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It has been shown previously that a naturally occurring mutation of the human LH/CG receptor (hLHR), which replaces L457 in helix III with arginine, results in a receptor that constitutively elevates basal cAMP but does not respond to human CG (hCG) with further cAMP production. In the present study, substitutions of L457 with several amino acids were examined. The constitutive activation of cAMP production was observed only when L457 was replaced with a positively charged residue. Although constitutive activation of the inositol phosphate pathway could not be detected when measuring inositol phosphate production, the use of a more sensitive reporter gene assay for protein kinase C activation revealed the constitutive activation of this pathway by the R- and K-substituted mutants. Therefore, L457 of the hLHR plays a key role in stabilizing the receptor in an inactive conformation. Molecular modeling shows that the insertion of R, K, or H at position 457 triggers the receptor transition toward an active state due to the proximity of an anionic amino acid, D578, in helix VI. These substitutions cause perturbations in helix III-helix VI and helix III-helix VII interactions that culminate in the opening of a solvent-accessible site in the cytosolic domains potentially involved in Gs recognition. Interestingly, L457R was completely unresponsive and the K- and H-substituted L457 hLHR mutants were significantly blunted in their cAMP responses to hCG stimulation. Cells expressing L457R were also unresponsive to hCG with regards to increased inositol phosphate production. Other substitutions of L457 were identified, though, that selectively permit the hormonal stimulation of only one of the two signaling pathways. These results suggest a pivotal role for L457 in hormone-stimulated signal transduction by the hLHR.

One of the hallmarks of the differentiation of granulosa cells is the estradiol and FSH/cAMP-dependent induction of the LH receptor (LHR). Previous studies using granulosa cells isolated from diethylstilbestrol-pretreated immature rats identified a novel cAMP-responsive element termed the SAS site (Sp1c adjacent site) in the promoter region of the rat (r) LHR gene. The studies presented herein show that there is an additional distal site located at nucleotide (nt) -933/-924 that appears to interact with the same transcription factor that binds to the promoter SAS site. Similar to the SAS site, the complex formed between granulosa cell nuclear extracts and this distal site is enhanced by cAMP treatment of the granulosa cells. The core sequence required for the formation of the DNA/protein complex at this distal rLHR site was determined to be AGTGG(A)GGGG. With the exception of adenine at -928, substitution of any residue within this sequence prevented formation of this complex. The core sequence of this distal site differs from that of the proximal SAS site, which is GGGGG, and hence the distal site has been termed a SAS-like site. Reporter gene assays using constructs containing the -2,109/-1 region of the rLHR demonstrate that mutation of the distal SAS-like site abolishes the cAMP-induced transcription of the rLHR gene in rat granulosa cells, underscoring the functional significance of this site. Given the lack of sequences in the 5'-flanking region of the rLHR gene consistent with known cAMP-responsive elements, the identification of the novel SAS and SAS-like sites in the rLHR gene provides important clues toward understanding the mechanisms by which the rLHR gene is induced by FSH/cAMP.

The expression of the lutropin receptor (LHR) in granulosa cells is a complex phenomenon under the hormonal control of FSH and estradiol. Using primary cultures of granulosa cells from immature female rats pretreated with diethylstilbestrol (a compound with estrogen-like activity), the role of FSH in LHR induction was studied. Previous studies from our laboratory have shown that FSH or 8-bromo-cAMP addition to these cells causes a marked increase in the rate of transcription of the rat LHR (rLHR) gene. The present studies were undertaken to compare the properties of the rLHR gene in undifferentiated vs. differentiated rat granulosa cells as a means of determining those elements that confer basal activity and cAMP responsiveness. Our studies show that the proximal 155 bp (relative to the translational initiation codon) of the 5'-flanking region of rLHR gene represent a minimal promoter that accounts for the basal expression of this receptor in rat granulosa cells. A major domain located between nucleotides (nt) -90 and -120, with another one possibly being between nt -120 and -155, induced activation of basal transcriptional activity. An inhibitory domain was observed to lie between nt -186 and -1375. Our data further show that multiple elements within the 2.1 kb of the 5'-flanking region of the rLHR gene are involved in the 8-bromo-cAMP-induced expression of the LHR gene. Of these, the three Sp1-binding sites within the proximal portion of the 5'-flanking region appear to be important for both basal as well as cAMP-induced rLHR gene transcription.

The LH/CG receptor (LHR) is a member of the family of G protein-coupled receptors and activates Gs when stimulated by LH or CG. Studies from other G protein-coupled receptors have implicated the carboxyl-terminal region of the third intracellular loop as being involved in the activation of G proteins. It has been suggested that the potential ability of this region to form an amphiphilic helix, with positively charged residues aligned to one face, may be important for this biological activity. To test whether the positively charged lysine residues, and thus an amphiphilic helix, in the carboxyl terminal region of the rat LHR (rLHR) are indeed important in the activation of Gs by the rLHR, a mutant rLHR was constructed in which lysines 541, 544, and 557 were simultaneously substituted with alanines. Clonal 293 cells expressing comparable numbers of cell-surface recombinant wild type rLHR or rLHR(K541,544,547A) were generated. Cells expressing the mutant receptor-bound human CG (hCG) with the same high affinity as those expressing the wild type rLHR. Since the numbers of receptors and binding affinities between the two cell lines were comparable, any changes in basal or hCG stimulated cAMP production could readily be interpreted as an alteration in the mutant receptor's ability to activate Gs. It was found, however, that basal cAMP production, the concentration of hCG required to elicit half-maximal cAMP production, and the maximal levels of cAMP produced in response to hCG were all unchanged in cells expressing rLHR(K541,544,547A).(ABSTRACT TRUNCATED AT 250 WORDS)

The LH/CG receptor is a G protein-coupled receptor present on gonadal cells whose levels are modulated by a number of hormones, growth factors, and second messenger analogs. With the recently cloned cDNA for the LH/CG receptor, it has been shown that changes in the levels of the cognate mRNA are involved, at least in part, in the observed changes in receptor density. In order to study the transcriptional regulation of the LH/CG receptor we have isolated a 2-kilobase region of the 5'-flanking region of the rat LH/CG receptor gene and subcloned nucleotide -1 (relative to the translational initiation codon) to -1370 into a luciferase reporter plasmid. We show here that this region of the LH/CG receptor gene is able to enhance luciferase activity in MA-10 cells, a line of Leydig tumor cells that normally express LH/CG receptors, as opposed to human kidney 293 cells, which do not. Furthermore, the addition of 8-bromo-cAMP to MA-10 cells, under conditions known to decrease LH/CG receptor numbers and receptor mRNA levels, decreases the relative luciferase activity to about 26% of control. This decrease in reporter gene activity is severely blunted in a subclone of MA-10 cells with a cAMP-resistant phenotype. Our studies show, for the first time, that sequence(s) present with 1370 base pairs of the translational start site of the rat LH/CG receptor gene are sufficient for conferring expression of this gene in Leydig cells and for the negative modulation of LH/CG receptor gene transcription by high concentrations of cAMP.

Using MA-10 Leydig tumor cells as a model system we have examined the possibility that the lutropin/choriogonadotropin (LH/CG)-induced down-regulation of the LH/CG receptor is accompanied by changes in LH/CG receptor mRNA. We show that LH or CG are indeed capable of reducing the levels of LH/CG receptor mRNA, but that the time course and magnitude of the reduction in receptor mRNA are such that this phenomenon cannot account entirely for the down-regulation of the receptor. In fact, we estimate that LH/CG can reduce the number of LH/CG receptors by at least 80% with little or no change in the levels of LH/CG receptor mRNA. These data are consistent with our previous hypothesis that the LH/CG-induced down-regulation of the LH/CG receptor is primarily due to an increase in the rate of degradation of the receptor that occurs as a result of the receptor-mediated endocytosis of LH/CG. Our studies also show that the LH/CG-induced down-regulation of the LH/CG receptor mRNA is mediated by cAMP. Thus, addition of 8-bromo-cAMP to MA-10 cells leads to a similar reduction in the levels of LH/CG receptor and receptor mRNA; while deglycosylated human CG, a hormone derivative that binds to the LH/CG receptor but has a reduced ability to stimulate cAMP synthesis, does not reduce the levels of LH/CG receptor mRNA. Last, human CG or 8-bromo-cAMP are unable to reduce LH/Cg receptor mRNA in a mutant MA-10 cell line that express a cAMP-resistant phenotype.

A perifusion system has been used to study the in vitro desensitization of isolated Leydig tumor cells. It was observed that the cells become refractory, as measured by decreased rates of steroidogenesis, during continuous perifusions with saturating concentrations of either human choriogonadotropin (CG), cholera toxin, or 8-bromo-cyclic AMP. Furthermore, an initial perifusion of the cells with either human CG, cholera toxin, or 8-bromo-cyclic AMP causes subsequent desensitization towards all three stimuli. Thus, each of these stimuli is equally effective in inducing a state of desensitization in these cells that is manifested by a steroidogenic lesion(s) distal to cyclic AMP formation. It was found that the post-cyclic AMP lesion(s) in the desensitized state occurs prior to the formation of pregnenolone. However, the decreased rates of steroidogenesis do not seem to arise from a depletion of intracellular cholesterol.

A perifusion system has been developed in which a dose-dependent response of isolated Leydig tumor cells to steroidogenic stimuli, as assessed by the rates of steroid production, can be measured as a function of time. In response to a continuous perifusion for 340 min with a saturating concentration of hCG, ovine LH (oLH), cholera toxin, or 8-Br-cAMP, there is a rapid increase in the rate of progesterone production, which reaches a maximum about 100 min after the onset of stimulus in the medium and then declines to a rate somewhat higher than basal. Thus cholera toxin and 8-Br-cAMP as well as hCG and oLH are able to desensitize the Leydig tumor cells to further stimulation by the same agent. Although a 10-min pulse of a saturating concentration of hCG yields the same steroidogenic response as that elicited by continuous perifusion with saturating hCG, a pulse of a saturating concentration of oLH yields a steroidogenic response only when oLH is maintained in the perifusate. These data establish a substantial difference in the actions of hCG and oLH. The results could be explained by the higher apparent affinity of hCG for the gonadotropin receptor, such that, upon removal of hormone from the perifusate, oLH would be more readily eluted from the receptor. These findings support the hypothesis that oLH and hCG exert their stimulatory effects only while bound to the cell surface. (Endocrinology 108: 632, 1981)

The diastereoisomers of adenosine 3',5'-cyclic phosphorothioate, (Sp)-cAMPS and (Rp)-cAMPS, have been previously shown to act as agonists and antagonists, respectively, in the activation of several mammalian cAMP-dependent protein kinases. In an effort to characterize further the involvement of cAMP in the activation of Leydig cell steroidogenesis by lutropin/choriogonadotropin (LH/CG), we examined the effects of these cyclic nucleotide analogues on a clonal strain of cultured murine Leydig tumor cells (designated MA-10). Our results show that (i) (Sp)-cAMPS activates and (Rp)-cAMPS inhibits the isolated cAMP-dependent protein kinase of the MA-10 cells; (ii) both analogues inhibit the isolated cAMP phosphodiesterase(s); (iii) (Sp)-cAMPS activates steroid biosynthesis in intact cells, but (Rp)-cAMPS does not; and (iv) (Rp)-cAMPS is a competitive inhibitor of the activation of steroidogenesis by (Sp)-cAMPS, 8-bromo-cAMP, human CG, cholera toxin, and forskolin. However, (Rp)-cAMPS is a more effective inhibitor when steroidogenesis is activated by (Sp)-cAMPS or 8-bromo-cAMP than when it is activated by human CG, cholera toxin, or forskolin. This difference appears to be related to the combined effects of (Rp)-cAMPS on the cAMP-dependent protein kinases and cAMP phosphodiesterase(s). We conclude that cAMP is a quantitatively important mediator of the activation of steroidogenesis by LH/CG even at low concentrations of hormone where an increase in steroid biosynthesis cannot be easily correlated with increased cAMP accumulation. Thus, our data indicate that if other second messengers are involved in the activation of steroidogenesis by LH/CG, they must do so by acting together with, rather than independently of, cAMP.

The present studies examined whether the increase in cell surface LH receptors in primary cultures of porcine granulosa cells after exposure to FSH or cholera toxin, agents that increase intracellular cAMP, is due to de novo synthesis of the receptor or to a cAMP-dependent translocation of an intracellular pool of LH receptors to the cell surface. LH receptor induction by FSH was fully inhibited by the addition of cycloheximide to the incubation media, but resumed after cycloheximide was removed. These data suggest that FSH-induced LH receptor appearance requires protein synthesis. However, to be confident that the inhibition of LH receptor appearance did not result from lack of transit of preformed receptors requiring a rapidly turning over pool of proteins, we assayed for possible latent receptors in the cell interior by extracting granulosa cells with Triton X-100. Under conditions which detected about 74% of LH receptors in cells exposed to cholera toxin, little [125]iodo-hCG-binding activity was detected in cells not exposed to a cAMP-promoting stimulus. These findings suggest that a preformed pool of LH receptors does not exist in untreated cells, and that the cAMP-mediated induction of LH receptors requires de novo synthesis of the receptor.

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.

The studies presented herein were designed to investigate the effects of mouse epidermal growth factor (mEGF) on steroid biosynthesis in a clonal strain of cultured murine Leydig tumor cells (designated MA-10). We show that in short-term incubations (up to 8 h), mEGF activates steroid biosynthesis without affecting cAMP levels. The maximal activation of steroid biosynthesis by mEGF (about 10-fold) is, however, much lower than the maximal activation detected with human choriogonadotropin (hCG) or cAMP analogues (about 1000-fold). We also show that mEGF has two (opposing) effects on the activation of steroidogenesis by hCG. Initially, it transiently attenuates the increase in intracellular cAMP and steroid biosynthesis provoked by submaximal concentrations of hCG. At later times, however, it potentiates the stimulatory effects of submaximal concentrations of hCG on steroid biosynthesis in a synergistic fashion. Last, we show that mEGF and submaximal concentrations of cAMP analogues also activate steroidogenesis in a synergistic fashion and that the degree of synergism attained with cAMP analogues plus mEGF is much higher than that attained with hCG plus mEGF. Taken together, our results show that mEGF (i) activates steroidogenesis without affecting cAMP levels and (ii) modulates the activation of steroidogenesis by the cAMP second messenger system.