Calcium Influx through L-type Channels Is Required for Selective Activation of Extracellular Signal-regulated Kinase by Gonadotropin-releasing Hormone

Self Cite

134

105

Specialized membrane microdomains known as lipid rafts are thought to contribute to G-protein coupled receptor (GPCR) signaling by organizing receptors and their cognate signaling molecules into discrete membrane domains. To determine if the GnRHR, an unusual member of the GPCR superfamily, partitions into lipid rafts, homogenates of ␣T3-1 cells expressing endogenous GnRHR or Chinese hamster ovary cells expressing an epitope-tagged GnRHR were fractionated through a sucrose gradient. We found the GnRHR and c-raf kinase constitutively localized to low density fractions independent of hormone treatment. Partitioning of c-raf kinase into lipid rafts was also observed in whole mouse pituitary glands. Consistent with GnRH induced phosphorylation and activation of c-raf kinase, GnRH treatment led to a decrease in the apparent electrophoretic mobility of c-raf kinase that partitioned into lipid rafts compared with unstimulated cells. Cholesterol depletion of ␣T3-1 cells using methyl-␤-cyclodextrin disrupted GnRHR but not c-raf kinase association with rafts and shifted the receptor into higher density fractions. Cholesterol depletion also significantly attenuated GnRH but not phorbol ester-mediated activation of extracellular signal-related kinase (ERK) and c-fos gene induction. Raft localization and GnRHR signaling to ERK and c-Fos were rescued upon repletion of membrane cholesterol. Thus, the organization of the GnRHR into low density membrane microdomains appears critical in mediating GnRH induced intracellular signaling.

Section: Cholesterol Depletion Uncouples Gnrhr-but Not Phorbolsupporting

confidence: 78%

Section: Gnrh Activation Of Erk and C-fos Expression Is Lost Withsupporting

confidence: 70%

Constitutive Localization of the Gonadotropin-releasing Hormone (GnRH) Receptor to Low Density Membrane Microdomains Is Necessary for GnRH Signaling to ERK

Navratil

Bliss

Berghorn

et al. 2003

Self Cite

134

105

“…The small G-protein gem (kir) has recently been reported to down-regulate L-type calcium channel function (24). The induction of gem by GnRH could contribute to the modulation of GnRH-induced gonadotropin release and MAP kinase activation, which both depend on L-channel activation (25). IB inhibits NFB signaling.…”

Section: Confirmation Of Gene Regulation By Real-time Pcr-mentioning

confidence: 99%

Gonadotropin-releasing Hormone Receptor-coupled Gene Network Organization

Wurmbach¹,

Yuen²,

Ebersole³

et al. 2001

169

146

An early gene cDNA microarray was developed to study genes that are regulated immediately following gonadotropin-releasing hormone (GnRH) receptor activation. 956 selected candidate genes were printed in triplicate, a t statistic-based regulation algorithm was used for data analysis, and the response to GnRH in a time course from 1 to 6 h was determined. Measurements were highly reproducible within arrays, between arrays, and between experiments. Accuracy and algorithm reliability were established by real-time polymerase chain reaction assays of 60 genes. Gene changes ranging from 1.3-to 31-fold on the microarray were confirmed by real-time polymerase chain reaction. Many of the genes were found to be highly regulated. The regulated genes identified were all elevated at 1 h of treatment and returned nearly or completely to baseline levels of expression by 3 h of treatment. This broad, robust, and transient transcriptional response to constant GnRH exposure includes modulators of signal transduction (e.g. Rgs2 and IB), cytoskeletal proteins (e.g. ␥-actin), and transcription factors (e.g. c-Fos, Egr1, and LRG21). The interplay of the activators, repressors, and feedback inhibitors identified embodies a combinatorial code to direct the activity of specific downstream secondary genes.

“…The major signal transduction pathways in cells expressing GnRH receptors are initiated by activation of phospholipase C. The consequent calcium (Ca 2ϩ ) mobilization and activation of protein kinase C (PKC) by GnRH are key elements in the hypothalamic control of gonadotropin secretion from the anterior pituitary gland (1,3,5). Activation of PKC and Ca 2ϩ mobilization during GnRH receptor stimulation are also responsible for mediating downstream signals leading to activation of extracellularly regulated mitogen-activated protein kinases (ERK1/2 MAPKs) that transmit signals from the cell surface to the nucleus to regulate transcriptional and other processes (7)(8)(9)(10)(11)(12)(13). However, the specific PKC isoforms that are involved in GnRH-induced ERK1/2 activation in GT1-7 cells are not known.…”

mentioning

confidence: 99%

Dependence of Gonadotropin-releasing Hormone-induced Neuronal MAPK Signaling on Epidermal Growth Factor Receptor Transactivation

Shah¹,

Soh²,

Catt³

2003

The hypothalamic decapeptide, gonadotropin-releasing hormone (GnRH), utilizes multiple signaling pathways to activate extracellularly regulated mitogen-activated protein kinases (ERK1/2) in normal and immortalized pituitary gonadotrophs and transfected cells expressing the GnRH receptor. In immortalized hypothalamic GnRH neurons (GT1-7 cells), which also express GnRH receptors, GnRH, epidermal growth factor (EGF), and phorbol 12-myristate 13-acetate (PMA) caused marked phosphorylation of ERK1/2. This action of GnRH and PMA, but not that of EGF, was primarily dependent on activation of protein kinase C (PKC), and the ERK1/2 responses to all three agents were abolished by the selective EGF receptor kinase inhibitor, AG1478. Consistent with this, both GnRH and EGF increased tyrosine phosphorylation of the EGF receptor. GnRH and PMA, but not EGF, caused rapid phosphorylation of the proline-rich tyrosine kinase, Pyk2, at Tyr 402 . This was reduced by Ca 2؉ chelation and inhibition of PKC, but not by AG1478. GnRH stimulation caused translocation of PKC␣ and -⑀ to the cell membrane and enhanced the association of Src with PKC␣ and PKC⑀, Pyk2, and the EGF receptor. The Src inhibitor, PP2, the C-terminal Src kinase (Csk), and dominant-negative Pyk2 attenuated ERK1/2 activation by GnRH and PMA but not by EGF. These findings indicate that Src and Pyk2 act upstream of the EGF receptor to mediate its transactivation, which is essential for GnRH-induced ERK1/2 phosphorylation in hypothalamic GnRH neurons.