G.S. Menzies

Non-genomic steroid receptors in the bovine ovary

Over the last few years, rapid and physiologically important non-genomic actions of all classes of steroid hormones have been described in many cell types. A putative non-genomic membrane progesterone receptor (NGPR) was the first, and so far the only, non-genomic steroid receptor cloned. Two homologous NGPR proteins have been identified in the human, and a similar protein in the bovine and rat. Various detection methods have been used to identify putative NGPRs in a range of tissues: however, different methods often yield quite different molecular weights, and probably detect distinct moieties. We describe some properties of the specific cell-surface membrane binding sites for [3H]-progesterone in enriched cell membrane preparations of bovine luteal and follicular cells. Similar binding sites were also detected in cell-membranes of some (but not all) bovine tissues. Western blots of detergent extracts of bovine luteal membranes identified a protein (85kDa) that reacted with an antiserum to the N-terminal peptide of porcine NGPR. Activity was low in native non-denatured extracts, but increased dramatically in a dose-dependent manner following pretreatment with the cholesterol-complexing agent, digitonin. This protein was co-precipitated by antisera to caveolin. In contrast, a specific monoclonal antibody to the ligand binding domain of the genomic progesterone receptor (Mab C262) detected two proteins (M(r), 55 and 60kDa) in luteal membrane detergent extracts. Immunostaining of these proteins by Mab C262 was abolished by digitonin concentration-dependent manner in non-denatured extracts. However, both proteins were unaffected by digitonin in fully denatured detergent extracts, suggesting that digitonin induced a conformational change in the native protein that prevented binding of Mab C262 to its epitope. Our data suggest the presence of a complex of two or more distinct membrane-associated progesterone-binding proteins in bovine luteal membranes. Moreover, their conformations are specifically affected by removal of bound cholesterol.

Stimulation of specific binding of [3H]-progesterone to bovine luteal cell-surface membranes: specificity of digitonin

Non-genomic actions of progesterone have been described in the ovary, and luteal membranes of several species have been shown to possess specific binding sites for [3H]-progesterone. However, binding of radiolabelled progesterone to luteal membranes was demonstrable only in the presence of digitonin. Digitonin is a non-ionic detergent which is thought to act by forming one-to-one complexes with certain sterols. It is also a cardiotonic agent, inhibiting (Na+-K+) ATPase activity by interaction with the extracellular (ouabain/K+) binding site. We therefore investigated which properties of digitonin were responsible for its stimulatory actions on progesterone binding to bovine luteal membranes. A range of compounds with detergent, cardiotonic and or cholesterol-complexing activities were tested for their effects on [3H]-progesterone binding to bovine luteal membrane fractions, and on haemolysis of rat erythrocytes. Stimulation of progesterone binding to luteal membranes was highly specific for digitonin, and a number of ionic and non-ionic detergents, cardenolides, saponins and cholesterol-complexing reagents tested failed either to stimulate [3H]-progesterone binding to bovine luteal membranes in the absence of digitonin, or to inhibit binding specifically in the presence of digitonin. When digitonin was first reacted with excess cholesterol or pregnenolone to form the respective digitonides, stimulatory activity was greatly reduced, suggesting that the ability of digitonin to interact with (an) endogenous steroid(s) may be important in its action. High performance liquid chromatography (HPLC)-mass spectrometry of commercially available digitonin preparations indicated the presence of numerous minor impurities in most commercial digitonin preparations. Three major UV-absorbing peaks were isolated and characterised by mass spectrometry: all stimulated progesterone binding to bovine luteal membrane receptors in a dose-dependent manner, though to differing extents. Our data suggest that the unique action of digitonin on luteal membrane progesterone receptors is not related to its detergent or cardiotonic properties, but appears to be related to its ability to complex with membrane sterols.

Specific, high-affinity binding sites for human luteinizing hormone (hLH) and human chorionic gonadotrophin (hCG) in Candida species

The presence of specific binding sites for [125I]-labelled hLH and hCG is described in Candida species. Binding was present in three strains of Candida albicans, and in Candida tropicalis, and was greatest in microsomes, though binding was also present in cytosol fractions. hLH and hCG mutually competed for these binding sites. Other hormones did not bind and did not compete for hLH binding sites. Scatchard plots showed two classes of binding sites, one with high affinity, low capacity and the other with lower affinity, high capacity binding in both microsomes and cytosol. This is the first report of specific binding sites for mammalian peptide hormones in a yeast.

Co-purification of a ribonuclease and human chorionic gonadotrophin β-core protein from human urine and displacement of 125I-human luteinizing hormone from Candida albicans binding sites by ribonucleases

An 18 kDa pregnancy urine protein preparation, purified to apparent electrophoretic homogeneity as judged by silver-staining of polyacrylamide gels, inhibited binding of 125I-hLH (human luteinizing hormone) to Candida albicans microsomes, reacted with monoclonal and polyclonal antibodies raised against human chorionic gonadotrophin (hCG) beta-core protein and exhibited ribonuclease (RNase) activity. Eleven of the 12 amino acids at the N-terminus of a protein in this preparation were identical to those of the N-terminus of human non-secretory ribonuclease. These results indicate co-purification of hCG beta-core with a RNase. An 18 kDa RNase was also purified from a commercial hCG preparation (Chorulon). However, no RNase activity was detected in a highly purified commercial preparation (Profasi). Three commercial RNase preparations displaced 125I-hLH from C. albicans binders at extremely low concentrations (< 0.001 microg/ml RNase) whereas only slight displacement of 125I-hLH from sheep luteal binding sites was observed with very high concentrations of the RNases (100 microg/ml RNase). The co-purification of hCG beta-core and RNase from pregnancy urine and the displacement of 125I-hLH from C. albicans binding sites by RNases may be related to the close relationship that has been identified between mammalian RNase inhibitors and the extracellular domain of gonadotrophin receptors. The presence of RNase in commercial preparations of gonadotrophins should be borne in mind during any investigations that involve impure preparations of these hormones.

Failure to detect gonadotrophin-releasing hormone receptors in human benign and malignant breast tissue and in MCF-7 and MDA-MB-231 cancer cells

We have measured the binding of radiolabelled analogues of gonadotrophin-releasing hormone (GnRH) to homogenates of human breast cancer and benign breast tissue, and to MCF-7 and MDA-MB-231 cell lines. Although incubation of breast cancer homogenates with the 125I-labelled GnRH agonist analogues, buserelin [(D-Ser tBU6)GnRH 1-9 ethylamide] and tryptorelin [(D-Trp6) GnRH 1-9 ethylamide] appeared to show significant though low, specific GnRH agonist binding in a high proportion of breast cancers (32/42 for buserelin; 15/32 for tryptorelin) and benign breast tissues (13/16 for buserelin; 10/12 for tryptorelin), after correction for displaceable binding in control assay tubes, GnRH agonist binding to breast tissue was no longer apparent. The lack of specific binding was not due to inactivation of GnRH agonist tracers, as > 86% of the unbound tracer was still capable of rebinding to fresh placental membranes after incubation with breast cancer homogenates. GnRH agonist did not bind to MCF-7 and MDA-MB-231 cells, however GnRH agonist tracer inactivation following exposure to these cells was very high. We have shown recently that human placental receptors bound salmon GnRH and chicken GnRH II as well as GnRH agonists, but not other isoforms of GnRH. However, no isoform of GnRH bound significantly to human breast tumour tissue. In summary, we could not confirm the presence of specific GnRH binding sites in homogenates and membranes from human breast tissues in this study. Low levels of apparently specific binding of GnRH agonist tracers could be accounted for entirely by displacement of tracer from assay tubes. Inability to demonstrate specific binding was not due to extensive inactivation of GnRH tracers (although this may be a factor in the failure to demonstrate GnRH binding to MCF-7 and MDA-MB-231 cell lines).

Inactivation of porcine corpus luteum lutropin receptors by a microsomal enzyme: Activation in ageing and regressing corpora lutea

The binding of 125I-labelled human choriogonadotropin (hCG) to homogenates of porcine corpora lutea showed a marked departure from ideal behaviour, due to a time- and temperature-dependent inactivation of both free hormone and unoccupied receptors. Occupied receptors were not affected. Lutropin (LH)-receptor-inactivating activity was detected after preincubation of homogenates, particulate fractions and microsomes, but little activity could be demonstrated in cytosol fractions. Inactivation was dependent on the temperature and pH of preincubation, and on tissue concentration: LH-receptor inactivation was first-order with respect to preincubation time. Lutropin-receptor-inactivating activity was low in early-luteal and mid-luteal phase in pig corpora lutea, but was increased significantly in late-luteal and regressing corpora lutea.