Binding and internalization of native gonadoliberin (GnRH) by anterior pituitary gonadotrophs of the rat

Summary To identify anterior pituitary cell types containing GnRH binding sites and to study the internalization process of this peptide by target cells under physiological conditions, autoradiography was performed on rat anterior pituitaries removed at specific time intervals (2–60 min) after intravenous injection of mono-radioiodinated ¹²⁵I-GnRH into intact males. At electron-microscopic level, gonadotrophs and lactotrophs appeared to contain silver grains. Concomitant administration of an excess of unlabeled GnRH with the radioiodinated hormone prevented this localization indicating the specificity of the reaction. The time-course study in gonadotrophs showed that 2 min after injection silver grains could be found over the plasma membrane, secretory granules and nuclear membrane. Similar results were observed 5 and 15 min after injection. Extensive label was observed over the nucleus and nuclear membrane 15 to 60 min after injection. The injection of a radioiodinated GnRH agonist [D-Trp⁶, Pro⁹ (Net), DesGly¹⁰]-GnRH produced comparable results. In contrast, the injection of ¹²⁵I-[D-pGlu¹, D-Phe², Trp^3,6]-GnRH, an antagonist of GnRH, produced positive labeling only at the plasma membrane without internalization. These results indicate that, after binding with receptors on the plasma membrane, GnRH is rapidly internalized, accumulating in secretory granules, and localizing over the nuclear membrane and later, in the nucleus. Association of radioactivity with secretory granules could be related to a specific action of GnRH at this level or to receptor recycling, and presence of label in the nucleus may be related to stimulation of neosynthesis of LH and GnRH receptors.     

Receptor-binding properties of gonadotropin-releasing hormone derivatives. Prolonged receptor occupancy and cell-surface localization of a potent antagonist analog

The receptor-binding properties and in vitro biological effects of a highly active gonadotropin-releasing hormone (GnRH) antagonist, [N-acetyl-D-p-chloro-Phe1,2D-Trp3,D-Lys6,D-Ala10]GnRH, were compared with those of the GnRH superagonist analog, [D-Ala6] des-Gly10-GnRH-N-ethylamide. In rat pituitary particles and isolated pituitary cells, the 125I-labeled GnRH antagonist showed saturable high-affinity binding (Ka v 8.4 +/- 1.4 X 10(9) M-1) to the same receptor sites which bound the GnRH agonist. The rate of dissociation of the receptor-bound antagonist from pituitary particles and cells was extremely slow in comparison with that of the agonist ligand. Also, dissociation of the antagonist analog was incomplete, with a residual fraction of tightly bound ligand that was proportional to the duration of preincubation. The [D-Lys6]GnRH antagonist prevented GnRH-induced luteinizing hormone release during static incubation and superfusion of cultured pituitary cells, but in contrast to the agonist did not cause desensitization of the gonadotroph. Although the antagonist caused a prolonged reduction in available GnRH receptor sites, this was attributable to persistent occupancy by the slowly dissociating ligand rather than to receptor loss. Autoradiographic analysis of [D-Lys6]GnRH-antagonist uptake by cultured pituitary cells revealed that the peptide remained bound at the cell membrane for up to 2 h, in contrast with the rapid endocytosis of GnRH agonists. The slow dissociation of receptor-bound antagonist was consistent with its ability to cause sustained blockade of GnRH actions, and its prolonged cell-surface location suggests that receptor activation is necessary to initiate the rapid internalization of hormone-receptor complexes that is a feature of the agonist-stimulated gonadotroph.     

Redistribution and recycling of internalized membrane in seminal vesicle secretory cells

This study was performed to clarify the fate of membrane constituents internalized from the apical domain in secretory cells, in particular their possible recycling and the compartments involved in it. Glycoproteins of the apical membrane of seminal vesicle secretory cells from guinea-pig were covalently labeled in vitro (0°C, 20 min) with ³H-galactose and the epithelium incubated for 15 min (37°C, first incubation) to allow endocytosis. The label which was not internalized was then exposed to enzymatic hydrolysis (0°C, 30 min) and the epithelium re-incubated to allow membrane movement for 15 and 30 min (37°C, 2nd incubation). After each step of the protocol, tissue pieces were fixed and processed for electron microscope autoradiography and the results studied by morphometric analysis. Following labeling, 99% of the silver grains were associated with the apical domain of the cell membrane (AD). After the 1st incubation at 37°C, 30° of the grains were inside the cells in association with the cytoplasmic vesicles (Cyt ves), secretory vacuoles (SV), Golgi vesicles (GV), Golgi cisternae (GC), multivesicular bodies (MVB), lysosomes (LYS), and the cell membrane basolateral domain (BLD). About 58% of non-internalized radioactivity was removed by hydrolysis. During the 2nd incubation at 37°C the concentration of label increased in BLD and LYS, decreased in SV and MVB, and fluctuated in GC, GV and AD. The distribution of grains observed at 15 min, as compared using the χ-square test, was highly significantly different from that expected without recycling. The results show that cell membrane glycoproteins internalized at the cell apex recycle back to the membrane apical domain and are consistent with the involvement of GC and SV in the recycling pathway. Membrane shuttle between the apical and basolateral domains of the cell membrane is also suggested by these observations.     

Binding and internalization of 125I-LDL in normal and mutant human fibroblasts. A quantitative autoradiographic study.

Using a quantitative EM autoradiographic technique, we have visualized the membrane binding and receptor-mediated uptake of low density lipoprotein (LDL) in human fibroblasts. The initial binding was restricted to the plasma membrane (2 h of incubation at 4 °C) and approx. 62% of the grains could be localized to coated pits in the plasma membrane. When the incubations were carried out at 37 °C, ¹²⁵I radioactivity was found both on the membrane and within the cell and predominantly localized on or within lysosomes. In cells from the patient J. D., a familial hypercholesterolemic homozygote with an internalization defect, initial binding of ¹²⁵I-LDL was restricted to the plasma membrane but not preferentially localized to coated segments of the plasma membrane. After incubation for 30 min at 37 °C, the membrane bound ¹²⁵I-LDL in J. D. cells was not internalized. These data confirm results obtained with ferritin-labeled LDL and illustrate the complementary application of two different morphologic probes, each of which offers special advantages for special problems.     

Interaction of fluorescent gonadotropin-releasing hormone with receptors in cultured pituitary cells

A fluorescent derivative of the gonadotropin-releasing hormone (GnRH) agonist analog, [D-Lys6]GnRH, was synthesized for receptor studies and shown to be biologically active. The rhodamine-derivatized peptide (Rh-GnRH) retained 40% of the receptor binding activity of [D-Lys6]GnRH, and 50% of the luteinizing hormone-releasing activity assayed in cultured pituitary cells. The fluorescent analog was employed to visualize the distribution of GnRH receptors in cultured pituitary cells, using the technique of video-intensified fluorescence microscopy. The binding of Rh-GnRH was confined to the large gonadotrophs which comprised 15% of the cell population. The specificity of the binding was shown by the absence of significant fluorescence in the presence of a 100-fold excess of [D-Lys6]GnRH, or when Rh-GnRH was incubated with choriocarcinoma, neuroblastoma, or 3T3 cell lines devoid of GnRH receptors. The interaction of Rh-GnRH with living pituitary cells was characterized by an initial diffuse distribution, followed by the formation of polar aggregates that later appeared to be internalized. These observations emphasize the value of fluorescent derivatives of GnRH for elucidating the course of the interaction with specific receptors on pituitary gonadotrophs. The initial results indicate that GnRH-receptor complexes undergo aggregation during stimulation of luteinizing hormone release, and are later internalized for subsequent degradation and/ or intracellular actions.     

Receptor-mediated uptake of GnRH agonist and antagonists by cultured gonadotropes: evidence for differential intracellular routing

The binding and uptake of the GnRH agonist D-Lys6-GnRH and of the antagonists [N-Ac-D-(pyro)-Cl-Phe1,2-D-Trp3-Lys6-D-Ala10]-GnRH and D-p-Glu1-D-Phe2-D-Trp3-D-Lys6-GnRH by dispersed pituitary gonadotropes was studied with electron microscopy. The peptides were coupled to colloidal gold markers with a diameter of 6 or 20 nm which were incubated separately or together for time periods between 15 and 180 min. Both antagonists could be found after 45 and 180 min at 37 degrees C in lysosomes as well as at the plasma membrane of gonadotropes. Co-incubation of both antagonists or of agonist and either antagonist resulted in uptake of the conjugates into separate lysosomes as well as mixed together into the same lysosome. Localization of the antagonists in structures associated with the Golgi apparatus was not observed at the time points studied. The results show that both GnRH agonist- and antagonist-conjugates are biologically active and that they are internalized by the gonadotropes via receptor mediated endocytosis. The failure to detect antagonist conjugates in the Golgi apparatus may indicate that passage through this organelle requires activation of the receptors by agonists and that the uptake of antagonist into lysosomes due to normal membrane protein turnover.     

Characterization of gonadotropin-releasing hormone (GnRH) binding to pituitary receptors in goldfish (Carassius auratus)

Goldfish pituitary gonadotropin-releasing hormone (GnRH) receptors were characterized by using a superagonist analog of teleost GnRH (tGnRH-A; [D-Arg6, Trp7, Leu8, Pro9-NHEt]-GnRH). Equilibrium binding of 125I-tGnRH-A to a goldfish pituitary membrane preparation was achieved after a 30-min incubation at 4 degrees C; binding was significantly reduced after increasing incubation temperature to 22 degrees C. Binding of the radioligand was a function of tissue concentration, with a linear correlation over the range of 0.5-2 pituitary per tube. Incubation of the pituitary membrane preparation with increasing concentrations of 125I-tGnRH-A indicated saturable binding at radioligand concentrations of 470 pM and above. The binding of 125I-tGnRH-A was found to be reversible after addition of the cold analog, and the dissociation curve could be resolved into two linear components; slower rates of dissociation of 125I-tGnRH-A were observed after the addition of excess unlabeled tGnRH than after the addition of tGnRH-A, indicating that the analog is more effective in displacing the label than the native peptide. Addition of the cold analog displaced bound 125I-GnRH-A, and Scatchard analysis suggested the presence of at least two classes of binding sites: a high-affinity/low-capacity site and a low-affinity/high-capacity site. Bound 125I-GnRH-A was displaced by tGnRH from both sites in parallel to that observed with tGnRH-A, indicating that both peptides bind to the same classes of binding sites; however, tGnRH-A had a greater affinity for the receptors than the native tGnRH. These results demonstrated the presence and provided characterization of GnRH receptors in goldfish pituitary.     

Surface interactions and intracellular localization of cationic ferritin: similarity to 125I-insulin in 3T3-L1 adipocytes

We previously reported that in 3T3-L1 adipocytes 125I-insulin associates preferentially with microvilli and coated pits at low temperatures and early times of incubation. At higher temperatures it is internalized through a series of membrane limited intracellular compartments. In the present study, we used a high resolution probe, cationic ferritin (CF), to track adsorptive endocytosis in the 3T3-L1 adipocyte. We find that CF initially associates with coated pits at 2 min of incubation at 37 degrees C. With further incubation at 37 degrees C CF is internalized and after 2 to 10 min of incubation is predominantly localized to coated and non-coated clear vesicles. Approximately 50% of the apparent coated vesicles seen near the plasma membrane on single thin sections are shown by serial sectioning to be true vesicles (i.e., without a surface connection). At later time points CF is localized predominantly to lysosomal structures and, to a much smaller extent, Golgi-related structures. The remarkable similarity between 125I-insulin and CF with respect to post-binding processing suggests that while the membrane receptor confers the initial specificity, post-binding events are common for different types of ligands after they bind to cell surfaces and are subject to adsorptive endocytosis.     

Redistribution and recycling of internalized membrane in seminal vesicle secretory cells

This study was performed to clarify the fate of membrane constituents internalized from the apical domain in secretory cells, in particular their possible recycling and the compartments involved in it. Glycoproteins of the apical membrane of seminal vesicle secretory cells from guinea-pig were covalently labeled in vitro (0 degrees C, 20 min) with 3H-galactose and the epithelium incubated for 15 min (37 degrees C, first incubation) to allow endocytosis. The label which was not internalized was then exposed to enzymatic hydrolysis (0 degrees C, 30 min) and the epithelium re-incubated to allow membrane movement for 15 and 30 min (37 degrees C, 2nd incubation). After each step of the protocol, tissue pieces were fixed and processed for electron microscope autoradiography and the results studied by morphometric analysis. Following labeling, 99% of the silver grains were associated with the apical domain of the cell membrane (AD). After the 1st incubation at 37 degrees C, 30% of the grains were inside the cells in association with the cytoplasmic vesicles (Cyt ves), secretory vacuoles (SV), Golgi vesicles (GV), Golgi cisternae (GC), multivesicular bodies (MVB), lysosomes (LYS), and the cell membrane basolateral domain (BLD). About 58% of non-internalized radioactivity was removed by hydrolysis. During the 2nd incubation at 37 degrees C the concentration of label increased in BLD and LYS, decreased in SV and MVB, and fluctuated in GC, GV and AD. The distribution of grains observed at 15 min, as compared using the chi-square test, was highly significantly different from that expected without recycling. The results show that cell membrane glycoproteins internalized at the cell apex recycle back to the membrane apical domain and are consistent with the involvement of GC and SV in the recycling pathway. Membrane shuttle between the apical and basolateral domains of the cell membrane is also suggested by these observations.     

Persistence of immunoreactive TRH and GnRH in long-term primary anterior pituitary cultures

Intact anterior pituitary tissue and primary anterior pituitary cultures were stained with 1:30,000 anti-TRH and 1:10,000 anti-GnRH using the peroxidase antiperoxidase immunocytochemical technique. Stains applied to serial ultrathin sections of intact pituitaries showed that TRH immunoreactivity could be localized in secretory granules of thyrotropes, gonadotropes and corticotropes whereas GnRH immunoreactivity was found only in gonadotropes and corticotropes. Long-term primary pituitary cultures were studied to remove the anterior pituitary cells from hypothalamic influences. In these cell populations both TRH and GnRH immunoreactivity persisted. In addition, quantification of the stained cells at the light microscopic level demonstrated that the volume fraction of TRH and GnRH immunoreactive cells remained constant up to 3 weeks of culture. Studies of serial ultrathin sections through cells from these cultures showed TRH or GnRH localized in secretory granules of cells that contained LH and ACTH, but not TSH. Both liquid and solid phase immunoabsorption specificity controls were used to validate the immunocytochemical stains. These studies suggest that the pituitary TRH and GnRH immunoreactivities may not be completely of hypothalamic origin, but may also be endogenous to a subpopulation of unique multihormonal pituitary cells.     

Quantitative determination of the intracellular fate of internalized plasma membrane in dissociated pituitary prolactin cells utilizing a radioiodinated cationic ferritin probe (CFI*) and electron microscopic autoradiography

Dissociated anterior pituitary cells derived from estrogen-treated female rats were incubated with radioiodinated cationic ferritin (CFI) for 2 min and subsequently in the absence of CFI for varying periods of time up to 3 hr in order to quantitate, using electron microscopic autoradiography, the distribution of retrieved plasma membrane in these cells. Following a 2-min incubation with CFI, autoradiographic grains were found to be associated almost exclusively with the plasma membrane. With increasing periods of incubation in the absence of CFI, grain-density analysis revealed increasing levels of CFI in multiple intracellular organelles. The levels of CFI were greatest for the lysosomes, intermediate for the mature secretory granules, and least for the Golgi cisternae and immature secretory granules. These findings are consistent with the idea that a portion of the retrieved plasma membrane is degraded in lysosomes and that the remainder is recycled to organelles comprising the secretory pathway to be reutilized in successive waves of the secretory cycle.     

Desensitization of pituitary gonadotropes by mediators of LH release

Desensitization of pituitary gonadotropes by exposure to 10 nM gonadotropin-releasing hormone (GnRH) for 6 h severely impaired the luteinizing hormone (LH) response to a second 3-h treatment with GnRH, and reduced the secretory responses to 50 microM arachidonic acid (AA), 100 nM tetradecanoyl phorbol-13-acetate (TPA), and AA + TPA. Pretreatment with AA blocked subsequent responses to AA but not to other secretagogues. Pretreatment with TPA attenuated the LH response to TPA, but not to GnRH, AA, and AA + TPA. After exposure to AA + TPA, all subsequent responses were abolished. Each of the secretagogues reduced GnRH receptor binding, but only GnRH-induced receptor loss and desensitization were reversed by simultaneous incubation with a GnRH antagonist. Similar results were obtained when 16-h pretreatment periods were used, or when the data were normalized for the concomitant reduction of cellular LH content. These findings indicate that GnRH-receptor loss and depletion of LH content are not the sole causes of GnRH-induced desensitization. Receptor uncoupling and impairment of AA- and protein kinase C-dependent pathways may also be involved in this process.     

Temporal behavior of abrin in the intoxication of chinese hamster cells (line CHO)

Abrin, a potent cytotoxin, was utilized as a probe to elucidate the mechanism by which external proteins are delivered to the cytoplasm of mammalian cells. Abrin bound rapidly to the surface receptors of the Chinese hamster cells (line CHO) and appeared to be internalized immediately without any significant lag. The maximum level of abrin internalization was achieved within eight minutes, based on both biochemical and electron microscopic autoradiographic studies with [¹²⁵|] abrin. About 10% of the silver grains of internalized [¹²⁵|] abrin were associated with vesicular structures, irrespective of the incubation time. Inhibition of protein synthesis began 30 minutes postincubation, and this latent period was not dependent on extracellular toxin concentration. SDS-polyacrylamide gel electrophoresis of the internalized [¹²⁵|] abrin indicated that internalized abrin molecules remained intact even after two hours of incubation.     

Solubilized active pituitary and ovarian gonadotropin-releasing hormone receptors retain binding properties for adenosine 3′, 5′-cyclic monophosphate derivatives

The non-denaturing zwitterionic detergent, (3 (3-cholamidopropyl)-dimethyl-ammonio)-1-propane sulfonate (CHAPS), has been used to solubilize membrane gonadotropin-releasing hormone (GnRH) receptors from rat ovaries. The solubilized receptors retain a high affinity (K_a = 1.85 ± 0.3 nM^?1), comparable to the affinity measured in membrane particles (K_a = 3.25 ± 0.7 nM^?1), and a preserved specificity for several analogs and fragments of GnRH. At millimolar concentrations, cyclic AMP derivatives inhibit [125_I] - GnRH analog binding to both membrane particles and soluble receptors from pituitary and ovary. These results support the hypothesis that cyclic AMP may play the role of an extracellular messenger by interacting with the GnRH receptor itself.     

Inhibition of coated pit formation in Hep2 cells blocks the cytotoxicity of diphtheria toxin but not that of ricin toxin

It has been recently shown (Larkin, J. M., M. S. Brown, J. L. Goldstein, and R. G. W. Anderson, 1983, Cell, 33:273-285) that after a hypotonic shock followed by incubation in a K+-free medium, human fibroblasts arrest their coated pit formation and therefore arrest receptor-mediated endocytosis of low density lipoprotein. We have used this technique to study the endocytosis of transferrin, diphtheria toxin, and ricin toxin by three cell lines (Vero, Wi38/SV40, and Hep2 cells). Only Hep2 cells totally arrested internalization of [125I]transferrin, a ligand transported by coated pits and coated vesicles, after intracellular K+ depletion. Immunofluorescence studies using anti-clathrin antibodies showed that clathrin associated with the plasma membrane disappeared in Hep2 cells when the level of intracellular K+ was low. In the absence of functional coated pits, diphtheria toxin was unable to intoxicate Hep2 cells but the activity of ricin toxin was unaffected by this treatment. By measuring the rate of internalization of [125I]ricin toxin by Hep2 cells, with and without functional coated pits, we have shown that this labeled ligand was transported in both cases inside the cells. Hep2 cells with active coated pits internalized twice as much [125I]ricin toxin as Hep2 cells without coated pits. Entry of ricin toxin inside the cells was a slow process (8% of the bound toxin per 10 min at 37 degrees C) when compared to transferrin internalization (50% of the bound transferrin per 10 min at 37 degrees C). Using the indirect immunofluorescence technique on permeabilized cells, we have shown that Hep2 cells depleted in intracellular K+ accumulated ricin toxin in compartments that were predominantly localized around the cell nucleus. Our study indicates that in addition to the pathway of coated pits and coated vesicles used by diphtheria toxin and transferrin, another system of endocytosis for receptor-bound molecules takes place at the level of the cell membrane and is used by ricin toxin to enter the cytosol.     

Effects of gonadotropin-releasing hormone (GnRH) on the cytodifferentiation of gonadotropes in rat adenohypophysial primordia in organ culture

The effects of gonadotropin-releasing hormone (GnRH) on the development of gonadotropes were investigated by the use of organ culture and by means of immunocytochemistry and radioimmunoassay. Pituitary primordia from rat fetuses were cultured in a medium with or without 10^-9 M GnRH during the first 24 h of culture. The ratio of the number of immunoreactive LH cells to the total number of cells in the explants derived from 13.5-day fetuses was increased by the GnRH treatment after 6 or 8 days of culture, while the total number of cells was not altered. LH released into the medium and LH content of explants were not affected by the GnRH treatment. Subsequent treatment with 10^-9 M GnRH for 4 h after 7 days of culture resulted in a marked release of LH, accompanying a significant decline in LH content, in both explants exposed or unexposed to the first GnRH treatment. However, the former explants contained a lower amount of LH than the latter explants. The present results indicate that pituitary primordia at 13.5 days of gestation are capable to respond to GnRH, and that GnRH is effective in stimulating the responsiveness of gonadotropes to GnRH during early pituitary cytodifferentiation.     

Transient receptor potential (TRP) channel function in the reproductive axis

Transient receptor potential (TRP) channels play important functional roles in the signal transduction machinery of hormone-secreting cells and have recently been implicated in reproductive physiology. While expression studies have demonstrated TRP channel expression at all levels of the hypothalamic–pituitary–gonadal (hpg) axis, functional details about TRP channel action at the level of the individual cells controlling reproduction are just beginning to emerge. Canonical TRP (TRPC) channels are prominently expressed in the reproductive center of the neuroendocrine brain, i.e. in kisspeptin and gonadotropin-releasing hormone (GnRH) neurons. Kisspeptin neurons are depolarized by leptin via activation of TRPC channels and kisspeptin depolarizes GnRH neurons through TRPC4 activation. Recent studies have functionally identified TRPC channels also in gonadotrope cells in the anterior pituitary gland, which secrete gonadotropins in response to GnRH and thus regulate gonadal function. TRP channel expression in these cells exhibits remarkable plasticity and depends on the hormonal status of the animal. Subsequent functional analyses have demonstrated that TRPC5 in gonadotropes contributes to depolarization of the plasma membrane upon GnRH stimulation and increases the intracellular Ca²⁺ concentration via its own Ca²⁺ permeability and via the activation of voltage-gated Ca²⁺ channels. However, conditional gene targeting experiments will be needed to unambiguously dissect the physiological role of TRPC channels in the different cell types of the reproductive axis in vivo.     

Autoradiographic study of binding and internalization of follicle-stimulating hormone in the mouse testis minces in vitro

The internalization of FSH-receptor complexes was demonstrated in mouse testis by means of light and electron microscopic autoradiography. Chopped testicular pieces were incubated with radioiodinated FSH (131I-NIADDK-rat FSH-I-4) for 10, 20, 60 and 180 min. After incubation the pieces were fixed with glutaraldehyde containing tannic acid, and embedded in Spurr's resin. Semithin and ultrathin sections were cut for light and electron microscopic autoradiography, respectively. In light microscopic autoradiographs, silver grains were preferentially localized over Sertoli cells, regardless of incubation time. Sixty to 70% of the total number of grains were located over Sertoli cells which account for only about 4% of the total cell population of the seminiferous tubules. The majority of these grains correspond to the specific FSH binding sites, because few grains remained after incubation with an excess amount of unlabeled FSH. In electron microscopic autoradiographs, the half-distance (HD) value for the 131I-labeled line source was about 216 nm in the present study. After 10 min of incubation, 56.6% of the total number of silver grains were located over the plasma membrane of Sertoli cells. In testicular pieces incubated for longer periods (20, 60 and 180 min), both the percentage and relative concentration of grains increased in the Golgi apparatus and lysosomes and decreased in the plasma membrane. These results suggest that [131I]iodo-FSH first binds to FSH receptors on the plasma membrane of Sertoli cells, then FSH-receptor complexes are internalized. The increase in the number of grains over the lysosomes following longer incubation, indicates that internalized [131I]iodo-FSH or FSH-receptor complexes are subjected to degradation.     

Localization of putative gonadotrophin releasing hormone receptor protein in the anterior pituitary

Specific binding of a fully biologically active 125I-gonadotrophin releasing hormone (GnRH) to isolated anterior pituitary cells is time dependent, saturable and the concentration dependent binding curves exhibit positive cooperativity. Binding to intact or solubilized plasma membranes and an affinity purified GnRH receptor protein reveals in all instances multiple high affinity binding sites. Thus, GnRH receptor protein appears to be an intrinsic constituent of the cell membrane, and perhaps, other membranous organelles. To investigate the latter, the binding of 125I-GnRH to various subcellular fractions was studied and its affinity and time requirements determined. GnRH binding to plasma membranes and secretory granules was to multiple high affinity sites, while that to nuclei and microsomes was to a single high affinity site. Binding was 1.83 +/- 0.07, 0.78 +/- 0.04, 0.31 +/- 0.03 and 0.27 +/- 0.03 fmol micrograms-1 protein for isolated plasma membranes, secretory granules, microsomes and nuclei, respectively, after 30 min incubation with 10(-9) M GnRH. The magnitude of binding to microsomes did not change during the incubation period. It did not show any decrease (p greater than 0.05) in isolated nuclei and plasma membranes, except for the 24 h time period, when a significant drop (p less than 0.001) was seen. Binding to the secretory granule fraction culminated at 15 min and then decreased (p less than 0.001) steadily to a non-detectable level at 24 h. Thus GnRH receptor protein or its portion may be an integral part of some membranous particles in the anterior pituitary cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stimulation of pituitary gonadotropin release does not require internalization of gonadotropin-releasing hormone

Binding of gonadotropin-releasing hormone (GnRH, pyro-Glu1-His2-Trp3-Ser4-Tyr5-Gly6-Leu7-Arg8-Pro9-Gly-NH210) to its plasma membrane receptor is the first step leading to the release of pituitary luteinizing hormone. As in the case of other plasma membrane receptors, patching, capping, and internalization of this hormone-receptor complex occurs rapidly following exposure of cultured pituitary cells to physiological levels of releasing hormone. In the present study we sought to determine whether gonadotropin release could occur under conditions which rigorously excluded internalization. A GnRH analog, D-Lys6-GnRH (to which a small quantity of [125I]iodoTyr5-D-Lys6-GnRH was added), was coupled by its epsilon-amino group with an N-hydroxysuccinimide ester then, through a 10-A spacer arm, to a cross-linked agarose matrix. Exposure of the product to proteases, soaps, detergents, solvents, chaotropic agents, or cell cultures resulted in dissociation of < 0.28% of biologically active releasing hormone. The apparent potency of the immobilized analog was one-fourth that of the free form and it was still capable of evoking a full luteinizing hormone secretory response. It can, therefore, be concluded that internalization of GnRH is not required for gonadotropin release.