Proteasome involvement in agonist-induced down-regulation of mu and delta opioid receptors.

This study investigated the mechanism of agonist-induced opioid receptor down-regulation. Incubation of HEK 293 cells expressing FLAG-tagged delta and mu receptors with agonists caused a time-dependent decrease in opioid receptor levels assayed by immunoblotting. Pulse-chase experiments using [(35)S]methionine metabolic labeling indicated that the turnover rate of delta receptors was accelerated 5-fold following agonist stimulation. Inactivation of functional G(i) and G(o) proteins by pertussis toxin-attenuated down-regulation of the mu opioid receptor, while down-regulation of the delta opioid receptor was unaffected. Pretreatment of cells with inhibitors of lysosomal proteases, calpain, and caspases had little effect on mu and delta opioid receptor down-regulation. In marked contrast, pretreatment with proteasome inhibitors attenuated agonist-induced mu and delta receptor down-regulation. In addition, incubation of cells with proteasome inhibitors in the absence of agonists increased steady-state mu and delta opioid receptor levels. Immunoprecipitation of mu and delta opioid receptors followed by immunoblotting with ubiquitin antibodies suggested that preincubation with proteasome inhibitors promoted accumulation of polyubiquitinated receptors. These data provide evidence that the ubiquitin/proteasome pathway plays a role in agonist-induced down-regulation and basal turnover of opioid receptors.

Pharmacological profiles of selective non-peptidic delta opioid receptor ligands.

Several non-peptidic opioids have been synthesized recently as part of a program to develop selective delta receptor agonists. In this study, the affinities of a set of compounds for cloned delta and mu opioid receptors expressed in HEK 293 cell lines were determined by competition analysis of [3H]bremazocine binding to membrane preparations. All compounds studied exhibited high affinity and selectivity, with apparent dissociation constants in the range of 0.6-1.7 nM for the delta opioid receptor and 240-1165 nM for the mu opioid receptor. We next sought to determine which domain of the delta receptor was critical for mediating the highly selective binding by analysis of ligand affinities for mu/delta receptor chimeras. Receptor binding profiles suggested that a critical site of receptor/ligand interaction was located between transmembrane domain 5 (TM5) and TM7 of the delta receptor. Substitution of tryptophan 284, located at the extracellular surface of TM6, with lysine, which is found at the equivalent position in the mu opioid receptor, led to a spectrum of effects on affinities, depending on the ligand tested. Affinities of SB 219825 and SB 222941 were particularly sensitive to the substitution, displaying a 50-fold and 70-fold decrease in affinity, respectively. Activities of the delta receptor-selective agonists were tested in two functional assays. Brief exposure of HEK 293 cells expressing delta opioid receptors with selective ligands induced phosphorylation of MAP kinase, although the non-peptidic ligands were less efficacious than the enkephalin derivative DADL (Tyr-D-Ala-Gly-Phe-D-Leu). Similarly, chronic exposure of HEK 293 cells expressing delta opioid receptors with selective, non-peptidic ligands, with the exception of SB 206848, caused receptor down-regulation, however, the SB compounds were less efficacious than DADL.

mu Opioid receptor: role for the amino terminus as a determinant of ligand binding affinity.

The importance of the amino-terminal domain of the mu opioid receptor (MOR) as a component of the high affinity ligand-binding pocket was evaluated. A deletion mutant lacking 64 amino acids from the amino-terminus of MOR (DeltaN64) was constructed and expressed in HEK 293 cells. The affinities of bremazocine and cyclazocine were similar for the truncated and full-length MORs. Affinities of the mu receptor antagonist, naloxone, and the mu receptor agonist, morphine, were decreased 3.5-fold and 6-fold, respectively, for the truncated receptor relative to the wild-type MOR. Similarly, the affinities of the opioid peptide agonists, DAMGO (Tyr-D-Ala-Gly-MePhe-Gly-ol), beta-endorphin, and DADL (Tyr-D-Ala-Gly-Phe-D-Leu), for the DeltaN64 receptor were decreased from 3- to 8-fold as a result of the deletion. In contrast, the affinities of the alkaloid agonists, methadone and fentanyl, and the peptide agonists, endomorphin 1 and endomorphin 2, for the truncated receptor relative to MOR were reduced dramatically by 20- to 60-fold. MOR is glycosylated when expressed in HEK 293 cells; however, analysis of N-glycosidase F-treated membranes indicated that N-glycan chains within the amino-terminal domain of MOR do not contribute significantly to ligand affinities. These results indicate that amino acid residues within the amino-terminal domain of MOR play a crucial role in the composition of the binding pocket for a select group of agonists.

Structure and regulation of opioid receptors.

Significant advances have been made in understanding the structure, function, and regulation of opioid receptors and endogenous opioid peptides since their discovery approximately 25 years ago. This review summarizes recent studies aimed at identifying key amino acids that confer ligand selectivity to the opioid receptors and that are critical constituents of the ligand binding sites. A molecular model of the delta receptor based on the crystal structure of rhodopsin is presented. Agonist-induced down regulation of opioid receptors is discussed, highlighting recent evidence for the involvement of the ubiquitin/proteasome system in this process.

Identification of a novel gene product preferentially expressed in rat lens epithelial cells.

PURPOSE: We searched for an mRNA that is differentially expressed in rat lens epithelial and fiber cells to use as a marker for differentiation. METHODS: An mRNA differential display method was used to identify genes with altered expression during differentiation of lens epithelial cells. RESULTS: One gene (partial sequence) was identified and confirmed to be expressed preferentially in undifferentiated rat lens epithelial cells. Among non-lens tissues, a trace amount was detected in cornea, but not in retina or eleven non-ocular tissues tested. This cloned partial gene sequence (322 base pairs) is at least 93% identical to overlapping rat and mouse EST sequences, and extends the sequence of both ESTs at the 5' end. CONCLUSIONS: A clone has been isolated that is preferentially expressed in lens epithelial cells. Expression of this gene was down-regulated when rat pup lens epithelial cell explants were induced to differentiate. The unusual pattern of expression suggests a novel function for this gene.

The role of GM1 ganglioside in regulating excitatory opioid effects.

Our studies with cultured cells have provided new insight into the particular role of GM1 in regulating excitatory opioid responses. GM1 is significantly elevated in chronic opioid-treated cells via Gs/adenylyl cyclase activation. Such GM1 elevation promotes coupling of opioid receptor with Gs, resulting in attenuation of inhibitory opioid effects and induction of a sustained excitatory response. Application of exogenous GM1, but not other gangliosides, induces excitatory opioid responses not only in neurons and neuroblastoma cells that bear intrinsic opioid receptors but also in nonneuronal cells that are transfected with delta-opioid receptor. The latter system provides evidence that allosteric binding of GM1 changes receptor conformation from a Gi-coupled to a Gs-coupled mode. This is supported by preliminary experiments with a mutated delta-opioid receptor.

Sensitivity of opioid receptor binding to N-substituted maleimides and methanethiosulfonate derivatives.

A series of N-substituted maleimides was shown to effectively inactivate bremazocine binding to delta opioid receptors. Apparent second order rate constants for inactivation increased with increasing size of the N-substituent: N-methyl < N-ethyl < N-butyl < N-phenylmaleimide. It is suggested that the positive chain length effect is attributed to nonpolar interactions with the receptor in the vicinity of the reactive group. Binding to mu and delta opioid receptors was equally sensitive to inactivation by (2-aminoethyl)methanethiosulfonate; the [2-(trimethylammonium)ethyl] and (2-sulfonatoethyl) derivatives were less active. Site-directed mutagenesis of the mu opioid receptor indicated that Cys159, Cys190, Cys235, Cys292, or Cys321, residing in transmembrane domain 3, 4, 5, 6, and 7, respectively, were not the site of modification.

Studies on inhibition of mu and delta opioid receptor binding by dithiothreitol and N-ethylmaleimide. His223 is critical for mu opioid receptor binding and inactivation by N-ethylmaleimide.

The sensitivity of mu and delta receptor binding to dithiothreitol and N-ethylmaleimide was examined to probe receptor structure and function. Binding to both receptor types was inhibited by dithiothreitol (IC50 values = 250 mM), suggesting the presence of inaccessible but critical disulfide linkages. mu receptor binding was inhibited with more rapid kinetics and at lower N-ethylmaleimide concentrations than delta receptor binding. Ligand protection against N-ethylmaleimide inactivation suggested that alkylation was occurring within, or in the vicinity of, the receptor binding pocket. Sodium ions dramatically affected the IC50 of N-ethylmaleimide toward both receptor types in a ligand-dependent manner. Analysis of receptor chimeras suggested that the site of N-ethylmaleimide alkylation on the mu receptor was between transmembrane domains 3 and 5. Substitution of cysteines between transmembrane domains 3 and 5 and elsewhere had no effect on receptor binding or sensitivity toward N-ethylmaleimide. Serine substitution of His223 in the putative second extracellular loop linking transmembrane domains 4 and 5 protected against N-ethylmaleimide inactivation. The H223S substitution decreased the affinity of bremazocine 25-fold, highlighting the importance of this residue for the formation of the high affinity bremazocine binding site in the mu opioid receptor.

Studies on mu and delta opioid receptor selectivity utilizing chimeric and site-mutagenized receptors.

Opioid receptors are members of the guanine nucleotide binding protein (G protein)-coupled receptor family. Three types of opioid receptors have been cloned and characterized and are referred to as the delta, kappa and mu types. Analysis of receptor chimeras and site-directed mutant receptors has provided a great deal of information about functionally important amino acid side chains that constitute the ligand-binding domains and G-protein-coupling domains of G-protein-coupled receptors. We have constructed delta/mu opioid receptor chimeras that were express in human embryonic kidney 293 cells in order to define receptor domains that are responsible for receptor type selectivity. All chimeric receptors and wild-type delta and mu opioid receptors displayed high-affinity binding of etorphine (an agonist), naloxone (an antagonist), and bremazocine (a mixed agonist/antagonist). In contrast, chimeras that lacked the putative first extracellular loop of the mu receptor did not bind the mu-selective peptide [D-Ala2,MePhe4,Gly5-ol]enkephalin (DAMGO). Chimeras that lacked the putative third extracellular loop of the delta receptor did not bind the delta-selective peptide, [D-Ser2,D-Leu5]enkephalin-Thr (DSLET). Point mutations in the putative third extracellular loop of the wild-type delta receptor that converted vicinal arginine residues to glutamine abolished DSLET binding while not affecting bremazocine, etorphine, and naltrindole binding. We conclude that amino acids in the putative first extracellular loop of the mu receptor are critical for high-affinity DAMGO binding and that arginine residues in the putative third extracellular loop of the delta receptor are important for high-affinity DSLET binding.

Cloning of a human cDNA encoding a putative nucleotide-binding protein related to Escherichia coli MinD.

A novel human cDNA encoding a putative nucleotide-binding protein (NBP) was obtained by screening a human SHSY5Y neuroblastoma library. The deduced protein contains 320 amino acids (aa) with a M(r) of 34,540. NBP displays sequence similarity with the product of the minD gene from Escherichia coli. MinD is involved in the proper placement of the division septum, and has ATPase activity. NBP and MinD contain consensus nucleotide (nt)-binding domains. The NBP mRNA is approx. 1500 nt in length and is expressed in several human cell lines and in all rat tissues examined, with the highest levels in lung and testis.

Sequence of the 5'-flanking region of the rat c-fos proto-oncogene.

The proto-oncogene c-fos is an immediate-early gene that becomes activated by a wide variety of extracellular stimuli. Since many studies regarding regulation of c-fos are conducted in vivo using rats, or with rat cell lines, we have cloned the rat c-fos gene in order to verify that the same regulatory elements that have been characterized in the human and mouse c-fos promoter are also present and functional in the rat. The nucleotide (nt) sequence of the 5'-flanking region of the rat c-fos gene displays remarkable similarity with the mouse and human c-fos genes (93 and 77% identity, respectively). cis-Acting regulatory elements, such as the sis-inducible element (SIE), serum-response element (SRE), AP-1-recognition site, calcium/cAMP-response element (Ca/CRE) and TATA box are present in the rat c-fos 5'-flanking region. A putative glucocorticoid-response element (GRE) is present 13 bp downstream from the AP-1-recognition site.

Regulation of calbindin-D28K gene expression in response to acute and chronic morphine administration.

The effect of acute and chronic morphine administration on calbindin-D28K (calbindin) gene expression has been studied. One group of adult male rats received a single injection of morphine (10 mg/kg, s.c.) or saline and were sacrificed 1 or 4 h later. Another group was injected with escalating doses of morphine sulfate twice daily for 15 days to induce tolerance and physical dependence. Rats were sacrificed 1 h after the last injection. In a third group, the effect of naloxone-precipitated withdrawal on gene expression in morphine-addicted rats was also analyzed 1 h after naloxone (1 mg/kg, i.p.). The cerebellum and remaining brain (minus the cerebellum) were removed, and total RNA was extracted and used for analysis. Calbindin mRNA levels in cerebellum were decreased to 30%-40% control at 1 and 4 h after a single morphine injection. Co-administration of the opiate antagonist, naloxone, reversed the effect of morphine. Tolerance developed to the acute effects in that levels were not altered significantly 1 h after morphine injection in chronically-treated rats. Unlike the cerebellum, calbindin mRNA in the remainder of the brain (minus the cerebellum) was unchanged 1 and 4 h following morphine administration to drug-naive rats, but was increased more than 2-fold compared to controls 1 h after morphine injection in chronically treated animals. Naloxone-precipitated withdrawal caused a small (20%) but significant decrease in calbindin mRNA in the cerebellum, with no change in the brain (minus the cerebellum).(ABSTRACT TRUNCATED AT 250 WORDS)

cis-acting elements in the 5'-untranslated region of rat testis proenkephalin mRNA regulate translation of the precursor protein.

We found that a rat testis cDNA library contained two types of proenkephalin cDNA, corresponding to somatic and germ cell-specific transcripts. The somatic form is identical in primary sequence to previously characterized transcripts from rat brain and heart. The longer germ cell-specific cDNA contains a novel 5' terminus derived from the somatic intron A. We found that germ cell-specific transcripts translate in vitro with significantly lower efficiency than do somatic-type transcripts. Deletion analysis indicated that translational inefficiency is mediated primarily by the presence of four upstream AUG codons followed by short open reading frames within the leader sequence. Secondary structure in the form of a stem-and-loop upstream of the initiator codon for proenkephalin was found to have a slightly inhibitory effect on translation. These results, coupled with the very low abundance of the somatic transcript, provide a possible explanation for the low levels of opioid peptides found in adult rat testis in spite of the abundant quantity of germ cell-specific proenkephalin transcript.

Human ribosomal protein L37a: cloning of the cDNA and analysis of differential gene expression in tissues and cell lines.

A cDNA corresponding to human ribosomal protein L37a (hL37a) was obtained by screening a SHSY5Y neuroblastoma library. The full-length cDNA contained 366 nucleotides (nt) in addition to a poly(A) tail. The pyrimidine-rich sequence, CTTTCT, that is common to many ribosomal protein-encoding cDNAs was present at the 5' terminus. The nt sequence displayed 85% identity with rat L37a (rL37a) cDNA. The predicted protein contains 92 amino acids with a M(r) of 10,277, is highly basic, and has 100% sequence identity with rL37a. A putative zinc-finger domain is present in the central region of the protein. Human lymphocytes and several human cell lines express hL37a mRNA at significantly higher levels than the rat cell lines and rat tissues tested. The hL37a gene does not contain introns.

The 'serotonin/norepinephrine link' beyond the beta adrenoceptor.

C6 rat glioma cells were utilized as a model system to probe the 'serotonin/norepinephrine link' at the level of preproenkephalin (PPE) gene expression. The beta adrenoceptor mediated increase in PPE mRNA was attenuated by the selective beta 1 adrenoceptor antagonist metoprolol which blocked the isoproterenol induced cyclic AMP generation by 97%. The subtype nonspecific antagonist propranolol blocked both the isoproterenol induced increase in cyclic AMP and the increase in the PPE mRNA steady-state levels. Serotonin (5-HT) had no effect on the density of beta adrenoceptors or their down-regulation by isoproterenol and did not alter the PPE gene expression in the absence of the beta signal. However, 5-HT significantly deamplified the beta signal mediated enhancement of the PPE mRNA thus indicating that the aminergic link occurs beyond the beta adrenoceptor.

Molecular cloning, sequence analysis and translation of proenkephalin mRNA from rat heart.

Proenkephalin mRNA is abundant in rat cardiac ventricles but surprisingly low levels of opioid peptides or precursor forms derived from proenkephalin are present in tissue extracts. Proenkephalin mRNA in rat heart was characterized at the molecular level with the use of cDNA sequencing, in vitro translation, and primer extension. Two positive proenkephalin cDNA clones were obtained by screening approx. 20,000 recombinant phages from a heart cDNA library. Sequence analysis of the cDNA clones indicated that the heart transcript was the same form as in rat brain, but differed from the germ cell-specific testis transcript that utilizes a different transcriptional start site. Heart proenkephalin cRNA translated efficiently, resulting in the synthesis of a 35 kDa protein that was immunoprecipitated by an antibody specific to the protein. The transcriptional initiation sites utilized in the heart were the same as in the brain, based on primer extension studies. These data suggest that the proenkephalin transcript found in abundance in rat heart is the same form as found in the brain, and differs from the testis-type transcript. We conclude that the scant level of proenkephalin-derived peptides in the heart is not due to an intrinsic inability of the proenkephalin transcript to translate.

Stimulation of c-fos and c-jun gene expression and down-regulation of proenkephalin gene expression in C6 glioma cells by endothelin-1.

The linkage between the transmembrane signal transduction system utilized by endothelin and alterations in gene expression has been investigated in C6 glioma cells. Treatment of C6 cells with endothelin-1 caused a rapid and transient 5-fold increase in c-fos and c-jun mRNA levels, followed by a decrease at 4 h. Dose-response studies indicated that 1 nM endothelin-1 caused half-maximal induction of c-fos mRNA 0.5 h after treatment and that maximal induction was elicited with a concentration of 10 nM. Actinomycin D totally abolished the rapid increase in c-fos mRNA caused by endothelin, indicating that the effect is at the transcriptional level. Endothelin-1 caused a decrease in proenkephalin mRNA to 50% of control levels at 4 h after treatment and had no effect on histone H4 mRNA over a 24 h period that was examined. These data indicate that receptor binding of endothelin-1 leads to rapid changes in the expression of immediate-early response genes which may cause more prolonged changes in the expression of AP-1 and/or CREB target genes in the nervous system.

Glucocorticoid-mediated down regulation of c-fos mRNA in C6 glioma cells: lack of correlation with proenkephalin mRNA.

We investigate the linkage between the transcriptional factor, c-fos, and expression of proenkephalin in rat C6 glioma cells. C6 cells contained abundant levels of c-fos mRNA. Treatment of cells with dexamethasone resulted in a 10-fold decline in c-fos transcripts and a small increase in proenkephalin mRNA. Combined exposure to dexamethasone and isoproterenol also induced a decrease in c-fos mRNA while proenkephalin mRNA increased 8-fold. Treatment of the C6 cells with phorbol 12-myristate 13-acetate caused a 13-fold increase in c-fos expression 0.5 h after administration and a decrease in proenkephalin mRNA. These data indicate that c-fos and proenkephalin mRNA are not regulated in a sequential, parallel manner, that newly synthesized c-fos is not the determining factor controlling proenkephalin gene regulation, and that c-fos expression is under negative control by glucocorticoids.

The measurement of complement fixation by autoantibodies directed against thyroid membrane antigens.

This paper describes the use of sensitized sheep red blood cells for the detection and titration of complement fixation by autoantibodies directed against human thyroid membranes in the serum of patients with autoimmune thyroid disease. Patients with elevated circulating levels of TPO antibodies and diagnosed as having autoimmune hypothyroidism (including Hashimoto's disease) or autoimmune hyperthyroidism (Graves' disease) were studied. Complement fixation titres were highest in those patients with autoimmune hypothyroidism compared with the autoimmune hyperthyroid group. Serum samples obtained from a group of patients with thyroid neoplasia and from normal healthy volunteers were negative in this test. The TPO antibody activity when "corrected" for its CF potency suggests that the autoantibodies found in autoimmune hypothyroidism are potentially more destructive than those found in the non-destructive autoimmune thyroid diseases.

Differential regulation of proenkephalin gene expression by estrogen in the ventromedial hypothalamus of male and female rats: implications for the molecular basis of a sexually differentiated behavior.

The ventrolateral aspect of the ventromedial hypothalamic nucleus (VL-VM) contains many estrogen-concentrating neurons which mediate estrogen facilitation of reproductive behavior. Previous studies have shown that estrogen treatment increases proenkephalin (PE) gene expression in neurons of the VL-VM in ovariectomized female rats, and that enkephalin peptides may stimulate lordosis behavior. To determine whether there is a sex difference in steroid hormone regulation of PE gene expression we have examined the effects of estrogen and testosterone on PE mRNA levels in male rats. Slot blot hybridization analysis of RNA isolated from the ventromedial hypothalamus indicated that estrogen treatment increased PE mRNA levels in the VL-VM of ovariectomized female rats (2.2-fold), but had no measurable effect on PE mRNA levels in gonadectomized males. Testosterone treatment of gonadectomized males also had no effect on PE gene expression. To determine whether the sex difference in estrogen-inducibility of PE gene expression is due to the developmental effects of gonadal steroids, we have investigated the effect of estrogen on PE mRNA levels in the VL-VM of neonatally androgenized female rats. Unlike the genetic male, the androgenized females responded to estrogen treatment with a female-typical increase in PE mRNA levels (1.7-fold). Further, although the androgenized rats clearly exhibited signs of defeminization, they did exhibit estrogen-facilitated lordosis behavior when tested with manual stimulation. The PE mRNA induction in estrogen-treated androgenized rats correlated well with the lordosis scores obtained by manual stimulation testing. These results indicate that estrogen regulation of PE gene expression in the VL-VM is sexually differentiated and support the hypothesis that the enkephalinergic neurons of the VL-VM are involved in the regulation of female reproductive behavior.