Degradation of the subunits of receptor-bound human choriogonadotropin by Leydig tumor cells

Biologically active, iodine-labeled derivatives of human choriogonadotropin in which all the iodine is localized either in the alpha or beta subunits have been prepared. It is found that upon binding to Leydig tumor cells these derivatives are ultimately degraded to 3'-monoiodotyrosine. A comparison of the rates of degradation of the derivatives labeled exclusively in the alpha or beta subunits show that the alpha subunit is degraded somewhat faster than the beta subunit. It was also found that NH4Cl, chloroquine and leupeptin inhibited the degradation of both subunits to the same extent. These results show that the Leydig tumor cells degrade both subunits of the receptor-bound human choriogonadotropin, and suggest that the two subunits are degraded by the same mechanism(s).     

Inhibition of the degradation of receptor-bound human choriogonadotropin by leupeptin.

The ability of leupeptin to block the degradation of receptor-bound human choriogonadotropin has been studied. It was found that this compound inhibited hormone degradation and intracellular cathepsin B activity in a parallel fasion, without affecting hormone-stimulated steroidogenesis.     

Internalization and recycling of lutropin receptors upon stimulation by porcine lutropin and human choriogonadotropin in porcine Leydig cells

Porcine lutropin (pLH) and human choriogonadotropin (hCG) recognize the same hormonal receptor and elicit the same steroidogenic response in porcine Leydig cells in primary culture. We compared the variation in the number of occupied and free receptors present on the cell surface under short-term stimulation by the two hormones at 35 degrees C. Both hormones produced a rapid dose-dependent decrease in the total number of the gonadotropin receptors present on the cell surface with a half-life of 8-10 min. This decrease was reversible upon hormone removal and receptors were recovered on the cell surface with the same half-life of 8-10 min. With pLH, the receptors were recycled in a free state, but in the presence of hCG the receptors were recycled in an occupied state. This difference could be related to the higher affinity of hCG for the receptor in 150 mM NaCl buffer (Ka = 1.6 X 10(9) for hCG and 1.5 X 10(7) for pLH) and higher stability to acid pH of the hCG-receptor complex (dissociation pK = 3.7 for hCG and 4.5 for pLH).     

Inhibition of the degradation of receptor-bound human choriogonadotropin by lysosomotropic agents, protease inhibitors, and metabolic inhibitors.

A previous report from this laboratory showed that binding of iodine-labeled human choriogonadotropin to Leydig tumor cells is not a reversible process (Ascoli, M., and Puett, D. (1978) J. Biol. Chem. 253, 4892--4899). Most of the cell-bound hormone was found to be degraded to 3'-monoiodotyrosine before being released from the cells, and the degradation process could be inhibited by the lysosomotropic agents NH4Cl, chloroquine, and Triton WR-1339. It is reported herein that the degradation of receptor-bound human choriogonadotropin is an energy-dependent process, which can be inhibited by compounds that interfere with glycolysis or oxidative phosphorylation (e.g. NaF, NaN3, NaCN, and 2-deoxyglucose). Hormone degradation is also inhibited by some protease inhibitors such as the chloromethyl ketones of lysine and phenylalanine, but not by specific trypsin inhibitors (e.g. p-aminobenzamidine and p-tosyl-L-arginine methyl ester). With the exception of NH4Cl, it was found that the compounds which inhibit hormone degradation also inhibit hormone-stimulated steroidogenesis. However, the present results involving dose dependency, and those given in the following paper (Ascoli, M. (1978) J. Biol. Chem. 253, 7839--7843), indicate that these two phenomena are not related.     

Demonstration of a direct effect of inhibitors of the degradation of receptor-bound human choriogonadotropin on the steroidogenic pathway.

Results presented in the previous paper (Ascoli, M., and Puett, D. (1978) J. Biol. Chem. 253, 7832--7838) show that the degradation of receptor-bound 125I-labeled human choriogonadotropin can be inhibited by chloroquine, protease inhibitors, and metabolic inhibitors. These compounds were also shown to inhibit gonadotropin-stimulated steroidogenesis. It is reported herein that these inhibitors also block the stimulation of steroidogenesis by both cholera toxin and 8-Br-adenosine 3':5'-monophosphate, thus showing that they are not specific for the hormonal stimuli. These results, taken together with previous observations that show that NH2Cl can block hormone degradation without inhibiting hormone-stimulated steroidogenesis, strongly suggest that the degradation of choriogonadotropin is not required for its stimulatory action on progesterone production.     

Removal of the surface-bound human choriogonadotropin results in the cessation of hormonal responses in cultured Leydig tumor cells

The relationship between the binding and internalization of human choriogonadotropin and the stimulation of cAMP and steroid production was studied in cultured Leydig tumor cells. It was found that removal of the surface-bound hormone results in a rapid cessation of both cAMP and steroid production. We propose that the surface-bound hormone is responsible for the activation of steroidogenesis and that hormone internalization is involved in the deactivation of this process.     

Internalization and recycling of receptor-bound gonadotropin-releasing hormone agonist in pituitary gonadotropes

The fate of cell surface gonadotropin-releasing hormone (GnRH) receptors on pituitary cells was studied utilizing lysosomotropic agents and monensin. Labeling of pituitary cells with a photoreactive GnRH derivative, [azidobenzoyl-D-Lys6]GnRH, revealed a specific band of Mr = 60,000. When photoaffinity-labeled cells were exposed to trypsin immediately after completion of the binding, the radioactivity incorporated into the Mr = 60,000 band decreased, with a concomitant appearance of a proteolytic fragment (Mr = 45,000). This fragment reflects cell surface receptors. Following GnRH binding, the hormone-receptor complexes underwent internalization, partial degradation, and recycling. The process of hormone-receptor complex degradation was substantially prevented by lysosomotropic agents, such as chloroquine and methylamine, or the proton ionophore, monensin. Chloroquine and monensin, however, did not affect receptor recycling, since the tryptic fragment of Mr = 45,000 was evident after treatment with these agents. This suggests that recycling of GnRH receptors in gonadotropes occurs whether or not the internal environment is acidic. Based on these findings, we propose a model describing the intracellular pathway of GnRH receptors.     

Internalization and degradation of receptor-bound interferon-gamma by murine macrophages. Demonstration of receptor recycling

Although the interferon-gamma (IFN-gamma) receptor on murine and human mononuclear phagocytes has been defined and partially characterized, very little data exists which describes the ultimate fate of receptor-bound ligand. The current studies were specifically designed to define the metabolic processes which act on murine recombinant IFN-gamma following its interaction with murine macrophages at physiologic temperatures. Ligand internalization was demonstrated by comparing binding of [125I]IFN-gamma to macrophages at 4 degrees C and 37 degrees C. When binding was carried out at 4 degrees C, 96% of the cell-associated [125I]IFN-gamma remained accessible at the plasma membrane and could be stripped from the cell by exposure to pronase. In contrast, at 37 degrees C, only 35% of the cell-associated radioactivity was pronase strippable. Macrophages degraded [125I]IFN-gamma into trichloroacetic acid-soluble material at 37 degrees C at a constant rate of 7000 molecules/cell/hr over a 12-hr time period. The amount of IFN-gamma degraded correlated with the amount of IFN-gamma bound to the cell surface. The receptor was neither up- nor down-regulated by ligand or by other agents known to regulate macrophage functional activity such as IFN-alpha, IFN-beta, lipopolysaccharide, or phorbol myristate acetate. The constant uptake of IFN-gamma by macrophages was due to the presence of an intracellular receptor pool (62% of the total receptor number) and to a mechanism of receptor recycling. Evidence for the latter was obtained using lysosomotropic agents which blocked degradation but not binding and internalization of ligand and caused the intracellular accumulation of receptor. By comparing the relationship between receptor occupancy and biologic response induction, two activation mechanisms became apparent. Induction of certain functions, such as H2O2 secretion, appeared to require only a single round of receptor occupancy. However, induction of more complex functions such as nonspecific tumoricidal activity appeared to require three to four rounds of receptor occupancy. These results thus support the concept that IFN-gamma internalization and receptor recycling are essential in the induction of nonspecific tumoricidal activity by macrophages.     

Immunoprecipitation of the lutropin/choriogonadotropin receptor from biosynthetically labeled Leydig tumor cells. A 92-kDa Glycoprotein

The structure of the lutropin/choriogonadotropin (LH/CG) receptor has been studied by immunoprecipitating the receptor from biosynthetically labeled cultured Leydig tumor cells (designated MA-10). This was performed by binding human choriogonadotropin (hCG) to the labeled cells, solubilizing the hormone-receptor complex, partially purifying the complex by lectin chromatography, and immunoprecipitating the complex with an antibody that recognizes receptor-bound hCG. The conditions used for the release of the radiolabeled receptor from the immunoprecipitate and the subsequent analysis of this material on sodium dodecyl sulfate gels allowed us to determine directly the structure of the free (not hormone-occupied) LH/CG receptor. From experiments using cells labeled with [35S]methionine and [35S]cysteine, we show that the LH/CG receptor is composed of a single polypeptide chain that migrates as a 92-kDa protein on sodium dodecyl sulfate gels whether analyzed in the absence or presence of reducing agents. Other studies presented demonstrate that the LH/CG receptor is a glycoprotein.     

Internalization and degradation of glucagon by human mononuclear cells

The processing of glucagon by circulating human mononuclear cells was examined. Glucagon bound to the membrane with a turnover time of 4.4 minutes per site after 15 minutes of incubation and 8 minutes per site after 90 minutes. The amount of intact intracellular hormone increased by 3-fold by 90 minutes suggesting a slowing of intracellular processing with prolonged incubation. Excess unlabelled insulin also slowed the processing of glucagon at the degradative step with no effect on binding or internalization of glucagon. Subcellular fractionation of the cells showed that most hormone accumulated in the 500xg pellet and in the 100,000xg supernatant. N-ethylmaleimide blocked intracellular glucagon degradation suggesting a role for intracellular sulfhydryl-dependent enzymes. Kinetic analysis of the dissociation of glucagon revealed a second order process with K values of 2.2 X 10(-2) fm-1 min-1 and 1.4 X 10(-2) fm-1 min-1 for dissociation from membranes and from membranes + intracellular sites, respectively. T 1/2 values were 6 min. for membrane dissociation and 9 min for membranes + cells. These findings suggest that glucagon interaction with mononuclear cells has characteristics similar to other receptor bound ligands including internalization processing and metabolism.     

On the mechanisms involved in the regulation of the cell-surface receptors for human choriogonadotropin and mouse epidermal growth factor in cultured Leydig tumor cells

The MA-10 cells are a clonal strain of mouse Leydig tumor cells that have receptors for human choriogonadotropin (hCG) and mouse epidermal growth factor (mEGF). Exposure of the cells to hCG results in a reduction in the number of surface hCG receptors, and little or no change in the number of surface mEGF receptors. On the other hand, exposure of the cells to mEGF results in a reduction in the number of both surface mEGF receptors and surface hCG receptors. In order to study these phenomena, we assumed that the number of surface receptors is determined by the rate at which receptors appear at the surface and by the rate of receptor internalization. When these rates were measured, we found that hCG and mEGF reduce their respective surface receptors by increasing the rate of receptor internalization, and that mEGF reduces the surface hCG receptors by decreasing the rate of appearance of the receptor.     

Fate determination of fetal Leydig cells

Leydig cell (LC) is one of the most important somatic cell types in testis, which localized in the interstitium between seminiferous tubules. The major function of Leydig cells is to produce steroid hormone, androgens. LC differentiation exhibits a biphasic pattern in rodent testes, which are divided into two different temporal mature populations, fetal Leydig cells (FLCs) and adult Leydig cells (ALCs). FLCs are transiently present in fetal testes and undergo involution or degeneration after birth. FLCs are completely devoid and replaced by ALCs in adult testes. Comparing to ALCs, FLCs display unique morphology, ultrastructure and functions. The origin of FLCs has been debated for many years, but it is still a mystery. Many factors have been reported regulating the specification, proliferation and differentiation of FLCs. FLCs degenerate in a few weeks postnatally, however, the underlying mechanism is still unknown. In this review, we will focus on the fate determination of FLCs, and summarize the resent progress on the morphology, ultrastructure, function, origin and involution of FLCs.     

Characterization of the expression products of recombinant human choriogonadotropin and subunits

Human choriogonadotropin (hCG) is a placental glycoprotein hormone composed of a 92-amino acid alpha subunit noncovalently linked to a 145-amino acid beta subunit. We report here the expression of biologically active hCG in mouse C127 cells transfected with expression vectors containing the DNA coding for both subunits. In addition, the same cell line was used to express the alpha subunit alone. The expression products were purified by affinity chromatography using specific monoclonal antibodies to hCG or its subunits. The system secreting biologically active hCG also produced a 10-fold or greater molar excess of free beta subunit. The dimeric hormone, as well as the excess beta subunit, resembles the standard urinary hCG and beta subunit by chemical and biological criteria. In contrast, when the vector encoding for the alpha subunit was expressed alone, the alpha subunit had a higher molecular weight than both standard alpha and the alpha found in the expressed dimeric hormone. The molecular weight difference between expressed alpha subunit and standard alpha was found to reside in the alpha peptide consisting of residues 52-91 which contained all of the carbohydrate of the alpha subunit. The N-asparagine-linked carbohydrate moieties in the recombinant alpha were found to be triantennary in contrast to biantennary in urinary alpha, and this hyperglycosylation was responsible for the higher molecular weight of the alpha subunit when it was expressed alone. We found no evidence of O-threonine glycosylation at position alpha 39 reported to be present in free forms of the alpha subunit; however, the companion paper (Corless, C.L., Bielinska, M., Ramabhadran, T. V., Daniels-McQueen, S. Otani, T., Reitz, B. A., Tiemeier, D. C., and Boime, I. (1987) J. Biol Chem. 262, 14197-14203) finds a small quantity of O-glycosylation. Since the excess beta subunit appears to be of normal size and contains the expected complement of sugars, only free alpha subunit seems to be a potential substrate for addition of extra sugar moieties. No large beta subunit forms have been found by others, while large alpha subunits have been described both clinically and in tissue culture systems. These observations imply that the conformation of the free alpha subunit, in the regions of the glycosylation recognition sites, allows easier access for glycosyltransferases than those same sites in the beta subunit. When alpha is combined with beta, the local structures around the alpha glycosylation sites are apparently altered so as to make the synthesis of triantennary chains less favorable.     

Studies of the specific role of the subunits of choriogonadotropin for biological, immunological and physical properties of the hormone. Digestion of choriogonadotropin and its isolated subunits with serine carboxypeptidase

The residues 90-92 can be split off from the C-terminal region of the isolated alpha-subunit of choriogonadotropin (residues 88--92: -Tyr-Tyr-His-Lys-Ser-OH) by means of serine carboxypeptidase (des-Lys91,Ser92-alpha-subunit; des-(90-92)-alpha-subunit). However, when choriogonadotropin is digested by serine carboxypeptidase, only the residues 143-145 (-Leu-Pro-Gln-OH) form the C-terminus of the beta-subunit are released (des-(143-145)-choriogonadotropin). Depending on the pH conditions, glutamine 145 and the residues 143-145, respectively, are liberated by digestion of the isolated beta-subunit (des-Gln145-beta-subunit and des-(143-145)-beta-subunit, respectively). The present study provides evidence that the C-termini of both the isolated subunits and those in choriogonadotropin are probably arranged on the surface of the molecules. The biological activity of des-(143-145)-choriogonadotropin is not significantly decreased. The immunological activity, however, is reduced when measured by complement fixation. In comparison to the native hormone, a four-fold amount of des-(143-145)-choriogonadotropin has to be applied to obtain highest complement fixation. The conformation of des-(143-145)-choriogonadotropin does not seem to differ from that of the native hormone, when estimated both by CD measurements and by Ans-choriogonadotropin fluorescence. The respective determinant therefore seems to depend, at least to some extent, on the sequence of the C-terminal region of the beta-subunit of the hormone; complement fixation, however, does not seem to be affected significantly, when the des-(143-145)-beta-subunit is compared with the native beta-subunit using an antiserum against the native beta-subunit. This provides evidence that this C-terminal determinant is possibly more immunogenic at the hormone than at the isolated beta-subunit. The biological activity of recombined choriogonadotropin in vivo as well as in vitro is markedly reduced when serine 92 is removed from the C-terminus of the alpha-subunit (des-Ser92,Lys91-alpha-native beta-subunit: 36% residual activity in vivo). Biological activity is lost when the residues 88-90 are removed by digestion of the des-Ser92,Lys91-alpha-subunit with carboxypeptidase A. Recombination products between a modified alpha-and the native beta-subunit show a reduced Anschoriogonadotropin fluorescence (des-Lys91,-Ser92-alpha + native beta-subunit: 52%; des-(88-92)-alpha- + native beta-subunit: 23%). The Ans-induced aggregation of choriogonadotropin, however, also takes place in those recombination products which display a low Ans-choriogonadotropin fluorescence, indicating that the reduction is probably not caused by a portion of the molecules losing their binding sites for Ans. Therefore the diminished Ans-choriogonadotropin fluorescence seems to signal small conformational changes. The CD spectra of the native and the des-(90-92)-alpha-subunit, however, seem not to differ significantly. It is shown that the release of amino acids from the C-terminus of the alpha-subunit causes a disturbance of the interaction between the subunits. This seems to prevent an effective conformational change of the beta-subunit which probably is a prerequisite for the binding of the hormone to the receptors of Leydig cells.     

Processing of human choriogonadotropin and its receptors by cultured pig Leydig cells. Role of cyclic AMP and protein synthesis

We have examined the process by which human choriogonadotropin/luteinizing hormone (hCG/LH) receptors are regulated in cultured porcine Leydig cells. Treatment of Leydig cells with human choriogonadotropin, cholera toxin, forskolin and cyclic 8-bromoAMP (8-BrcAMP) produced a loss of surface receptors without modification of the binding affinity. This negative regulation of the number of receptors mediated by maximal concentrations of hCG was higher than that induced by the other agents. The extent of receptor loss in cells treated with increasing concentrations of hCG was highly correlated with their capacity to stimulate cAMP production. However, there was little correlation between down-regulation and cAMP production of these cells treated by hCG plus forskolin or cholera toxin plus forskolin, where a synergistic cAMP production was obtained. Following exposure of Leydig cells to both hCG and 8-BrcAMP, the surface receptor disappearance began after an initial lag period of about 6-8 h. Thereafter a 50% loss of surface receptor was observed in the next 8-h incubation. Monensin with hCG shortens this lag period before initiation of receptor loss. Kinetic studies with 125I-hCG, in the presence or absence of monensin, showed that the half-life of the receptor-bound hormone complexes at the cell surface was 10.5 h and 8 h respectively. Therefore, the steady state of the surface receptor during the lag phase of 8 h is probably related to recycling of internalized receptors and/or translocation of performed receptors. Cycloheximide and actinomycin D inhibit hCG-mediated and 8-BrcAMP-mediated down-regulation. Cycloheximide lengthens ligand-receptor complexes at the surface by slowing down the rate of internalization (half-life of 20 h), but this mechanism is not enough per se to explain the effect of cycloheximide. Pulses of hCG or 8-BrcAMP for 4 h and 8 h sufficed to induce nearly maximal down-regulation. However, it was possible to attenuate this triggering effect by adding cycloheximide after pulse of the cells. Thus, even after removal of the triggering agent (hCG or 8-BrcAMP), the loss of surface receptor could be triggered by a protein-sensitive signal. Taken as a whole these results indicate that a coordinated interaction is involved in the cell-surface hCG/LH receptor regulation. The apparent steady state of the number of receptors during the first hours of stimulation passed through a reuptake of internalized receptors.(ABSTRACT TRUNCATED AT 400 WORDS)     

Lutropin is processed much more rapidly than human choriogonadotropin by porcine Leydig cells in primary culture

Lutropin (LH) and human choriogonadotropin (hCG) share the same receptor and stimulate testosterone production in porcine Leydig cells in primary culture. Cells were pulsed with [125I]LH or [125I]hCG. During the chase, more than 80% of cell-bound LH consisted in internalized material which was degraded and excreted (half-time : 25 min) NH4Cl largely inhibited this degradation. On the contrary, hCG remained essentially bound to the cell surface and was not degraded by the cells with or without NH4Cl up to 160 min.     

The inositol phosphate/diacylglycerol pathway in MA-10 Leydig tumor cells. Activation by arginine vasopressin and lack of effect of epidermal growth factor and human choriogonadotropin

It is now well established that mouse epidermal growth factor (mEGF) modulates the hormonal responsiveness of MA-10 Leydig tumor cells but does not affect cell multiplication. The studies presented herein are the first in a series of experiments designed to characterize the intracellular signaling systems activated by mEGF and their possible roles in mediating the diverse biological actions of this growth factor in MA-10 cells. We show that (i) MA-10 cells express a hormone-sensitive inositol phosphate/diacylglycerol pathway that can be stimulated with arginine vasopressin (AVP), (ii) mEGF does not activate this pathway, and (iii) activation of this pathway with arginine vasopressin does not mimic the biological actions of mEGF. Other data presented show that lutropin/choriogonadotropin, the principal endocrine regulators of Leydig cell function, also do not stimulate the inositol phosphate/diacylglycerol pathway in MA-10 cells.