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Molecular Endocrinology, doi:10.1210/me.2002-0284
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Molecular Endocrinology 17 (6): 1075-1084
Copyright © 2003 by The Endocrine Society

Phosphoinositide 3-Kinase Activity Is Required for Biphasic Stimulation of Cyclic Adenosine 3',5'-Monophosphate by Relaxin

Bao T. Nguyen1, Luping Yang1, Barbara M. Sanborn and Carmen W. Dessauer

Department of Integrative Biology and Pharmacology (B.T.N., L.Y., C.W.D.) and Department of Biochemistry and Molecular Biology (B.M.S.), University of Texas Health Science Center at Houston, Houston, Texas 77030

Address all correspondence and requests for reprints to: Carmen W. Dessauer, University of Texas Health Science Center at Houston, 6431 Fannin Street, Houston, Texas 77030. E-mail: Carmen.W.Dessauer{at}uth.tmc.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The G protein-coupled receptors LGR7 and LGR8 have recently been identified as the primary receptors for the polypeptide hormone relaxin and relaxin-like factors. RT-PCR confirmed the existence of mRNA for both LGR7 and LRG8 in THP-1 cells. Whole cell treatment of THP-1 cells with relaxin produced a biphasic time course in cAMP accumulation, where the first peak appeared as early as 1–2 min with a second peak at 10–20 min. Selective inhibitors for phosphoinositide 3-kinase (PI3K), such as wortmannin and LY294002, showed a dose-dependent inhibition of relaxin-mediated increases in cAMP, specific for the second peak of the relaxin time course. Adenylyl cyclase activation by relaxin in purified plasma membranes from THP-1 cells was not inhibited by LY294002, consistent with a mechanism involving direct stimulation by a G{alpha}s-coupled relaxin receptor. However, reconstitution of membranes with cytosol from THP-1 cells enhanced adenylyl cyclase activity and restored LY294002 sensitivity. In addition, relaxin increased PI3K activity in THP-1 cells. Neither the effects of relaxin nor the inhibition of relaxin by LY294002 was mediated by the activity of phosphodiesterases. Taken together, we show that PI3K is required for the biphasic stimulation of cAMP by relaxin in THP-1 cells and present a novel signal transduction pathway for the activation of adenylyl cyclase by a G protein-coupled receptor.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
RELAXIN, a 6-kDa protein, is classically regarded as a reproductive hormone in a number of mammalian species, with many important implications for the regulation of pregnancy, including modulation of uterine activity and tissue remodeling (1). In addition to its effects on female reproductive tissues, relaxin is also produced in the male prostate, released into seminal fluid (2, 3), and may enhance sperm motility (1). Moreover, relaxin elicits remarkable pleiotropic characteristics in diverse nonreproductive systems with potential physiological and clinical significance that include decreasing fibrosis (4, 5, 6, 7), inhibition of histamine release (8), stimulation of angiogenesis (9), modulation of blood pressure (10), enhancement of neuropeptide release (1), regulation of heart rate (11), and association with congestive heart failure (12).

Despite the existence of compelling evidence for important roles of relaxin in physiological conditions, until recently little was known about the intracellular signaling pathways initiated by relaxin. Many earlier studies showed that relaxin increased cAMP and activated protein kinase A (PKA) in several cell types and tissues, similar to classical G protein-coupled receptor (GPCR) activation of Gs (1, 13, 14, 15, 16). In contrast, relaxin induced tyrosine phosphorylation of an approximately 220-kDa protein in human uterine fibroblasts (16, 17, 18, 19), potentially implicating a receptor tyrosine kinase as a relaxin target. Likewise, Bartsch et al. (20) reported that tyrphostins (tyrosine kinase inhibitors) inhibited relaxin-stimulated cAMP response. Zhang et al. (21) recently demonstrated that relaxin activates the MAPK pathway in human endometrial and THP-1 monocytic cells. Finally, relaxin activates the nitric oxide (NO) pathway and increases intracellular cGMP levels in human vascular smooth muscle and breast cancer cells (22, 23).

Recently, LGR7 and LGR8, formerly orphan GPCRs, were characterized as receptors for relaxin (24). These receptors stimulated cAMP production when overexpressed in HEK293 cells. LGR8, but not LGR7, is also a receptor for the relaxin-like factor INSL3 (25) and again gives rise to increased cAMP upon stimulation.

Previous studies have addressed the fact that GPCR agonists can activate phosphoinositide 3-kinase (PI3K), particularly receptors that couple to Gi and Gq (26, 27). More recently, a Gs-coupled receptor has also been shown to activate PI3K (28). PI3Ks are lipid kinases that phosphorylate the 3'-OH group of the inositol ring in membrane phospholipids to generate intracellular second messengers (reviewed in Refs. 29, 30, 31). The preferred inositol-containing substrate in intact cells is phosphatidylinositol 4,5-bisphosphate which is converted to phosphatidylinositol 3,4,5-triphosphate. PI3Ks play important roles in gene transcription, cytoskeletal remodeling, mitogenic signaling, and metabolic control. The downstream events initiated by PI3Ks are reminiscent of some of the pleiotropic effects of relaxin, many of which are not easily explained by a simple increase in cAMP. We were thus prompted to examine PI3K involvement in relaxin-mediated signaling pathways.

In this study, we employed a human monocytic cell line THP-1, which has a robust cAMP response upon relaxin stimulation, as a model system to further investigate the signaling mechanisms initiated by relaxin. Our results indicate relaxin receptors LGR7 and LGR8 are expressed in THP-1 cells. We show that relaxin-stimulated cAMP accumulation is biphasic and demonstrate that PI3K activity is required for the production of the second wave of cAMP by relaxin both in vivo and in vitro.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Using RT-PCR, we show that THP-1 cells express mRNA encoding the relaxin receptors LGR7 and LGR8 (Fig. 1Go). The human myometrial cell line PHM1–41 has been used extensively to examine relaxin-mediated responses and also expressed LGR7. A substantially weaker signal was consistently detected with LGR8 in this cell line (Fig. 1Go).



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  		Figure 1. LGR7 and LGR8 Are Present in THP-1 and PHM1–41 Cells

Poly (A)+ RNA from cultured THP-1 cells (lanes 1 and 5) and PHM1–41 cells (lanes 2 and 6) were reverse transcribed and then amplified using primers for LGR7 (lanes 1–4) or LGR8 (lanes 5–8). PCR products were electrophoresed on a 1.5% agarose gel, stained with 0.5 µg/ml ethidium bromide, and visualized under UV light. Size markers are indicated on the left-hand side of the gel. Negative controls with (lanes 3 and 7) and without (lanes 4 and 8) Taq polymerase are shown.

 
Relaxin Increases cAMP with a Biphasic Time Course
THP-1 cells have been used successfully in a number of laboratories to study relaxin-mediated responses because of their large increases in cAMP accumulation upon treatment with relaxin as compared with other cells and tissues (16, 32, 33). We also observed a robust 8-fold stimulation of cAMP accumulation by relaxin in THP-1 cells that was highly synergistic with low levels of forskolin (Fig. 2AGo). In these experiments, forskolin concentrations were chosen that generate only a 2-fold increase in cAMP to enhance the synergistic effect. A similar synergy of relaxin with forskolin was described earlier for THP-1 and rat myometrial cells using human and porcine relaxin, respectively (14, 16).



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  		Figure 2. Relaxin-Mediated Increase in cAMP in THP-1 Cells

A, Duplicate samples (3 x 105 cells per treatment) were pretreated in the presence of PDE inhibitor (50 µM IBMX) for 15 min, and then treated with the vehicle, 0.5 µg/ml relaxin (R), 0.5 µg/ml relaxin + 2 µM forskolin (R + F), or 2 µM forskolin (F) for 20 min. Reactions were stopped upon addition of HCl and total cAMP accumulation was measured by enzyme immunoassay as described in Materials and Methods. Data are expressed as the mean ± SE from a single experiment and are representative of five different experiments. Significant differences shown between groups are identical to those obtained using pooled data (P < 0.05, n = 5) and are designated by different lowercase letters (ANOVA and Duncan’s test). B, Duplicate samples (3 x 105 cells per treatment) were pretreated in the presence (A and B) or absence (C) of 50 µM IBMX for 15 min and then treated with vehicle (t = 0) or 0.5 µg/ml relaxin for various time intervals. Passages of early (A, passage 5), middle (B, passage 21), and late (C, passage 33) THP-1 cells were used. Data from each panel are expressed as the mean ± SE from a single experiment and are representative of at least two different experiments for each panel.

 
The time course for cAMP accumulation in THP-1 cells was biphasic with an early peak occurring at approximately 1 min and a second wave of cAMP at 10–20 min (Fig. 2BGo). The time course changed slightly in these cells with increasing passage number; however, we consistently observed a biphasic pattern using acetylated EIA detection methods for cAMP both in the presence and absence of phosphodiesterase (PDE) inhibitors. Similar time courses have also been measured using [3H]adenine incorporation methods (data not shown).

PI3K Inhibitors Partially Block Relaxin Stimulation
The cAMP time course is consistent with two potential pathways for relaxin action. Previous reports have suggested that stimulation of cAMP by relaxin is sensitive to tyrosine kinase inhibitors (20, 34). We found that inhibitors of PI3K also inhibited relaxin-stimulated cAMP accumulation. The specific PI3K inhibitor (LY294002) partially blocked relaxin-mediated increases in cAMP in THP-1 cells, giving rise to a 47 ± 9% inhibition of relaxin stimulation in six separate experiments (Fig. 3AGo). However, pretreatment with LY294002 had no effect on the stimulation of adenylyl cyclase (AC) by forskolin or isoproterenol (a ß-adrenergic agonist) (Fig. 3AGo). cAMP accumulation was measured after 20 min of relaxin stimulation in these initial experiments to measure the second wave of cAMP production. The partial block of relaxin-stimulated cAMP accumulation was also observed with the more general PI3K inhibitor (wortmannin) (Fig. 3BGo). LY294002 exhibited a dose-dependent inhibition with an IC50 of 8 µM (Fig. 3CGo), similar to the IC50 for inhibition of the purified PI3K enzyme by LY294002 (1.4 µM) (35). Both LY294002 (Fig. 3DGo) and wortmannin (data not shown) preferentially inhibited the second wave of cAMP accumulation. The first peak of cAMP production was inhibited by approximately 30%, whereas the second wave of cAMP production was inhibited by more than 70% by LY294002.



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  		Figure 3. Inhibitors of PI3K Inhibit Relaxin-Mediated Increases in cAMP in THP-1 Cells in Vivo

THP-1 cells (duplicate samples of 3 x 105 cells per treatment) were pretreated in the presence of 50 µM IBMX with or without PI3K inhibitors [A; 50 µM LY294002 (LY); B, 100 nM wortmannin] for 15 min, followed by treatment with vehicle, 0.5 µg/ml relaxin, 0.5 µg/ml relaxin + 1 µM forskolin, 1 µM forskolin, or 1 µM isoproterenol (Iso) for 20 min. Data are expressed as the mean ± SE from a single experiment and are representative of three different experiments for each inhibitor. Significant differences (*, P < 0.05) within a group are designated (t test). C, Dose-dependent inhibition by LY294002 was measured as described above. Cells, pretreated with 50 µM IBMX and the indicated concentrations of LY294002 for 15 min, were assayed with vehicle or 0.5 µg/ml relaxin for 20 min. D, Time course of relaxin inhibition by LY294002 was measured by pretreatment with 50 µM IBMX with or without 50 µM LY294002 for 15 min followed by treatment with 0.5 µg/ml relaxin for the indicated times. Top panel, Time course of cAMP accumulation. Bottom panel, Percent inhibition by LY294002. Data in C and D are expressed as the mean ± SE from a single experiment and are representative of two different experiments.

 
We postulate that the partial block elicited by the PI3K inhibitors is a consequence of the stimulation of multiple pathways by relaxin, only one of which is sensitive to these inhibitors. Activation of G{alpha}s and its subsequent activation of AC occurs very rapidly (32) and is consistent with the peak at 1–2 min. This mechanism is insensitive to the effects of PI3K inhibitors as demonstrated for stimulation of cAMP production by the ß-adrenergic receptor (Fig. 3AGo). However, the second wave of cAMP is not consistent with the effects observed with most Gs-coupled receptors (32, 36). The second pathway leading to an increase in cAMP requires PI3K activity. Additional evidence for these two mechanisms was derived from in vitro studies of relaxin with purified plasma membrane preparations from THP-1 cells.

Reconstitution of Relaxin Signaling Requires Membrane and Cytosolic Components
Relaxin has previously been shown to generate a 25% and 40% increase in AC activity in isolated plasma membrane preparations from nonpregnant rat myometrium and THP-1 cells, respectively (20, 37). Our membrane preparations showed a 1.5-fold stimulation of cAMP production by relaxin that was relatively insensitive to the PI3K inhibitor, LY294002 (Fig. 4AGo). PI3K is mainly cytosolic, and one potential mechanism of activation is translocation to the membrane by protein-protein interactions (29). The presence of PI3K was therefore not expected in our membrane preparations. The increase in cAMP in purified plasma membranes was most likely due to an activation of G{alpha}s by the LGR7 and/or LGR8 relaxin receptor. However, if unstimulated cytosol derived from THP-1 cells was added back to the membrane preparations, the relaxin-mediated stimulation of cAMP production was enhanced and LY294002 sensitivity was restored (Fig. 4AGo). In this system, inhibitors of PI3K only partially blocked the increase in cAMP production by relaxin as was observed in whole cells (Fig. 3Go). In contrast, PI3K inhibitors have no significant effect on isoproterenol-stimulated cAMP production in membranes alone or in membranes reconstituted with cytosol (Fig. 4BGo). Hence LGR7 and/or LGR8 receptors mediate a mechanism distinct from that of ß-adrenergic receptors. The IC50 for inhibition by LY294002 in the reconstitution system upon relaxin stimulation was approximately 3 µM (Fig. 4CGo), similar to that observed in whole cells (Fig. 3CGo).



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  		Figure 4. PI3K Inhibitor LY294002 Inhibits Relaxin-Mediated Increases in cAMP in Vitro

Purified plasma membranes from THP-1 cells (60 µg) were pretreated with 50 µM IBMX in the presence or absence of 100 µM LY294002 for 20 min. Adenylyl cyclase activity was measured upon stimulation with vehicle, 1 µg/ml of relaxin (A), or 100 nM isoproterenol (Iso) (B) for 20 min at 30 C. Where indicated, cytosol (0.3 mg/ml final) was added to plasma membranes before pretreatment with inhibitors and then assayed as before. Significant differences (*, P < 0.05) within a group are designated (t test). C, Dose-dependent inhibition by LY294002 of reconstituted membranes and cytosol was measured as described in panel A. Data for panels A and C are expressed as the mean ± SE and are representative of two different experiments. Data presented in panel B are the average of two experiments each performed in duplicate.

 
Relaxin Increases PI3K Activity
PI3K activity was required not only for biphasic cAMP accumulation by relaxin; it was also stimulated upon relaxin treatment in THP-1 cells. We assessed PI3K activity using an in vitro lipid kinase assay. Briefly, cells (passage nos. 14–25) were stimulated with relaxin for 5 min, lysed, and subjected to immunoprecipitation using an antibody against phosphotyrosine. The immunoprecipitates were then assayed for lipid kinase activity. Using phosphatidylinositol as the substrate, relaxin stimulated a significant (P < 0.05) increase in the lipid kinase activity of PI3K (1.45 ± 0.03 fold; n = 6) (Fig. 5Go). Pretreatment with wortmannin for 30 min nearly eliminated the relaxin-stimulated increase in PI3K activity. Basal activity of THP-1 cells was unaffected by wortmannin. In addition, we also observed a 2.1 ± 0.6-fold increase in PI3K activity upon longer stimulation with relaxin (20 min; passage no. 40) (data not shown).



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  		Figure 5. Relaxin Stimulates PI3K Activity

THP-1 cells (passage nos. 14–25) were starved in 1% dextran-coated charcoal-stripped FBS for 24 h and pretreated with wortmannin (100 nM) or vehicle control [dimethylsulfoxide (DMSO)] for 30 min at 37 C. Cells were then stimulated with 1 µg/ml relaxin (R) for 5 min, subjected to centrifugation, and lysed upon addition of lysis buffer. PI3K was immunoprecipitated from cell lysates using a monoclonal antibody directed against phosphotyrosine as described in Materials and Methods. Subsequently, an in vitro lipid kinase assay was performed using phosphatidylinositol (PI) and [{gamma}-32P]ATP as substrate. The product, 32P-labeled phosphatidylinositol 3-phosphate (PIP), was then separated by TLC and visualized and quantitated by autoradiography. Results are expressed as fold activation over control from several independent experiments (n = 3–6), each performed in duplicate or triplicate (upper graph). Significant differences between groups are identical to those obtained from individual experiments (P < 0.05; ANOVA analysis and Duncan’s test) and are designed by different lowercase letters. Shown in the lower panel is a representative autoradiograph of a TLC plate from a single experiment in which the product PIP is visualized.

 
Relaxin-Stimulated Increase in cAMP Is Not Directly Mediated by a PDE
Bartsch et al. (20) originally proposed that relaxin stimulated a tyrosine kinase receptor, which in turn led to the inhibition of a PDE, possibly PDE8, thus resulting in the elevation of cAMP. We have used two PDE inhibitors to determine whether the PI3K-dependent pathway stimulated by relaxin requires inhibition of a PDE. If this is the case, inhibition of PDE should attenuate the ability of relaxin to increase cAMP. Isobutylmethylxanthine (IBMX) is a general PDE inhibitor for most classes of PDEs but not PDE8 (38, 39). Dipyridamole inhibits the cGMP-dependent PDEs and PDE8A and B (IC50 = 5–40 µM) (38, 39). The inclusion of the PDE inhibitor IBMX did not alter the biphasic nature of the cAMP time course upon relaxin stimulation in THP-1 cells (Fig. 2BGo). Although IBMX significantly increased basal and relaxin-stimulated cAMP as expected, the inhibition of PDE activity is not the mechanism of the PI3K-required second wave of cAMP accumulation. Relaxin-mediated stimulation of cAMP remained sensitive to LY294002 (30–50% inhibition) in the presence of dipyridamole and a wide range of IBMX concentrations (Fig. 6Go). In addition, if relaxin increased cAMP solely by decreasing PDE8 activity, an inhibitor of PDE8 should also increase cAMP. No elevation of cAMP by dipyridamole was observed; rather we generally observed an inhibition of both basal and relaxin-mediated stimulation. This decrease in cAMP may reflect the nonspecific nature of dipyridamole, which also has effects on nucleotide transport and metabolism (40, 41).



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  		Figure 6. Relaxin-Mediated Increases in cAMP and Inhibition by LY294002 Are Independent of PDE Inhibitors

THP-1 cells (duplicate samples of 3 x 105 cells per treatment) were pretreated in the presence of increasing concentrations of IBMX (A) or 250 µM dipyridamole (B) for 15 min before treatment with relaxin for 20 min (A) or as indicated (B). The PI3K inhibitor (50 µM LY294002; LY) was added for 15 min before stimulation with relaxin where indicated. C, Fold activation of treatments with 0.5 µg/ml relaxin or 0.5 µg/ml relaxin + 50 µM LY294002 as compared with basal treatment in the presence or absence of 50 µM LY294002 was measured over the indicated range of IBMX concentrations. Data are expressed as the mean ± SE from a single experiment and are representative of at least two different experiments.

 
On the basis of these data, we suggest a working model for relaxin-mediated cAMP accumulation in THP-1 cells (Fig. 7Go). Specific aspects of this model and potential downstream signaling components are discussed below.



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  		Figure 7. Illustration of the Proposed Model for Relaxin-Mediated Increases in cAMP

See text for further description.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
The molecular mechanisms by which relaxin elicits downstream effects, including how it elevates cAMP, have been unclear. The recent identification of LGR7 and LGR8 as G protein-coupled relaxin receptors (24) points to a direct mechanism for cAMP production via G{alpha}s stimulation of AC. However, a number of studies have implicated tyrosine kinases in the action of relaxin. Relaxin increased tyrosine phosphorylation of unidentified proteins in human uterine fibroblasts and atrial myocytes (42). Tyrosine kinase inhibitors attenuated the ability of relaxin to increase cAMP in THP-1 cells (20), human endometrial stroma cells (20), and human myometrium (34) and inhibited relaxin-stimulated nitrite accumulation in vascular smooth muscle cells (23). Taken together, these data suggest that relaxin may activate multiple pathways by a common receptor.

The mRNA for LGR7 and LGR8 is present in THP-1 cells. The time course of cAMP stimulation by relaxin is biphasic in these cells with an early peak detected at 1 min and a later wave of cAMP accumulation at 10–20 min. This same biphasic time course was observed in many of the initial relaxin studies in uterine strips and myometrial cells, often as a predominantly delayed signal at 15–20 min with a slight but reproducible early peak (13, 14). This is in stark contrast to time courses of cAMP accumulation observed with agonists of the ß-adrenergic receptor and other Gs-coupled receptors in myometrial and S49 lymphoma cells (14, 32). For example, isoproterenol produces a sharp increase in cAMP production between 1 and 5 min in a large number of cell types, including THP-1 cells (data not shown). cAMP levels then either decrease or plateau, depending on the presence and concentration of PDE inhibitors.

The biphasic cAMP time course suggests that at least two pathways may be acting to increase cAMP accumulation upon stimulation with relaxin: a G{alpha}s membrane delimited pathway, and a second somewhat delayed pathway. Inhibitors of PI3K activity block cAMP accumulation but only with a maximum inhibition of approximately 50%. No inhibitory effect was observed upon activation with a ß-adrenergic receptor agonist or a direct adenylyl cyclase activator, forskolin. More importantly, the inhibition was dose-dependent and was more prominent for the second wave of cAMP production.

To further explore the role of PI3K, we have performed reconstitution experiments with cytosolic and purified plasma membrane preparations from THP-1 cells. Relaxin stimulation increased cAMP production 1.5-fold in purified plasma membrane preparations that were insensitive to an inhibitor of PI3K, consistent with a direct activation of AC by G{alpha}s. However, reconstitution of plasma membranes with cytosol further increased stimulation by relaxin and resulted in a dose-dependent partial inhibition by LY294002, consistent with a second pathway containing at least one cytosolic component. Stimulation of cAMP production by isoproterenol was unaffected by LY294002 upon reconstitution of membranes with cytosol.

Our model for relaxin stimulation of cAMP is presented in Fig. 7Go and involves an activation of PI3K by relaxin. Stimulation of THP-1 cells with relaxin significantly increased PI3K activity. There are several distinct classes of phosphoinositide kinases. p110{gamma} belongs to class Ib and is the main member of the subfamily that is sensitive to hormones and neurotransmitters interacting with GPCRs (27). Gß{gamma} complexes interact directly with p110{gamma} to activate this enzyme (43). However, members of the class Ia and class II families may also be regulated by G proteins (via either G{alpha}- or Gß{gamma}-subunits) by other less defined mechanisms (27). Further studies are required to identify a specific isoform of PI3K involved in this pathway.

Neither the biphasic nature of relaxin stimulation nor the sensitivity to PI3K inhibitors was mediated by PDEs as originally proposed (20). The fold increase for relaxin stimulation of cAMP and inhibition by LY294002 remained constant over a 20-fold concentration range of cAMP with increasing concentrations of IBMX (Fig. 6Go). Dipyridamole, an inhibitor of PDE8, also had no effect on the inhibition by PI3K inhibitors; however, this drug has many side effects. Even in the absence of a good PDE8 inhibitor, we can rule out PDE8 involvement with the following kinetic argument. Due to the low Michaelis-Menten constant (Km) of PDE8, the enzyme is essentially fully active at low cAMP concentrations, and therefore the amount of cAMP hydrolyzed by PDE8 remains essentially constant over a large range of cAMP. Hence, at higher cAMP concentrations, the fraction of cAMP hydrolyzed by PDE8 is decreased. Any inhibition of PDE8 by relaxin would produce a greater effect at low concentrations of cAMP; this was not observed in THP-1 cells.

We propose a bifurcated pathway for cAMP generation that involves the activation of both G{alpha}s and PI3K. There are few examples of stimulation of an effector molecule by two pathways generated from a single Gs-coupled receptor. Both types I and II AC can be regulated by direct binding of G{alpha}s- and Gß{gamma}-subunits, but in the case of type II AC it is unclear whether this occurs in vivo by activation of the same receptor or by activation of both Gs- and Gi-coupled receptors (44, 45). L-type Ca2+ channels can also be regulated by Gs-coupled receptors via two distinct pathways involving G{alpha}s activation of PKA and Gß{gamma} activation of protein kinase C (PKC) (46). It is also possible that LGR7 and/or LGR8 can activate another heterotrimeric G protein in addition to Gs. However, in THP-1 cells, pertussis toxin treatment has no effect on the fold stimulation of cAMP accumulation by relaxin, ruling out effects of Gi, Go, or Gz (20). Although relaxin stimulates PKA activation in PHM1–41 cells or rat myometrium, relaxin has never been observed to increase Ca2+ release or stimulate phospholipase Cß, typical of Gq-mediated effects (Refs. 47 and 48 and data not shown). Finally, we cannot rule out stimulation of G12 or G13 although these G proteins are often associated with cytoskeletal reorganization and focal adhesion assembly not typically observed upon relaxin treatment (49). Therefore, at present our model includes only activation of Gs. Whether relaxin utilizes G{alpha}s or Gß{gamma} to activate PI3K remains to be determined.

It is unclear what downstream events lead from PI3K activation to stimulation of AC. It has been suggested that PI3K activity may serve to compartmentalize Gs signaling in some cell types (50). This could be one potential mechanism for enhancement. Alternatively, phosphatidylinositol 3,4,5-triphosphate generated by PI3K may activate downstream components that serve to enhance AC activity. Studies employing relatively specific chemical inhibitors of PI3K and more recent studies using dominant-negative PI3K molecules have identified several downstream enzymes that are activated by PI3K; these enzymes include phosphoinositide-dependent protein kinase, protein kinase B (Akt), p70 ribosomal S6-kinase, protein kinase C{epsilon} and -{zeta} (PKC{epsilon} and PKC{zeta}), and ERK 1/2 (31, 51, 52). Relaxin is unable to activate Akt in normal endometrial cells (21) or in THP-1 cells (data not shown). However, relaxin can activate MAPK in THP-1 cells, endometrial cells, and pulmonary and cardiac smooth muscle cells (21). MAPK activation is often downstream of PI3K activation for a number of Gs-, Gq-, and Gi-coupled receptors (52, 53). However, it is unclear how increased ERK activation can lead to increased AC activity. One intriguing downstream effector of PI3K is PKC{zeta}, which is a direct activator of type V AC and displays synergy with both forskolin and G{alpha}s stimulation of AC (54). Further studies are required to determine the role of PKC{zeta} or any other PKC in the regulation of cAMP by relaxin.

Relaxin clearly increases cAMP and activates protein kinase A in a number of tissues and cell lines. However, additional pathways are also activated in response to relaxin, including increased tyrosine phosphorylation, ERK phosphorylation, and NO synthase expression and the subsequent generation of cGMP. It is possible that in addition to cAMP generation, stimulation of PI3K by relaxin is involved in these pathways as well. By stimulation of two distinct signal transduction pathways, relaxin treatment may have pleiotropic downstream effects, many of which may have useful clinical applications.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Materials
Forskolin, IBMX, isoproterenol, wortmannin, and phosphatidylinositol were obtained from Sigma (St. Louis, MO); LY 294002 was obtained from Calbiochem (La Jolla, CA). Cell culture reagents were obtained from Life Technologies, Inc. (Gaithersburg, MD). cAMP enzyme immunoassay kit was purchased from Assay Designs, Inc. (Ann Arbor, MI). Porcine relaxin was purified as described previously (14). Avian myeloblastosis virus (AMV) reverse transcriptase was obtained from Promega Corp. (Madison, WI). Taq DNA polymerase was obtained from Roche Molecular Biochemicals (Indianapolis, IN). ATP ([{gamma}32P], 6000 Ci/mmol) was purchased from NEN Life Science Products (Boston, MA).

Cell Culture
The human monocytic cell line THP-1 was cultured at 37 C and 5% CO2 in RPMI 1640 containing 2 mM L-glutamine, 10% fetal bovine serum (FBS), 2 mM L-glutamine, 50 U/ml penicillin, and 50 µg/ml streptomycin. Cells were used at a density of approximately 1 x 106 cells/ml. Culture of the immortalized pregnant human myometrial cell line PHM1–41 has been described previously (55).

Assay for Relaxin-Mediated Increase in cAMP Production
Cultured cells were washed with prewarmed PBS and resuspended with cell suspension media (RPMI 1640 + 50 mM HEPES, pH 7.4) at 37 C. Cells were pretreated with PDE inhibitor (50 µM IBMX) for 15 min, and then treated with 0.5 µg/ml relaxin for 20 min, except where indicated. Treatments were terminated by addition of 1 N HCl (0.1 N HCl final). Total cAMP (intra- and extracellular) was detected by enzyme immunoassay. Data represent mean ± SE and were analyzed by one-way ANOVA (where indicated) and Duncan’s modified multiple range test and t test.

RT-PCR Analysis
Poly(A)+ RNA was isolated from human THP-1 and PHM1–41 cells, and then reverse transcribed and amplified by PCR as previously described (24). Briefly, 1 x 107 cells were harvested by centrifugation at 1500 rpm for 5 min and washed twice with cold PBS and resuspended in lysis buffer provided by Fast Track 2.0 kit (Invitrogen, San Diego, CA). Poly (A)+ RNA was isolated with the FastTrack 2.0 kit according to the manufacturer’s protocol. For cDNA synthesis, 2 µg of Poly(A)+ RNA and 2.5 mM oligo (deoxythymidine) were heated to 65 C for 5 min, chilled on ice for 5 min, and centrifuged briefly. The following components were added to the annealed oligo deoxythymidine/template: AMV buffer, 0.2 mM deoxynucleotide triphosphate mix, 1 U/µl RNAsin Ribonuclease inhibitor, nuclease-free water, and 1 U/µl AMV reverse transcriptase followed by incubation at 42 C for 1.5 h. PCRs were carried out in a final volume of 20 µl containing 5 µl of cDNA, 125 µM deoxynucleotide triphosphate mix, 500 µM primers, PCR buffer, and 2.5 U of Taq DNA polymerase. PCR conditions were: 95 C for 90 sec followed by 35 cycles (94 C for 30 sec and 68 C for 3 min) and final extension (72 C for 5 min). PCR products were electrophoresed on a 1.5% agarose gel, stained with 0.5 µg/ml ethidium bromide, and visualized under UV light. Primer sets used for amplification of LGR7 and LGR8 were as described (24), with the exception of the LGR8 forward primer: 5'-CACAGAGAGCACAGCAGAATGGCT.

Preparation and Reconstitution of Plasma Membrane and Cytosol
THP-1 cells were pelleted, washed twice with cold buffer A (137 mM NaCl, 5.36 mM KCl, 1.1 mM KH2PO4, 1.08 mM Na2HPO4, final pH 7.2), and resuspended in buffer B (150 mM NaCl, 20 mM HEPES, 1 mM EDTA, and 1 mM benzamidine, final pH 7.4). Cells were disrupted by nitrogen cavitation (pressurized at 500 pounds per square inch for 25 min on ice). Unbroken cells and nuclei were removed by centrifugation (2600 rpm for 5 min). Half of the supernatant was subjected to centrifugation at 21,000 rpm for 30 min at 4 C to obtain the cytosolic fraction. The other half of the supernatant was centrifuged at 25,000 rpm for 35 min at 4 C to isolate plasma membrane by sucrose sedimentation at the 23%/43% interface (56). To measure adenylyl cyclase activity, plasma membranes (60 µg) were pretreated with 50 µM IBMX in the presence or absence of LY294002 for 20 min at 30 C. Vehicle or 1 µg/ml relaxin was then added and the reaction started upon addition of 100 µM ATP and assay buffer components as described previously (57). Reactions were terminated after 20 min incubation at 30 C by addition of 1 N HCl (0.1 N final). cAMP was detected by enzyme immunoassay and expressed as picomoles/minute/milligram of membrane protein. Reconstitution experiments were performed by addition of cytosol (0.3 mg/ml) to purified plasma membranes and treated as described above.

PI3K Assay
PI3K activity in immune complexes was assayed as described previously (58). Briefly, THP-1 cells (1 x 107 cells per reaction) were starved in phenol red-free RPMI with 1% charcoal-stripped FBS for 24 h and then stimulated with 1 µg/ml of relaxin for 20 min at 37 C. Cell lysates were prepared in lysis buffer (50 mM Tris-HCl, pH 7.5; 150 mM NaCl; 20 mM tetrasodium pyrophosphate; 10 mM EDTA; 10% glycerol; 1% Triton X-100; 200 mM NaF; 4 mM Na3VO4; 1 mM phenylmethylsulfonyl fluoride; 5 µg/ml leupeptin), and proteins were immunoprecipitated with a phosphotyrosine antibody. Immunoprecipitates were washed sequentially in buffer A (50 mM Tris-HCl, pH 7.5; 150 mM NaCl; 1% Triton X-100; and 100 µM Na3VO4), buffer B (100 mM Tris-HCl, pH 7.5; 500 mM LiCl; and 100 µM Na3VO4), buffer C (10 mM Tris-HCl, pH 7.5; 100 mM NaCl; 1 mM EDTA; and 100 µM Na3VO4), and then pellets were dissolved in TNE buffer (10 mM Tris-HCl, pH 7.5; 100 mM NaCl; and 1 mM EDTA). Sonicated phosphatidylinositol (20 µg) was added to the immunoprecipitates, and the kinase reaction was started by addition of 30 µCi [{gamma}-32P]ATP. The reaction was performed at room temperature for 20 min and stopped by addition of a chloroform/methanol mixture, followed by lipid extraction. The phospholipids in the organic phase were recovered and spotted onto silica gel thin-layer chromatography (TLC) plate precoated with 1% KOH-oxalate. Migration was performed in CH3OH-CHCl3-H2O-25% NH4OH (45:35:7:3). The product (phosphatidylinositol 3-phosphate) was then visualized and quantitated using a Storm PhosphorImager (Molecular Dynamics, Sunnyvale, CA).


   ACKNOWLEDGMENTS
 
We thank Dr. Chun-Ying Ku for her help with THP-1 and PHM1–41 cell culture; Dr. G. Marshall for providing THP-1 cells; and Dr. Jeffrey Frost, Lyudmila Zilberman, and Dr. Victoria Knutson for providing technical advice.


   FOOTNOTES
 
This work was supported by the Texas Advanced Research Program Project No. 011618-0059-1999 (to C.W.D.) and in part by NIH Grant HD-09618 (to B.M.S.).

1 B.T.N. and L.Y. contributed equally to this work. Back

Abbreviations: AC, Adenylyl cyclase; AMV, avian myeloblastosis virus; FBS, fetal bovine serum; GPCR, G protein-coupled receptor; IBMX, isobutylmethylxanthine; PI3K, phosphoinositide 3-kinase; PDE, phosphodiesterase; PKA, protein kinase A; PKC, protein kinase C; TLC, thin-layer chromatography.

Received for publication August 15, 2002. Accepted for publication February 17, 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Sherwood OD 1994 Relaxin. In: Knobil E, Neill JD, eds. The physiology of pregnancy. New York: Raven Press; 861–1009
  2. Bani D 1997 Relaxin: a pleiotropic hormone. Gen Pharmacol 28:13–22[Medline]
  3. Gunnersen JM, Crawford RJ, Tregear GW 1995 Expression of the relaxin gene in rat tissues. Mol Cell Endocrinol 110:55–64[CrossRef][Medline]
  4. Samuel CS, Sakai LY, Amento EP 2001 Relaxin modulates fibrillin-2, but not fibrillin-1, gene expression in human dermal fibroblasts. In: Tragear GW, Ivell R, Bathgate RA, Wade JD, eds. Relaxin 2000. Amsterdam: Kluwer Academic Publishers; 389–392
  5. Unemori EN, Pickford LB, Salles AL, Piercy CE, Grove BH, Erikson ME, Amento EP 1996 Relaxin induces an extracellular matrix-degrading phenotype in human lung fibroblasts in vitro and inhibits lung fibrosis in a murine model in vivo. J Clin Invest 98:2739–2745[Medline]
  6. Garber SL, Mirochnik Y, Brecklin CS, Unemori EN, Singh AK, Slobodskoy L, Grove BH, Arruda JA, Dunea G 2001 Relaxin decreases renal interstitial fibrosis and slows progression of renal disease. Kidney Int 59:876–882[CrossRef][Medline]
  7. Williams EJ, Benyon RC, Trim N, Hadwin R, Grove BH, Arthur MJ, Unemori EN, Iredale JP 2001 Relaxin inhibits effective collagen deposition by cultured hepatic stellate cells and decreases rat liver fibrosis in vivo. Gut 49:577–583[Abstract/Free Full Text]
  8. Bani D, Ballati L, Masini E, Bigazzi M, Sacchi TB 1997 Relaxin counteracts asthma-like reaction induced by inhaled antigen in sensitized guinea pigs. Endocrinology 138:1909–1915[Abstract/Free Full Text]
  9. Unemori EN, Lewis M, Constant J, Arnold G, Grove BH, Normand J, Deshpande U, Salles A, Pickford LB, Erikson ME, Hunt TK, Huang X 2000 Relaxin induces vascular endothelial growth factor expression and angiogenesis selectively at wound sites. Wound Repair Regen 8:361–370[CrossRef][Medline]
  10. Yang RH, Bunting S, Wyss JM, Berecek KH, Zhang L, Jin H 1995 Pressor and bradycardic effects of centrally administered relaxin in conscious rats. Am J Hypertens 8:375–381[CrossRef][Medline]
  11. Kakouris H, Eddie LW, Summers RJ 1992 Cardiac effects of relaxin in rats. Lancet 339:1076–1078[CrossRef][Medline]
  12. Dschietzig T, Richter C, Bartsch C, Laule M, Armbruster FP, Baumann G, Stangl K 195 2001 The pregnancy hormone relaxin is a player in human heart failure. FASEB J 15:2187–2195[Abstract/Free Full Text]
  13. Sanborn BM, Weisbrodt NW, Kuo HS, Sherwood OD 1980 The interaction of relaxin with the rat uterus. I. Effect of cyclic nucleotide levels and spontaneous contractile activity. Endocrinology 106:1210–1215[Abstract/Free Full Text]
  14. Hsu CJ, McCormack SM, Sanborn BM 1985 The effect of relaxin on cyclic AMP concentrations in rat myometrial cells in culture. Endocrinology 116:2029–2035[Abstract/Free Full Text]
  15. Meera P, Anwer K, Monga M, Oberti C, Stefani E, Toro L, Sanborn BM 1995 Relaxin stimulates myometrial calcium-activated potassium channel activity via protein kinase A. Am J Physiol 269:C312–C317
  16. Parsell DA, Mak JY, Amento EP, Unemori EN 1996 Relaxin binds to and elicits a response from cells of the human monocytic cell line, THP-1. J Biol Chem 271:27936–27941[Abstract/Free Full Text]
  17. Palejwala S, Stein DE, Weiss G, Monia BP, Tortoriello D, Goldsmith LT 2001 Relaxin positively regulates matrix metalloproteinase expression in human lower uterine segment fibroblasts using a tyrosine kinase signaling pathway. Endocrinology 142:3405–3413[Abstract/Free Full Text]
  18. Palejwala S, Tseng L, Wojtczuk A, Weiss G, Goldsmith LT 2002 Relaxin gene and protein expression and its regulation of procollagenase and vascular endothelial growth factor in human endometrial cells. Biol Reprod 66:1743–1748[Abstract/Free Full Text]
  19. Osheroff PL, King KL 1995 Binding and cross-linking of 32P-labeled human relaxin to human uterine cells and primary rat atrial cardiomyocytes. Endocrinology 136:4377–4381[Abstract]
  20. Bartsch O, Bartlick B, Ivell R 2001 Relaxin signalling links tyrosine phosphorylation to phosphodiesterase and adenylyl cyclase activity. Mol Hum Reprod 7:799–809[Abstract/Free Full Text]
  21. Zhang Q, Liu SH, Erikson M, Lewis M, Unemori E 2002 Relaxin activates the MAP kinase pathway in human endometrial stromal cells. J Cell Biochem 85:536–544[CrossRef][Medline]
  22. Bani D, Masini E, Bello MG, Bigazzi M, Sacchi TB 1995 Relaxin activates the L-arginine-nitric oxide pathway in human breast cancer cells. Cancer Res 55:5272–5275[Abstract/Free Full Text]
  23. Bani D, Failli P, Bello MG, Thiemermann C, Bani ST, Bigazzi M, Masini E 1998 Relaxin activates the L-arginine-nitric oxide pathway in vascular smooth muscle cells in culture. Hypertension 31:1240–1247[Abstract/Free Full Text]
  24. Hsu SY, Nakabayashi K, Nishi S, Kumagai J, Kudo M, Sherwood OD, Hsueh AJ 2002 Activation of orphan receptors by the hormone relaxin. Science 295:671–674[Abstract/Free Full Text]
  25. Kumagai J, Hsu SY, Matsumi H, Roh JS, Fu P, Wade J, Bathgate RA, Hsueh AJ 2002 INSL3/Leydig insulin-like peptide activates the LGR8 receptor important in testis descent. J Biol Chem 277:31283–31286[Abstract/Free Full Text]
  26. Yart A, Roche S, Wetzker R, Laffargue M, Tonks N, Mayeux P, Chap H, Raynal P 2002 A function for phosphoinositide 3-kinase ß lipid products in coupling ß {gamma} to Ras activation in response to lysophosphatidic acid. J Biol Chem 277:21167–21178[Abstract/Free Full Text]
  27. Curnock AP, Logan MK, Ward SG 2002 Chemokine signalling: pivoting around multiple phosphoinositide 3-kinases. Immunology 105:125–136[CrossRef][Medline]
  28. Colombo F, Noel J, Mayers P, Mercier I, Calderone A 2001 ß-Adrenergic stimulation of rat cardiac fibroblasts promotes protein synthesis via the activation of phosphatidylinositol 3-kinase. J Mol Cell Cardiol 33:1091–1106[CrossRef][Medline]
  29. Wymann MP, Pirola L 1998 Structure and function of phosphoinositide 3-kinases. Biochim Biophys Acta 1436:127–150[Medline]
  30. Corvera S, Czech MP 1998 Direct targets of phosphoinositide 3-kinase products in membrane traffic and signal transduction. Trends Cell Biol 8:442–446[CrossRef][Medline]
  31. Cantley LC 2002 The phosphoinositide 3-kinase pathway. Science 296:1655–1657[Abstract/Free Full Text]
  32. Barber R 1986 Discrimination between intact cell desensitization and agonist affinity changes. Mol Cell Endocrinol 46:263–270[CrossRef][Medline]
  33. Bathgate RA, Samuel CS, Burazin TC, Layfield S, Claasz AA, Reytomas IG, Dawson NF, Zhao C, Bond C, Summers RJ, Parry LJ, Wade JD, Tregear GW 2002 Human relaxin gene 3 (H3) and the equivalent mouse relaxin (M3) gene. Novel members of the relaxin peptide family. J Biol Chem 277:1148–1157[Abstract/Free Full Text]
  34. Kuznetsova L, Plesneva S, Derjabina N, Omeljaniuk E, Pertseva M 1999 On the mechanism of relaxin action: the involvement of adenylyl cyclase signalling system. Regul Pept 80:33–39[CrossRef][Medline]
  35. Vlahos CJ, Matter WF, Hui KY, Brown RF 1994 A specific inhibitor of phosphatidylinositol 3-kinase, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002). J Biol Chem 269:5241–5248[Abstract/Free Full Text]
  36. Bhalla RC, Sanborn RM, Korenman SG 1972 Hormonal interactions in the uterus: inhibition of isoproterenol-induced accumulation of adenosine 3':5'-cyclic monophosphate by oxytocin and prostaglandins. Proc Natl Acad Sci USA 69:3761–3764[Abstract/Free Full Text]
  37. Sanborn BM, Kuo HS, Weisbrodt NW, Sherwood OD 1982 Effect of porcine relaxin on cyclic nucleotide levels and spontaneous contractions of the rat uterus. In: Anderson RR, ed. Relaxin. New York: Plenum Press; 273–287
  38. Soderling SH, Bayuga SJ, Beavo JA 1998 Cloning and characterization of a cAMP-specific cyclic nucleotide phosphodiesterase. Proc Natl Acad Sci USA 95:8991–8996[Abstract/Free Full Text]
  39. Hayashi M, Matsushima K, Ohashi H, Tsunoda H, Murase S, Kawarada Y, Tanaka T 1998 Molecular cloning and characterization of human PDE8B, a novel thyroid-specific isozyme of 3',5'-cyclic nucleotide phosphodiesterase. Biochem Biophys Res Commun 250:751–756[CrossRef][Medline]
  40. Thorn JA, Jarvis SM 1996 Adenosine transporters. Gen Pharmacol 27:613–620[Medline]
  41. Connolly GP, Duley JA 2000 Ecto-nucleotidase of cultured rat superior cervical ganglia: dipyridamole is a novel inhibitor. Eur J Pharmacol 397:271–277[CrossRef][Medline]
  42. Palejwala S, Stein D, Wojtczuk A, Weiss G, Goldsmith LT 1998 Demonstration of a relaxin receptor and relaxin-stimulated tyrosine phosphorylation in human lower uterine segment fibroblasts. Endocrinology 139:1208–1212[Abstract/Free Full Text]
  43. Stephens L, Smrcka A, Cooke FT, Jackson TR, Sternweis PC, Hawkins PT 1994 A novel phosphoinositide 3 kinase activity in myeloid-derived cells is activated by G protein ß {gamma} subunits. Cell 77:83–93[CrossRef][Medline]
  44. Tang W-J, Gilman AG 1991 Type-specific regulation of adenylyl cyclase by G protein ß{gamma} subunits. Science 254:1500–1503[Abstract/Free Full Text]
  45. Nielsen MD, Chan GC, Poser SW, Storm DR 1996 Differential regulation of type I and type VIII Ca2+-stimulated adenylyl cyclases by Gi-coupled receptors in vivo. J Biol Chem 271:33308–33316[Abstract/Free Full Text]
  46. Zhong J, Dessauer CW, Keef KD, Hume JR 1999 Regulation of L-type Ca2+ channels in rabbit portal vein by G protein {alpha}s and ß{gamma} subunits. J Physiol 517:109–120[Abstract/Free Full Text]
  47. Anwer K, Hovington JA, Sanborn BM 1989 Antagonism of contractants and relaxants at the level of intracellular calcium and phosphoinositide turnover in the rat uterus. Endocrinology 124:2995–3002[Abstract/Free Full Text]
  48. Dodge K, Sanborn BM 1998 Evidence for inhibition by protein kinase A of receptor/Gq/phospholipase C coupling by a mechanism not involving PLC2. Endocrinology 139:2265–2271[Abstract/Free Full Text]
  49. Dhanasekaran N, Dermott JM 1996 Signaling by the G12 class of G proteins. Cell Signal 8:235–245[CrossRef][Medline]
  50. Jo SH, Leblais V, Wang PH, Crow MT, Xiao RP 2002 Phosphatidylinositol 3-kinase functionally compartmentalizes the concurrent G(s) signaling during ß2-adrenergic stimulation. Circ Res 91:46–53[Abstract/Free Full Text]
  51. Sajan MP, Standaert ML, Bandyopadhyay G, Quon MJ, Burke Jr TR, Farese RV 1999 Protein kinase C-{zeta} and phosphoinositide-dependent protein kinase-1 are required for insulin-induced activation of ERK in rat adipocytes. J Biol Chem 274:30495–30500[Abstract/Free Full Text]
  52. Liebmann C 2001 Regulation of MAP kinase activity by peptide receptor signalling pathway: paradigms of multiplicity. Cell Signal 13:777–785[CrossRef][Medline]
  53. Schwindinger WF, Robishaw JD 2001 Heterotrimeric G-protein ß{gamma}-dimers in growth and differentiation. Oncogene 20:1653–1660[CrossRef][Medline]
  54. Kawabe J, Iwami G, Ebina T, Ohno S, Katada T, Ueda Y, Homcy CJ, Ishikawa Y 1994 Differential activation of adenylyl cyclase by protein kinase C isoenzymes. J Biol Chem 269:16554–16558[Abstract/Free Full Text]
  55. Monga M, Ku CY, Dodge K, Sanborn BM 1996 Oxytocin-stimulated responses in a pregnant human immortalized myometrial cell line. Biol Reprod 55:427–432[Abstract]
  56. Seibold A, Williams B, Huang ZF, Friedman J, Moore RH, Knoll BJ, Clark RB 2000 Localization of the sites mediating desensitization of the ß(2)-adrenergic receptor by the GRK pathway. Mol Pharmacol 58:1162–1173[Abstract/Free Full Text]
  57. Dessauer CW 2002 Kinetic analysis of the action of P-site analogs. Methods Enzymol 345:112–126[Medline]
  58. Chakravorty A, Joslyn MI, Davis JS 1993 Characterization of insulin and insulin-like growth factor-I actions in the bovine luteal cell: regulation of receptor tyrosine kinase activity, phosphatidylinositol-3-kinase, and deoxyribonucleic acid synthesis. Endocrinology 133:1331–1340[Abstract/Free Full Text]



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