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M Muyan

Publications and source records attributed to M Muyan.

11 recordsLinked to original sources
PubMed2001

Fusion estrogen receptor proteins: toward the development of receptor-based agonists and antagonists.

Estrogen-induced signaling mediated by estrogen receptors (ERs) is also affected by aberrant ERs that act as constitutively active or dominant negative modulators. Variant ERs can contribute to carcinogenesis and to the loss of estrogen responsiveness, rendering antiestrogen therapy ineffective. Determining target gene response during co-synthesis of different ER species is difficult, because dimers formed in the presence of more than one ER species are a heterogenous population of homo- or heterodimers. We engineered a homofusion ERalpha as a prototype single-chain receptor by genetically conjugating two ER monomers into a covalently fused single-chain protein to obtain a homogeneous population. This permits analysis of symmetrical or asymmetrical mutations that simulate variant homo- and heterodimers. Although a monomer, the homofusion receptor exhibited similar biochemical and functional properties to the dimeric ERalpha. We used activation function-2 (AF2) defective mutants as a model in either one or both receptor domains for a dominant-negative phenotype by suppressing the reporter activity induced by the WT receptor. When co-expressed with ERalpha, the fusion variant deficient in both AF2 functions suppressed the reporter activity effectively induced by ERalpha. These results show the utility of fusion receptors as models for generation of receptor-based agonists and antagonists.

Animals↗
PubMed1998

Sequence requirements for estrogen receptor binding to estrogen response elements.

The estrogen receptor (ER) is a transcription factor that binds to a specific DNA sequence found in the regulatory regions of estrogen-responsive genes, called the estrogen response element (ERE). Many genes that contain EREs have been identified, and most of these EREs contain one or more changes from the core consensus sequence, a 13-nucleotide segment with 10 nucleotides forming an inverted repeat. A number of genes have multiple copies of these imperfect EREs. In order to understand why natural EREs have developed in this manner, we have attempted to define the basic sequence requirements for ER binding. To this end, we measured the binding of homodimeric ER to a variety of nonconsensus EREs. We discovered that an ERE containing even a single change from the consensus may be unable to bind ER. However, an ERE with two changes from the consensus may be capable of binding avidly to ER in the context of certain flanking sequences. We found that changes in the sequences flanking a nonconsensus ERE can greatly alter ER-ERE affinity, either positively or negatively. Careful study of sequences flanking a series of EREs made it possible to develop rules that predict whether ER binds to a given natural ERE and also to predict the relative amounts of binding when comparing two EREs.

Animals↗
PubMed1998

Dissociation of early folding events from assembly of the human lutropin beta-subunit.

The human LH of the anterior pituitary is a member of the glycoprotein hormone family that includes FSH, TSH, and placental CG. All are noncovalently bound heterodimers that share a common alpha-subunit and beta-subunits that confer biological specificity. LHbeta and CGbeta share more than 80% amino acid sequence identity; however, in transfected Chinese hamster ovary (CHO) cells, LHbeta assembles with the alpha-subunit more slowly than does hCGbeta, and only a fraction of the LHbeta synthesized is secreted, whereas CGbeta is secreted efficiently. To understand why the assembly and secretion of these related beta-subunits differ, we studied the folding of LHbeta in CHO cells transfected with either the LHbeta gene alone, or in cells cotransfected with the gene expressing the common alpha-subunit, and compared our findings to those previously seen for CG. We found that the rate of conversion of the earliest detectable folding intermediate of LH, pbeta1, to the second major folding form, pbeta2, did not differ significantly from the pbeta1-to-pbeta2 conversion of CGbeta, suggesting that variations between the intracellular fates of the two beta-subunits cannot be explained by differences in the rates of their early folding steps. Rather, we discovered that unlike CGbeta, where the folding to pbeta2 results in an assembly-competent product, apparently greater than 90% of the LH pbeta2 recovered from LHbeta-transfected CHO cells was assembly incompetent, accounting for inefficient LHbeta assembly with the alpha-subunit. Using the formation of disulfide (S-S) bonds as an index, we observed that, in contrast to CGbeta, all 12 LHbeta cysteine residues formed S-S linkages as soon as pbeta2 was detected. Attempts to facilitate LH assembly with protein disulfide isomerase in vitro using LH pbeta2 and excess urinary alpha-subunit as substrate were unsuccessful, although protein disulfide isomerase did facilitate CG assembly in this assay. Moreover, unlike CGbeta, LHbeta homodimers were recovered from transfected CHO cells. Taken together, these data suggest that differences seen in the rate and extent of LH assembly and secretion, as compared to those of CG, reflect conformational differences between the folding intermediates of the respective beta-subunits.

Animals↗
PubMed1998

The carboxyl-terminal region is a determinant for the intracellular behavior of the chorionic gonadotropin beta subunit: effects on the processing of the Asn-linked oligosaccharides.

The placental hormone human CG (hCG) consists of two noncovalently linked alpha- and beta-subunits similar to the other glycoprotein hormones LH, FSH, and TSH. These heterodimers share a common alpha subunit but differ in their structurally distinct beta subunits. The CGbeta subunit is distinguished among the beta subunits by the presence of a C-terminal extension with four serine-linked oligosaccharides (carboxyl terminal peptide or CTP). In previous studies we observed that deleting this sequence decreased assembly of the truncated CGbeta subunit (CGbeta114) with the alpha-subunit and increased the heterogeneity of the secreted forms of the uncombined subunit synthesized in transfected Chinese hamster ovary (CHO) cells. The latter result was attributed to alterations in the processing of the two N-linked oligosaccharides. To examine at what step this heterogeneity occurs, the CGbeta and CGbeta114 genes were transfected into wild-type and mutant CHO cell lines that are defective in the late steps of the N-linked carbohydrate-processing pathway. We show here that removal of the CTP alters the processing of the core mannosyl unit of the subunit to complex forms at both glycosylation sites and that the oligosaccharides contain polylactosamine. Although it has been presumed that there is little intramolecular interaction between the CTP and the proximal domains of the subunit, our data suggest that the CTP sequence participates in the folding of the newly synthesized subunit, which is manifest by the posttranslational changes observed here.

Amino Sugars↗
PubMed1997

Design of stable biologically active recombinant lutropin analogs.

Glycoprotein hormones are noncovalent heterodimers comprised of a common alpha subunit and a hormone-specific beta subunit. Secretion and biologic action of these hormones are dependent on the formation of the heterodimer. The human LH beta subunit is unique among the other beta subunits in that it assembles inefficiently with the alpha subunit. To bypass this rate-limiting step, we constructed the LH single chains where the carboxy terminus of beta was fused to the amino terminus of alpha subunit through a linker. Compared to the human LH heterodimer, the extent of secretion was greater for the tethers although the rate was dependent on the nature of the linker. The LH single chains were biologically active even though there was loss of recognition by a LH-specific monoclonal antibody. This suggests that receptor binding of the single chains is not impaired by changes in the heterodimeric configuration resulting from tethering the subunits. In addition, single chains exhibited a remarkably greater in vitro stability than the heterodimer, implying that these analogs will be useful as diagnostic reagents and that their purification will be facilitated.

Animals↗
PubMed1996

The carboxy-terminal region of the beta-subunits of luteinizing hormone and chorionic gonadotropin differentially influence secretion and assembly of the heterodimers.

One of the major structural differences between the LH beta and CG beta subunits is the carboxy-terminal region: beyond amino acid 114, LH beta has a hydrophobic heptapeptide stretch, while CG beta contains a 31-amino acid hydrophilic carboxy-terminal peptide (CTP) that is O-glycosylated. The CG beta subunit is secreted quantitatively as a monomer and assembles efficiently whereas secretion and assembly of LH beta is inefficient. We previously implicated the carboxy-terminal heptapeptide as a determinant for the different intracellular behavior manifested by the LH beta subunit compared with the CG beta subunit. Here we tested the function of the heptapeptide and CTP domains by fusing them to their counterparts at amino acid 114 of CG beta or LH beta subunits. The secretion and assembly of these chimeras were examined in transfected Chinese hamster ovary cells. Removal of the heptapeptide enhanced the amount of LH beta subunit secreted 4-fold compared with intact LH beta. Fusion of this heptapeptide to CG beta 114, i.e. CG beta lacking the CTP, decreased the amount of secreted subunit 2-fold compared with wild type human CG beta. Similar experiments reveal that although deleting the CTP from the CG beta subunit did not significantly alter secretion, the combination efficiency of the truncated subunit was reduced to 60%. Perturbing the native carboxy-terminal sequence of either subunit increased the heterogeneity of the secreted forms. This result suggests that these regions are also involved in the posttranslational processing of the asparagine-linked oligosaccharides of the beta-subunits. Fusion of the LH beta heptapeptide to the truncated CG beta subunit decreased combination with the alpha-subunit. These data further support the hypothesis that the carboxy-terminal regions of LH beta and CG beta subunits play a role in the intracellular behavior of the corresponding heterodimers.

Animals↗
PubMed1994

Secretion of lutropin and follitropin from transfected GH3 cells: evidence for separate secretory pathways.

Although lutropin (LH) and follitropin (FSH) are synthesized in the same pituitary gonadotropes, their secretion patterns in response to several experimental paradigms are not the same. Previous studies showing differences in secretion kinetics and the magnitude of hormone release by secretagogues imply differences in mechanisms for the storage and release of these hormones. To examine the secretory fate of LH and FSH, the genes encoding the common alpha-subunit and the corresponding beta-subunits were transfected in rat somatotrope-derived GH3 cells, which contain regulated and constitutive secretory pathways. The use of a gene transfer/heterologous cell system avoids physiological variations and functional heterogeneity of gonadotropes. Pulse-labeling and subsequent chase experiments demonstrated that although one third of newly synthesized FSH enters a regulated pathway, the majority is released constitutively. This contrasts with LH, which is mainly secreted through a regulated pathway. Although stored LH and FSH are released from GH3 cells in response to both KCl and forskolin, the magnitude of FSH release by secretagogues is smaller than that of LH. In Chinese hamster ovary cells, which are devoid of regulated secretory pathway and lack secretory granules, the mature forms of LH and FSH are neither stored nor released by secretagogues. These observations indicate that the intracellular mechanisms for the storage and release of LH and FSH differ and suggest that primary secretion of LH and FSH is via the regulated and constitutive pathways, respectively.

Animals↗
PubMed1993

Modulation of gonadotropin-releasing hormone-stimulated luteinizing hormone release in cultured male equine anterior pituitary cells by gonadal steroids.

The objective of the present study was to determine whether the testicular steroids, i.e., testosterone (T), dihydrotestosterone (DHT), estradiol (E2), estrone (E1), and estrone sulfate (E1SO4), play a physiological role in regulating LH release in the male horse by direct actions at the anterior pituitary gland. Enzymatically dispersed anterior pituitary cells from stallions (n = 4) or geldings (n = 3) were cultured for 48 h in alpha-modified Eagle's medium containing 10% steroid-free horse medium. To determine the effects of the steroids on the LH response to GnRH, the cells were incubated for 24 h in fresh media with or without 10(-10) M E2 or 10(-8) M T or DHT followed by a 4-h incubation +/- GnRH (10(-11) to 10(-7) M). Media and cells were analyzed for LH by RIA. In the stallion, GnRH increased LH release (p < 0.001) in a dose-dependent manner (ED50 GnRH = 4.5 x 10(-9) M), and this response was unaltered by T or DHT but greatly enhanced by E2 (p < 0.001). E2 lowered the ED50 for GnRH to 5 x 10(-10) M and increased the maximum LH response to GnRH by 350%. The LH release in response to a constant dose of 1 nM GnRH was unaltered by varying doses of T, DHT, or E1SO4 (10(-11) to 10(-7) M).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Testosterone suppresses 8-bromo-adenosine 3',5'-monophosphate and gonadotropin-releasing hormone-stimulated luteinizing hormone subunit synthesis.

The major objective of this study was to determine the effects of testosterone (T) on 8-bromo-cAMP (8-br-cAMP)- and GnRH-stimulated LH subunit polypeptide synthesis and glycosylation in cultured male anterior pituitary cells. The anterior pituitaries from 1-week castrate male rats were enzymatically dispersed and incubated for 48 h in steroid-free medium, followed by a 48-h incubation with or without 10 nM T. The cells were then incubated for 12 h in medium containing [35S]methionine ([35S]Met) and [3H]glucosamine ([3H]Gln) with or without 1 mM 8-br-cAMP or 1 nM GnRH, with or without 10 nM T. Incorporation of radiolabeled precursors into LH subunits was determined by specific immunoprecipitation of the LH dimer with subsequent analysis of the individual LH alpha- and beta-subunits by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. LH content was quantified by RIA (iLH). Both 8-br-cAMP and GnRH stimulated iLH release. T suppressed basal and GnRH-induced iLH secretion, whereas it enhanced iLH release stimulated by 8-br-cAMP. Both 8-br-cAMP and GnRH stimulated total (cell plus medium) [35S]Met and [3H]Gln incorporation into LH alpha and LH beta, and these responses were suppressed by T. Basal [35S]Met incorporation into the LH subunits was inhibited by T, whereas T had no effect on basal levels of [3H]Gln incorporation. Neither T nor GnRH altered [3H]Gln cell uptake or incorporation into total proteins, whereas 8-br-cAMP increased these responses. There were no treatment effects on [35S]Met cell uptake or incorporation into total proteins. These results suggest that 8-br-cAMP, similar to GnRH, stimulates both polypeptide synthesis and glycosylation of the LH alpha- and beta-subunits and that T suppresses these responses to 8-br-cAMP and GnRH in a similar fashion. These data indicate that cAMP is involved in mediating the actions of GnRH on LH subunit biosynthesis and that the inhibition of LH subunit polypeptide synthesis and glycosylation by T involves postreceptor events that are regulated by cAMP-dependent mechanisms.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1991

Direct effects of free and conjugated steroids on GnRH stimulated LH release in cultured equine anterior pituitary cells.

Enzymatically dispersed anterior pituitary cells from donor mares were cultured for 48 h in alpha-modified Eagles' medium containing 10% steroid-free horse serum. The cells were then incubated for 24 h in fresh medium oestrogen followed by a 4-h incubation with or without GnRH. Media and cell extracts were analyzed for LH by radioimmunoassay. In the first series of experiments, pituitary cells from Day-3 dioestrous mares were preincubated with ethanol (control) or different concentrations of E2 (10(-11) to 10(-7) M) for 24 h prior to a 4-h incubation without (basal) or with 1.0 nM GnRH. E2 increased (P less than 0.001) GnRH-stimulated LH release with a maximal response obtained at 10(-10) M E2 with an ED50 of 3 x 10(-11) M E2. Also E2 enhanced greatly the responsiveness of the pituitary to varying concentrations of GnRH by decreasing the minimum effective dose and ED50 by 93% and 78%, respectively, and by increasing the maximum response to GnRH by 250%. E2 had no effect on basal LH secretion. In a second experiment, the effects of E2, E1 and E1S on basal and GnRH stimulated LH release were evaluated. Although E1 increased GnRH-stimulated LH release, the maximally effective dose of E1 to enhance LH release and the ED50 for E1 were 100 times greater than those found for E2. E1S had no oestrogenic activity even at concentrations as high as 100 nM.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗