Dual internalization pathway for lutropin and choriogonadotropin in porcine Leydig cells in primary culture

The role of the high affinity receptor in the internalization of porcine lutropin (pLH) and human choriogonadotropin (hCG) by porcine Leydig cells in primary culture during short-term stimulation by the two hormones was investigated. The fate of the hormones was followed either by electron microscopy (with colloidal gold-labeled hormones) or by measurement of the cellular distribution of [125I]pLH and [125I]hCG. With both techniques, the internalization of pLH was found to be one order of magnitude greater than hCG, though the recycling rate of the high affinity receptors was the same with both hormones. However, when the cell surface was progressively depleted of its high affinity receptors by preincubation with increasing doses of hCG or pLH, the internalization of [125I]pLH remained high and largely independent of the number of high affinity receptors still available on the cell surface, while that of [125I]hCG was found to be proportional to this number. The endocytosis of [125I]pLH could only be inhibited by the simultaneous presence of micromolar concentrations of unlabeled pLH, hCG or alpha or beta subunits of ovine LH (oLH). The intact alpha-hCG subunit and the deglycosylated alpha-oLH subunit were less potent, while beta-hCG and deglycosylated beta-oLH had no significant effect. These results could be explained by the existence of a "carrier" or "scavenger" receptor for LH, but with a low affinity (congruent to 3.10(6) M-1) and present in excess on the cell surface as compared to the high affinity receptor. The possible physiological significance of this receptor is discussed.     

Gonadotropin receptor complexes and free receptors in porcine Leydig cell cultures during recovery from hCG stimulation

Free and occupied gonadotropin receptors were studied in vitro in porcine Leydig cells culture maintained in chemically defined medium. Free receptors were evaluated by the binding capacity for 125I-hCG. hCG bound molecules (or hCG receptor complexes) were evaluated using immunocytochemical visualization on fixed cells. Exposure to hCG for 16 hours (.5 to 50 ng/ml) induced the disappearance of free receptors. After removal of the hormone, the return to control levels was observed at 48 and 72 hours. Visualization of hCG bound at the cell surface indicates that, following continuous exposure to gonadotropins for 48 hours, hCG molecules are still present on the cell. Following short-time exposure (1 h) to hCG and 48 hrs washing the number of stained cells is very close to the initial value suggesting that the occupied sites (at 48 hours) represent the initial hormone receptor complexes. These results indicate that, during prolonged incubation, hCG binding is not reversible, that the half-life of some of the complexes at the cell surface is very long and that the receptors recovery is slow and is probably the result of a de novo synthesis.     

Conformational restrictions of the sheep testicular receptor discriminates pituitary lutropin and placental gonadotropins

A membrane preparation from the testis of maturing Dorset-Leicester-Suffolk sheep, capable of discriminating pituitary LH (lutropin) from placental gonadotropins human choriogonadotropin (hCG) and equine choriogonadotropin is described. Maximum binding of 125I-oLH (ovine lutropin) to the testicular receptors occurred at 4 degrees C in a rapid manner, attaining equilibrium in 12-16 h. Under such optimal conditions, only unlabeled ovine LH or the structurally identical bovine LH effectively competed for receptor occupation. Other highly purified pituitary LH preparations from rat and human pituitaries were weakly (4-10%) active in displacement assays. Purified hCG or equine choriogonadotropin, which were highly potent in rat testicular LH receptor assays, could not compete with 125I-oLH for binding to the sheep LH receptor at 4 degrees C. Thus, the sheep testicular LH receptor was highly specific in recognizing pituitary LH conformation. The presence of an ovine/bovine LH alpha- or beta-subunit in recombinants with hCG subunit counterparts was required to generate an effective conformation capable of receptor recognition. Chemically deglycosylated hCG, containing 75% less carbohydrate and which showed greater binding to other LH receptors, failed to recognize sheep LH receptor, suggesting that excess carbohydrate in hCG was not a factor in hindering binding of the native placental hormone. Scatchard analysis using 125I-hCG/125I-oLH revealed that there were separate sites with similar affinities but vastly different capacities. The hCG binding sites, which could also be effectively occupied by oLH, were less than 10% of oLH binding sites. Thus, the Dorset-Leicester-Suffolk sheep testicular receptor provides an important and unique in vitro test system to distinguish pituitary LH from placental LH-like hormones. We infer that temperature-dependent conformational restrictions of the sheep testicular LH receptor are involved in recognizing differences in these highly similar and structurally homologous hormones.     

Analysis of the steady state binding, internalization, and degradation of human interferon-alpha2

Scatchard analyses of the equilibrium binding of radiolabeled human interferon-alpha2 (huIFN-alpha2) to Madin-Darby bovine kidney cells previously exposed to subsaturating concentrations of IFN-alpha showed approximately a 50% decrease in the number of cell surface receptors and no change in the apparent dissociation constant, Kd, compared with cells not exposed to interferon. The steady state equations describing the interaction of polypeptide ligands with cell surface receptors under physiological conditions (Wiley, H.S., and Cunningham, D.D. (1981) Cell 25, 433-440) have allowed us to determine, under steady state conditions, the rate of insertion of receptors into the cell membrane, the endocytic rate constant of occupied receptors, the rate constant of turnover of unoccupied receptors, and the rate of hydrolysis of internalized ligand. Our results indicate that occupied and unoccupied interferon receptors are cleared from the cell surface at approximately the same rate. This suggests that the down-regulation of the huIFN-alpha2 receptor on Madin-Darby bovine kidney cells by huIFN-alpha2 differs from that of several other surface receptors for polypeptide hormones and growth factors analyzed on cultured cells in that the binding of huIFN-alpha2 to its receptor does not increase the rate of receptor endocytosis.     

Evidence for the rapid internalization and recycling of lutropin receptors in rat testis Leydig cells.

A study into the binding of 125I-human chorionic gonadotropin (hCG) to the lutropin (LH) receptor in rat testis Leydig cells, and subsequent internalization of the hormone-receptor complex, has been carried out. The results show that there is rapid internalization of the hormone-receptor complex; 240 receptors/cell (from a total of approx. 4000 receptors/cell) were internalized each minute in the first hour after exposure to hCG. Radioactivity was released from the cell 1 h after internalization and was found to be associated with highly degraded hCG. The endocytic process was found to have two temperature-sensitive steps. At 4 degrees C, movement of the hormone-receptor complex inside the cell did not occur, and at 21 degrees C hormone accumulated within the cytoplasm but was not degraded or released from the cell. At 34 degrees C, internalization, degradation and loss of the degraded hormone from the cell occurred. These processes appeared to reach a steady state after 2 h. Even though there is rapid internalization of the hormone-receptor complex following exposure to hCG, the binding sites on the cell surface were maintained for at least 4 h. The number of binding sites on the cell surface was not decreased by a protein synthesis inhibitor but was reduced to undetectable levels by monensin. This compound inhibits acidification of endocytic vesicles, which is known to be an important prerequisite to receptor cycling. It is concluded that, in the rat testis Leydig cells, following binding of hCG to the LH receptor there is rapid internalization of the complex and that recycling of the receptor occurs to the cell surface. This process may be essential in maintaining the capacity of the Leydig cell to bind fresh hormone.     

A constitutively active mutant of the human lutropin receptor (hLHR-L457R) escapes lysosomal targeting and degradation

Using biochemical and imaging approaches, we examined the postendocytotic fate of the complex formed by human choriogonadotropin (hCG) and a constitutively active mutant of the human lutropin receptor (hLHR-L457R) found in a boy with precocious puberty and Leydig cell hyperplasia. After internalization, some of the complex formed by the hLHR-wild type (hLHR-wt) and hCG recycles to the cell surface, and some is found in lysosomes where the hormone is degraded. In contrast, the complex formed by the hLHR-L457R and hCG is not routed to the lysosomes, most of it is recycled to the cell surface and hormone degradation is barely detectable. For both, hLHR-wt and -L457R, there is an hCG-induced loss of cell surface receptors that accompanies internalization but this loss cannot be prevented by leupeptin. The removal of recycling motifs of the hLHR by truncation of the C-terminal tail at residue 682 greatly enhances the lysosomal accumulation of the hormone-receptor complexes formed by the hLHR-wt or the L457R mutant, the degradation of the internalized hormone, and the loss of cell surface receptors. The degradation of the hormone internalized by these mutants as well as the loss of cell surface receptors is largely prevented by leupeptin. These results highlight a previously unrecognized complexity in the postendocytotic trafficking of the hLHR and document a clear difference between the properties of the constitutively active mutant and the agonist-activated hLHR-wt. This lack of lysosomal degradation of the L457R mutant could contribute to its constitutive activity by prolonging the duration of signaling.     

Fast renal trapping of porcine Luteinizing Hormone (pLH) shown by <Superscript>123</Superscript>I-scintigraphic imaging in rats explains its short circulatory half-life

Background  

Sugar moieties of gonadotropins play no primary role in receptor binding but they strongly affect their circulatory half-life and consequently their in vivo biopotencies. In order to relate more precisely hepatic trapping of these glycoproteic hormones with their circulatory half-life, we undertook a comparative study of the distribution and elimination of porcine LH (pLH) and equine CG (eCG) which exhibit respectively a short and a long half-life. This was done first by following half-lives of pLH in piglets with hepatic portal circulation shunted or not. It was expected that such a shunt would enhance the short half-life of pLH. Subsequently, scintigraphic imaging of both ¹²³I-pLH and ¹²³I-eCG was performed in intact rats to compare their routes and rates of distribution and elimination.     

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)     

Identification of LH/hCG receptors in rabbit uterus

Luteinizing hormone (LH) is believed to act via specific receptors to control gonadal steroidogenesis and reproductive processes. Recently A. J. Ziecik, P. D. Stanchev, and J. E. Tilton (Endocrinology 119:1159, 1986) reported surprisingly that LH/hCG receptors were present in porcine uterus, a tissue not known to be a target for LH action. We report herein the identification of high-affinity LH receptors in the rabbit uterus. Uteri from adult New Zealand white rabbits were homogenized in Tris-HCl, 0.25 M sucrose. After filtration and sequential centrifugation, a partially purified pellet containing receptors was obtained. This preparation was incubated with a trace (1300 cpm) (50 pg) 125I-labeled chorionic gonadotropin and with various unlabeled protein hormones. Receptor bound was separated from free hormone by centrifugation at 1000 g. Affinity was estimated by Woolf plot analysis. Specific binding sites for LH/hCG were identified. The following Kd's were calculated: human LH, 1.6 X 10(-11); hCG, 0.5 X 10(-11); human TSH, 1.3 X 10(-9); and human FSH, 7.85 X 10(-9). The reaction of human FSH and TSH with the receptor is best explained by LH contamination of these hormones. A similar preparation of rat liver showed that no binding sites were present. Rabbit ovarian LH receptors had a Kd slightly higher at 4.1 X 10(-11) than that of the uterine LH receptors. Rabbit ovarian receptors were present at 2.27 X 10(-13) M/mg protein compared to uterine receptors at 4.65 X 10(-15) M/mg protein. We conclude specific- and high-affinity binding sites (receptors) for LH are present in the rabbit uterus. The function of these receptors remains unknown.     

Down-regulation of high affinity interleukin 2 receptors in a human tumor T cell line. Interleukin 2 increases the rate of surface receptor decay

We have described a human tumor T cell line, IARC 301, which constitutively expresses high affinity interleukin 2 (IL2) receptors, and showed that after binding to its receptors, IL2 is endocytosed and degraded. Here we present evidence that IL2 down-regulates its own high affinity receptors. Within 1 h, IL2 induces a 60% decrease in surface receptor expression. In order to maintain this down-regulation, IL2 concentration must be high enough for the receptors to be saturated throughout the incubation. The effect of IL2 on the kinetics of receptor internalization was investigated with two approaches. First, the initial rate of IL2 internalization was measured, and no difference could be detected whether the receptors were saturated with IL2 or only partially occupied. Second, the initial rate of surface receptor decay was followed and found to be significantly decreased in the presence of IL2. Although the half-life of IL2 receptors is very short in the absence of IL2, t 1/2 approximately 65 min, suggesting that these receptors are constantly endocytosed, it can still be reduced to t 1/2 approximately 25 min when the receptors are saturated with ligand. This suggests that occupied receptors are internalized faster than and independently from free receptors. The difference in internalization rates can explain the observed receptor down-regulation.     

The porcine LH/hCG receptor. Characterization and purification

Porcine luteal LH/hCG receptor (LH/hCG R) was solubilized with 70-80% recovery from the crude plasma membrane fraction by Triton X-100 in the presence of 25% glycerol and protease inhibitors. The solubilized receptor maintained 90% of original activity at -60 degrees C for 90 days. Equilibrium association constant (Ka) values of 1.92, 2.22, and 2.03 X 10(10) M-1 were observed for the whole homogenate, plasma membrane fraction, and solubilized LH/hCG R preparations, respectively. The specific binding capacity for the same fractions were 49, 70, 55 fmol/mg protein, respectively. Complexes of LH/hCG R and Triton X-100 were resolved into two components with approximate Mr = 2.7 X 10(5) and 5.4 X 10(5) by gel filtration on Sepharose 6B and two glycoprotein components by chromatography on concanavalin A-Sepharose. Solubilized porcine LH/hCG R was purified by two cycles of affinity chromatography on highly purified hCG-Sepharose with an overall recovery of 30-35% of the initial activity in the Triton extract. Purified porcine LH/hCG R had a specific binding capacity of 2300 pmol/mg protein and a Ka = 1.5 X 10(10) M-1. Silver staining of sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels demonstrated that the major protein in porcine LH/hCG R preparations has Mr = 68,000. A weakly staining band at Mr = 45,000 was also observed in the purified receptor preparation. Analysis of iodinated purified LH/hCG R by autoradiography has confirmed these results. Porcine LH/hCG R was purified 40,000-fold by this method.     

Kinetic and autoradiographic studies on binding of hCG to the testicular LH receptors of neonatal rats

The properties of hCG binding to LH receptors of the neonatal (5-day-old) rat testis were analysed and compared with those of the adult testis. The equilibrium association constants (Ka) of hCG-binding were similar at both ages, 2-4 X 10(10) M-1. In contrast, kinetic binding studies revealed that the association and dissociation rate constants of hCG binding were more rapid in the neonatal testis. Likewise, it was observed that the progression from loose (easily dissociable) to tight (non-dissociable) binding was less complete in the young than in the adult testis. Autoradiography of 125I-labelled hCG binding to interstitial cell suspensions at the two ages showed that the gonadotrophin binding per Leydig cell was about 50% lower in the neonatal testis. Conversely, since the surface area of adult Leydig cells was about 4-fold larger, the receptor density appeared to be higher in the neonatal Leydig cells. The rapid recovery of LH receptors after hCG stimulation, typical of the neonatal cells, was due to rapid replenishment of binding in the cells initially occupied by the injected hormone, rather than to an hCG-induced increase of Leydig cell number. Finally, in-vivo experiments with cycloheximide revealed that the rapid recovery of LH receptors was dependent on protein synthesis. These differences in the kinetics of neonatal testicular LH receptor turnover may be involved in the unique functional features of the fetal-neonatal growth phase of rat testicular Leydig cells.     

Purification and characterization of lutropin receptor from membranes of pig follicular fluid

Membranes derived from free floating granulosa cells in porcine ovarian follicular fluid were used as a starting material for structural characterization of both LH/hCG and FSH receptors. The receptors were highly hormone-specific and showed single classes of high-affinity binding sites (Kd = 19-74 pM). Their molecular weights as determined by affinity cross-linking with their respective 125I-ligands were similarly 70,000. The membrane-localized receptors could be solubilized with reduced Triton X-100 in the presence of 20% glycerol with good retention of hormone binding activity. The Triton extracts of membranes also showed hormone specificity and equilibrium binding constants similar to the membrane receptors (Kd = 32-48 pM). Affinity chromatography on divinylsulfonyl-Sepharose-oLH columns was utilized to purify the solubilized LH/hCG receptor to a specific activity of 2000 pmol/mg of protein. The purified receptor exhibited a high specificity for hCG and hLH but not for hFSH nor bTSH. The purified receptor was iodinated and visualized to be composed of a major protein of Mr approximately 70,000 and other minor proteins of molecular weights ranging from 14,000 to 40,000. Except for the Mr 14,000 protein, all other protein species bound to the concanavalin A-Sepharose column. The data suggest that the ovarian LH/hCG and FSH receptors are structurally similar and consist of a single polypeptide chain, as recently documented for the LH/hCG receptor (Loosefelt et al., 1989; McFarland et al., 1989).     

Effect of activating and inactivating mutations on the phosphorylation and trafficking of the human lutropin/choriogonadotropin receptor

The effects of several mutations of the human LH receptor (hLHR) on the phosphorylation, internalization, and turnover of the cell surface receptor were examined. Three gain-of-function mutations associated with Leydig cell hyperplasia (L457R and D578Y) and one associated with Leydig cell adenomas (D578H), one signaling-impaired mutation associated with Leydig cell hypoplasia (I625K), and two laboratory designed signaling-impaired mutations (D405N and Y546F) were used. The signaling-impaired mutations showed a reduction in human CG (hCG)-induced receptor phosphorylation and internalization. Mutation of the phosphorylation sites of these loss-of-function mutants had little or no additional effect on internalization. Cotransfection with G protein-coupled receptor kinase-2 (GRK2) rescued the hCG-induced phosphorylation and internalization of the signaling-impaired mutations but only if the phosphorylation sites were intact. Overexpression of arrestin-3 rescued the rate of internalization regardless of whether or not the phosphorylation sites were intact. Only two of the three constitutively active mutants displayed an increase in basal phosphorylation. Although they all failed to respond to hCG with increased receptor phosphorylation, they all internalized hCG faster than wild-type hLHR (hLHR-wt). Mutation of the phosphorylation sites of these constitutively active mutants lengthened the half-time of internalization of hCG toward that of hLHR-wt. Overexpression of arrestin-3 had little or no effect on the already short half-time of internalization of hCG mediated by these mutants. The data obtained with the signaling-impaired and phosphorylation-deficient mutants of the hLHR support a model whereby receptor phosphorylation and activation play a redundant role in the internalization of hCG. The results obtained with the constitutively active mutants suggest that, when occupied by hCG, these mutants assume a conformation that bypasses many of the steps (i.e. activation, phosphorylation, and/or arrestin binding) involved in internalization.     

Purification and partial characterization of rat ovarian lutropin receptor

Lutropin (LH) receptor was solubilized from pseudopregnant rat ovaries and purified by two cycles of affinity chromatography on human choriogonadotropin (hCG)-Affi-Gel 10. The purified receptor preparation contained a single class of high-affinity 125I-hCG binding sites with an equilibrium dissociation constant (Kd) of 5.1 X 10(-10) M (at 20 degrees C) and had a specific hormone binding capacity of 7920 pmol/mg of protein. The purified receptor migrated as a single 90-kDa band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under both nonreducing and reducing conditions. Affinity cross-linking of the purified receptor to 125I-hCG produced a 130-kDa complex. Hormone-binding ability of the purified 90-kDa polypeptide was demonstrated also by ligand blotting. The purified receptor was electroblotted onto nitrocellulose after sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions followed by incubation with 125I-hCG. Autoradiography revealed labeling of a 90-kDa band. This labeling was displaced by unlabeled hCG and human LH but not by human follitropin or rat prolactin. In addition, LH receptors of bovine corpora lutea and mouse Leydig tumor cells were shown by ligand blotting to contain a 90-kDa hormone binding unit, suggesting that LH receptor structure is well conserved among mammalian species. The purified rat ovarian LH receptor bound to immobilized wheat germ agglutinin, implying that the receptor is a glycoprotein. These results demonstrate that the hormone-binding unit of rat ovarian LH receptor is a 90-kDa membrane glycopolypeptide.     

Functional states of the luteinizing hormone/choriogonadotropin-receptor complex in rat Leydig cells

Three classes of gonadotropins with different ratios of stimulating to binding activities (S/B ratio) in rat Leydig cells have been identified. An S/B ratio of 1 was observed for rat luteinizing hormone (LH), porcine LH, and equine choriogonadotropin (CG) (class I), whereas ovine and equine LH exhibited and S/B ratio of 10-20 (class II) and human CG (hCG) (class III) an S/B ratio of 60. We coined the term "superactivity" to designate this particular behavior. This phenomenon was further studied by comparing the competitive activities of porcine LH (pLH) and hCG in radioreceptor assays using rat Leydig cell membranes and either radiolabeled oLH or hCG as the tracer, in the presence or absence of 150 mM NaCl. At equilibrium, both native hormones were equipotent in competing with 125I-oLH binding, but hCG was 4-fold more potent than pLH when 125I-hCG was used. Moreover, the binding rates of both hormones were considerably diminished in the presence of NaCl, but hCG binding at equilibrium was not affected, whereas that of oLH was almost completely abolished. From these results and previous data on the binding and internalization of these hormones, we suggest the existence of two interconvertible functional states of the hormone-receptor complex: (formula; see text). The equilibrium constant k3/k4 would be extremely high for hCG and lower and lower for the hormones in class II and class I, respectively. The equilibrium constant k1/k2 would be the one affected by the presence of NaCl and seems to be similar for all the hormones tested. The normal activity or superactivity of gonadotropins would thus be primarily dependent on the equilibrium between HR1 and HR2.     

Synchronization of ovulation in cyclic gilts with porcine luteinizing hormone (pLH) and its effects on reproductive function

The overall objective was to evaluate the use of porcine luteinizing hormone (pLH) for synchronization of ovulation in cyclic gilts and its effect on reproductive function. In an initial study, four littermate pairs of cyclic gilts were given altrenogest (15 mg/d for 14 d). Gilts received 500 microg cloprostenol (Day 15), 600 IU equine chorionic gonadotropin (eCG) (Day 16) and either 5mg pLH or saline (Control) 80 h after eCG. Blood samples were collected every 4h, from 8h before pLH/saline treatment to the end of estrus. Following estrus detection, transcutaneous real-time ultrasonography and AI, all gilts were slaughtered 6d after the estimated time of ovulation. Peak plasma pLH concentrations (during the LH surge), as well as the amplitude of the LH surge, were greater in pLH-treated gilts than in the control (P=0.01). However, there were no significant differences between treatments in the timing and duration of estrus, or the timing of ovulation within the estrous period. In a second study, 45 cyclic gilts received altrenogest for 14-18d, 600 IU eCG (24h after last altrenogest), and 5mg pLH, 750 IU human chorionic gonadotropin (hCG), or saline, 80 h after eCG. For gilts given pLH or hCG, the diameter of the largest follicle before the onset of ovulation (mean+/-S.E.M.; 8.1+/-0.2 and 8.1+/-0.2mm, respectively) was smaller than in control gilts (8.6+/-0.2mm, P=0.05). The pLH and hCG groups ovulated sooner after treatment compared to the saline-treated group (43.2+/-2.5, 47.6+/-2.5 and 59.5+/-2.5h, respectively; P<0.01), with the most synchronous ovulation (P<0.01) in pLH-treated gilts. Embryo quality (total cell counts and embryo diameter) was not significantly different among groups. In conclusion, pLH reliably synchronized ovulation in cyclic gilts without significantly affecting embryo quality.     

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.     

Biological function of the LH receptor is associated with slow receptor rotational diffusion

The biological activity of luteinizing hormone (LH) receptors can be affected by modifications to the receptor's amino acid sequence or by binding of hormone antagonists such as deglycosylated hCG. Here we have compared rotational diffusion of LH receptors capable of activating adenylate cyclase with that of non-functional hormone-occupied receptors at 4 degrees C and 37 degrees C using time-resolved phosphorescence anisotropy techniques. Binding of hCG to the rat wild-type receptor expressed on 293 cells (LHR-wt cells) or to the LH receptor on MA-10 cells produces functional receptors which exhibit rotational correlation times longer than 1000 micros. However, modification of the LH receptor by substitution of Lys583-->Arg (LHR-K583R) results in a receptor that is non-functional and which has a significantly shorter rotational correlation time of 130+/-12 micros following binding of hCG. When these receptors are treated with deglycosylated hCG, an inactive form of hCG, the rotational correlation times for the LH receptors on LHR-wt and MA-10 cells are also shorter, namely 64+/-8 and 76+/-14 micros, respectively. Finally, a biologically active truncated form of the rat LH receptor expressed in 293 cells (LHR-t631) has slow rotational diffusion, greater than 1000 micros, when occupied by hCG and a significantly shorter rotational correlation time of 103+/-12 micros when occupied by deglycosylated hCG. The effects of rat LH binding to LH receptors on these various cell lines were similar to those of hCG although the magnitude of the changes in receptor rotational diffusion were less pronounced. We suggest that functional LH receptors are present in membrane complexes that exhibit slow rotational diffusion or are rotationally immobile. Shorter rotational correlation times for non-functional hormone-receptor complexes may reflect the absence of essential interactions between these complexes and other membrane proteins.     

Regulation by phorbol esters of asialoglycoprotein and transferrin receptor distribution and ligand affinity in a hepatoma cell line

We have investigated the simultaneous regulation of cell surface distribution and ligand binding of the asialoglycoprotein (ASGP) receptor and the transferrin receptor in a hepatoma cell line by phorbol esters. One hour exposure to phorbol esters causes a redistribution of both receptors to the cell interior as shown by radioligand binding at 4 degrees C and selective immunoprecipitation from the plasma membrane. This effect is temperature- and dose-dependent and is not seen with 4-alpha-phorbol, an inactive tumor promoter. The mechanism and kinetics of the ASGP receptor response to phorbol esters appears to differ from that of the transferrin receptor in this cell line. Within the first 10 min there is a decrease in binding of iodinated ligands for both receptors to the HepG2 cell surface. For the transferrin receptor this results from a net internalization of receptor molecules from the plasma membrane pool, while for the ASGP receptor this decrease is accounted for by a 3.5-fold reduction in ligand binding affinity (6.6 X 10(-8) M to 24.0 X 10(-8) M), with essentially no change in the number of ASGP receptors recoverable from the plasma membrane pool by immunoprecipitation. The altered affinity of the ASGP-R is transient; the Kd returns to control levels by 20 min of continued exposure to the agent. The transferrin receptor shows no change in binding affinity during the course of exposure to phorbol esters. ASGP receptors in cells exposed to phorbol esters for 1 h maintain their competence to deliver exogenous ligand to intracellular sites of degradation and to participate in the recycling pathway of receptor-mediated endocytosis, although at a lower rate than in control cells. We conclude that under identical conditions phorbol esters modulate the binding capacity of two receptors at the cell surface by separate mechanisms. Furthermore, the transient nature of the altered ASGP-R binding affinity suggests that at least two mechanisms, receptor redistribution as well as decreased binding affinity, are operative in the modulation of ASGP-R cell surface binding during the first hour of exposure to the phorbol esters.