Degradation Of Receptor Protein Research Articles

Artemisinin disrupts androgen responsiveness of human prostate cancer cells by stimulating the 26S proteasome-mediated degradation of the androgen receptor protein.

Androgen receptor (AR) expression and activity is highly linked to the development and progression of prostate cancer and is a target of therapeutic strategies for this disease. We investigated whether the antimalarial drug artemisinin, which is a sesquiterpene lactone isolated from the sweet wormwood plant Artemisia annua, could alter AR expression and responsiveness in cultured human prostate cancer cell lines. Artemisinin treatment induced the 26S proteasome-mediated degradation of the receptor protein, without altering AR transcript levels, in androgen-responsive LNCaP prostate cancer cells or PC-3 prostate cancer cells expressing exogenous wild-type AR. Furthermore, artemisinin stimulated AR ubiquitination and AR receptor interactions with the E3 ubiquitin ligase MDM2 in LNCaP cells. The artemisinin-induced loss of AR protein prevented androgen-responsive cell proliferation and ablated total AR transcriptional activity. The serine/threonine protein kinase AKT-1 was shown to be highly associated with artemisinin-induced proteasome-mediated degradation of AR protein. Artemisinin treatment activated AKT-1 enzymatic activity, enhanced receptor association with AKT-1, and induced AR serine phosphorylation. Treatment of LNCaP cells with the PI3-kinase inhibitor LY294002, which inhibits the PI3-kinase-dependent activation of AKT-1, prevented the artemisinin-induced AR degradation. Furthermore, in transfected receptor-negative PC-3 cells, artemisinin failed to stimulate the degradation of an altered receptor protein (S215A/S792A) with mutations in its two consensus AKT-1 serine phosphorylation sites. Taken together, our results indicate that artemisinin induces the degradation of AR protein and disrupts androgen responsiveness of human prostate cancer cells, suggesting that this natural compound represents a new potential therapeutic molecule that selectively targets AR levels.

Benzo(a)pyrene triggers desensitization of \u03b22-adrenergic pathway

Exposure to environmental polycyclic aromatic hydrocarbons (PAHs), such as benzo(a)pyrene (B(a)P), has been linked to several health-threatening risks. PAHs were also shown to hinder adrenergic receptor (ADR) responses. As we previously demonstrated that B(a)P can directly interact with the β2ADR, we investigated here whether B(a)P could decrease β2ADR responsiveness by triggering receptor desensitization phenomena. We firstly showed that exposure to B(a)P reduced β2ADR-mediated epinephrine-induced induction of NR4A gene mRNAs and of intracellular cAMP. Analysis of β2ADR protein expression demonstrated that B(a)P rapidly decreased membrane expression of β2ADR with a subsequent degradation of receptor protein. B(a)P exposure concomitantly rapidly increased the β2ADR mRNA levels. The use of the β-blockers, propranolol and ICI 118.551, demonstrated the involvement of β2ADR itself in this increase. However, sustained exposure to B(a)P induced a diminution of β2ADR mRNA steady-state as a result of the acceleration of its degradation. Together, these results show that, beside the well-known activation of the aryl hydrocarbon receptor, PAH deleterious effects may involve the dysfunction of adrenergic responses through, in part, the desensitization of β2ADR. This may be taken in consideration when β2-agonists/antagonists are administered in patients exposed to important concentrations of PAHs, e.g. in cigarette smokers.

Estrogen receptor degradation: a CUE for endocrine resistance?

Despite the undoubted success of adjuvant endocrine therapies that target the estrogen receptor pathway, not all women with estrogen receptor-positive breast cancer respond to these therapies, and many who initially respond will subsequently relapse. Deregulation of various aspects of estrogen receptor signaling has been highlighted as a mechanism of resistance and as a basis for alternative therapeutic approaches. However, a recent publication refocuses attention on the estrogen receptor itself by showing that the ubiquitin-binding CUE domain-containing protein 2 is a regulator of estrogen receptor protein degradation and a marker of endocrine resistance in breast cancer.

Estrogen receptor-α: molecular mechanisms and interactions with the ubiquitin proteasome system.

Estrogen receptor-α (ERα) is a protein with a long history of study that precedes the advent of modern molecular biology. Over the course of 50years, ERα has been increasingly recognized as a prominent model for the study of the mechanism of gene transcription in vertebrates. It also serves as a regulatory molecule for numerous physiological and disease states. Several fundamental insights have been made using ERα as a model protein, from the discovery that endocrine hormones elicit gene transcription to our understanding of the relationship between ERα-mediated transcription and transcription factor degradation by the ubiquitin proteasome system (UPS). Understanding of receptor protein degradation developed alongside other aspects of its molecular biology, from early observations in the 1960s that ERα is degraded on hormone treatment to the current understanding of ERα transcriptional regulation by the UPS. Here, we present the concept of ERα turnover from the perspective of the historical development of this notion and highlight some of the latest discoveries regarding this process. We discuss the logic and significance of ERα degradation pathways in the context of cell and whole-organism homeostasis.

Inhibition of squalene synthase upregulates PCSK9 expression in rat liver

Proprotein convertase subtilisin/kexin type 9 (PCSK9) plays a critical role in cholesterol metabolism by enhancing the degradation of the LDL receptor protein in the liver. It has previously been shown that administration of zaragozic acid A (ZA), a potent inhibitor of squalene synthase, also significantly increases the rate of degradation of hepatic LDL receptor protein. Thus, we decided to determine whether ZA administration might act to up regulate hepatic expression of the rat PCSK9 gene. Administration of ZA resulted in increased PCSK9 mRNA and protein levels in rat liver surprisingly in concert with an increase in hepatic LDL receptor mRNA levels, LDL receptor protein turnover, and decreased serum cholesterol levels. These observations suggest an involvement of PCSK9 in hepatic LDL receptor protein degradation and perhaps, in increasing the rate of LDL receptor cycling resulting in lower serum cholesterol levels in response to cholesterol biosynthesis inhibitors.

The NEDD8 pathway is required for proteasome-mediated degradation of human estrogen receptor (ER)-alpha and essential for the antiproliferative activity of ICI 182,780 in ERalpha-positive breast cancer cells.

Steroid hormone receptors, including estrogen receptor-alpha (ERalpha), are ligand-activated transcription factors, and hormone binding leads to depletion of receptor levels via preteasome-mediated degradation. NEDD8 (neural precursor cell-expressed developmentally down-regulated) is an ubiquitin-like protein essential for protein processing and cell cycle progression. We recently demonstrated that ubiquitin-activating enzyme (Uba)3, the catalytic subunit of the NEDD8-activating enzyme, inhibits ERalpha transcriptional activity. Here we report that Uba3-mediated inhibition of ERalpha transactivation function is due to increased receptor protein turnover. Coexpression of Uba3 with ERalpha increased receptor degradation by the 26S proteasome. Inhibition of NEDD8 activation and conjugation diminished polyubiquitination of ERalpha and blocked proteasome-mediated degradation of receptor protein. The antiestrogen ICI 182,780 is known to induce ER degradation. In human MCF7 breast cancer cells modified to contain a disrupted NEDD8 pathway, ICI 182,780 degradation of ERalpha was impaired, and the antiestrogen was ineffective at inhibiting cell proliferation. This study provides the first evidence linking nuclear receptor degradation with the NEDD8 pathway and the ubiquitin-proteasome system, suggesting that the two pathways can act together to modulate ERalpha turnover and cellular responses to estrogens. Based on our observation that an intact NEDD8 pathway is essential for the antiproliferation activity of the ICI 182,780 in ERalpha positive breast cancer cells, we propose that disruptions in the NEDD8 pathway provide a mechanism by which breast cancer cells acquire antiestrogen resistance while retaining expression of ERalpha.

Expression of a novel member of sorting nexin gene family, SNX-L, in human liver development

The sorting nexin (SNX) protein family is implicated in the regulation of receptor degradation and membrane traffic in the cell. With the aim of identifying novel genes involved in receptor degradation and recycling, we have cloned a new member of the sorting nexin gene family, human sorting nexin L, SNX-L (or SNX21). This gene includes 4 exons and 3 introns, and is located on chromosome 20q12–13.1 region, encompassing 8 kb. The full-length cDNA of SNX-L is 1811 bp, with an open reading frame of 1092 bp. The protein consists of 364 amino acids and encodes a 40 kDa protein. The SNX-L protein has a common PX domain shared by all SNX family members. The similarity of SNX-L PX domain to the PX consensus sequence is over 40%. PX domains have been shown to associate with specific phospholipids and membrane compartments. Expression analysis of SNX-L mRNA indicates that SNX-L is distinctly and highly expressed in fetus liver, but only weakly expressed in brain, muscle (skeleton muscle, smooth muscle, and cardiac muscle), kidney, and adrenal gland. Strong liver expression of SNX-L is maintained from 12 to 25 weeks during human fetus development, suggesting that SNX-L may be a regulatory gene involved in receptor protein degradation during embryonic liver development.

Evidence for enhanced ubiquitin-mediated proteolysis of the chicken progesterone receptor by progesterone

Genomic actions of progesterone are mediated via A and B isoforms of the progesterone receptor (PR). One major factor controlling PR level is progesterone causing negative autoregulation (down-regulation) of the receptor protein. In this work we studied the mechanism whereby progesterone exerts its effects on PR level in the chicken oviduct. We found that progesterone does not markedly regulate PR mRNA expression. Furthermore, we demonstrate here for the first time that PR is a target for ubiquitylation and that the proportion of ubiquitylated PR is increased by progesterone treatment. Our data suggest that ligand-induced down-regulation of PR involves enhanced degradation of receptor protein by ubiquitin-proteasome system in vivo.

Inhibitors of 3-Hydroxy-3-methylglutaryl Coenzyme A Reductase Unmask Transcriptional Regulation of Hepatic Low-Density Lipoprotein Receptor Gene Expression by Dietary Cholesterol

The mechanism by which dietary cholesterol regulates expression of the hepatic low-density lipoprotein (LDL) receptor was investigated. In a previous study (Arch. Biochem. Biophys.325, 242–248, 1996), we demonstrated that dietary cholesterol reduces the rate of LDL receptor protein degradation without affecting steady-state levels of receptor protein. In view of these findings, it was expected that dietary cholesterol would decrease the rate of transcription of the hepatic LDL receptor gene, resulting in lower mRNA levels and lower rates of synthesis of LDL receptor protein. Surprisingly, neither the rate of transcription nor the level of LDL receptor mRNA was reduced in response to dietary cholesterol, even though hepatic cholesterol levels were increased twofold. This suggests that under normal conditions, dietary cholesterol does not affect LDL receptor gene expression at the level of transcription. In contrast, feeding 2% cholesterol to rats fed a diet supplemented with 0.04% lovastatin significantly decreased hepatic LDL receptor mRNA levels and transcription rates. These results suggest that lovastatin unmasks transcriptional regulation of the hepatic LDL receptor by dietary cholesterol. The levels of the mature nuclear forms of sterol response element binding proteins-1 and -2 were unaffected despite significant changes in hepatic cholesterol levels, mRNA levels, and transcription rates caused by lovastatin treatment. This suggests that the observed changes in transcription rates may not be mediated by these proteins in rat liver.

Role of the amino terminus of the third intracellular loop in agonist-promoted downregulation of the alpha2A-adrenergic receptor.

A prominent feature of long-term regulation of the alpha2A-adrenergic receptor (alpha2AAR) is a loss of cellular receptors over time (downregulation). The molecular determinants of downregulation were sought by targeting regions of the receptor involved in G protein coupling and phosphorylation. Mutated receptors, consisting of chimeric substitutions of analogous beta2-adrenergic receptor (beta2AR) and serotonin 5-hydroxytryptamine1A (5-HT1A) receptor sequence into the second intracellular loop (ICL2) (residues 113-149), the amino terminus (residues 218-235) and carboxy terminus (residues 355-371) of ICL3, and a deletion of the beta-adrenergic receptor kinase (betaARK) phosphorylation sites in the third intracellular loop (ICL3) (residues 293-304), were expressed in Chinese hamster ovary (CHO) cells. Wild-type alpha2AAR underwent 31% +/- 3% downregulation after 24 h of exposure to 100 microM epinephrine. Loss of downregulation was observed with some mutants, but this was not related to functional coupling to inhibitory or stimulatory guanine nucleotide regulatory binding proteins (Gi or GS) or to phosphorylation. Rather, any mutant with a substitution of the amino terminus of ICL3 (regardless of whether the substitution was with beta2AR or 5-HT1A sequence) resulted in upregulation. Studies with an inhibitor of protein synthesis indicated that the primary mechanism of downregulation of the alpha2AAR is agonist-promoted degradation of receptor protein which requires a destabilization sequence in the amino terminus of ICL3. Thus, in contrast to other G protein-coupled receptors, in which G protein coupling or phosphorylation are critical for long-term agonist regulation, the alpha2AAR has a specific structural domain distinct from these other functional regions that serves to direct agonist-promoted downregulation.

Chronic effects of catecholamines on the beta 2-adrenoreceptor system in cultured human airway epithelial cells.

Chronic catecholamine treatment induces beta-adrenergic receptor (beta AR) downregulation, i.e., a loss of total cell receptors. In the human respiratory tract, the mechanism(s) underlying beta AR downregulation remains poorly understood. The present study, therefore, examined the effects of 24 h of exposure to isoproterenol (Iso; 10 nM or 1 microM) on beta AR density and the rate of beta AR degradation, steady-state beta 2-adrenergic receptor (beta 2 AR) mRNA levels, and the content of Gs alpha and Gi alpha proteins in cultured human bronchial epithelial cells (i.e., the BEAS-2B cell line). beta AR density assessed by binding with [125I]iodopindolol decreased in a dose-dependent fashion with 24 h of Iso exposure. With Iso (1 microM), beta AR density decreased by approximately 82%. In contrast, forskolin (100 microM) and dibutyryl adenosine 3',5'-cyclic monophosphate (1 mM), agents that also increase adenosine 3',5'-cyclic monophosphate (cAMP) levels, had no significant effect on beta AR density. Iso exposure also elicited a concomitant decrease in Iso-stimulated cAMP but had no significant effect on the content of the G proteins G alpha i2 and Gs alpha assessed by immunoblotting and toxin-catalyzed ADP ribosylation. Of note, Iso exposure (1 microM) had no effect on steady-state levels of beta 2 AR mRNA measured both by Northern analysis and by reverse transcriptase-polymerase chain reaction. However, beta AR half-life assessed in the presence of the protein synthesis inhibitor cycloheximide was reduced by approximately 60% in Iso-treated cells (i.e., from 37 h in control to 16 h in 1 microM Iso). These results suggest that, in human airway epithelial cells, beta 2 AR downregulation 1) is not primarily driven by intracellular cAMP levels, 2) is not associated with significant decreases in steady-state levels of beta 2 AR mRNA, and 3) is largely posttranslationally regulated by increases in the rate of receptor protein degradation.

Mechanisms of β-Adrenergic Receptor Desensitization and Resensitization

Desensitization represents the summation of several different processes, including receptor phosphorylation, receptor sequestration (defined as the agonist-induced translocation of receptor away from the plasma membrane), enhanced degradation of intracellular messengers, and degradation of receptor protein. Rapid receptor desensitization, however, appears to be mediated by uncoupling of the receptor from its respective G protein, a consequence of receptor phosphorylation. In the case of the β2-adrenergic receptor (β2AR), phosphorylation by two distinct classes of serine-threonine kinases leads to receptor desensitization. The first class, the second-messenger—dependent kinases—cyclic adenosine monophosphate (CAMP)-dependent protein kinase (PKA) and protein kinase C—phosphorylate and directly uncouple β2AR from Gs. Because these kinases phosphorylate receptors in an agonist-independent manner, this process permits cross-talk between receptor families. The second class of enzymes, the G protein-coupled receptor kinases (GRKs), plays a highly specialized role in receptor desensitization because only agonist-occupied receptors serve as substrates for these enzymes. In the case of GRKs, the very signal that promotes activation of the G protein and the effector (that is, ligand binding) also promotes the desensitization of that specific receptor. Once uncoupled from the G protein, receptor function can be restored only by receptor dephosphorylation, a process termed resensitization. The phosphatases and regulatory mechanisms involved in this resensitization process have only recently begun to be elucidated and are included in this chapter.

Inhibitors of Cholesterol Biosynthesis Increase Hepatic Low-Density Lipoprotein Receptor Protein Degradation

Inhibitors of cholesterol biosynthesis are believed to lower serum cholesterol levels by enhancing the removal of serum low-density lipoprotein (LDL) by increasing hepatic LDL receptor function. Thus, the effects of several different inhibitors of cholesterol biosynthesis were examined for their effects on the expression of the hepatic LDL receptor in rats. We found that administration of inhibitors of 3- hydroxy-3-methylglutaryl–coenzyme A reductase such as lovastatin, pravastatin, fluvastatin, and rivastatin resulted in increased hepatic LDL receptor mRNA levels. Surprisingly, these agents failed to increase levels of immunoreactive LDL receptor protein in rat liver even when the dose and length of treatment were increased. Treatment of rats with zaragozic acid A, an inhibitor of squalene synthase, caused even greater increases in hepatic LDL receptor mRNA levels, but did not increase levels of immunoreactive protein. Further investigation revealed that the rate of degradation of the hepatic LDL receptor was increased in rats given inhibitors of cholesterol biosynthesis. The greatest increase in the rate of degradation was seen in animals treated with zaragozic acid A which caused the largest increase in hepatic LDL receptor mRNA levels. In contrast, hepatic LDL receptor protein was stabilized in cholesterol-fed rats. It appears that increased potential for LDL receptor protein synthesis, reflected in increased mRNA levels, is offset by a corresponding increase in the rate of receptor protein degradation resulting in constant steady-state levels of hepatic LDL receptor protein. These findings are suggestive of increased cycling of the hepatic LDL receptor. This postulated mechanism can provide for enhanced hepatic uptake of lipoproteins without increasing steady-state levels of LDL receptor protein.

Cyclic AMP-Mediated Desensitization of D1 Dopamine Receptor-Coupled Adenylyl Cyclase in NS20Y Neuroblastoma Cells

Cyclic AMP-mediated desensitization of D1 dopamine receptor-coupled adenylyl cyclase was investigated using NS20Y neuroblastoma cells. Pretreatment of the cells for 24 h with 8-(4-chlorophenylthio)-adenosine-3′:5′-cyclic monophosphate (CPT-cAMP), a membrane-permeable analog of cAMP, resulted in an ∼90% reduction of the maximum dopamine-stimulated adenylyl cyclase activity. In addition, there was a twofold reduction in the potency of dopamine for stimulating cAMP production that was not dependent on the concentration of Mg2+ in the assay. These effects of CPT-cAMP pretreatment were time dependent, showing a t1/2 of about 3 h and a maximum reduction after about 8 h. Receptor-binding activity, as measured using the D2-selective antagonist [3H]SCH-23390, also declined following CPT-cAMP pretreatment with a t1/2 of about 5 h and a maximum reduction of about 70% after 20 h. Saturation analysis indicated that the loss in radioligand binding was due to a reduction in maximum binding capacity (Bmax) with no alteration in receptor affinity (KD). The EC50 of CPT-cAMP for producing enzyme desensitization and D1 receptor downregulation was determined to be about 30 μM with a maximal response occurring at 1 mM. These regulatory effects of CPT-cAMP were pharmacologically specific as other analogs of cAMP, such as dibutryl-cAMP, 8-bromo-cAMP, and Sp-cAMPS, were capable of inducing D1 receptor desensitization and downregulation, whereas treatment of the cells with the cAMP antagonist Rp-cAMPS had no effect. Conversely, Rp-cAMPS was capable of blocking the regulatory effects of CPT-cAMP but was apparently without effect in blocking dopamine-induced desensitization. In addition to desensitization and receptor downregulation, CPT-cAMP treatment was found to promote functional uncoupling of the receptor as suggested by a loss of high-affinity agonist binding observed in dopamine/[3H]SCH-23390 competition assays following desensitization. Removal of CPT-cAMP resulted in recovery to control activities within 24 h. This recovery could be completely blocked through inhibition of cellular protein synthesis using cycloheximide. These data indicate that increasing the intracellular levels of cAMP can promote desensitization of the D1 receptor/adenylyl cyclase system and can induce a downregulatlon of D1 receptor-binding activity, presumably involving degradation of receptor protein.

Amino-terminal polymorphisms of the human beta 2-adrenergic receptor impart distinct agonist-promoted regulatory properties.

We have recently delineated three naturally occurring polymorphisms of the human beta 2-adrenergic receptor caused by missense mutations encoding for amino acids 16 and 27 of the extracellular N-terminus of the receptor. We have studied the functional consequences of these polymorphisms by site-directed mutagenesis and the recombinant expression of these receptors in Chinese hamster fibroblasts. The polymorphisms consist of substitutions of Gly for Arg at amino acid 16 (Arg16-->Gly), Glu for Gln at amino acid 27 (Gln27-->Glu), and a combination of both substitutions. All three mutated receptors displayed normal agonist binding and functional coupling to Gs, resulting in the stimulation of adenylyl cyclase activity. However, these mutations markedly altered the degree of agonist-promoted downregulation of receptor expression. After 24-h exposure to 10 microM isoproterenol, wild-type beta 2AR underwent a 26 +/- 3% reduction in receptor density. In contrast, Arg16-->Gly underwent a 41 +/- 3% reduction. Gln27-->Glu, on the other hand, was found to be completely resistant to downregulation. Arg16-->Gly+Gln27-->Glu also underwent an increased downregulation compared to wild-type beta 2AR (39 +/- 4%). The rates of new receptor synthesis after irreversible alkylation were not different between these receptors, nor were the rates of agonist-promoted receptor internalization to the intracellular pool. Gln27-->Glu cellular mRNA minimally increased during agonist exposure, and wild-type beta 2AR and the other mutated receptor mRNAs did not change, which infer that the aberrant downregulation patterns of these polymorphisms may be due to the altered degradation of receptor protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential heat stress stability of epidermal growth factor receptor and erbB‐2 receptor tyrosine kinase activities

The epidermal growth factor (EGF) and erbB-2 receptors are structurally related membrane-bound tyrosine kinases. While these proteins exhibit close sequence homology, 50% overall and 80% in the tyrosine kinase domains, they respond very differently to heat stress. In NIH-3T3 or NR6 cells transfected with wild-type EGF-R and incubated at 37 degrees C or heat shocked at 46 degrees C, EGF binds to its receptor and stimulates receptor autophosphorylation to equivalent extents. At 46 degrees C, however, the basal tyrosine kinase activity of the wild-type erbB-2 receptor is rapidly lost. When cells containing chimeric receptors composed of the EGF-R extracellular domain and intracellular domain of erbB-2 were heat stressed, 125I-EGF bound to the receptors, but did not stimulate receptor autophosphorylation. The decline in EGF-stimulated chimeric erbB-2 receptor autophosphorylation is dependent on the length of heat shock, with nearly 100% of the kinase activity lost after 60 min at 46 degrees C. The loss of chimeric receptor erbB-2 kinase activity is not due to degradation of receptor protein, nor is it attributable to a specific transmembrane domain from either the EGF or erbB-2 receptors. Sensitivity of erbB-2 to heat stress is also not a result of denaturation of this receptor's carboxy-terminal domain. Insertion of the erbB-2 tyrosine kinase domain into the EGF-R confers heat stress sensitivity to the resultant chimeric receptor. Thus, although the EGF-R and erbB-2 kinase domains show a high degree of homology, the secondary/tertiary structures of these domains would seem to be stabilized in distinct manners.

Characterization of a temperature-sensitive mutation in the hormone binding domain of the human estrogen receptor. Studies in cell extracts and intact cells and their implications for hormone-dependent transcriptional activation.

A previous report from this laboratory (Reese, J.C., and Katzenellenbogen, B. S. (1991) J. Biol. Chem. 266, 10880-10887) identified an estrogen receptor (ER) mutant which had a similar binding affinity for estradiol as wild-type ER but displayed a dose-response shift for estradiol in transactivation studies. In this study, we have utilized hormone binding, DNA binding, and gene transfer experiments to further characterize this mutant, which contains an alanine substitution for a cysteine at amino acid 447 in the hormone binding domain of the receptor. Hormone binding studies indicate that the C447A receptor is a temperature-sensitive mutant, whose instability is only apparent at elevated temperatures, and that ligand can stabilize the mutant receptor. Western blot analysis reveals that the temperature-sensitive loss of hormone binding is not attributable to a degradation of receptor protein, but rather is an inactivation of the receptor's hormone binding ability. In addition to the loss in the hormone binding capacity of the C447A mutant, this mutant shows a temperature-sensitive loss in the DNA binding ability of the receptor. Transactivation profiles of the mutant and wild-type receptors demonstrate that incubation of transfected cells with increasing concentrations of estradiol at more ambient temperatures shifts the mutant receptor's dose-response curves to the left, converging on the wild-type curve. Hence, these transactivation studies reveal that the dose-response shift observed for this mutant in cells reflects the measured instability of the hormone binding and DNA interaction of the C447A mutant that can be demonstrated in vitro. In addition, this temperature-sensitive ER mutant is of interest in that its DNA binding is now ligand-dependent with the result that transcriptional activation now parallels receptor occupancy by ligand, which is similar to other steroid hormone receptors.

Defect of epidermal 12(S)-hydroxyeicosatetraenoic acid receptors in psoriasis.

12-hydroxyeicosatetraenoic acid (12-HETE) is assumed to play a central role in the pathophysiology of psoriasis. Since its effects in skin are mediated by specific high-affinity receptors, we studied the receptor characteristics in cultured epidermal cells from involved and apparently healthy skin of psoriasis patients by radioligand binding assay. Involved and uninvolved psoriatic epidermal cells showed a fourfold decrease in the number of 12-HETE binding sites as compared with normal healthy individuals and patients with atopic dermatitis, while receptor affinity remained unchanged. The decrease in receptor number was evident in psoriatic cells even in long-term culture and was not due to receptor down-regulation, defective response to interferon gamma or to protease degradation of receptor protein. The decrease in the number of 12-HETE receptors detectable even in clinically normal psoriatic skin functionally leads to diminished 12-HETE uptake and may thus represent a primary central molecular defect in the pathophysiology of the disease.

Reconstitution of galactosyl receptor inactivation in permeabilized rat hepatocytes is ATP-dependent

A subpopulation of galactosyl receptors (GalRs) on isolated rat hepatocytes undergo a reversible inactivation and reactivation process during constitutive recycling (McAbee, D. D., and Weigel, P. H. (1988) Biochemistry 27, 2061-2069). Here, we report the reconstitution of this GalR inactivation in digitonin-permeabilized rat hepatocytes. Permeabilization of freshly isolated cells at 4 degrees C with 0.002% (w/v digitonin releases cytosol containing 35-40% of the total cellular protein, 10-15% of a lysosomal marker, and 5-10% of an early endosomal marker. Incubation of permeabilized cells with cytosol at 37 degrees C results in a time-dependent reduction of total 125I-asialoorosomucoid binding activity, which proceeds with first order kinetics (t 1/2 = 11.3 min). Only half of the total cellular GalRs are affected; maximal GalR activity loss, obtained by 30 min, is 50.5 +/- 9.5% (n = 21) of the control (4 degrees C) value. Increasing the digitonin concentration up to 0.055% does not increase the extent of inactivation. Permeabilized cells with reduced GalR activity were assessed for GalR protein content by Western blot analysis and by binding of anti-GalR antibody. The results show that the reduced 125I-asialoorosomucoid binding is due to GalR inactivation rather than receptor protein degradation. GalR inactivation does not occur in the absence of cytosol or in the presence of dialyzed cytosol. The cytosol also loses its GalR inactivating ability in the presence of an ATP-depleting system. GalR inactivation in the absence of cytosol is achieved by incubating permeabilized washed cells at 37 degrees C with ATP but not with ADP, AMP, or other NTPs. The rate and extent of inactivation are dependent on the ATP concentration. Half-maximal and maximal GalR inactivation are obtained at 0.3 and 3.0 mM ATP, respectively. In the presence of cytosol, permeabilized hepatocytes could replenish cytosolic ATP by oxidative phosphorylation. As a result, similar levels of GalR inactivation were obtained with 500-fold lower ATP concentrations. We conclude that ATP is the only cytosolic component necessary for GalR inactivation in permeabilized rat hepatocytes.