High efficiency method for gene transfer in normal pituitary gonadotropes: adenoviral-mediated expression of G protein-coupled receptor kinase 2 suppresses luteinizing hormone secretion.

The level of LH secretion is determined by both alterations in gonadotrope responsiveness and alterations in GnRH secretion. The molecular mechanisms underlying gonadotrope responsiveness are unknown, but may include G protein-coupled receptor kinases (GRKs). Typically, GRKs phosphorylate the intracellular regions of seven-transmembrane receptors permitting beta-arrestin to bind, which prevents receptor activation of its G protein. Previously, we reported that heterologous expression of GRK2, -3, and -6 in GnRH receptor-expressing COS cells by complementary DNA transfection suppressed GnRH-stimulated inositol trisphosphate production, and that coexpression of GRK2 and beta-arrestin-2 was more inhibitory than either expressed alone. Here, we have investigated the effect of GRK2 on GnRH-stimulated LH secretion using adenovirus-mediated gene transfer in normal pituitary gonadotropes. Pituitary cells were infected with adeno-GRK2 or adeno-beta-galactosidase constructs at a multiplicity of infection of 60 (number of viral particles per cell). Seventy-two hours later, GRK2 expression was measured by enzyme-linked immunosorbent assay, and GnRH-stimulated LH secretion (10(-7) M GnRH-A for 90 min) was assayed by RIA. Adeno-beta-galactosidase infected 96-99% of the cells based on X-Gal staining. Uninfected and adeno-beta-galactosidase-infected cells exhibited endogenous GRK immunoreactivity of about 0.5 (OD405), and LH secretion of 14.8-17.7 ng/ml. Adeno-GRK2-infected cells showed a GRK2 immunoreactivity of about 2.5 (OD405) and LH secretion of 2.5 ng/ml. Therefore, adeno-GRK2 infection resulted in a 5-fold increase in the GRK2 OD405 value, which was accompanied by an 80-85% decrease in GnRH-stimulated LH secretion. GnRH-stimulated inositol trisphosphate production by gonadotropes also was inhibited, suggesting a site of action for GRK2 at phospholipase Cbeta or earlier in the signal transduction pathway. The significance of these findings is 2-fold: 1) adenoviral-mediated gene transfer permits investigation of the regulatory role of gene products in the cell of interest, the gonadotrope, rather than in heterologous cell systems; and 2) additional, stronger evidence is provided that supports a role for GRKs in setting the responsiveness of GnRH receptor signaling.

Potential regulatory roles for G protein-coupled receptor kinases and beta-arrestins in gonadotropin-releasing hormone receptor signaling.

GnRH stimulates gonadotropin secretion, which desensitizes unless the releasing hormone is secreted or administered in a pulsatile fashion. The mechanism of desensitization is unknown, but as the GnRH receptor is G protein coupled, it might involve G protein-coupled receptor kinases (GRKs). Such kinases phosphorylate the intracellular regions of seven-transmembrane receptors, permitting beta-arrestin to bind, which prevents the receptor from activating G proteins. Here, we tested the effect of GRKs and beta-arrestins on GnRH-induced inositol trisphosphate (IP3) production in COS cells transfected with the GnRH receptor complementary DNA. GRK2, -3, and -6 overexpression inhibited IP3 production by 50-75% during the 30 sec of GnRH treatment. Coexpression of GRK2 and beta-arrestin-2 suppressed GnRH-induced IP3 production more than that of either alone. Immunocytochemical staining of rat anterior pituitary revealed that all cells expressed GRK2, -3, and -6; all cells also expressed the beta-arrestins. Western blots on cytosolic extracts of rat pituitaries revealed the presence of GRK2/3 and beta-arrestin-1 and -2. The expression of GRKs and beta-arrestins by gonadotropes and their inhibition of GnRH-stimulated IP3 production in COS-1 cells expressing the GnRH receptor suggest a potential regulatory role for the GRK/beta arrestin paradigm in GnRH receptor signaling.

Identification by polymerase chain reaction of the G protein-coupled receptor kinases expressed in the mouse gonadotropic alpha T3-1 cell line.

G protein-coupled receptor kinases (GRK 1-6) stimulate short-term desensitization (< 5 min) by phosphorylating G-protein coupled receptors, and also participate in receptor sequestration, which may relate to intermediate-term desensitization (30-60 min). The existence of such kinases and hence a potential role for them in gonadotrope/GnRH receptor desensitization was investigated using the PCR to identify GRKs in messenger RNA (mRNA) from the mouse alpha T3-1 gonadotrope cell line. The 150-bp complementary DNAs amplified by PCR from the kinase catalytic domain were cloned and sequenced. Seventeen of 42 clones were receptor kinases based on high nucleotide identities of 85-100% and amino acid identities of 97-100% with rat GRK2 and 3, and with human GRK6. Among the eight GRK3 clones was one differing from rat GRK3 by a single nucleotide and seven differing by six; no amino acid difference resulted from the nucleotide differences. Of the five GRK2 clones, one sequence was identical with rat GRK2, but four sequences differed by three nucleotides and one amino acid. Among four GRK6 sequences, one showed 15 nucleotide differences from human GRK6 (with no amino acid differences), and three had 16 nucleotide and one amino acid differences. For each of the three GRKs found, the most closely related isoform is assumed to be the mouse homolog of rat GRK2 and GRK3, and human GRK6, whereas the others are assumed to be previously undescribed isoforms or subtypes of GRK2, 3, and 6. Immunocytochemical staining using antibodies to GRK2, 3, and 6 confirmed their presence in alpha T3-1 cells. The function of these GRKs in alpha T3-1 cells is unknown, but they may be involved in short-term desensitization of the gonadotrope/GnRH receptor or perhaps, more likely, the sequestration of this receptor during intermediate-term desensitization.

Potential role for a regulator of G protein signaling (RGS3) in gonadotropin-releasing hormone (GnRH) stimulated desensitization.

The cellular and molecular mechanisms of gonadotrope desensitization are unknown but transduction of the GnRH signal is known to involve sequentially the GnRH receptor, Gq alpha protein, phospholipase C beta-1, inositol-1,4,5-trisphosphate (IP3), and intracellular Ca+2 release. Here, we report the results of studies of a new family of proteins known as regulators of G protein signaling (RGS) that recently have been implicated in desensitization of several ligand induced processes. Using DNA-mediated transfection, we co-expressed the GnRH receptor and RGS1,2,3, or 4 in COS-1 cells. Control cells and those expressing RGS1,2, and 4 produced five fold increases in IP3 levels during the 30 sec after treatment with GnRH. In contrast, RGS3 expression suppressed by 75% the GnRH-induced IP3 responses. RGS3 was shown to bind Gq alpha protein in a model in vitro system: recombinant RGS3-glutathione-S-transferase (GST) fusion protein bound five-fold more 35S-met labeled Gq alpha protein than did with GST alone, suggesting that the mechanism of RGS3 action is attenuation of Gq alpha protein activation of phospholipase C. RGS3 mRNA and protein were observed to be expressed endogenously in the gonadotropic alpha T3-1 cell line. These results suggest a potential role for RGS3 in modulating the LH secretory responsiveness of the pituitary gonadotrope to GnRH.

A regulator of G protein signaling, RGS3, inhibits gonadotropin-releasing hormone (GnRH)-stimulated luteinizing hormone (LH) secretion.

BACKGROUND: Luteinizing hormone secreted by the anterior pituitary gland regulates gonadal function. Luteinizing hormone secretion is regulated both by alterations in gonadotrope responsiveness to hypothalamic gonadotropin releasing hormone and by alterations in gonadotropin releasing hormone secretion. The mechanisms that determine gonadotrope responsiveness are unknown but may involve regulators of G protein signaling (RGSs). These proteins act by antagonizing or abbreviating interaction of Galpha proteins with effectors such as phospholipase Cbeta. Previously, we reported that gonadotropin releasing hormone-stimulated second messenger inositol trisphosphate production was inhibited when RGS3 and gonadotropin releasing hormone receptor cDNAs were co-transfected into the COS cell line. Here, we present evidence for RGS3 inhibition of gonadotropin releasing hormone-induced luteinizing hormone secretion from cultured rat pituitary cells. RESULTS: A truncated version of RGS3 (RGS3T = RGS3 314-519) inhibited gonadotropin releasing hormone-stimulated inositol trisphosphate production more potently than did RSG3 in gonadotropin releasing hormone receptor-bearing COS cells. An RSG3/glutathione-S-transferase fusion protein bound more 35S-Gqalpha than any other member of the G protein family tested. Adenoviral-mediated RGS3 gene transfer in pituitary gonadotropes inhibited gonadotropin releasing hormone-stimulated luteinizing hormone secretion in a dose-related fashion. Adeno-RGS3 also inhibited gonadotropin releasing hormone stimulated 3H-inositol phosphate accumulation, consistent with a molecular site of action at the Gqalpha protein. CONCLUSIONS: RGS3 inhibits gonadotropin releasing hormone-stimulated second messenger production (inositol trisphosphate) as well as luteinizing hormone secretion from rat pituitary gonadotropes apparently by binding and suppressing the transduction properties of Gqalpha protein function. A version of RGS3 that is amino-terminally truncated is even more potent than intact RGS3 at inhibiting gonadotropin releasing hormone-stimulated inositol trisphosphate production.

Epitope-tagged gonadotropin-releasing hormone receptors heterologously-expressed in mammalian (COS-1) and insect (Sf9) cells.

The molecular cloning and nucleotide sequencing of the gonadotropin-releasing hormone (GnRH) receptor represented an enhanced step in the experimental effort to understand this key molecule in the reproductive process at a cell and molecular level. A subsequent step in this broad effort is heterologous expression of the receptor in model cell systems for studies of signal transduction and desensitization, processes that may require immunologic detection of the receptor. Therefore, the GnRH receptor was tagged at its N-terminus using recombinant DNA procedures with the HA-1 epitope that is bound by a monoclonal antibody (12CA5). COS-1 cells expressing this receptor bound [(125)I]D-Ala6-desGly10-GnRH ethylamide (GnRH-A) with the expected high affinity (IC(50) = 0.47 nM), and were immunocytochemically stained by the 12CA5 antibody. Signal transduction was demonstrated by GnRH-induced [(3)H]inositol phosphate accumulation in receptor-expressing COS-1 cells. Western blotting of COS-1 cell membranes expressing the receptor revealed protein bands at 67, 57, and 32 kDa. Immunoprecipitation occurred when the solubilized receptor from COS-1 cell membranes was reacted with 12CA5 antibody and anti-mouse IgG Sepharose, and the presence of the receptor demonstrated either by its binding of [(125)I]GnRH-A or by its detection on Western blots. Desensitization of inositol 1,4,5-trisphosphate (IP(3)) production by N-epitope-tagged GnRH receptor expressing COS-1 cells was evoked by a five min GnRH pretreatment; [(32)P]i labeling of such cells during desensitization followed by immunoprecipitation of the N-epitope-tagged receptor was not associated with receptor phosphorylation. Finally, the epitope tagged receptor was expressed in the high-yield baculovirus/insect Sf9 cell system: the membrane receptor bound [(125)I]GnRH-A with slightly lowered affinity (IC(50) = 1.4 nM), and in Western blots yielded protein bands of 32, 56/57, 69, and 120/140 kDa. The development and validation of these heterologous systems will permit the study of several GnRH receptor-mediated processes that are poorly understood.

Rat gonadotropin-releasing hormone (GnRH) receptor: tissue expression and hormonal regulation of its mRNA.

The binding of gonadotropin-releasing hormone (GnRH) to its receptor in the anterior pituitary gland is the key molecular interaction regulating the reproductive process of mammals. Here, we report the isolation of a cDNA representing this receptor from rat anterior pituitary and the regulation of expression of its mRNA. The rat GnRH receptor cDNA was composed of 2909 nucleotides and encoded a protein containing 327 amino acids having a seven transmembrane topology. Northern blot analysis on RNA from rat pituitary, ovary and testis showed four different transcripts (5.0, 4.5, 2.5 and 1.3 kb) of which the 5.0 kb form was most abundant. The levels of expression of the transcripts were found to be highest in the pituitary followed by the ovary and the testis (about 40% and 5% compared to pituitary, respectively). Using the more sensitive reverse transcriptase/PCR technique, we also detected GnRH receptor mRNA in the adrenal and the hypothalamus. Measurement of pituitary GnRH receptor mRNA levels (the 5.0 kb form) during the estrous cycle showed the lowest levels at estrus (1.0-fold), a 2.2 +/- 0.57 (mean +/- SEM) -fold increase at diestrus I, a 3.5 +/- 0.41-fold increase at diestrus II, a 2.6 +/- 0.34-fold increase on the morning of proestrus, and a 1.9 +/- 0.25-fold on the afternoon of proestrus. Removal of the ovaries led to a 2.7 +/- 0.29-fold increase in GnRH receptor mRNA levels in the pituitary gland; treatment of ovariectomized rats with estrogen resulted in a significant decrease in GnRH receptor mRNA levels. Our studies demonstrate ovarian regulation of GnRH receptor mRNA expression in the anterior pituitary gland.

Dual intracellular pathways in gonadotropin releasing hormone (GNRH) induced desensitization of luteinizing hormone (LH) secretion.

The mechanisms of GnRH-induced desensitization of LH secretion are poorly understood. Protein kinase C (PKC) and protein kinase A (PKA) desensitize some receptors of the 7-membrane type, and the GnRH receptor has consensus phosphorylation sites for PKC in the first and third intracellular loops, and a site for PKA in the first intracellular loop. In the first set of experiments we determined whether synthetic peptides representing the three intracellular loops of the receptor could be phosphorylated in vitro by purified PKC and PKA. As compared with a model substrate peptide for PKC, the third intracellular loop was phosphorylated 74% and the first intracellular loop 21%; PKA-phosphorylated the first intracellular loop peptide 17% as well as a model peptide substrate. In the second set of experiments, we used phorbol 12-myristate 13 acetate (PMA), an established PKC stimulator, and cholera toxin (CTX), established to activate the Gs protein and presumed to activate PKA, to treat cultured rat pituitary cells followed by LH measurements. Treatment with both drugs severely impaired GnRH-stimulated LH secretion whereas neither drug alone reduced LH secretion. Dibutyryl cAMP did not duplicate the effects of cholera toxin suggesting that the CTX action could not be explained by an increase in cAMP. These results suggest that more than one intracellular signaling pathway requires activation in order to induce desensitization; one pathway involves PKC and the other involves a pathway stimulated by cholera toxin, presumably Gs protein, which does not involve PKA.

Mutation of Escherichia coli cytosine deaminase significantly enhances molecular chemotherapy of human glioma.

Combined treatment using adenoviral (Ad)-directed enzyme/prodrug therapy and radiation therapy has the potential to become a powerful method of cancer therapy. We have developed an Ad vector encoding a mutant bacterial cytosine deaminase (bCD) gene (AdbCD-D314A), which has a higher affinity for cytosine than wild-type bCD (bCDwt). The purpose of this study was to evaluate cytotoxicity in vitro and therapeutic efficacy in vivo of the combination of AdbCD-D314A with the prodrug 5-fluorocytosine (5-FC) and ionizing radiation against human glioma. The present study demonstrates that AdbCD-D314A infection resulted in increased 5-FC-mediated cell killing, compared with AdbCDwt. Furthermore, a significant increase in cytotoxicity following AdbCD-D314A and radiation treatment of glioma cells in vitro was demonstrated as compared to AdbCDwt. Animal studies showed significant inhibition of subcutaneous or intracranial tumor growth of D54MG glioma xenografts by the combination of AdbCD-D314A/5-FC with ionizing radiation as compared with either agent alone, and with AdbCDwt/5-FC plus radiation. The results suggest that the combination of AdbCD-D314A/5-FC with radiation produces markedly increased cytotoxic effects in cancer cells in vitro and in vivo. These data indicate that combined treatment with this novel mutant enzyme/prodrug therapy and radiotherapy provides a promising approach for cancer therapy.

A gonadotropin-releasing hormone (GnRH) receptor specific for GnRH II in primates.

Mammalian gonadotropin-releasing hormone (GnRH I) is a hypothalamic decapeptide that stimulates gonadotropic hormone secretion upon interaction with its membrane receptors (type I) on pituitary cells, thereby governing reproductive processes. A second releasing hormone (GnRH II) expressed in mammals was shown earlier to be expressed in nonmammals and to have its own receptor. Here we demonstrate that a second receptor (type II) gene is present in the human genome, and report the cloning and characterization of its cDNA from monkeys. The cDNA encodes a G-protein-coupled/7 transmembrane receptor having a C-terminal cytoplasmic tail; it resembles more closely the type II receptors of amphibians and fish (approximately 55% identity) than it does the type I receptor of humans (approximately 39%). The GnRH type II receptor proved to be experimentally functional with, and specific for, GnRH II. GnRH receptor type II RNA is expressed ubiquitously in human tissues. This is the first report of a GnRH type II receptor in mammals. Its identification will permit exploration of its role in regulating gonadotropin secretion, female sexual behavior, and tumor cell growth.

Angiotensin II type-1 receptor subtype cDNAs: differential tissue expression and hormonal regulation.

A rat angiotensin, type 1A (AT1A) receptor cDNA was cloned recently and shown to be a member of the 7-transmembrane, G-protein coupled family of receptors. Here, we report the cloning, sequencing, and expression of a previously unsuspected second form of the type 1 receptor (AT1B) in the rat which exhibits high similarity with the AT1A receptor relative to amino acid sequence (95% identity), binding of angiotensin II analogs, and utilization of Ca+2 as its intracellular second messenger. The adrenal and pituitary gland express primarily AT1B mRNA whereas vascular smooth muscle and lung express primarily AT1A mRNA. Estrogen treatment suppressed AT1B but not AT1A mRNA levels in the pituitary gland. Thus, the unexpected existence of two putative AT1 receptor genes appears to be related to the differential regulation of their expression rather than to different functional properties of the encoded receptor proteins.

Cloning, sequencing, and expression of human gonadotropin releasing hormone (GnRH) receptor.

Gonadotropin releasing hormone is a hypothalamic decapeptide that stimulates the release of gonadotropic hormones from the anterior pituitary gland. Therapeutically, the human pituitary GnRH receptor is the target of agonists used in the suppression of prostate cancer. Here we report the isolation of a cDNA representing this receptor. It encodes a protein with a transmembrane topology similar with that of other G protein-coupled, 7-transmembrane receptors. Binding studies of the cloned receptor demonstrate high affinity and pharmacological properties similar with the native human pituitary GnRH receptor. Northern blot and reverse transcriptase/PCR analysis revealed that its mRNA is expressed in pituitary, ovary, testis, breast, and prostate but not in liver and spleen. Availability of a human GnRH receptor cDNA should permit the design of improved analogs for therapeutic applications.