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.     

The C-terminal tail of the rat lutropin/choriogonadotropin (CG) receptor independently modulates human (h)CG-induced internalization of the cell surface receptor and the lysosomal targeting of the internalized hCG-receptor complex

The analysis of 21 progressive truncations of the C-terminal tail of the rat LH/CG receptor (rLHR) revealed the presence of a region delineated by residues 628-649 that, when removed, enhanced the degradation of the internalized human (h)CG. The analysis of these truncations also revealed the presence of a region delineated by residues 624-631 that, when removed, enhanced the rate of internalization of hCG. Since there is little overlap between these two regions, we conclude that the structural features of the rLHR that mediate internalization and degradation of the internalized hormone are different. Detailed analyses of cells expressing a truncation at Y637 (designated rLHR-t637) showed that the enhanced degradation of hCG observed in the these cells is due to an increase in the rate of transfer of the internalized hCG-rLHR complex from the endosomes to the lysosomes rather than to the enhanced dissociation of the hCG-rLHR complex in the lysosomes.     

Human chorionic villous macrophages as a fetal biological shield from maternal chorionic gonadotropin

In addition to its most well characterized biological role in the rescue and maintenance of corpus luteum function, human chorionic gonadotropin (hCG) also stimulates the onset of fetal gonadal steroidogenesis. However, excess hCG is teratogenic to fetal gonadal tissues, and therefore hCG must be tightly regulated. Although there is an anatomical barrier between the fetal vessels and maternal blood, other mechanisms may regulate hCG levels. In the present study, we investigated whether human chorionic villous macrophages degraded maternal hCG. Isolated human macrophages incorporated and degraded hCG in a time-dependent manner. Human placental villous macrophages and phorbol myristate acetate (PMA)-treated THP-1 cells expressed the gene encoding an exon 9-deleted form of the luteinizing hormone/chorionic gonadotropin (LH/CG) receptor; expression of the full-length receptor was not determined. While both PMA-treated or untreated THP-1 cells could uptake hCG into their cytoplasms, hCG degradation and excretion of its byproducts only progressed in PMA-treated THP-1 cells. In conclusion, hCG internalization and degradation are different processes in macrophages that protect fetal gonadogenesis from excess hCG. The exon 9-deleted LH/CG receptor, but not the full-length receptor, is involved in the degradation of cytoplasmic hCG by organ-specific, dominant–negative interactions.     

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.     

Class A scavenger receptor-mediated adhesion and internalization require distinct cytoplasmic domains

Class A scavenger receptors (SR-A) are transmembrane glycoproteins that mediate both ligand internalization and cell adhesion. Previous studies have identified specific amino acids in the cytoplasmic tail of SR-A that regulate receptor internalization; however, the role of cytoplasmic domains in regulating cell adhesion has not been addressed. To investigate the role of cytoplasmic domains in SR-A-mediated adhesion and to address whether SR-A-mediated adhesion and internalization require distinct cytoplasmic domains, different SR-A constructs were stably expressed in human embryonic kidney (HEK 293) cells. Deleting the entire cytoplasmic tail (SR-A Delta 1-55) greatly reduced receptor protein abundance. Retaining the six amino acids proximal to the membrane (SR-A Delta 1-49) restored receptor protein abundance. Although SR-A Delta 1-49 localized to the cell surface, cells expressing this receptor failed to internalize the ligand acetylated low density lipoprotein. Replacing the cytoplasmic tail of SR-A with that of the transferrin receptor (TfR/SR-A) resulted in retention of the chimeric receptor in the endoplasmic reticulum suggesting a specific role for the membrane-proximal amino acids in trafficking SR-A from the endoplasmic reticulum to the Golgi. Like SR-A expressing cells, cells expressing SR-A Delta 1-49 displayed increased spreading and adhesion, demonstrating that the membrane-proximal amino acids were sufficient for SR-A-mediated cell adhesion. Together, our results indicate a critical role for the membrane-proximal amino acids in SR-A trafficking and demonstrate that SR-A-mediated adhesion and internalization require distinct cytoplasmic domains.     

Evidence for dual coupling of the murine luteinizing hormone receptor to adenylyl cyclase and phosphoinositide breakdown and Ca2+ mobilization. Studies with the cloned murine luteinizing hormone receptor expressed in L cells.

The murine receptor for luteinizing hormone (LHR) was cloned and expressed in L cells. This LHR (mature protein of 674 amino acids) is very similar to that of the rat (same length, 36 amino acid differences) but differs significantly more from that of man (673 amino acids, 109 differences). Expression of the murine LHR in L cells led to the appearance of binding sites for human chorionic gonadotropin (hCG) with a Kd of 150 pM and an LH- and hCG-stimulable adenylyl cyclase activity (EC50 = 50-100 pM hCG). Upon labeling pools of phosphoinositides with [3H]myo-inositol, L cells expressing the murine LHR responded to hCG with an increase in their rate of phosphoinositide hydrolysis (EC50 = 2,400 pM hCG). This was accompanied by an increase in intracellular Ca2+ [( Ca2+]i), as determined by the Fura2 method. This increase in [Ca2+]i in response to hCG was dependent on the LHR, for HCG did not affect [Ca2+]i in L cells not expressing the LHR. The effect was not due to the cAMP-forming activity of the LH receptor, for neither forskolin nor prostaglandin E1, which both increase cAMP levels in L cells, had a similar effect in either control or LHR-expressing cells and isoproterenol had no effect in L cells expressing a functionally active hamster beta-adrenergic receptor. The effect was also not due to overexpression of a Gs-coupled receptor, for L cells expressing 8-fold higher levels of the human V2 vasopressin receptor did not mimic the Ca(2+)-mobilizing response of the LH receptor. We conclude that the LH receptor has the capability of activating two intracellular signaling pathways: one leading to stimulation of adenylyl cyclase and resulting in increases in cAMP and a second leading to stimulation of phospholipase C and resulting in formation of inositol phosphates and elevations in [Ca2+]i. These data correlate positively with and provide a mechanistic explanation for previous reports on the ability of hCG to mobilize phosphoinositides and increasing [Ca2+]i in luteal and granulosa cells (e.g. Davis, J. S., West, L. A., and Farese, R. V. (1984) J. Biol. Chem. 259, 15028-15034).     

Pleiotropic effects of substitutions of a highly conserved leucine in transmembrane helix III of the human lutropin/choriogonadotropin receptor with respect to constitutive activation and hormone responsiveness

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.     

Activation of the luteinizing hormone/choriogonadotropin hormone receptor promotes ADP ribosylation factor 6 activation in porcine ovarian follicular membranes.

Previously we demonstrated in a cell-free ovarian follicular plasma membrane model that agonist-dependent desensitization of the luteinizing hormone/choriogonadotropin receptor (LH/CG R) is GTP-dependent, mimicked by the addition of ADP-ribosylation factor (ARF) nucleotide binding site opener, which acts as a guanine nucleotide exchange factor for ARFs 1 and 6, and selectively inhibited by synthetic N-terminal ARF6 peptides. We therefore sought direct evidence that activation of the LH/CG R promotes activation of ARF1 and/or ARF6. Using a classic ARF activation assay, the cholera toxin-catalyzed ADP-ribosylation of G alpha(s), results show that LH/CG R activation stimulates an ARF protein by a brefeldin A-independent mechanism. Synthetic N-terminal inhibitory ARF6 but not ARF1 peptide blocks LH/CG R-stimulated ARF activity. LH/CG R activation also promotes the binding of a photoaffinity GTP analog to a protein that migrates on one- and two-dimensional polyacrylamide gel electrophoresis with ARF6. These results suggest that ARF6 is the predominant ARF activated by the LH/CG R. To activate ARF6, the LH/CG R does not appear to signal through the C-terminal regions of G alpha(i) or G alpha(q) or through the second or third intracellular loops or the N terminus of the cytoplasmic tail of the LH/CG R. Although exogenous recombinant ARNO promotes only a small increase in ARF6 activation in the presence of activated LH/CG R, hCG-stimulated ARF6 activation is reduced to basal levels by catalytically inactive ARF nucleotide binding-site opener. These results provide direct evidence that LH/CG R activation leads to the activation of membrane-delimited ARF6.     

Multiple inhibitory effects of a luteinizing hormone-releasing hormone agonist on hCG-dependent steroidogenesis and on FSH-dependent responses in ovarian cells in vivo and in vitro

[125I] labelled [D-Leu6, des-Gly-NH10(2)] LH-RH ethylamide (LH-RHa), when injected into immature female rats, bound specifically not only to the pituitary but also to the ovaries. LH-RHa inhibited hCG-stimulated progesterone production and ovarian weight augmentation in hypophysectomized immature female rats in vivo. FSH-induced ovarian hCG receptors and ovarian weight gain in diethylstilbestrol (DES)-treated hypophysectomized immature female rats were also suppressed by LH-RHa. Progesterone production by rat luteal cells in vitro was inhibited by LH-RHa. LH-RHa did not change the affinity or population of LH/hCG receptor in porcine granulosa cells in short term incubation. However, LH-RHa inhibited induction of LH/hCG receptor stimulated by FSH and insulin in long term culture of porcine granulosa cells. LH-RHa delayed hCG-stimulated cyclic AMP accumulation in porcine granulosa cells. These findings suggest that LH-RHa inhibits hCG-stimulated cyclic AMP accumulation and subsequent progesterone production as well as FSH-stimulated LH/hCG receptor induction by acting directly on ovarian cells.     

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.     

Characterization of the carboxyl-terminal domain of the rat glucose-dependent insulinotropic polypeptide (GIP) receptor. A role for serines 426 and 427 in regulating the rate of internalization.

Glucose-dependent insulinotropic polypeptide (GIP) is a gastrointestinal hormone involved in the regulation of insulin secretion. In non-insulin-dependent diabetes mellitus insulin responses to GIP are blunted, possibly due to altered signal transduction or reduced receptor number. Site-directed mutagenesis was used to construct truncated GIP receptors to study the importance of the carboxyl-terminal tail (CT) in binding, signaling, and receptor internalization. Receptors truncated at amino acids 425, 418, and 405, expressed in COS-7 or CHO-K1 cells, exhibited similar binding to wild type receptors. GIP-dependent cAMP production with the 405 mutant was decreased in COS-7 cells. Maximal cAMP production in CHO-K1 cells was reduced with all truncated forms. Binding was undetectable with a receptor truncated at amino acid 400; increasing tail length by adding 5 alanines restored binding and signaling. Mutants produced by alanine scanning of residues 394-401, adjacent to transmembrane domain 7, were all functional. CT truncation by 30 or more amino acids, mutation of serines 426/427, singly or combined, or complete CT serine knockout all reduced receptor internalization rate. The majority of the GIP receptor CT is therefore not required for signaling, a minimum chain length of approximately 405 amino acids is needed for receptor expression, and serines 426 and 427 are important for regulating rate of receptor internalization.     

Eleven amino acids (Lys-201 to Lys-211) and 9 amino acids (Gly-222 to Leu-230) in the human thyrotropin receptor are involved in ligand binding.

Our previous studies involving chimeric thyrotropin-lutropin/choriogonadotropin (TSH-LH/CG) receptors suggest that multiple segments spanning the entire extracellular domain of the human TSH receptor contribute to the TSH binding site. Nevertheless, the mid-region (segment C, amino acid residues 171-260) of the receptor extracellular domain is particularly important in TSH binding. In the present studies, we constructed seven new chimeric receptors in order to analyze segment C in further detail. Seven small segments spanning segment C of the TSH receptor were replaced with the counterpart of the rat LH/CG receptor. These mutant receptors were stably introduced into Chinese hamster ovary cells and were tested for hormone binding and cAMP responsiveness to hormone stimulation. The results indicate that 11 amino acids of the TSH receptor (Lys-201 to Lys-211) and the corresponding region of the LH/CG receptor (Thr-202 to Ile-212) are important for specific TSH and human CG binding, respectively. In addition, nine amino acids of the TSH receptor (Gly-222 to Leu-230) are also involved in TSH binding. A further conclusion from these data is that TSH and human CG bind to partially overlapping sites on their respective receptor molecules.     

A novel cytoplasmic tail MXXXL motif mediates the internalization of prostate-specific membrane antigen

Prostate-specific membrane antigen (PSMA) is a transmembrane protein expressed at high levels in prostate cancer and in tumor-associated neovasculature. In this study, we report that PSMA is internalized via a clathrin-dependent endocytic mechanism and that internalization of PSMA is mediated by the five N-terminal amino acids (MWNLL) present in its cytoplasmic tail. Deletion of the cytoplasmic tail abolished PSMA internalization. Mutagenesis of N-terminal amino acid residues at position 2, 3, or 4 to alanine did not affect internalization of PSMA, whereas mutation of amino acid residues 1 or 5 to alanine strongly inhibited internalization. Using a chimeric protein composed of Tac antigen, the alpha-chain of interleukin 2-receptor, fused to the first five amino acids of PSMA (Tac-MWNLL), we found that this sequence is sufficient for PSMA internalization. In addition, inclusion of additional alanines into the MWNLL sequence either in the Tac chimera or the full-length PSMA strongly inhibited internalization. From these results, we suggest that a novel MXXXL motif in the cytoplasmic tail mediates PSMA internalization. We also show that dominant negative micro2 of the adaptor protein (AP)-2 complex strongly inhibits the internalization of PSMA, indicating that AP-2 is involved in the internalization of PSMA mediated by the MXXXL motif.     

Function of the cytoplasmic tail of human calcitonin receptor-like receptor in complex with receptor activity-modifying protein 2

Receptor activity-modifying protein 2 (RAMP2) enables calcitonin receptor-like receptor (CRLR) to form an adrenomedullin (AM)-specific receptor. Here we investigated the function of the cytoplasmic C-terminal tail (C-tail) of human (h)CRLR by co-transfecting its C-terminal mutants into HEK-293 cells stably expressing hRAMP2. Deleting the C-tail from CRLR disrupted AM-evoked cAMP production or receptor internalization, but did not affect [¹²⁵I]AM binding. We found that CRLR residues 428-439 are required for AM-evoked cAMP production, though deleting this region had little effect on receptor internalization. Moreover, pretreatment with pertussis toxin (100 ng/mL) led to significant increases in AM-induced cAMP production via wild-type CRLR/RAMP2 complexes. This effect was canceled by deleting CRLR residues 454-457, suggesting Gi couples to this region. Flow cytometric analysis revealed that CRLR truncation mutants lacking residues in the Ser/Thr-rich region extending from Ser⁴⁴⁹ to Ser⁴⁶⁷ were unable to undergo AM-induced receptor internalization and, in contrast to the effect on wild-type CRLR, overexpression of GPCR kinases-2, -3 and -4 failed to promote internalization of CRLR mutants lacking residues 449-467. Thus, the hCRLR C-tail is crucial for AM-evoked cAMP production and internalization of the CRLR/RAMP2, while the receptor internalization is dependent on the aforementioned GPCR kinases, but not Gs coupling.     

内皮素—1对大鼠排卵前卵泡颗粒细胞产生孕酮的影响

本文用离体细胞体外孵育法研究了内皮素－１（ＥＴ）对大鼠排卵前卵泡颗粒细胞孕酮生成的影响及其作用机理。结果发现,ＥＴ能显著抑制ｈＣＧ刺激下的孕酮产生,抑制作用在浓度为１０－８ｍｏｌ／Ｌ时,即有显著意义（Ｐ＜０．０５,ｎ＝６）,至１０－７ｍｏｌ／Ｌ时则有非常显著的意义（Ｐ＜０．０１,ｎ＝６）;不同浓度ＥＴ（１０－７—１０－７ｍｏｌ／Ｌ）,对颗粒细胞基础孕酮的产生无明显影响。进一步研究表明,ＥＴ对ｈＣＧ刺激下孕酮生成的抑制作用,在用免抗人内皮素抗血清（ＥＴ－Ａ）１：１０００及ｃＡＭＰ后能明显被逆转。实验中还观察到,ＥＴ使颗粒细胞ＬＨ／ｈＣＧ受体数下降,亲和力降低。本文结果提示,ＥＴ可能为卵巢内的一种局部调节肽,通过作用于ＥＴ受体,干扰ＬＨ／ｈＣＧ受体功能和ｃＡＭＰ生成而抑制颗粒细胞孕酮的产生。     

Deletions in the cytoplasmic domain of the polymeric immunoglobulin receptor differentially affect endocytotic rate and postendocytotic traffic

We have examined the function of the cytoplasmic domain of the polymeric immunoglobulin receptor (pIg-R) by producing two separate deletions in the cytoplasmic domain of the pIg-R, expressing the mutant receptors in polarized MDCK cells, and analyzing each for their effects on receptor and ligand traffic. Deletion of the C-terminal 30 amino acids (726-755) reduces the rate of internalization of receptor-bound ligand from the basolateral surface. However, this mutation has no effect on delivery of receptor from the Golgi to the basolateral surface or the post-endocytotic traffic of receptor and ligand. Mutation of a tyrosine at position 734 to serine produces a receptor with a similar phenotype. If residues 670-707 are deleted from the middle of the cytoplasmic domain, both basolateral delivery and internalization are unaffected. However, unlike wild type, after endocytosis from the basolateral surface, both receptor and ligand are largely degraded. We reported previously that deletion of the entire cytoplasmic domain prevents the basolateral delivery of newly synthesized receptor (Mostov, K.E., de Bruyn Kops, A., and Deitcher, D.L. (1986) Cell 47, 359-364). In contrast, the mutants reported here are delivered to the basolateral surface, suggesting that only residues 653-669 and/or 708-725 are necessary for basolateral delivery. Thus, different deletions in the cytoplasmic domain of the pIg-R can produce mutant receptors which alter different aspects of receptor traffic.     

The internalization signal in the cytoplasmic tail of lysosomal acid phosphatase consists of the hexapeptide PGYRHV.

Lysosomal acid phosphatase (LAP) is rapidly internalized from the cell surface due to a tyrosine-containing internalization signal in its 19 amino acid cytoplasmic tail. Measuring the internalization of a series of LAP cytoplasmic tail truncation and substitution mutants revealed that the N-terminal 12 amino acids of the cytoplasmic tail are sufficient for rapid endocytosis and that the hexapeptide 411-PGYRHV-416 is the tyrosine-containing internalization signal. Truncation and substitution mutants of amino acid residues following Val416 can prevent internalization even though these residues do not belong to the internalization signal. It was shown recently that part of the LAP cytoplasmic tail peptide corresponding to 410-PPGY-413 forms a well-ordered beta turn structure in solution. Two-dimensional NMR spectroscopy of two modified LAP tail peptides, in which the single tyrosine was substituted either by phenylalanine or by alanine, revealed that the tendency to form a beta turn is reduced by 25% in the phenylalanine-containing peptide and by approximately 50% in the alanine-containing mutant peptide. Our results suggest, that in the short cytoplasmic tail of LAP tyrosine is required for stabilization of the right turn and that the aromatic ring system of the tyrosine residue is a contact point to the putative cytoplasmic receptor.     

The luteinizing hormone receptor activates phospholipase C via preferential coupling to Gi2.

Binding of lutropin/choriogonadotropin (LH/CG) to its cognate receptor results in the activation of adenylyl cyclase and phospholipase C. This divergent signaling of the LH receptor is based on the independent activation of distinct G protein subfamilies, i.e. , Gs, Gi, and potentially also Gq. To examine the selectivity of LH receptor coupling to phospholipase C beta-activating G proteins, we used an in vivo reconstitution system based on the coexpression of the LH receptor and different G proteins in baculovirus-infected insect cells. In this paper, we describe a refined expression strategy for the LH receptor in insect cells. The receptor protein was inserted into the cell membrane at an expression level of 0.8 pmol/mg of membrane protein. Sf9 cells expressing the LH receptor responded to hCG challenge with a concentration-dependent accumulation of intracellular cAMP (EC50 = 630 nM) but not of inositol phosphates, whereas stimulation of the histamine H1 receptor in Sf9 cells led to increased phospholipase C (PLC) activity. Immunoblotting experiments using G protein-specific antisera revealed the absence of quantitative amounts of alpha i in Sf9 cells, whereas alpha s and alpha q/11 were detected. We therefore attempted to restore the hCG-dependent PLC activation by infection of Sf9 cells with viruses encoding the LH receptor and different G protein alpha subunits. HCG stimulation of cells coexpressing the LH receptor and exogenous alpha i2 resulted in stimulation of PLC activity. In cells coinfected with an alpha i3-baculovirus, hCG challenge led to a minor activation of PLC, whereas no hCG-dependent PLC stimulation was observed in cells coexpressing alpha i1. Most notably, coinfection with baculoviruses encoding alpha q or alpha 11 did not reproduce the PLC activation by the LH receptor. Thus, the murine LH receptor activates adenylyl cyclase via Gs and PLC via selective coupling to Gi2.     

Intraovarian thrombin and activated protein C signaling system regulates steroidogenesis during the periovulatory period

In addition to its role in blood coagulation, thrombin directly stimulates protease-activated receptors (PAR) or interacts with thrombomodulin (THBD) to activate membrane-bound protein C which stimulates PAR1 and PAR4 receptors to promote downstream pleiotropic effects. Our DNA microarray, RT-PCR, and immunostaining analyses demonstrated ovarian expression of THBD, activated protein C (APC) receptor [endothelial protein C receptor (EPCR)], as well as PAR1 and PAR4 receptors in mice. After treatment of gonadotropin-primed immature mice with an ovulatory dose of human chorionic gonadotropin (hCG) (a LH surrogate), major increases in the expression of THBD, EPCR, PAR1, and PAR4 were detected in granulosa and cumulus cells of preovulatory follicles. Immunoassay analyses demonstrated sustained increases in ovarian prothrombin and APC levels after hCG stimulation. We obtained luteinizing granulosa cells from mice treated sequentially with equine CG and hCG. Treatment of these cells with thrombin or agonists for PAR1 or PAR4 decreased basal and forskolin-induced cAMP biosynthesis and suppressed hCG-stimulated progesterone production. In cultured preovulatory follicles, treatment with hirudin (a thrombin antagonist) and SCH79797 (a PAR1 antagonist) augmented hCG-stimulated progesterone biosynthesis, suggesting a suppressive role of endogenous thrombin in steroidogenesis. Furthermore, intrabursal injection with hirudin or SCH79797 led to ipsilateral increases in ovarian progesterone content. Our findings demonstrated increased ovarian expression of key components of the thrombin-APC-PAR1/4 signaling system after LH/hCG stimulation, and this signaling pathway may allow optimal luteinization of preovulatory follicles. In addition to assessing the role of thrombin and associated genes in progesterone production by the periovulatory ovary, these findings provide a model with which to study molecular mechanisms underlying thrombin-APC-PAR1/4 signaling.