Gonadotropin-releasing Hormone Receptor Protein Research Articles

Solubilization of pituitary GnRH binding sites by means of a zwitterionic detergent

Solubilization in an active form of the pituitary plasma membrane protein carrying binding sites for gonadotropin-releasing hormone (GnRH) was possible by using as detergent CHAPS (3[(3-cholamidopropyl)dimethylammonio]-l-propane sulphonate), which is a zwitterionic derivative of cholic acid. In contrast, the use of the non-ionic detergent Triton X-100 resulted in the loss of GnRH binding sites. CHAPS at a concentration of 5–10 mM allowed the solubilization of up to 80% of available sites, and remaining non-solubilized sites retained unaltered binding properties. Characterization of solubilized binding sites of GnRH was achieved by using as radioactive ligand and standard a stable GnRH analog, [ D-Trp 6,(NEt)Pro 9,desGly 10]-GnRH. Maximum binding of GnRH to solubilized binding sites was possible in the presence of a very low amount of CHAPS in the incubation medium. Scatchard analysis of classical saturation experiments indicated the presence of a single class of high affinity, low capacity binding sites. The mean value for the affinity constant K A was 0.36±0.07 × 10 10 M -1 ( n = 5) for solubilized GnRH binding sites, when analysed in the presence of 2 mM CHAPS; when solubilized GnRH binding sites were analysed in the absence of CHAPS, significantly higher K A values were observed, ranging from 1.2 to 5.9 × 10 10 M -1. Parallel analysis of the plasma membranes prior to solubilization indicated a mean K A value of 0.53±0.13 × 10 10 M -1 ( n = 5). The binding specificity of the pituitary GnRH binding sites as seen by evaluating cross-reactions with a panel of agonists and antagonists of GnRH was not altered by solubilization. Gel filtration on Sephadex G-25 of solubilized binding sites preincubated with radioiodinated GnRH analog demonstrated the presence of a large molecular weight complex. In conclusion, CHAPS appears to be quite suitable for the solubilization of the binding site moiety of the pituitary GnRH receptor with good retention of the binding characteristics, thus offering a new possibility for the purification and characterization of the GnRH receptor protein.

Peptide-receptor protein relationships: Steroid feedback in GnRH stimulation of the anterior pituitary

Gonadotropin releasing hormone receptor protein (GnRH·RP) purifled to homogeneity has several binding sites for its effector hormone, as evidenced from the concentration dependence binding curve. Binding of a fully biologically active iodinated GnRH decreased ( P < 0.001) by pretreatment of immobilized receptor protein with physiological concentrations of both main sex steroids, testosterone and estradiol 17-β. With increasing concentrations of the steroids the binding of 125I-GnRH decreased biphasically for testosterone and multiphasically for estradiol 17-β. The findings are conducive to an allosteric receptor model.

Peptide—receptor protein relationships: importance in estradiol feedback

The gonadotropin releasing hormone (GnRH) acts to release bovine luteinizing hormone (LH) [I] as well as follicle-stimulating hormone (FSH) [2] in vivo and stimulates the release of LH from bovine anterior pituitary tissue slices during superfusion in vitro in a dose-dependent manner [ 11. Fully biologically active ‘251-labeled GnRH binds to isolated anterior pituitary cells in a dose-dependent and saturable manner and the concentration dependence curve (physiological concentrations of GnRH) exhibits positive cooperativity [3,4]. Similarly, GnRH binding in isolated anterior pituitary plasma membranes, solubilized plasma membranes and purified GnRH receptor protein (GnRH.RP) is to multiple binding sites [5]. GnRH.RP purified to homogeneity aggregates in solution upon binding of GnRH present in physiological concentrations I51. The concentration-dependence binding curves of labeled GnRH to isolated anterior pituitary cells of different origin suggested involvement of sex steroids [3,4]. The apparent effect of a predominantly estrogenic environment was inducible by the pretreatment of steer anterior pituitary plasma membranes in vitro with physiological concentrations of estradiol 17-p [3,4]. Here, the modulation of GnRH binding by estradiol 17-P at the level of GnRH.RP is investigated since steroids were implicated in normal LH response to GnRH in the bovine [ 1,6] as well as other species [7,8] and in the feedback control of preovulatory LH surge, in general.

BINDING OF GONADOTROPHIN RELEASING HORMONE (GnRH) BY BOVINE ANTERIOR PITUITARY PLASMA MEMBRANES AND PURIFIED GnRH RECEPTOR PROTEIN (GnRH.R)

[125I]-(GnRH) concentration dependence binding curves using isolated bovine anterior pituitary plasma membranes, intact and solubilized, and purified GnRH receptor protein, are compared. In all instances the concentration dependence binding curves had a stepwise character here interpreted as multisigmoid, with several steep increases and plateaus. These curves are compatible with the existence of several binding sites for GnRH. Purification of the GnRH receptor protein (GnRH.R) resulted in about 500 000-fold increase of binding activity and yielded a single protein species on polyacrylamide gel electrophoresis in SDS, of estimated molecular weight 60 000. Similarity of GnRH binding by the purified protein with that of intact and solubilized plasma membranes suggested that a single protein was responsible for the binding in each instance. Thus heterogeneity of GnRH binding is likely attributable to phase transitions of a single receptor protein into different allosteric forms. The data suggest that positive cooperativity is involved in the studied system.

Uremic Sex

Gonadotropin-releasing hormone (GnRH) is briefly discussed. Luteinizing hormone (LH) secretory response to GnRH in vitro was obtained even in calcium-free medium. It is concluded that calcium is not an absolute requirement for the GnRH-stimulated LH release. In the absence of calcium, however, there was no response of LH to nonspecific stimuli such as enhanced potassium concentration. It is proposed that GnRH receptor protein exists in allosteric forms with different binding activities and that molecular rearrangement of the protein may modifiy the properties of the plasma membrane enough to change its permeability to ionic environment and secretory products. Therefore, the receptor protein may have a regulatory role independent of the cyclic adenosine monophosphate system.