Biomedical subjects

K M Menon

Publications and source records attributed to K M Menon.

At least 19 recordsLinked to original sources

PubMed2001

Retention of glucose on oligosaccharide chains linked to the LH/hCG receptor prevents cell surface expression.

In the first step of asparagine-linked oligosaccharide chain maturation, terminal glucose residues are removed from the high mannose oligosaccharide core by glucosidases I and II. The role that glucose residues play in trafficking the luteinizing hormone/human chorionic gonadotropin (LH/hCG) receptor from the endoplasmic reticulum to the cell surface was investigated. Glucosidases I and II were inhibited by incubating 293 T cells transiently transfected with LH/hCG receptor cDNA with 5 mM 1-deoxynojirimycin (DNJ). DNJ treatment resulted in a marked reduction in cell surface [(125)I]hCG binding. Similar results were obtained from glucosidase I-deficient Lec 23 Chinese hamster ovarian (CHO) cells and wild-type CHO cells that were transiently transfected with LH/hCG receptor cDNA. Immunoprecipitation followed by Western blotting of transfected 293 T cells incubated in the presence or absence of 5 mM DNJ revealed that there is substantially less receptor in DNJ-treated cells than in control cells. These results show that the removal of glucose residues is necessary for trafficking the LH/hCG receptor to the cell surface.

1-Deoxynojirimycin↗

PubMed2001

Palmitoylation of the luteinizing hormone/human chorionic gonadotropin receptor regulates receptor interaction with the arrestin-mediated internalization pathway.

The luteinizing hormone/human chorionic gonadotropin receptor (LH/hCGR) undergoes palmitoylation at cysteine residues 621 and 622 located in the carboxyl terminal tail of the receptor. This study examined the biological function of palmitoylation with respect to its effect on receptor internalization. Coexpression of wild-type (WT) or C621/622G mutant receptors with arrestin-2 increased receptor internalization in 293T cells. Furthermore, measurements of rate enhancement upon overexpression of arrestin indicate that the palmitoylation deficient mutant receptor is more prone to utilizing the arrestin mediated internalization pathway than the WT receptor. Coexpression of G-protein-coupled receptor kinase 4 (GRK4) with wild type receptor resulted in an increase in internalization, while coexpression with the mutant receptor did not result in further enhancement of internalization. Additionally, 293T cells expressing mutant receptor were responsive to hCG with respect to production of inositol phosphates. Taken together, these results suggest that the palmitoylation state of the receptor governs internalization by regulating the accessibility of the receptor to the arrestin-mediated internalization pathway.

Arrestin↗

PubMed2001

Regulation of high density lipoprotein receptor messenger ribonucleic acid expression and cholesterol transport in theca-interstitial cells by insulin and human chorionic gonadotropin.

The synthesis of androgens by theca-interstitial cells is stimulated by LH and the insulin/insulin-like growth factor I (IGF-I) system. An essential element of the steroidogenesis is the uptake of plasma cholesterol and transformation to steroid hormones. In the rat, the uptake of cholesterol by the theca-interstitial cells is mediated by the high density lipoprotein receptor. The goal of the present study was to examine whether insulin has any effect on cholesterol delivery into theca-interstitial cells. The effects of insulin and hCG on the expression of the high density lipoprotein receptor (SR-BI) messenger RNA (mRNA) and intracellular cholesterol levels were examined in rat theca-interstitial cells under in vivo and in vitro conditions. Twenty-five-day-old rats were treated with insulin, hCG, or insulin followed by hCG. The expression of SR-BI mRNA was then examined in ovaries enriched in theca-interstitial cell population by Northern blot analysis. Treatment with insulin increased the expression of SR-BI mRNA over that in controls treated with saline. hCG administration also increased the expression of SR-BI mRNA. A combination of insulin followed by hCG produced an even greater increase in SR-BI mRNA expression. Measurements of cellular cholesterol in the ovarian tissue showed an increase in total and free cholesterol levels in response to insulin treatment. As expected, administration of hCG produced a depletion of cellular cholesterol, and the depletion was even more pronounced in response to treatment with insulin and hCG. The effect of insulin and hCG on SR-SBI mRNA expression was then examined under in vitro conditions using primary cultures of theca-interstitial cells. Treatment with insulin produced an increase in SR-BI mRNA expression. As the cultured theca-interstitial cells were not able to maintain hCG receptors, hCG addition produced no increase in SR-BI mRNA expression. However, in the presence of insulin, these cells were able to maintain hCG receptors and readily responded to hCG to increase SR-BI mRNA expression. Although insulin alone produced a modest increase in total and free cholesterol levels, in the presence of insulin, hCG produced the expected depletion of cellular cholesterol content. The present study shows that insulin has a stimulatory effect on the expression of high density lipoprotein receptors in theca-interstitial cells, suggesting that one of the actions of insulin is to increase intracellular cholesterol, which is subsequently mobilized for androgen biosynthesis in theca-interstitial cells.

Animals↗

PubMed2000

Regulatory role of the 3' untranslated region of luteinizing hormone receptor: effect on mRNA stability.

Posttranscriptional regulation of luteinizing hormone receptor (LHR) mRNA has a significant role in regulating cell surface receptor expression during ovarian cycle. In order to gain insight into the mechanism of posttranscriptional regulation, the cis-acting elements in the 3' untranslated region (3' UTR) of LHR mRNA were examined by transfection studies followed by measurements of the receptor expression and receptor mRNA half-life. The results show that the inhibitory effect exerted by the 3.51 kb 3' UTR of the LHR resides in the distal 1.98 kb region. Half-life measurement of LHR mRNA showed that the inhibitory effect of the 3' UTR was due to a decrease in receptor mRNA stability. Deletion studies revealed that the entire 1.98 kb region is required for LHR mRNA destabilization. Although the 3.51 kb 3' UTR of LHR contains 11 AUUUA motifs, their removal had no effect on mRNA stability or receptor expression. Thus, although AUUUA motifs have been implicated in mRNA stability, these motifs do not appear to play an inhibitory role in LHR expression. The 3' UTR of LHR was also able to decrease the expression of a reporter gene indicating that the inhibitory effect of 3' UTR is not unique to the open reading frame of LHR. The present studies show that the distal 1.98 kb portion of the 3' UTR exerts an inhibitory effect on the expression of LHR by decreasing the receptor mRNA half-life. The inhibitory effect of 3' UTR might play a role in the maintenance of the steady state levels of the receptor mRNA under different physiological states.

3' Untranslated Regions↗

PubMed1999

Sequence-specific binding of a hormonally regulated mRNA binding protein to cytidine-rich sequences in the lutropin receptor open reading frame.

In previous studies, a lutropin receptor mRNA binding protein implicated in the hormonal regulation of lutropin receptor mRNA stability was identified. This protein, termed LRBP-1, was shown by RNA gel electrophoretic mobility shift assay to specifically interact with lutropin receptor RNA sequences. The present studies have examined the specificity of lutropin receptor mRNA recognition by LRBP-1 and mapped the contact site by RNA footprinting and by site-directed mutagenesis. LRBP-1 was partially purified by cation-exchange chromatography, and the mRNA binding properties of the partially purified LRBP-1 were examined by RNA gel electrophoretic mobility shift assay and hydroxyl-radical RNA footprinting. These data showed that the LRBP-1 binding site is located between nucleotides 203 and 220 of the receptor open reading frame, and consists of the bipartite polypyrimidine sequence 5'-UCUC-X(7)-UCUCCCU-3'. Competition RNA gel electrophoretic mobility shift assays demonstrated that homoribopolymers of poly(rC) were effective RNA binding competitors, while poly(rA), poly(rG), and poly(rU) showed no effect. Mutagenesis of the cytidine residues contained within the LRBP-1 binding site demonstrated that all the cytidines in the bipartite sequence contribute to LRBP-1 binding specificity. Additionally, RNA gel electrophoretic mobility supershift analysis showed that LRBP-1 was not recognized by antibodies against two well-characterized poly(rC) RNA binding proteins, alphaCP-1 and alphaCP-2, implicated in the regulation of RNA stability of alpha-globin and tyrosine hydroxylase mRNAs. In summary, we show that partially purified LRBP-1 binds to a polypyrimidine sequence within nucleotides 203 and 220 of lutropin receptor mRNA with a high degree of specificity which is indicative of its role in posttranscriptional control of lutropin receptor expression.

3' Untranslated Regions↗

PubMed1999

Evidence that constitutively active luteinizing hormone/human chorionic gonadotropin receptors are rapidly internalized.

The luteinizing hormone/human chorionic gonadotropin (LH/hCG) receptor, which belongs to the family of G-protein coupled receptors, plays an important role in gonadal steroidogenesis. Substitution of aspartic acid 556 of the LH/hCG receptor with glycine (D556G) creates a constitutively active receptor that activates adenylyl cyclase in the absence of hormone. To examine receptor internalization, human embryonic kidney cells (293 T) expressing wild type (WT) or D556G mutant receptors were incubated with [125I]hCG and subsequently analyzed for cell surface bound and internalized radioactivity. Comparison of the rate constants of internalization of the D556G mutant and WT receptors revealed that the rate of internalization of the D556G mutant was five times greater than that of the WT receptor. Although the D556G receptor internalizes [125I]hCG rapidly, a corresponding increase in [125I]hCG degradation was not seen. The internalization of another constitutively active LH/hCG receptor (aspartic acid 556 to tyrosine) was also greater than that of the WT receptor. Internalization of receptor bound [125I]hCG was inhibited by a hypertonic sucrose solution, confirming that the ligand enters the cell by receptor-mediated endocytosis. Furthermore, the constitutively active D556G and D556Y LH/hCG receptors utilize the arrestin dependent internalization pathway. These results suggest that the active state conformation of the constitutively active receptor is conducive to rapid internalization.

Arrestins↗

PubMed1999

Glucocorticoids stimulate the accumulation of lipids in the rat corpus luteum.

We investigated the physiological basis for the trophic effect of glucocorticoids in rat corpora lutea in the absence of pituitary gonadotropins. Immature (Day 29) Sprague-Dawley rats were given eCG and hCG to induce the development of corpora lutea and were hypophysectomized on Day 32. Beginning on Day 40, rats received twice-daily s.c. injections of either dexamethasone (dex; 200 microg/rat/day) or vehicle (controls) and then were killed on Day 44. Plasma 20alpha-dihydroprogesterone, a major steroid produced by the corpora lutea, was higher (p </= 0.01) in dex-treated than in control rats (44.5 +/- 2.3 vs. 23.0 +/- 5.6 ng/ml). Dexamethasone treatment increased lipid droplets and lipid in the corpora lutea as revealed by electron microscopy and oil red O staining. Cholesterol esters were higher in corpora lutea of dex-treated rats compared to controls (14.8 +/- 1.1 vs. 2.2 +/- 0.5 microg/mg corpora lutea wet tissue, respectively; p </= 0.05). Another group of hypophysectomized rats was treated with either a high or a lower dosage of corticosterone, both of which caused an elevation to > 2-fold of plasma 20alpha-dihydroprogesterone concentration compared to controls. Glucocorticoid receptor protein (about 92 kDa) was detected in both luteal and nonluteal ovarian tissues in this animal model. These effects of glucocorticoids and the presence of the glucocorticoid receptor raise the possibility of a physiological role for glucocorticoids in the rat corpus luteum.

Animals↗

PubMed1998

Identification of a hormonally regulated luteinizing hormone/human chorionic gonadotropin receptor mRNA binding protein. Increased mrna binding during receptor down-regulation.

To elucidate the molecular events associated with the regulation of luteinizing hormone/human chorionic gonadotropin (LH/hCG) receptor mRNA stability during hCG-induced receptor down-regulation, we have identified an LH/hCG receptor-specific mRNA binding protein. Proteins were isolated from control and down-regulated rat ovary and were incubated with in vitro transcribed RNAs corresponding to the full-length LH/hCG receptor, as well as 5'- and 3'-truncated receptor forms. Resultant ribonucleoprotein complexes were analyzed by RNA gel mobility shift. A prominent Mr 50,000 ribonucleoprotein complex was identified with the following characteristics: 1) specificity for LH/hCG receptor open reading frame sequences located between nucleotides 102 and 282; 2) lack of competition by nonspecific RNAs; 3) a 3-fold increase in RNA binding activity during hCG-induced receptor down-regulation; and 4) limited tissue expression. This report describes the first evidence of an LH/hCG receptor mRNA binding protein, which we term LRBP-1, for luteinizing hormone receptor RNA binding protein-1. This protein is a candidate for a trans-acting factor involved in the hormonal regulation of LH/hCG receptor mRNA stability in rat ovary.

Animals↗

PubMed1998

In situ hybridization of high density lipoprotein (scavenger, type 1) receptor messenger ribonucleic acid (mRNA) during folliculogenesis and luteinization: evidence for mRNA expression and induction by human chorionic gonadotropin specifically in cell types that use cholesterol for steroidogenesis.

The present studies were undertaken to examine the expression of the high density lipoprotein (HDL) receptor, SR-B1 messenger RNA (mRNA) in ovarian cell types during folliculogenesis and luteinization using in situ hybridization histochemistry and to examine its hormonal regulation using Northern blots. For the in situ study for HDL receptor mRNA localization, 21-day-old rats were treated with 50 IU PMSG, and ovaries were collected 0, 24, and 56 h postinjection. At 56 h, animals were treated with a single dose of hCG, and ovaries were subsequently collected at 6-, 12-, 24-, and 72-h and 5-day intervals. In addition, on day 4 of pseudopregnancy, a second dose of 50 IU hCG or saline was administered, and ovaries were collected at 12, 24, and 48 h to determine the induction of the expression of HDL receptor mRNA. The results of in situ hybridization histochemistry showed that in the immature ovary, HDL receptor mRNA is associated with theca interna and interstitial cells (stroma). The mRNA expression in these cell types increased with PMSG treatment, but no signal was detected in the granulosa cells. Northern blot analysis also showed a marked increase in mRNA content in thecal and interstitial cells during follicular development. During luteinization, the intensity of the signal began to appear in the luteinized granulosa cells. With the completion of luteinization, the signal in the corpus luteum tissue became more intense. Further treatment with hCG increased the HDL receptor mRNA content compared with that in the saline-treated control. These results demonstrate that the cholesterol-using cell types of the ovary, namely the interstitial cells, thecal cells, and fully luteinized granulosa cells are endowed with the HDL receptor mRNA, which provides credence to the functional significance of the role of HDL receptor SR-B1 in cholesterol transport and ovarian steroidogenesis.

Animals↗

PubMed1997

Role of palmitoylation of conserved cysteine residues of luteinizing hormone/human choriogonadotropin receptors in receptor down-regulation.

The conserved cysteine residues 621 and 622 of luteinizing hormone/human chorionic gonadotropin receptors were converted to serine (C621S, C622S, C621/622S) and glycine residues (C621/C622G) by site directed mutagenesis. The wild type and mutant receptor cDNAs were cloned into the mammalian expression vector (PCMV4) and human embryonic kidney cells (293 cells) were transiently transfected with these constructs. Equilibrium binding studies with [(125)I]hCG (human chorionic gonadotropin) showed that the mutant and wild type receptors expressed on the cell surface exhibited similar K(d). The effect of mutation of the conserved cysteine residues on the ability of the receptors to undergo ligand-induced down-regulation was then tested. In vitro exposure of cells expressing the wild type receptor to a saturating concentration of human chorionic gonadotropin (100 ng/ml) for 24 h resulted in modest down-regulation of receptors. The palmitoylation deficient mutants, C621S, C622S, C621/622S and C621/622G, showed increased down-regulation compared with the wild type receptor. The extent of down-regulation of the mutant receptors correlated with increased internalization of the receptor. Additionally, the G protein coupling efficiency of the palmitoylation deficient mutants was not different from the wild type since the EC(50)s for cyclic AMP (cAMP) production were identical in both groups. These studies demonstrate that palmitoylation deficient mutants are more prone to ligand-induced receptor down-regulation. Furthermore, abrogation of palmitoylation by mutagenesis showed no effect on the efficiency of the palmitoylation deficient mutants to couple to Gs protein.

Amino Acid Sequence↗

PubMed1997

Post-translational processing in the Golgi plays a critical role in the trafficking of the luteinizing hormone/human chorionic gonadotropin receptor to the cell surface.

Point mutations in the luteinizing hormone/human chorionic gonadotropin (LH/hCG) receptor have been shown to cause constitutive activation which results in precocious puberty in affected males. We introduced one of these mutations, Asp-556 --> Gly, into the rat LH/hCG receptor and demonstrated that the mutant receptor constitutively activated adenylate cyclase in transfected 293 T cells. The cell surface expression of the mutant receptor was lower than that of the wild type receptor. Pulse-chase studies showed that the 73-kDa precursor of both the mutant and wild type receptors was synthesized at comparable efficiencies. However, post-translational processing of the mutant receptor to the mature 92-kDa form, which has N-linked complex type oligosaccharide chains, was impaired. Sensitivity of the mutant receptor to peptide-N-glycanase F and endoglycosidase H, and insensitivity to sialidase indicated that the 73-kDa species represents the high mannose form that has not yet been trafficked through the medial and trans Golgi. Additionally, although the wild type receptor was palmitoylated, the mutant receptor was not. Although the high mannose 73-kDa species is capable of binding LH/hCG, our results show that post-translational processing in the Golgi is required for the mature 92-kDa receptor to reach the cell surface.

Amidohydrolases↗

PubMed1997

Transforming growth factor-beta negatively modulates proliferation and c-fos expression of the human endometrial adenocarcinoma cell line HEC-1-A.

Growth factor regulation of normal and cancerous cell proliferation has been well-documented and may be mediated by proto-oncogene activity. The purpose of this study was to assess changes in proliferation and mitogen-induced c-fos mRNA expression of an endometrial carcinoma cell line, HEC-1-A, in response to TGF-beta, a potent growth-inhibitory peptide. HEC-1-A cells were incubated in the presence or absence of TGF-beta. Mitogen-stimulated cells were additionally treated with epidermal growth factor (EGF). Changes in proliferation were measured by [3H]thymidine uptake assays. Alterations in EGF-induced c-fos expression following TGF-beta pretreatment were assessed by Northern blot using a 32P-labeled human c-fos probe. Finally, chloramphenicol acetyltransferase assays were performed to evaluate c-fos promoter activity in response to treatment conditions. Basal and EGF-stimulated proliferation was inhibited by TGF-beta in a dose- and time-dependent manner. TGF-beta also reversibly decreased EGF-induced c-fos mRNA expression in a dose- and time-dependent manner. Sequences in the c-fos promoter that were stimulated by EGF showed suppressed activity when preincubated with TGF-beta. These results show that TGF-beta negatively modulates EGF-induced c-fos expression, which may be related to the observed inhibition of carcinoma cell proliferation.

Adenocarcinoma↗

PubMed1997

Epidermal growth factor activates protein kinase C in the human endometrial cancer cell line HEC-1-A.

Previous studies from this laboratory have shown that epidermal growth factor (EGF) and the tumor promoter, phorbol myristate acetate (PMA), are mitogenic in the endometrial cancer cell line HEC-1-A. Since the effects of EGF have been shown to be mediated by the protein kinase C (PKC) pathway transduction system, we examined the possibility that the EGF-responsive signal in the endometrial cancer cell line HEC-1-A involves protein kinase C activation. HEC-1-A cells were grown to confluency in 100-mm dishes and maintained in a serum-free medium for 24 hr prior to treatment. The cells were treated with EGF at varying time intervals (0.25 to 60 min) and concentrations (0.1 to 200 ng/ml). The cells were then lysed, homogenized, and centrifuged at 105,000g for 1 hr at 4 degrees C. The supernatant was chromatographed on DEAE-Sephacel columns. The membranous pellet was resuspended in 5 ml of lysis buffer containing 1% Nonidet P-40 and also chromatographed on DEAE-Sephacel columns separately. The eluates were collected and assayed for protein kinase C activity by determining the amount of 32P transferred from [gamma-32P]ATP onto histones in the presence of the phospholipids, phosphatidylserine, and diolein. Our results show that the cytoplasmic and membrane fraction of the HEC-1-A cell line contained phosphotransferase activity which displayed kinetic characteristics typical of the protein kinase C enzyme. The optimal incubation time for protein kinase C activation in the cytosol by EGF was 5 min (30-fold stimulation). The protein kinase C activity was increased when the cell lines were incubated with increasing concentrations of EGF. Enzyme saturation was seen at a concentration of 10 ng/ml of EGF (4.5-fold stimulation). Western blot analysis confirmed the presence of the PKC enzyme in the cytosol and membranes of our cancer cell line. These results suggest that EGF, at least partially, exerts its effects on the endometrial adenocarcinoma cell line by activating protein kinase C through increased breakdown of phosphatidyl inositol (PI). The PI cascade appears to be an important signal transduction system mediating the growth stimulatory effects of EGF on endometrial carcinoma.

Blotting, Western↗

PubMed1996

3' untranslated region-mediated regulation of luteinizing hormone/human chorionic gonadotropin receptor expression.

Multiple luteinizing hormone/human chorionic gonadotropin (LH/hCG) receptor mRNAs (6.7, 4.4, 2.6, and 1.8 kb) have been identified in the rat ovary. Our laboratory has previously cloned a 3.5 kb cDNA that corresponds to the 3' untranslated region (3' UTR) of the 6.7 kb transcript, the major LH/hCG receptor mRNA in rat ovary. In order to determine the effects of the 3' UTR on receptor expression, we have constructed cDNAs corresponding to the open reading frame of LH/hCG receptor or luciferase, plus these constructs with the addition of the 3' UTRs associated with the short (4.4 kb) and long (6.7 kb) LH/hCG receptor transcripts, and measured receptor or luciferase expression in 293 cells transformed with large T antigen (293T). Ligand binding analysis with 125I-hCG revealed that the 3' UTR inhibited receptor expression, which occurs through posttranscriptional events. First, the 3' UTRs reduced receptor mRNA half-life in actinomycin D-arrested cells, as compared to the open reading frame alone. Second, LH/hCG receptor mRNAs with the long 3' UTR associated with significantly fewer ribosomes. The effect of the LH/hCG receptor 3' UTRs on luciferase expression was also determined. The short 3' UTR increased luciferase activity, whereas the long 3' UTR decreased luciferase expression. Thus, the short 3' UTR exerts opposite effects on receptor and luciferase expression. However, sequences in the long 3' UTR are sufficient to inhibit both receptor and luciferase expression in 293T cells.

Animals↗

PubMed1996

IRS-I expression on the luteinized rat ovary: IGF-I and cyclic AMP effects on IRS-I tyrosine phosphorylation.

The expression of insulin receptor substrate-I (IRS-I) mRNA was demonstrated in rat luteal cells by Northern blot analysis, in situ hybridization as well as by reverse transcriptase polymerase chain reaction. Western blot with a polyclonal anti IRS-I antibody showed the presence of a 183 kDa protein which corresponds to the size of IRS-I reported in other tissues. Further studies were performed to determine whether human chorionic gonadotropin (hCG) can interact with the insulin-like growth factor-I (IGF-I) signaling pathway to increase tyrosine phosphorylation of IRS-I. While hCG alone was ineffective in stimulating the phosphorylation of IRS-I, IGF-I mediated phosphorylation of IRS-I was increased by prior exposure to hCG. These results were further confirmed by the immunoprecipitation of IRS-I from the lysate of hCG- and IGF-I-treated luteal cell cultures followed by Western blotting with anti-phosphotyrosine antibody. Similarly, pretreatment with forskolin also increased IGF-I stimulated IRS-I phosphorylation. The increased tyrosine phosphorylation of IRS-I seen in response to IGF-I stimulation following treatment with either hCG or forskolin was not due to an increase in IRS-I content. Furthermore, IGF-I receptor tyrosine kinase activity was not affected by forskolin, suggesting that the increase in IRS-I tyrosine phosphorylation was not the result of an increase in its activity. Thus, we conclude that hCG/LH and IGF-I signaling pathways 'cross-talk' to increase the levels of IRS-I tyrosine phosphorylation. The observed increase in IRS-I tyrosine phosphorylation may be the result of an increase in the stability of the phosphorylated form of IRS-I.

Animals↗

PubMed1995

cAMP and PMA enhance the effects of IGF-I in the proliferation of endometrial adenocarcinoma cell line HEC-1-A by acting at the G1 phase of the cell cycle.

The present study was undertaken to determine whether endometrial cancer cell line HEC-1-A differ from nontransformed cells, in that the cAMP and protein kinase C pathways may enhance IGF-I effects in mitogenesis by acting at the G1 phase of the cell cycle instead of G0. Immunofluorescence staining of HEC-1-A cells using the proliferating cell nuclear antigen (PCNA) monoclonal antibody and flow cytometric analysis determined that HEC-1-A cells do not enter the G0 phase of the cell cycle when incubated in a serum-free medium. Approximately 51% of the cells were in G1, 12% were in S and 37% in G2 phase of the cell cycle prior to treatment. Forskolin and phorbol-12-myristate 13-acetate (PMA) were used to stimulate cAMP production and protein kinase C activity, respectively. IGF-I, forskolin and PMA each increased (P < 0.01) [3H]-thymidine incorporation in a dose and time dependent manner. The interaction of forskolin and PMA with IGF-I was then determined. Cells preincubated with forskolin or PMA followed by incubation with IFG-I incorporated significantly more (P < 0.01) [3H]-thymidine into DNA than controls or any treatment alone. It is concluded that forskolin and, to a lesser extent, PMA exert their effect at the G1 phase of the cycle to enhance IGF-I effects in cell proliferation.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1995

Identification of constitutively activating mutation of the luteinising hormone receptor in a family with male limited gonadotrophin independent precocious puberty (testotoxicosis).

A family of male limited gonadotrophin independent precocious puberty was examined for activating mutation of the LH receptor. A transition of A to G in nucleotide 1733 of the human LH receptor gene was identified in all affected males and in an unaffected carrier female. The mutation was shown by identifying a new restriction site created by the mutation. This mutation appears to be a common feature of the disorder, as it has been reported previously in unrelated families. Therefore, the presence of this new restriction site can serve as a diagnostic tool in males at risk before the onset of symptoms, as well as identifying carrier females.

Adenylyl Cyclases↗

PubMed1994

Palmitoylation of luteinizing hormone/human choriogonadotropin receptors in transfected cells. Abolition of palmitoylation by mutation of Cys-621 and Cys-622 residues in the cytoplasmic tail increases ligand-induced internalization of the receptor.

We have examined whether the two cysteine residues (621 and 622) of the carboxyl-terminal cytoplasmic domain of the rat luteinizing hormone (LH/hCG) receptor are potential sites for palmitoylation. The full-length LH/hCG receptor cDNA was cloned into an expression vector (hCGR-pCMV4). A human embryonic kidney cell line expressing large T antigen (293T cells) was transiently transfected with hCGR-pCMV4 by the calcium phosphate precipitation technique. The functional expression of the receptor was confirmed by 35S-labeled cysteine incorporation into the receptor as well as by 125I-human chorionic gonadotropin (hCG) binding. The transfected cells were then labeled with [3H]palmitic acid, and the labeled receptors purified on hCG-Affi-Gel matrix and subjected to SDS-polyacrylamide gel electrophoresis and autoradiography. The hCGR-pCMV4-transfected cells incorporated [3H]palmitic acid into a 92-kDa band corresponding to the mature form of the LH/hCG receptor; this band was absent in cells transfected with vector alone. Site-directed mutagenesis of either cysteine 621 or 622 to serine residue was partially inhibitory, whereas mutation of both cysteine residues (621 and 622) completely abolished palmitoylation. Scatchard analyses revealed that the mutant and wild type receptors have similar affinities for 125I-hCG. The biological function of palmitoylation was then examined in the transfected cells. The results showed that although the intracellular trafficking of the receptor and the ability to stimulate cyclic AMP production were unaffected, the rate of ligand-induced internalization of the receptor was higher in palmitoylation-deficient mutants compared to the wild type receptors. The first order rate constants of internalization of the mutant receptors were over 2-fold higher than the wild type. Intracellular degradation of the receptor-bound ligand was also higher in the mutants. These studies suggest that the native LH/hCG receptor is palmitoylated at cysteine residues 621 and 622, creating a membrane-anchoring site at the putative cytoplasmic domain. This palmitic acid-mediated anchoring decreases the ligand-induced receptor internalization thereby prolonging the retention of the ligand-bound receptor on the cell surface.

Amino Acid Sequence↗