Constitutive localization of the gonadotropin-releasing hormone (GnRH) receptor to low density membrane microdomains is necessary for GnRH signaling to ERK

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 alpha 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 alpha T3-1 cells using methyl-beta-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.     

Arrestin-mediated ERK activation by gonadotropin-releasing hormone receptors: receptor-specific activation mechanisms and compartmentalization

Activation of seven-transmembrane region receptors typically causes their phosphorylation with consequent arrestin binding and desensitization. Arrestins also act as scaffolds, mediating signaling to Raf and ERK and, for some receptors, inhibiting nuclear translocation of ERK. GnRH receptors (GnRHRs) act via Gq/11 to stimulate the phospholipase C/Ca2+/protein kinase C (PKC) cascade and the Raf/MEK/ERK cassette. Uniquely, type I mammalian GnRHRs lack the C-tails that are found in other seven-transmembrane region receptors (including nonmammalian GnRHRs) and are implicated in arrestin binding. Here we have compared ERK signaling by human GnRHRs (hGnRHRs) and Xenopus GnRHRs (XGnRHRs). In HeLa cells, XGnRHRs underwent rapid and arrestin-dependent internalization and caused arrestin/green fluorescent protein (GFP) translocation to the membrane and endosomes, whereas hGnRHRs did not. Internalized XGnRHRs were co-localized with arrestin-GFP, whereas hGnRHRs were not. Both receptors mediated transient ERK phosphorylation and nuclear translocation (revealed by immunohistochemistry or by imaging of co-transfected ERK2-GFP), and for both, ERK phosphorylation was reduced by PKC inhibition but not by inhibiting epidermal growth factor receptor autophosphorylation. In the presence of PKC inhibitor, Deltaarrestin-(319-418) blocked XGnRHR-mediated, but not hGnRHR-mediated, ERK phosphorylation. When receptor number was varied, hGnRHRs activated phospholipase C and ERK more efficiently than XGnRHRs but were less efficient at causing ERK2-GFP translocation. At high receptor number, XGnRHRs and hGnRHRs both caused ERK2-GFP translocation to the nucleus, but at low receptor number, XGnRHRs caused ERK2-GFP translocation, whereas hGnRHRs did not. Thus, experiments with XGnRHRs have revealed the first direct evidence of arrestin-mediated (probably G protein-independent) GnRHR signaling, whereas those with hGnRHRs imply that scaffolds other than arrestins can determine GnRHR effects on ERK compartmentalization.     

Signaling complexes associated with the type I gonadotropin-releasing hormone (GnRH) receptor: colocalization of extracellularly regulated kinase 2 and GnRH receptor within membrane rafts

Our previous work demonstrated that the type I GnRH receptor (GnRHR) resides exclusively and constitutively within membrane rafts in alphaT3-1 gonadotropes and that this association was necessary for the ability of the receptor to couple to the ERK signaling pathway. G(alphaq), c-raf, and calmodulin have also been shown to reside in this compartment, implicating a raft-associated multiprotein signaling complex as a functional link between the GnRHR and ERK signaling. In the studies reported here, we used subcellular fractionation and coimmunoprecipitation to analyze the behavior of ERKs with respect to this putative signaling platform. ERK 2 associated partially and constitutively with low-density membranes both in alphaT3-1 cells and in whole mouse pituitary. Cholesterol depletion of alphaT3-1 cells reversibly blocked the association of both the GnRHR and ERKs with low-density membranes and uncoupled the ability of GnRH to activate ERK. Analysis of the kinetics of recovery of ERK inducibility after cholesterol normalization supported the conclusion that reestablishment of the association of the GnRHR and ERKs with the membrane raft compartment was not sufficient for reconstitution of signaling activity. In alphaT3-1 cells, the GnRHR and ERK2 coimmunoprecipitated from low-density membrane fractions prepared either in the presence or absence of detergent. The GnRHR also partitioned into low-density, detergent-resistant membrane fractions in mouse pituitary and coimmunoprecipitated with ERK2 from these fractions. Collectively, these data support a model in which coupling of the GnRHR to the ERK pathway in gonadotropes involves the assembly of a multiprotein signaling complex in association with specialized microdomains of the plasma membrane.     

SET Protein Interacts with Intracellular Domains of the Gonadotropin-releasing Hormone Receptor and Differentially Regulates Receptor Signaling to cAMP and Calcium in Gonadotrope Cells

In mammals, the receptor of the neuropeptide gonadotropin-releasing hormone (GnRHR) is unique among the G protein-coupled receptor (GPCR) family because it lacks the carboxyl-terminal tail involved in GPCR desensitization. Therefore, mechanisms involved in the regulation of GnRHR signaling are currently poorly known. Here, using immunoprecipitation and GST pull-down experiments, we demonstrated that SET interacts with GnRHR and targets the first and third intracellular loops. We delineated, by site-directed mutagenesis, SET binding sites to the basic amino acids ⁶⁶KRKK⁶⁹ and ²⁴⁶RK²⁴⁷, located next to sequences required for receptor signaling. The impact of SET on GnRHR signaling was assessed by decreasing endogenous expression of SET with siRNA in gonadotrope cells. Using cAMP and calcium biosensors in gonadotrope living cells, we showed that SET knockdown specifically decreases GnRHR-mediated mobilization of intracellular cAMP, whereas it increases its intracellular calcium signaling. This suggests that SET influences signal transfer between GnRHR and G proteins to enhance GnRHR signaling to cAMP. Accordingly, complexing endogenous SET by introduction of the first intracellular loop of GnRHR in αT3-1 cells significantly reduced GnRHR activation of the cAMP pathway. Furthermore, decreasing SET expression prevented cAMP-mediated GnRH stimulation of Gnrhr promoter activity, highlighting a role of SET in gonadotropin-releasing hormone regulation of gene expression. In conclusion, we identified SET as the first direct interacting partner of mammalian GnRHR and showed that SET contributes to a switch of GnRHR signaling toward the cAMP pathway.     

Pulsatile and Sustained Gonadotropin-releasing Hormone (GnRH) Receptor Signaling: DOES THE ERK SIGNALING PATHWAY DECODE GnRH PULSE FREQUENCY?*

Gonadotropin-releasing hormone (GnRH) acts via G-protein-coupled receptors on gonadotrophs to stimulate synthesis and secretion of luteinizing hormone and follicle-stimulating hormone. It is secreted in pulses, and its effects depend on pulse frequency, but decoding mechanisms are unknown. Here we have used an extracellular signal regulated kinase-green fluorescent protein (ERK2-GFP) reporter to monitor GnRH signaling. GnRH caused dose-dependent ERK2-GFP translocation to the nucleus, providing a live-cell readout for activation. Pulsatile GnRH caused dose- and frequency-dependent ERK2-GFP translocation. These responses were rapid and transient, showed only digital tracking, and did not desensitize under any condition tested (dose, frequency, and receptor number varied). We also tested for the effects of cycloheximide (to prevent induction of nuclear-inducible MAPK phosphatases) and used GFP fusions containing ERK mutations (D319N, which prevents docking domain-dependent binding to MAPK phosphatases, and K52R, which prevents catalytic activity). These manipulations had little or no effect on the translocation responses, arguing against a role for MAPK phosphatases or ERK-mediated feedback in shaping ERK activation during pulsatile stimulation. GnRH also caused dose- and frequency-dependent activation of the α-gonadotropin subunit-, luteinizing hormone β-, and follicle-stimulating hormone β- luciferase reporters, and the latter response was inhibited by ERK1/2 knockdown. Moreover, GnRH caused frequency-dependent activation of an Egr1-luciferase reporter, but the response was proportional to cumulative pulse duration. Our data suggest that frequency decoding is not due to negative feedback shaping ERK signaling in this model.     

Identification of Ser153 in ICL2 of the gonadotropin-releasing hormone (GnRH) receptor as a phosphorylation-independent site for inhibition of Gq coupling

Type I gonadotropin-releasing hormone (GnRH) receptor (GnRHR) is unique among mammalian G-protein-coupled receptors (GPCRs) in lacking a C-terminal tail, which is involved in desensitization in GPCRs. Therefore, we searched for inhibitory sites in the intracellular loops (ICLs) of the GnRHR. Synthetic peptides corresponding to the three ICLs were inserted into permeabilized alphaT3-1 gonadotrope cells, and GnRH-induced inositol phosphate (InsP) formation was determined. GnRH-induced InsP production was potentiated by ICL2 > ICL3 but not by the ICL1 peptides, suggesting they are acting as decoy peptides. We examined the effects of six peptides in which only one of the Ser or Thr residues was substituted with Ala or Glu. Only substitution of Ser153 with Ala or Glu ablated the potentiating effect upon GnRH-induced InsP elevation. ERK activation was enhanced, and the rate of GnRH-induced InsP formation was about 6.5-fold higher in the first 10 min in COS-1 cells that were transfected with mutants of the GnRHR in which the ICL2 Ser/Thr residues (Ser151, Ser153, and Thr142) or only Ser153 was mutated to Ala as compared with the wild type GnRHR. The data indicate that ICL2 harbors an inhibitory domain, such that exogenous ICL2 peptide serves as a decoy for the inhibitory site (Ser153) of the GnRHR, thus enabling further activation. GnRH does not induce receptor phosphorylation in alphaT3-1 cells. Because the phosphomimetic ICL2-S153E peptide did not mimic the stimulatory effect of the ICL2 peptide, the inhibitory effect of Ser153 operates through a phosphorylation-independent mechanism.     

Chimeric GnRH-LH receptors and LH receptors lacking C-terminus palmitoylation sites do not localize to plasma membrane rafts

Luteinizing hormone and gonadotropin releasing hormone receptors (LHR and GnRHR, respectively) are G protein-coupled receptors with important functions in reproduction. We have developed chimeric GnRHR-LHR that contain the full GnRHR coupled to various forms of the LH receptor C-terminus to explore the role of the LH receptor C-terminus in raft localization of the receptor and signaling. Addition of the full-length LHR C-terminus to GnRHR resulted in localization of the resting chimeric receptor in the bulk membrane rather than plasma membrane rafts as has been reported for the wild-type GnRHR [A. Navratil, S. Bliss, K. Berghorn, J. Haughian, T. Farmerie, J. Graham, C. Clay, M. Roberson, Constitutive localization of the gonadotropin-releasing hormone (GnRH) receptor to low density membrane microdomains is necessary for GnRH signaling to ERK, J. Biol. Chem. 278 (2003) 31593-31602]. With truncation of the LHR C-terminus, approximately 3% of chimeric receptors appeared in low density membrane fractions. Palmitoylation of sites on the LHR C-terminus appears important for raft localization. Mutations to C-terminus palmitoylation sites eliminated translocation of LH receptors from the bulk membrane to rafts upon binding of hCG although these mutant receptors retained the ability to signal via cAMP.     

Differential desensitization and internalization of three different bullfrog gonadotropin-releasing hormone receptors

We previously demonstrated the presence of three distinct types of the gonadotropin-releasing hormone receptor (GnRHR) in a bullfrog (denoted bfGnRHR-1, bfGnRHR-2, and bfGnRHR-3). The bfGnRHRs exhibited differential tissue distribution and ligand selectivity. In the present study, we demonstrated the desensitization and internalization kinetics of these receptors in both transiently-transfected HEK293 cells and retrovirus-mediated stable cells. The time-course accumulation of the inositol phosphate in response to GnRH revealed that bfGnRHR-1 and -2 were rapidly desensitized, whereas bfGnRHR-3 was slowly desensitized. A comparison of the internalization kinetics revealed the most rapid rate and highest extent of internalization of bfGnRHR-2 among the three receptors. Interestingly, the mechanisms that underlie the receptor internalization appear to differ from each other. Internalization of bfGnRHR-1 was dependent on both dynamin and beta-arrestin, whereas those of bfGnRHR-2 and -3 were dependent on dynamin, but not on arrestin. These results, therefore, suggest that differential regulatory mechanisms for desensitization and internalization of the GnRHR are involved in diverse cellular and physiological responses to GnRH stimulation.     

Cytoskeletal reorganization dependence of signaling by the gonadotropin-releasing hormone receptor

Activation of classical G protein-coupled receptors (GPCRs) like the mammalian gonadotropin-releasing hormone receptor (GnRHR) typically stimulates heterotrimeric G protein molecules that subsequently activate downstream effectors. Receptor activation of heterotrimeric G protein pathways primarily controls intermediary cell metabolism by elevation or diminution of soluble cytoplasmic second messenger molecules. We have demonstrated here that stimulation of the GnRHR also results in a dramatic change in both cell adhesion and superstructural morphology. Gonadotropin-releasing hormone (GnRH) receptor activation rapidly increases the capacity of HEK293 cells expressing the GnRHR to remain matrix-adherent in the face of fluid insults. Coinciding with this profound elevation in matrix adherence, we demonstrated a GnRH-induced alteration in both cell morphology and the de novo generation of polymerized actin structures. GnRH induction of cytoskeletal remodeling was correlated with significant increases in the tyrosine phosphorylation status of a series of cytoskeletal associated proteins, e.g. focal adhesion kinase (FAK), c-Src, and microtubule-associated protein kinase (MAPK or ERK1/2). The activation of the distal downstream effector ERK1/2 was demonstrated to be sensitive to the disrupters of cytoskeletal rearrangement, cytochalasin D and latrunculin B. In addition to the sensitivity of ERKs to cytoskeletal integrity, GnRH-induced FAK and c-Src kinase activation were sensitive to these agents and the fibronectin-integrin antagonistic RGDS peptide. Activation of ERK was dependent on its protein-protein assembly with FAK and c-Src at focal adhesion complexes. Induction of the cell remodeling event leading to this signaling complex assembly occurred primarily via GnRHR activation of the monomeric G protein Rac but not RhoA. These findings demonstrated a clear divergence of GnRHR signaling via the Rac monomeric G protein focal adhesion signaling complex assembly and cytoskeletal remodeling independent of the classical heterotrimeric G protein-controlled phospholipase C-beta pathway.     

Systematic Quantification of Negative Feedback Mechanisms in the Extracellular Signal-regulated Kinase (ERK) Signaling Network

Cell responses are actuated by tightly controlled signal transduction pathways. Although the concept of an integrated signaling network replete with interpathway cross-talk and feedback regulation is broadly appreciated, kinetic data of the type needed to characterize such interactions in conjunction with mathematical models are lacking. In mammalian cells, the Ras/ERK pathway controls cell proliferation and other responses stimulated by growth factors, and several cross-talk and feedback mechanisms affecting its activation have been identified. In this work, we take a systematic approach to parse the magnitudes of multiple regulatory mechanisms that attenuate ERK activation through canonical (Ras-dependent) and non-canonical (PI3K-dependent) pathways. In addition to regulation of receptor and ligand levels, we consider three layers of ERK-dependent feedback: desensitization of Ras activation, negative regulation of MEK kinase (e.g. Raf) activities, and up-regulation of dual-specificity ERK phosphatases. Our results establish the second of these as the dominant mode of ERK self-regulation in mouse fibroblasts. We further demonstrate that kinetic models of signaling networks, trained on a sufficient diversity of quantitative data, can be reasonably comprehensive, accurate, and predictive in the dynamical sense.     

Involvement of Protein Kinase D1 in Signal Transduction from the Protein Kinase C Pathway to the Tyrosine Kinase Pathway in Response to Gonadotropin-releasing Hormone

The receptor for gonadotropin-releasing hormone (GnRH) belongs to the G protein-coupled receptors (GPCRs), and its stimulation activates extracellular signal-regulated protein kinase (ERK). We found that the transactivation of ErbB4 was involved in GnRH-induced ERK activation in immortalized GnRH neurons (GT1–7 cells). We found also that GnRH induced the cleavage of ErbB4. In the present study, we examined signal transduction for the activation of ERK and the cleavage of ErbB4 after GnRH treatment. Both ERK activation and ErbB4 cleavage were completely inhibited by YM-254890, an inhibitor of G_q/11 proteins. Down-regulation of protein kinase C (PKC) markedly decreased both ERK activation and ErbB4 cleavage. Experiments with two types of PKC inhibitors, Gö 6976 and bisindolylmaleimide I, indicated that novel PKC isoforms but not conventional PKC isoforms were involved in ERK activation and ErbB4 cleavage. Our experiments indicated that the novel PKC isoforms activated protein kinase D (PKD) after GnRH treatment. Knockdown and inhibitor experiments suggested that PKD1 stimulated the phosphorylation of Pyk2 by constitutively activated Src and Fyn for ERK activation. Taken together, it is highly possible that PKD1 plays a critical role in signal transduction from the PKC pathway to the tyrosine kinase pathway. Activation of the tyrosine kinase pathway may be involved in the progression of cancer.     

Differential regulation of metabotropic glutamate receptor 5-mediated phosphoinositide hydrolysis and extracellular signal-regulated kinase responses by protein kinase C in cultured astrocytes

The metabotropic glutamate receptor 5 (mGluR5) exhibits a rapid loss of receptor responsiveness to prolonged or repeated agonist exposure. This receptor desensitization has been seen in a variety of native and recombinant systems, and is thought to result from receptor-mediated, protein kinase C (PKC)-dependent phosphorylation of the receptor, uncoupling it from the G protein in a negative feedback regulation. We have investigated the rapid PKC-mediated desensitization of mGluR5 in cortical cultured astrocytes by measuring downstream signals from activation of mGluR5. These include activation of phosphoinositide (PI) hydrolysis, intracellular calcium transients, and extracellular signal-regulated kinase 2 (ERK2) phosphorylation. We present evidence that PKC plays an important role in rapid desensitization of PI hydrolysis and calcium signaling, but not in ERK2 phosphorylation. This differential regulation of mGluR5-mediated responses suggests divergent signaling and regulatory pathways which may be important mechanisms for dynamic integration of signal cascades.     

Receptor Level Mechanisms Are Required for Epidermal Growth Factor (EGF)-stimulated Extracellular Signal-regulated Kinase (ERK) Activity Pulses

In both physiological and cell culture systems, EGF-stimulated ERK activity occurs in discrete pulses within individual cells. Many feedback loops are present in the EGF receptor (EGFR)-ERK network, but the mechanisms driving pulsatile ERK kinetics are unknown. Here, we find that in cells that respond to EGF with frequency-modulated pulsatile ERK activity, stimulation through a heterologous TrkA receptor system results in non-pulsatile, amplitude-modulated activation of ERK. We further dissect the kinetics of pulse activity using a combination of FRET- and translocation-based reporters and find that EGFR activity is required to maintain ERK activity throughout the 10–20-minute lifetime of pulses. Together, these data indicate that feedbacks operating within the core Ras-Raf-MEK-ERK cascade are insufficient to drive discrete pulses of ERK activity and instead implicate mechanisms acting at the level of EGFR.     

Binding of agonist but not antagonist leads to fluorescence resonance energy transfer between intrinsically fluorescent gonadotropin-releasing hormone receptors

We have used spot fluorescence photobleaching recovery methods to measure the lateral diffusion of GnRH receptor (GnRHR) fused at its C terminus to green fluorescent protein (GFP) after binding of either GnRH agonists or antagonist. Before ligand binding, GnRHR-GFP exhibited fast rates of lateral diffusion (D = 18 +/- 2.8 x 10(-10)cm2 x sec(-1)) and high values for fractional fluorescence recovery (%R) after photobleaching (73 +/- 1%). Increasing concentrations of agonists, GnRH or D-Ala6-GnRH, caused a dose-dependent slowing of receptor lateral diffusion as well as a decreased fraction of mobile receptors. Increasing concentrations of the GnRH antagonist Antide slowed the rate of receptor diffusion but had no effect on the fraction of mobile receptors, which remained high. To determine whether the decrease in %R caused by GnRH agonists was due, in part, to increased receptor self-association, we measured the fluorescence resonance energy transfer efficiency between GnRHR-GFP and yellow fluorescent protein-GNRHR: There was no energy transfer between GnRHR on untreated cells. Treatment of cells with GnRH agonists led to a concentration-dependent increase in the energy transfer between GnRH receptors to a maximum value of 16 +/- 1%. There was no significant energy transfer between GnRH receptors on cells treated with Antide, even at a concentration of 100 nM. These data provide direct evidence that, before binding of ligand, GnRHR exists as an isolated receptor and that binding of GnRH agonists, but not antagonist, leads to formation of large complexes that exhibit slow diffusion and contain receptors that are self-associated.     

Constitutively active Gq impairs gonadotropin-releasing hormone-induced intracellular signaling and luteinizing hormone secretion in LbetaT2 cells

Chronic GnRH treatment causes homologous desensitization by reducing GnRH receptor and Gq/11 expression and by down-regulating protein kinase C (PKC), cAMP, and calcium-dependent signaling. It also causes heterologous desensitization of other Gq-coupled receptors, but the mechanisms involved remain elusive. In this study, we investigated the effect of constitutive activation of Gq signaling on GnRH-induced signaling and LH secretion. We show that adenoviral expression of a constitutively active mutant Gq(Q209L) results in a state of GnRH resistance but does not alter GnRH receptor expression. We observed that Gq(Q209L) reduced expression of phospholipase C (PLC)beta1, a target of Gq in these cells, but not PLCbeta3 or PLCgamma1. Downstream of PLCbeta1, expression of novel PKC isoforms (delta and epsilon) was reduced. Adenoviral expression of a kinase-inactive, dominant-negative version of PKCdelta impaired GnRH activation of ERK, but not induction of c-Fos and LHbeta proteins, indicating that the novel PKCs signal to the ERK cascade. Despite reductions in PLCbeta1, calcium responses to GnRH were elevated in Gq(Q209L)-infected cells due to increased calcium influx through L-type calcium channels. Paradoxically, downstream calcium-dependent signaling and LH secretion were impaired. Taken together, these data demonstrate that prolonged activation of the Gq pathway desensitizes GnRH-induced signaling by selectively down-regulating the PLC-PKC-Ca2+ pathway, leading to reduced LHbeta synthesis and LH secretion.     

Involvement of both G(q/11) and G(s) proteins in gonadotropin-releasing hormone receptor-mediated signaling in L beta T2 cells

The hypothalamic hormone gonadotropin-releasing hormone (GnRH) stimulates the synthesis and release of the pituitary gonadotropins. GnRH acts through a plasma membrane receptor that is a member of the G protein-coupled receptor (GPCR) family. These receptors interact with heterotrimeric G proteins to initiate downstream signaling. In this study, we have investigated which G proteins are involved in GnRH receptor-mediated signaling in L beta T2 pituitary gonadotrope cells. We have shown previously that GnRH activates ERK and induces the c-fos and LH beta genes in these cells. Signaling via the G(i) subfamily of G proteins was excluded, as neither ERK activation nor c-Fos and LH beta induction was impaired by treatment with pertussis toxin or a cell-permeable peptide that sequesters G beta gamma-subunits. GnRH signaling was partially mimicked by adenoviral expression of a constitutively active mutant of G alpha(q) (Q209L) and was blocked by a cell-permeable peptide that uncouples G alpha(q) from GPCRs. Furthermore, chronic activation of G alpha(q) signaling induced a state of GnRH resistance. A cell-permeable peptide that uncouples G alpha(s) from receptors was also able to inhibit ERK, c-Fos, and LH beta, indicating that both G(q/11) and G(s) proteins are involved in signaling. Consistent with this, GnRH caused GTP loading on G(s) and G(q/11) and increased intracellular cAMP. Artificial elevation of cAMP with forskolin activated ERK and caused a partial induction of c-Fos. Finally, treatment of G alpha(q) (Q209L)-infected cells with forskolin enhanced the induction of c-Fos showing that the two pathways are independent and additive. Taken together, these results indicate that the GnRH receptor activates both G(q) and G(s) signaling to regulate gene expression in L beta T2 cells.