affinity binding sites. The soluble receptor retained the binding specificity for different peptides and long C-terminal fragments of NPY exhibited by membrane preparations. Gel filtration of solubilized receptors resulted in a single peak of specific PYY binding activity corresponding to Mr = 350,000 whereas affinity labeling revealed a major band of Mr = 60,000. Since this binding activity was inhibited by guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) the Y2 receptor is probably solubilized as a receptor complex containing a G-protein along with the ligand binding protein. Y2 receptor binding sites from kidney tubular membranes were purified to homogeneity by a three-step procedure employing Mono S cation-exchange adsorption, affinity chromatography on wheat germ lectin-agarose beads, and affinity chromatography on NPY-Affi-Gel. Electrophoresis and silver staining of the final receptor preparation revealed a single protein with Mr = 60,000 whereas gel filtration showed a single peak at approximately Mr = 60,000. The purified protein can be affinity labeled with [125I-Tyr36]PYY, indicating that the Mr = 60,000 protein contains the ligand binding site of the Y2 receptor, and this binding is not affected by GTP gamma S. Scatchard transformation of binding data for the purified Y2 receptors was compatible with a single class of binding sites with Kd = 76 pM. The purified Y2 receptors retain their binding properties with regard to affinity and specificity for different members of the pancreatic polypeptide-fold peptide family. The specific activity of purified Y2 receptors was calculated to approximately 14.7 nmol of ligand binding/mg of receptor protein, which is consistent with the theoretical value (16.6 nmol/mg) for a pure Mr = 60,000 protein binding one PYY molecule. Purification to homogeneity thus reveals the Y2 receptor as an Mr = 60,000 glycoprotein.     

Transformation (4S to 5S) of the nuclear estrogen receptor is reversible but not accompanied by a change in the affinity for DNA

The nuclear estrogen receptor from calf uterus was used to investigate the possible relationship between receptor transformation (4S to 5S) and receptor activation (DNA binding). Receptors extracted from nuclei after exposure of uterine tissue tc [3H]estradiol sedimented at 5.2S, the characteristic value of the transformed receptor. After storage at -20 degrees C the receptor sedimented at 4.0S, indicating conversion of the 5S form into the non-transformed 4S form. Upon reincubation at 28 degrees C the 4S form transformed into the 5S form following second-order kinetics. The rate constant obtained was 4.3 x 10(7) M-1 min-1, a value identical to that reported for the cytosol receptor. These data show that receptor transformation is reversible. Molybdate (10-50 mM) was not able to prevent receptor transformation in the nuclear extract, but was inhibitory in cytosol. This suggests that molybdate does not prevent receptor transformation, but rather inhibits disaggregation of the 8S oligomer into the 4S monomer. In DNA-binding assays (DNA-cellulose or nuclei) the non-transformed (4S) and transformed (5S) states of the nuclear estrogen receptors displayed identical affinities for DNA. The present data show that 4S to 5S transformation of nuclear receptors follows a readily reversible process, but this process is not an essential step for the exposure of the receptors' DNA-binding site. Although the physiological function of the 5S form remains unclear it may be important for the recognition of specific gene regulatory sites.     

Macropinocytosis of the PDGF β-receptor promotes fibroblast transformation by H-RasG12V

Receptor tyrosine kinase (RTK) signaling is frequently increased in tumor cells, sometimes as a result of decreased receptor down-regulation. The extent to which the endocytic trafficking routes can contribute to such RTK hyperactivation is unclear. Here, we show for the first time that fibroblast transformation by H-RasG12V induces the internalization of platelet-derived growth factor β-receptor (PDGFRβ) by macropinocytosis, enhancing its signaling activity and increasing anchorage-independent proliferation. H-RasG12V transformation and PDGFRβ activation were synergistic in stimulating phosphatidylinositol (PI) 3-kinase activity, leading to receptor macropinocytosis. PDGFRβ macropinocytosis was both necessary and sufficient for enhanced receptor activation. Blocking macropinocytosis by inhibition of PI 3-kinase prevented the increase in receptor activity in transformed cells. Conversely, increasing macropinocytosis by Rabankyrin-5 overexpression was sufficient to enhance PDGFRβ activation in nontransformed cells. Simultaneous stimulation with PDGF-BB and epidermal growth factor promoted macropinocytosis of both receptors and increased their activation in nontransformed cells. We propose that H-Ras transformation promotes tumor progression by enhancing growth factor receptor signaling as a result of increased receptor macropinocytosis.     

Characterization of an altered membrane form of the beta-adrenergic receptor produced during agonist-induced desensitization

Incubation of 1321N1 human astrocytoma cells with 1 microM isoproterenol rapidly results in the conversion of a portion of the beta-adrenergic receptors to a membrane form that can be separated from markers for the plasma membrane by sucrose density gradient or differential centrifugation. This "light peak" form of the receptor reaches a maximal level within 10 min of incubation of cells with catecholamine. Two types of experiments suggest that the early phase of catecholamine-induced desensitization of the beta-adrenergic receptor-linked adenylate cyclase can be separated into at least two reactions. First, the agonist-induced loss of catecholamine-stimulated adenylate cyclase activity precedes the appearance of beta-adrenergic receptors in the light peak fraction by 1-2 min. Second, pretreatment of cells with concanavalin A prior to induction of desensitization blocks the formation of the light peak form of beta-adrenergic receptors without blocking the "uncoupling" reaction as measured by catecholamine-stimulated adenylate cyclase activity. Specificity for the reaction that converts beta-adrenergic receptors to the light peak form is indicated by the lack of a catecholamine-induced alteration in the sucrose density gradient distribution of muscarinic cholinergic receptors, adenylate cyclase or the guanine nucleotide-binding proteins, Ns and Ni. The light peak of beta-adrenergic receptors migrates at a density similar to that of at least a portion of the activity of galactosyltransferase, a marker for Golgi. Enzyme marker activities for lysosomes and endoplasmic reticulum are not associated with this population of beta-adrenergic receptors. Taken together, these and other data suggest that incubation of 1321N1 cells with isoproterenol results in a rapid uncoupling of beta-adrenergic receptors from adenylate cyclase which is followed by a change in the membrane form of the receptor. This latter step most likely represents internalization of receptors into a vesicular form which may then serve as the precursor state from which receptors are eventually lost from the cell.     

Failure of the bovine papillomavirus to transform mouse embryo fibroblasts with a targeted disruption of the insulin-like growth factor I receptor genes.

Mouse embryo cells with a targeted disruption of the insulin-like growth factor I receptor (IGF-IR) genes (R- cells) are refractory to transformation by the simian virus 40 large T antigen and/or an activated and overexpressed Ras, both of which readily transform cells from wild-type littermate embryos and other 3T3-like cells. R- cells are also refractory to transformation induced by overexpressed epidermal growth factor receptor and platelet-derived growth factor receptor beta. Since the platelet-derived growth factor receptor beta is required for transformation by bovine papillomavirus, we inquired whether the IGF-IR was also required for transformation by bovine papillomavirus E5 oncoprotein. We show here that R- cells are refractory to transformation by E5; reintroduction into R- cells of a human IGF-IR restores the susceptibility to transformation.     

Continuous annular chromatography: General characterization and application for the isolation of recombinant protein drugs

Isolation of recombinant protein drugs from cell culture supernatant is usually performed in batch mode, even if the fermentation process itself is continuous. As a novel approach, continuous separation techniques like continuous annular chromatography (CAC) can be used for continuous isolation. The potential of CAC for industrial application is demonstrated by continuous isolation of rFVIII from cell culture supernatant in pilot scale (i.e., 144-288 l/day). Thirty-fold concentration can be achieved at 94% yield, while purity is increased 3-5-fold. For this a batch direct feed ion exchange chromatography method was adapted to a commercial preparative CAC system (P-CAC). A headspace loading technique was used to maximize the concentration factor, while buffer incompatibility problems were addressed by a specifically modified inlet geometry. To allow sterile on-line coupling to FVIII-producing perfusion fermenters, an autoclavable pilot scale P-CAC prototype was developed. General characterization of P-CAC revealed a current limitation of the technology, i.e., variations in the outlet flow rates of up to +/-20%. These flow variations are shown to be caused mainly by a nonuniform annular resin bed and in turn result in "peak wobbling," i.e., the slight variation of peak position (up to +/-4 degrees ) and shape (e.g., A(s) = 0.9-1.4) as a specific function of column position. Some additional peak broadening, although less significant, is caused by a "peak oscillation" effect that results from the necessary segmentation of flow into discrete outlets. Both effects are only measurable if peaks are either monitored continuously or at least measured at multiple column positions. For isolation processes, these nonideal flow phenomena mean that more outlet streams have to be collected in order to achieve maximum yield and thus the achievable concentration factor is somewhat lower than the theoretical maximum.     

Ligand-independent oncogenic signaling by the epidermal growth factor receptor: v-ErbB as a paradigm

Relay of information from the extracellular environment into the cell often results from a peptide growth factor binding to its cognate cell surface receptor; this event is an integral mechanism by which many cellular functions occur, including cell growth, motility, and survival. In recent years, however, this requirement for ligand binding has been shown to be surpassed by several distinct mechanisms, including cell surface receptor cross-talk (e.g., between epidermal growth factor receptor [EGFR] and G-coupled receptors), receptor-extracellular matrix interactions (e.g., EGFR: integrin complexes), and finally by structural mutations within the receptor itself. While all of these pathways result in so-called ligand-independent signaling by the EGF receptor, to date, only structural mutations in the receptor have been shown to result in qualitative changes in downstream targets of the receptor, which specifically result in oncogenic signaling, transformation, and tumorigenicity. In this review, we describe aspects of the known signaling properties of the retroviral oncogene v-ErbB as a model of ligand-independent oncogenic signaling, and compare these properties to results emerging from ongoing studies on structurally related EGF receptor mutants originally identified in human tumors. A better understanding of the signaling pathways used by these uniquely oncogenic receptor tyrosine kinase mutants may ultimately reveal new targets for the development of novel therapeutics selective for the inhibition of tumor cell growth.