Rat gonadotropin-releasing hormone receptor expressed in insect cells induces activation of adenylyl cyclase

Delahaye, Richard; Manna, Pulak R.; Bérault, Annette; Berreur-Bonnenfant, J.; Berreur, P.; Counis, Raymond

doi:10.1016/s0303-7207(97)00194-9

Cited by 25 publications

(6 citation statements)

References 46 publications

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“…Although our findings are consistent with other studies on gonadotropes (51, 52), they differ from results with the recombinant rat GnRH receptor heterologously expressed in Sf9 insect cells (53) or in COS-7 cells (54). It is noteworthy, however, that in transfected somatotropic GH 3 (12,29) as well as in COS-7 cells (54) GnRH-initiated cAMP formation increases over at least 4 orders of magnitude, whereas in the latter cell model, agonist-induced IP formation is accomplished within 2 log orders of GnRH concentrations.…”

Section: Discussionsupporting

confidence: 79%

Gonadotropin-releasing Hormone Receptor Initiates Multiple Signaling Pathways by Exclusively Coupling to Gq/11Proteins

Grosse

Schmid

Schöneberg

et al. 2000

Journal of Biological Chemistry

146

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The agonist-bound gonadotropin-releasing hormone (GnRH) receptor engages several distinct signaling cascades, and it has recently been proposed that coupling of a single type of receptor to multiple G proteins (G q , G s , and G i ) is responsible for this behavior. GnRH-dependent signaling was studied in gonadotropic ␣T3-1 cells endogenously expressing the murine receptor and in CHO-K1 (CHO#3) and COS-7 cells transfected with the human GnRH receptor cDNA. In all cell systems studied, GnRH-induced phospholipase C activation and Ca 2؉ mobilization was pertussis toxin-insensitive, as was GnRH-mediated extracellular signal-regulated kinase activation. Whereas the G i -coupled m2 muscarinic receptor interacted with a chimeric G s protein (G s i5) containing the C-terminal five amino acids of G␣ i2 , the human GnRH receptor was unable to activate the G protein chimera. GnRH challenge of ␣T3-1, CHO#3 and of GnRH receptor-expressing COS-7 cells did not result in agonist-dependent cAMP formation. GnRH challenge of CHO#3 cells expressing a cAMP-responsive elementdriven firefly luciferase did not result in increased reporter gene expression. However, coexpression of the human GnRH receptor and adenylyl cyclase I in COS-7 cells led to clearly discernible GnRH-dependent cAMP formation subsequent to GnRH-elicited rises in [Ca 2؉ ] i . In ␣T3-1 and CHO#3 cell membranes, addition of [␣-32 P]GTP azidoanilide resulted in GnRH receptor-dependent labeling of G␣ q/11 but not of G␣ i , G␣ s or G␣ 12/13 proteins. Thus, the murine and human GnRH receptors exclusively couple to G proteins of the G q/11 family. Multiple GnRH-dependent signaling pathways are therefore initiated downstream of the receptor/G protein interface and are not indicative of a multiple G protein coupling potential of the GnRH receptor.

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Section: Discussionsupporting

confidence: 79%

Gonadotropin-releasing Hormone Receptor Initiates Multiple Signaling Pathways by Exclusively Coupling to Gq/11Proteins

Grosse

Schmid

Schöneberg

et al. 2000

Journal of Biological Chemistry

146

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“…The possibility that GnRH could signal through the cAMP pathway has been the object of a huge controversy. The most recent data, however, suggest that the GnRH receptor has the potentiality to couple with a G␣s (40,41) or may indirectly increase cAMP production via the Ca 2ϩ /calmodulin kinasemediated activation of adenylate cyclase I (42). Recent studies from our laboratory indicate that both GnRH and CTX are capable of inducing NOS I in primary cultures of normal rat pituitary cells.…”

Section: Discussionmentioning

confidence: 99%

Isolation and Characterization of a Rat Nitric Oxide Synthase Type I Gene Promoter that Confers Expression and Regulation in Pituitary Gonadotrope Cells

Bachir¹,

Laverrière²,

Counis³

2001

Endocrinology

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Nitric oxide synthase type I (NOS I) is expressed and up-regulated in rat pituitary gonadotrophs. Using rapid amplification of cDNA ends-PCR, 2 major transcripts with 5' ends corresponding to exon 1a but truncated of its first 369 or 384 nucleotides, indicative of two pituitary-specific transcription start sites, were identified. By chromosome walking, we isolated 5'-upstream of this truncated exon termed 1p, a novel -1653/+384-bp genomic region. Transient transfections, using the gonadotrope-derived alpha T3-1 and L beta T2 cell lines and the full-length or 5'-deleted sequences fused to a luciferase reporter gene, demonstrated that cell-specific positive and negative regions were present especially within the -246/-73 region, whereas the +12/+384 region was crucial for transcription. Moreover, in L beta T2 cells, the luciferase activity was increased by GnRH, with the full-length sequence being the most efficient and the -73/+60 region corresponding to the essential zone. The latter region was also crucial for cholera toxin-induced activation. Interestingly, GnRH and cAMP effects were not additive, implying a convergent step in the transduction cascade. These data provide evidence for the presence of several elements controlling NOS I expression in gonadotrophs and demonstrate that GnRH, the prime regulator of gonadotrope function, and cAMP may induce the transcription of NOS I in these cells.

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“…Thus, both agonists and antagonists ultimately reduce the circulating levels of gonadotropins and gonadal steroids. In heterologous expression systems, GnRH has been shown to stimulate cAMP accumulation via GnRH receptors (GnRH-Rs) expressed stably in GGH 3 cells (10) or transiently in COS-7 (11) and Sf9 cells (12). At the pituitary, GnRH acts via G protein-coupled receptors (GPCRs) that act via G q/11 to stimulate PLC, thereby causing an IP 3 -mediated mobilization of Ca 2ϩ from intracellular stores.…”

mentioning

confidence: 99%

Signaling and Antiproliferative Effects Mediated by GnRH Receptors After Expression in Breast Cancer Cells Using Recombinant Adenovirus

Everest¹,

Hislop²,

Harding³

et al. 2001

Endocrinology

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GnRH receptors (GnRH-Rs) are found in human cancers, including those of the breast, and GnRH can inhibit the growth of cell lines derived from such cancers. Although pituitary and extrapituitary GnRH-R transcripts appear identical, their functional characteristics may differ. Most extrapituitary GnRH-Rs have low affinity for GnRH analogs and may not activate PLC or discriminate between agonists and antagonists in the same way as pituitary GnRH-Rs. Here we have assessed whether GnRH-Rs expressed exogenously in breast cancer cells differ from those in gonadotropes. We found no evidence for endogenous GnRH-Rs in MCF7 cells, but after infection with adenovirus expressing the GnRH-R (Ad GnRH-R) at a multiplicity of infection of 10 or greater, at least 80% expressed GnRH-Rs. These had high affinity (K(d) for [(125)I]buserelin, 1.4 nM) and specificity (rank order of potency, buserelin>GnRH>>chicken GnRH-II) and mediated stimulation of [(3)H]IP accumulation. Increasing viral titer [from multiplicity of infection, 3-300] increased receptor number (10,000-225,000 sites/cell) and [(3)H]IP responses. GnRH stimulated ERK2 phosphorylation in Ad GnRH-R-infected cells, and this effect, like stimulation of [(3)H]IP accumulation, was blocked by GnRH-R antagonists. GnRH also inhibited [(3)H]thymidine incorporation into Ad GnRH-R-infected cells (but not control cells). This effect was mimicked by agonist analogs and inhibited by two antagonists. Thus, when exogenous GnRH-Rs are expressed at density comparable to that in gonadotropes, they are functionally indistinguishable from the endogenous GnRH-Rs in gonadotropes, and increasing expression of high affinity GnRH-Rs can dramatically enhance the direct antiproliferative effect of GnRH agonists on breast cancer cells.

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Rat gonadotropin-releasing hormone receptor expressed in insect cells induces activation of adenylyl cyclase

Cited by 25 publications

References 46 publications

Gonadotropin-releasing Hormone Receptor Initiates Multiple Signaling Pathways by Exclusively Coupling to Gq/11Proteins

Gonadotropin-releasing Hormone Receptor Initiates Multiple Signaling Pathways by Exclusively Coupling to Gq/11Proteins

Isolation and Characterization of a Rat Nitric Oxide Synthase Type I Gene Promoter that Confers Expression and Regulation in Pituitary Gonadotrope Cells

Signaling and Antiproliferative Effects Mediated by GnRH Receptors After Expression in Breast Cancer Cells Using Recombinant Adenovirus

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