Biomedical subjects

M Ascoli

Publications and source records attributed to M Ascoli.

At least 19 recordsLinked to original sources

PubMed1992

Truncation of the cytoplasmic tail of the lutropin/choriogonadotropin receptor prevents agonist-induced uncoupling.

An agonist-induced change in the functional properties of a constant number of receptors seems to be a ubiquitous phenomenon involved in the regulation of cell surface receptors. Although the mechanisms responsible for this phenomenon (called uncoupling or desensitization) have been studied in detail using beta 2-adrenergic receptors it is unclear if the models derived from these studies are applicable to other members of the family of G protein-coupled receptors. Since it has been shown previously that truncation of the C-terminal cytoplasmic tail of the beta 2-adrenergic receptor results in a delay in the onset of agonist-induced uncoupling (Bouvier, M., Hausdorff, W.P., De Blasi, A., O'Dowd, B.F., Kobilka, B.K., Caron , M.G., and Lefkowitz, R.J. (1988) Nature 333, 370-373), we now present experiments designed to test the effects of a similar truncation of the lutropin/choriogonadotropin (LH/CG) receptor on its functional properties. The results presented herein show that (i) clonal lines of human embryonic kidney cells stably transfected with cDNAs encoding for the wild-type (rLHR-wt) or a mutant receptor truncated at amino acid residue 631 (rLHR-t631) express functional LH/CG receptors as judged by their ability to bind hCG and to respond to it with increased cAMP accumulation; (ii) a preincubation of the cells expressing rLHR-wt with hCG leads to a reduction in the ability of hCG to activate adenylylcyclase; and (iii) this reduction is severely blunted in cells expressing rLHR-t631. These results demonstrate that the C-terminal cytoplasmic tail of the LH/CG receptor is necessary for agonist-induced uncoupling.

Animals↗

PubMed1992

Epidermal growth factor, a phorbol ester, and 3',5'-cyclic adenosine monophosphate decrease the transcription of the luteinizing hormone/chorionic gonadotropin receptor gene in MA-10 Leydig tumor cells.

In a recent series of experiments we have shown that the previously recognized ability of mouse epidermal growth factor (mEGF), cAMP, or phorbol 12-myristate 13-acetate (PMA) to reduce the density of LH/CG receptors in MA-10 cells is secondary to a reduction in receptor messenger RNA (mRNA). As a follow-up to these studies we now present experiments designed to determine if the reduction in LH/CG receptor mRNA is due to a decrease in transcription of the receptor gene and/or an increase in the rate of degradation of the mRNA. The potential effects of mEGF, cAMP, or PMA on the degradation of the LH/CG receptor mRNA were measured in MA-10 cells treated with Actinomycin D or in kidney cells permanently transfected with the LH/CG receptor complementary DNA driven by a heterologous promoter. Both experimental strategies revealed that none of these compounds increase the rate of degradation of the receptor mRNA. If anything, a stabilizing effect was noted. The potential effects of mEGF, cAMP, or PMA on the transcription of the LH/CG receptor gene in MA-10 cells were measured using nuclear run-off assays. All three compounds induced a rapid decrease in the transcription of the LH/CG receptor gene. The time course of these effects is similar, and by 2 h all of the stimuli had decreased transcription to 15-30% of control. Our studies show that the mEGF-, cAMP- and PMA-induced down-regulation of the LH/CG receptor in MA-10 cells is primarily (if not entirely) due to a decrease in the transcription of the receptor gene.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1992

Identification and characterization of a luteinizing hormone/chorionic gonadotropin (LH/CG) receptor precursor in a human kidney cell line stably transfected with the rat luteal LH/CG receptor complementary DNA.

It is well established that the LH/CG receptor expressed in gonadal cells is an 85- to 92-kilodalton (kDa) glycoprotein. Additionally, however, a number of reports have noted the existence of other putative receptor species, but few attempts have been made to characterize these variant receptor species. A cell line [293L(wt1)] had previously been isolated which expresses large numbers of high affinity cell surface LH/CG receptors. Visualization of the LH/CG receptor species expressed in these cells and in rat luteal cells using ligand blots revealed 85- and 90-kDa LH/CG receptors, respectively, while immunoblots revealed another 68-kDa glycoprotein receptor in both cell types. The presence of both the 85- and 68-kDa receptor species was confirmed using immunoprecipitation and affinity purification of metabolically labeled 293L(wt1) cells. Enzymatic deglycosylations established that the 85-kDa receptor is a sialoprotein, while the 68-kDa species contains exposed high mannose residues. Protease digestion before LH/CG receptor immunoprecipitations localized the 85-kDa receptor on the plasma membrane, while the 68-kDa receptor was shown to be located intracellularly. Pulse-chase experiments were then used to positively establish that the 68-kDa receptor protein is actually a precursor of the 85-kDa LH/CG receptor species.

Animals↗

PubMed1992

The 5'-flanking region of the rat luteinizing hormone/chorionic gonadotropin receptor gene confers Leydig cell expression and negative regulation of gene transcription by 3',5'-cyclic adenosine monophosphate.

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.

Animals↗

PubMed1992

The gonadotrophin receptors: insights from the cloning of their cDNAs.

The cloning and expression of the cDNA's for the gonadotrophin hormone receptors has confirmed that these receptors are each composed of a single polypeptide which can both bind hormone and activate adenylyl cyclase when occupied with agonist. Although some studies by others have suggested that the LH/CG receptor and the FSH receptor are composed of multiple subunits (Ascoli and Segaloff 1989; Reichert and Dattatreyamurty 1989; Shin and Ji 1985a, b, and c; Smith et al. 1985; Smith et al. 1986), biochemical studies on the LH/CG receptor have shown that it is composed of a single polypeptide with a molecular weight of 93,000 when analyzed on sodium dodecyl sulfate gels in the presence or absence of disulfide reducing agents (Ascoli and Segaloff 1989). As the LH/CG receptor has been shown to be readily proteolyzed into smaller-sized fragments (Ascoli and Segaloff 1986; Ascoli and Segaloff 1989), it is reasonable to postulate that the FSH receptor may be similarly susceptible to proteolysis, and that this may account for the discrepant reports on its structure. Clearly, the molecular cloning and functional expression of the cDNA's for the rat ovarian LH/CG receptor (McFarland et al. 1989) and the rat testicular receptor (Sprengel et al. 1990) demonstrate conclusively that the gonadotrophin receptors are indeed single polypeptides. As shown schematically in Fig. 5.12, the gonadotrophin hormone receptors (together with the thyrotrophin receptor, see Frazier et al. 1990; Libert et al. 1989; Nagayami et al. 1989; Parmentier et al. 1989) define a unique subclass of G protein-coupled receptors. Thus, in addition to the seven membrane-spanning domains which appear to be the hallmark of G protein-coupled receptors, the gonadotrophin receptors (and thyrotrophin receptor) also contain large extracellular domains. These extracellular domains are composed of a repeating leucine-rich motif which is found in a number of widely-diverse proteins, collectively termed leucine-rich glycoproteins. We have shown that the extracellular domain of the LH/CG receptor is entirely responsible for binding hormone with high affinity. This is in contrast to the rhodopsin-like G protein-coupled receptors which have relatively small amino-terminal extracellular domains and in which the ligands interact directly with amino acids within the transmembrane helices. As with other G protein-coupled receptors, however, it is thought that the gonadotrophin receptors interact with and activate Gs through residues located within the cytoplasmic loops and the amino-terminal region of the cytoplasmic tail.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗

PubMed1991

Attenuation of cAMP-mediated responses in MA-10 Leydig tumor cells by genetic manipulation of a cAMP-phosphodiesterase.

In order to assess the effect of increased cAMP degradation on the responsiveness on an endocrine cell, we have obtained stable transfectants of MA-10 Leydig tumor cells that overexpress a mammalian cAMP-phosphodiesterase. Two novel cell lines, designated MA-10(P+8) and MA-10(P+29), that express high levels of the transfected enzyme were characterized. Although the basal levels of cAMP in the mutant cell lines are comparable to those of the wild-type cells, the increase in cAMP accumulation elicited by human choriogonadotropin (hCG) is severely blunted. Further studies with MA-10(P+29) show that the ability of hCG to stimulate adenylyl cyclase activity is normal. The failure of MA-10(P+29) cells to accumulate cAMP in response to hCG can be correlated with a similar reduction in hCG-stimulated steroidogenesis. On the other hand, the maximal steroidogenic response of MA-10(P+29) cells to dibutyryl cAMP, a cAMP analogue that is fairly resistant to phosphodiesterase degradation, is normal. We also show that the ability of these cells to respond to hCG with increased cAMP accumulation and steroid synthesis can be restored with a specific phosphodiesterase inhibitor. These results demonstrate that overexpression of a cAMP-phosphodiesterase in MA-10 cells limits the levels of cAMP attained under hCG stimulation and supresses the steroidogenic response of these cells to hCG. Since gonadotropins increase the cAMP-phosphodiesterase activity in their target cells, these findings also provide evidence that this regulation plays a major role in the modulation of cell responsiveness. Last, these new cell lines should be valuable in the study of the actions of cAMP because they express a conditional and reversible cAMP-resistant phenotype.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1991

Lutropin/choriogonadotropin down-regulates its receptor by both receptor-mediated endocytosis and a cAMP-dependent reduction in receptor mRNA.

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.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1991

Epidermal growth factor and phorbol esters reduce the levels of the cognate mRNA for the LH/CG receptor.

The LH/CG receptor of MA-10 Leydig tumor cells can be down-regulated by LH/CG, mouse epidermal growth factor (mEGF), cAMP analogues and phorbol esters. In a recent publication we showed 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, and occurs with little or no change in the levels of LH/CG receptor mRNA. Although relatively unimportant for the LH/CG-induced down-regulation of the LH/CG receptor, we also found that LH/CG can in fact elicit a reduction in LH/CG receptor mRNA and that this reduction is mediated by cAMP. These findings raised the possibility that the ability of other compounds to down-regulate the LH/CG receptor may be accompanied by decreases in LH/CG receptor mRNA. In an attempt to answer this question, we examined the possibility that the mEGF- or phorbol ester-induced down regulation of the LH/CG receptor is due to changes in LH/CG receptor mRNA. The experiments presented herein show that this is indeed the case.

Animals↗

PubMed1991

The slow rate of internalization of deglycosylated human chorionic gonadotropin is not due to its inability to stimulate cyclic adenosine monophosphate accumulation.

Using a clonal strain of cultured Leydig tumor cells (designated MA-10), we have compared the internalization and degradation of human CG (hCG) and deglycosylated hCG. Deglycosylated hCG is a derivative of hCG which retains the ability to bind to the LH/CG receptor, but is unable to activate adenylyl cyclase and thus stimulate cAMP production. A comparison of the fates of the receptor-bound hormones during a single round of endocytosis showed that deglycosylated hCG is internalized slower than hCG. It was also noted that the rate of degradation of the internalized deglycosylated hCG is slower than that of hCG. Two sets of experiments were performed which led to the conclusion that the slower internalization of deglycosylated hCG is not due to its inability to stimulate cAMP production. First, the rate of internalization of deglycosylated hCG in MA-10 cells is not increased in the presence of a cAMP analog. Second, there is little or no difference in the rate of internalization of hCG in a subclone of MA-10 cells [designated MA-10(K3)] that express a cAMP-resistant phenotype.

Cell Line↗

PubMed1991

Multiple luteinizing hormone/chorionic gonadotropin receptor messenger ribonucleic acid transcripts.

It has previously been shown that multiple messenger RNA (mRNA) species can be identified in gonadal tissues by probes specific for the LH/CG receptor. Here we show that the sizes and relative abundancies of gonadal LH/CG receptor transcripts are quite variable between such closely related species as rat and mouse. These patterns of LH/CG receptor mRNAs are yet different from that observed in human embryonic kidney 293 cells that have been transfected with a cDNA encoding for the rat luteal LH/CG receptor. In spite of the diversity in the number and sizes of LH/CG receptor mRNA transcripts, however, our data also show that the size of the cell surface receptor expressed in these three cells/tissues is identical. We further show that the most abundant LH/CG receptor mRNA present in MA-10 cells, a clonal strain of cultured Leydig tumor cells, is a 1.2 kilobase transcript which encodes for a truncated version of the LH/CG receptor corresponding to the extracellular hormone-binding domain. It does not appear, however, that this transcript is translated into a functional protein.

Animals↗

PubMed1990

Anti-phosphotyrosine immunoprecipitation of phosphatidylinositol 3' kinase activity in different cell types after exposure to epidermal growth factor.

In previous studies from this laboratory, it was shown that mouse epidermal growth factor (mEGF) or insulin increased the labeling of phosphaditylinositol-3,4-bisphosphate (PI-3,4-P2) in MA-10 cells prelabeled with different radioactive precursors (Pignataro, O.P., and Ascoli, M. (1990) J. Biol. Chem. 265, 1718-1723 and Mol. Endocrinol. (1990) 4, 758-765). In order to further characterize this phenomenon we sought to determine if we could use anti-phosphotyrosine antibodies to immunoprecipitate a phosphatidylinositol (PI) kinase activity from MA-10 cells treated with mEGF or insulin. Our data indicate that this is indeed the case, and that the PI kinase precipitated is a PI-3' kinase. A second cell type, A431 cells, in which we were unable to detect an increase in PI-3,4-P2 labeling when stimulated with mEGF or insulin, was also studied. It was found that, as in MA-10 cells, A431 cells also contain an immunoprecipitable PI-3' kinase activity that is increased in response to mEGF or insulin.

Animals↗

PubMed1990

Epidermal growth factor increases the labeling of phosphatidylinositol 3,4-bisphosphate in MA-10 Leydig tumor cells.

Previous studies from this laboratory have shown that mouse epidermal growth factor (mEGF) modulates the hormonal responsiveness of MA-10 Leydig tumor cells without affecting cell multiplication. In an attempt to characterize the intracellular signaling systems activated by mEGF in this cell type, we examined its effects on the labeling of phosphatidylinositols in cells that had been preincubated with different radioactive precursors. Here we report that exposure of MA-10 cells to mEGF, but not other ligands that affect their differentiated function, results in an increase in the labeling of an unusual phosphatidylinositol that does not appear to be present in unstimulated cells. This phosphatidylinositol has been identified as phosphatidylinositol 3,4-bisphosphate.

Animals↗

PubMed1990

Structure of the lutropin/choriogonadotropin receptor.

In summary, the LH/CG receptor is a single polypeptide which contains a large hydrophilic domain that is situated extracellularly, attached to a region that spans the plasma membrane seven times, the carboxy-terminal region being intracellular. This topology was predicted by the amino acid sequence and has been confirmed by our immunofluorescence studies. The extracellular domain, which is related to a family of leucine-rich glycoproteins, is presumably involved in binding the large glycoprotein hormones hCG and LH. The carboxy-terminal half of the receptor, which is related to the family of rhodopsinlike receptors, is (by analogy with these receptors) presumably involved in the coupling of the receptor to the G protein. Our transfection studies confirm that this single polypeptide is capable of binding hormone and activating adenylyl cyclase. Therefore, not only is the structure of the LH/CG receptor unique compared to other cell surface receptors characterized to date, but also its structure suggests that the mechanism of the translation of hormone binding to G protein coupling in this receptor is different from other G protein-coupled receptors whose ligands are much smaller and intercalcate among the transmembrane helices. We predict that, due to the homology among the glycoprotein hormones, the structures of the FSH and TSH receptors share extensive amino acid and structural homology with the LH/CG receptor. Last, our newly acquired knowledge about the structure of the LH/CG receptor, and the development of a cDNA and antibodies for this receptor, should enable more detailed studies on the function and regulation of the LH/CG receptor, not previously possible.

Amino Acid Sequence↗

PubMed1990

Epidermal growth factor desensitizes the gonadotropin-responsive adenylyl cyclase in membranes isolated from MA-10 Leydig tumor cells and luteinized rat ovaries.

In a previous publication we showed that addition of mouse epidermal growth factor (mEGF) to MA-10 Leydig tumor cells or rat luteal cells leads to an attenuation of the elevated rate of cAMP accumulation provoked by subsequent addition of hCG. Those studies also suggested that this was due to a decrease in the hCG-activated adenylyl cyclase activity, but formal proof of this hypothesis was not presented. The experiments presented herein were conducted to investigate the mechanisms responsible for this phenomenon and show that our initial suggestion was correct, because we can show that mEGF attenuates the hCG-activated adenylyl cyclase in membranes from MA-10 cells or luteinized rat ovaries. More importantly, however, the establishment of a cell-free system in which mEGF attenuates adenylyl cyclase allows us to conduct detailed investigations on the molecular mechanisms that underly this phenomenon. Using this system we have been able to show that the ability of mEGF to attenuate adenylyl cyclase activity 1) is specific for the hCG-activated enzyme, since it cannot be detected when the enzyme is activated with other ligands, such as vasoactive intestinal peptide or isoproterenol; 2) does not appear to be due to a reduction in the activity of the catalytic subunit of adenylyl cyclase or the stimulatory GTP-binding protein (Gs), because mEGF does not affect the stimulation of adenylyl cyclase by other effectors, and it has little or no effect on the ability of Gs to restore isoproterenol sensitivity to the adenylyl cyclase of S49 cyc- membranes; and 3) optimal expression of the mEGF effect can only be obtained in the presence of ATP or GTP.

Adenosine Triphosphate↗

PubMed1990

Reduced gonadotropin responses in a novel clonal strain of Leydig tumor cells established by transfection of MA-10 cells with a mutant gene of the type I regulatory subunit of the cAMP-dependent protein kinase.

Although it is clear that cAMP is an important mediator of the actions of LH/CG in Leydig cells, recent studies from several laboratories have shown that the functions of Leydig cells can also be modulated by hormones and growth factors that do not appear to use cAMP as a second messenger. Thus, in order to increase our understanding of the importance of cAMP as a modulator of the functions of Leydig cells we have used a genetic approach to establish permanent cell lines that express a cAMP-resistant phenotype. MA-10 cells, a clonal strain of cultured Leydig tumor cells that express many of the characteristics of normal Leydig cells, were transfected with an expression vector controlled by the metallothionein promoter and encoding for a mutant form of the regulatory subunit of the type I cAMP-dependent protein kinase. Three stable transfectants that display a Zn+2-dependent decrease in cAMP-dependent protein kinase activity were established. Further characterization of one of the transfectants (designated MA-10(K3)) revealed a parallel reduction in the ability of cAMP and human CG to induce cell rounding, to increase steroid synthesis, or to induce c-fos mRNA. Our initial studies on these mutant cells have already provided novel information about the actions of human CG. These cell lines will also be valuable for further studies on the signaling systems that mediate hormone action in Leydig cells.

Animals↗

PubMed1990

Studies with insulin and insulin-like growth factor-I show that the increased labeling of phosphatidylinositol-3,4-bisphosphate is not sufficient to elicit the diverse actions of epidermal growth factor on MA-10 Leydig tumor cells.

In a recent publication we showed that addition of mouse epidermal growth factor (mEGF) to MA-10 Leydig tumor cells rapidly leads to an increase in the incorporation of [3H]inositol-derived radioactivity into an unusual lipid that was identified as phosphatidylinositol-3,4-bisphosphate (PI-3,4-P2). Other ligands that are known to bind to MA-10 cells, such as hCG and arginine vasopressin, however, did not elicit this effect. Inasmuch as mEGF modulates the differentiated functions of MA-10 cells in a number of ways, our findings raised the possibility that PI-3,4-P2 may be an intracellular mediator of these actions of mEGF. In an attempt to answer this question, we set out to determine if other ligands increase the labeling of PI-3,4-P2 in MA-10 cells prelabeled with [3H]inositol, and if such ligands mimic the diverse biological actions of mEGF on these cells. The experiments presented herein show that insulin, insulin-like growth factor-I, and transforming growth factor-alpha increase the labeling of PI-3,4-P2 in MA-10 cells, but only transforming growth factor-alpha mimics the actions of mEGF on the differentiated functions of MA-10 cells. We conclude that an increase in the labeling of PI-3,4-P2 is not sufficient to elicit these actions of mEGF.

Animals↗

PubMed1989

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.

Animals↗

PubMed1989

Regulation of the differentiated functions of Leydig tumor cells by epidermal growth factor.

The three effects of mEGF on MA-10 Leydig tumor cells that have been discussed here are summarized in TABLE 7. The earliest effect of mEGF on MA-10 cells can be detected within 5 min of addition of mEGF and it lasts for about 60 min. During this time mEGF transiently attenuates hCG-stimulated adenylate cyclase activity. Although the magnitude of this effect is small, it can be correlated with a transient attenuation of the hCG-provoked increase in steroid synthesis. At longer times (i.e., 1-8 h) mEGF activates steroid synthesis by a "cAMP-independent pathway" and it potentiates (in a synergistic fashion) the activation of steroidogenesis by hCG, other compounds that activate adenylate cyclase activity, and cAMP analogues. At even longer times (i.e., 8-48 h) mEGF down-regulates the LH/CG receptors and by doing so, limits the steroidogenic response of the cells to hCG. From a biochemical point of view, our data provide an excellent example of those actions of growth factors that are unrelated to the control of cell multiplication, and of the complexity involved even when dealing with a single cell type and a single growth factor. Admittedly we know very little about the molecular basis of the phenomena described herein. Current work in our laboratory, however, is aimed at filling this gap. Among all the questions that we can address, we believe that it is particularly important to characterize the intracellular signaling system(s) activated by mEGF and to determine if a single signaling system is responsible for the diverse biological actions of mEGF in MA-10 cells. From a physiological point of view, our data may also prove important to the understanding of the regulation of testicular functions. There is increasing evidence for the production of EGF (or related peptides such as transforming growth factor alpha) in several tissues, including the testes and ovaries. These findings, together with the results summarized here suggest that EGF (or related peptides) act within the testes in a paracrine, or autocrine fashion and that they may have important modulatory effects on the activation of Leydig cell steroidogenesis by gonadotropins.

Adenylyl Cyclases↗