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Originally published In Press as doi:10.1074/jbc.M304273200 on June 5, 2003

J. Biol. Chem., Vol. 278, Issue 34, 31593-31602, August 22, 2003
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Constitutive Localization of the Gonadotropin-releasing Hormone (GnRH) Receptor to Low Density Membrane Microdomains Is Necessary for GnRH Signaling to ERK*

Amy M. Navratil {ddagger}, Stuart P. Bliss §, Kathie A. Berghorn §, James M. Haughian {ddagger}, Todd A. Farmerie {ddagger}, James K. Graham {ddagger}, Colin M. Clay {ddagger}  and Mark S. Roberson §

From the {ddagger}Department of Biomedical Sciences, Colorado State University, Fort Collins, Colorado 80523 and the §Department of Biomedical Sciences, Cornell University, Ithaca, New York 14853

Received for publication, April 23, 2003


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Over the past several years it has become evident that the specificity and fidelity of intracellular signaling is partially achieved through compartmentalization of interacting proteins into discrete subcellular domains. In this regard, the plasma membrane is no exception. Of particular interest are discrete microdomains consisting of tightly packed sphingolipids and cholesterol in the exoplasmic leaflet (1). Due to this unique lipid character, these membrane microdomains are characterized by resistance to solubilization in low ionic strength detergents and a low buoyant density in sucrose gradients as compared with the "bulk" plasma membrane (2). Although related in regard to lipid composition, two distinct subsets of membrane microdomains have been described based on different morphological and biochemical characteristics. The first of these, caveolae, are flask-shaped invaginations of the plasma membrane that are defined by the presence of the marker protein caveolin (3, 4). In contrast, lipid rafts lack caveolin and are not topologically distinct from the plasma membrane (1, 5). Although the molecular determinants that dictate inclusion or exclusion of proteins from lipid rafts or caveolae are not fully understood, it is evident that many of these raft-associated or caveolar proteins represent components of established intracellular signaling cascades such as G-proteins, Ras, and Src family kinases (4, 6). Thus, membrane microdomains may represent a form of signaling platform that organizes efficient and specific signal transduction by facilitating interactions among co-segregated proteins such as membrane receptors and their cognate signaling proteins. Within these domains, further specificity of signaling is likely mediated by scaffolding proteins, such as caveolins and 14-3-3 proteins, which bring sequential members of a signaling cascade into direct proximity (7, 8).

Consistent with the presence of G-proteins in lipid microdomains, several members of the superfamily of G-protein-coupled receptors (GPCR)1 have been shown to partition into lipid rafts and caveolae (9–13). Often, the translocation of GPCR into membrane microdomains appears to require ligand activation; however, {beta}-adrenergic receptor subtypes have been found distributed between both caveolin containing and bulk plasma membrane fractions (10, 14). Herein we have sought to determine the membrane localization of the mammalian type I gonadotropin releasing hormone receptor (GnRHR). An atypical member of the rhodopsin-like family of GPCR, the GnRHR is located on the cell surface of pituitary gonadotropes (14–16). The binding of the hypothalamic decapeptide GnRH to the pituitary GnRHR not only stimulates but is obligatory for the synthesis and secretion of luteinizing hormone (LH) (15, 16). In the absence of GnRH input to the pituitary gland, LH production and, consequently, gonadal function in mammals ceases (17, 18). Thus, the GnRHR represents the site that mediates the primary stimulatory input to gonadotrope cells. Upon ligand activation, it is well established that agonist-occupied GnRHR couples to G{alpha}q/11 leading to stimulation of phospholipase C, formation of inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG), elevation of intracellular free calcium and activation of one or more isoforms of protein kinase C (PKC) (19, 20). These early events underlie GnRH activation of multiple mitogen-activated protein kinase (MAPK) signaling cascades including p38 MAPK, c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) (21). ERK activation by GnRH is dependent on both PKC and calcium influx via L-type voltage-gated calcium channels and proceeds through a c-raf kinase-dependent mechanism (21, 22).

Although classified as a member of the rhodopsin class of GPCR, the GnRHR displays several unique structural characteristics of which perhaps the most striking is an extremely short C-terminal cytoplasmic domain of only 1–2 amino acids (23, 24). In more prototypical GPCRs, this domain is quite extensive and contains phosphorylation sites for G-protein-coupled receptor kinases, second messenger-regulated kinases (such as protein kinase A) and casein kinases. In some GPCRs, these phosphorylation events allow for interaction with {beta}-arrestins and subsequent receptor deactivation and internalization (25, 26). Consistent with the lack of an extensive C-terminal tail, the GnRHR does not appear to undergo arrestin-dependent internalization or desensitization (27–31). In fact, the GnRHR has been considered as a naturally occurring internalization resistant mutant (28). Thus, the GnRHR is both structurally and functionally unusual.

Given its unique structural and functional attributes we sought to assess the membrane distribution of the mammalian GnRHR. Herein, we find that unlike other GPCRs, such as the muscarinic acetylcholine receptor and B2 bradykinin receptor that are uniformly distributed throughout the plasma membrane and localize to caveolae only in the presence of ligand (11, 13), the GnRHR constitutively resides in a low density membrane microdomain in both the gonadotrope-derived {alpha}T3-1 cell line that expresses the endogenous GnRHR gene (32) and Chinese hamster ovary (CHO) cells. We also find that c-raf kinase, a target of GnRHR signaling, is constitutively, but not exclusively, localized to low density membrane fractions. Furthermore, disruption of raft organization by cholesterol depletion attenuates GnRHR activation of ERK and induction of c-fos gene expression suggesting that the sublocalization of GnRHR and c-raf kinase to membrane microdomains is functionally significant.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—The C-terminal anti-GnRHR, anti-caveolin-1, anti-c-Fos, anti-G{alpha}q/11, anti-c-raf kinase, anti-b-raf kinase, anti-p-ERK, and anti-ERK-1 antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). The antibody against H+-ATPase was a generous gift from Dr. Klaus Bayenbach (Cornell University). The anti-flotillin-1 and pan-caveolin antibodies were purchased from BD Transduction Laboratories (San Jose, CA). The anti-tubulin antibody and methyl-{beta}-cyclodextrin (CD) were obtained from Sigma. The anti-HA antibody was purchased from Roche Applied Science (Indianapolis, IN). Anti-goat, anti-rabbit, and anti-mouse secondary antibodies were from Pierce, Santa Cruz Biotechnology, or Bio-Rad. [3H]inositol (14 Ci/mmol) was obtained from Amersham Biosciences. D-Ala6-desGly10-GnRH-Pro9-ethylamine ([D-Ala6]GnRH) was purchased from Sigma.

Cell Culture—{alpha}T3-1 cells were maintained in high glucose DMEM from Mediatech (Herndon, VA) containing 2 mM glutamine, 100 units of penicillin/ml, 100 µg streptomycin/ml, 5% fetal bovine serum, and 5% horse serum. CHO cells were maintained in high glucose DMEM containing 2 mM glutamine, 100 units of penicillin/ml, 100 µg of streptomycin/ml, 10% fetal bovine serum, and 1x nonessential amino acids from Invitrogen. All cells were grown in 5% CO2 at 37 °C in a humidified environment.

Construction of Hemagglutinin-tagged GnRH Receptor Attached to Green Fluorescent Protein (GFP)—The construction of the GnRHR-GFP fusion cDNA has been described (33). A 2-step PCR procedure was used to attach the HA sequence (YDYDVPDYA) immediately adjacent to the initiation codon of the GnRHR-GFP fusion protein. Appropriate placement of the HA tag was confirmed by sequencing.

Preparation of Cholesterol-loaded CD (CLCD)—Cholesterol (200 mg) was dissolved in 1 ml of chloroform. In a separate beaker, 1 g of CD was dissolved in 2 ml of methanol. A 0.45-ml aliquot of the cholesterol solution was added to the CD solution, stirred, and then placed under a stream of nitrogen gas until the chloroform and methanol evaporated. The resulting crystals were allowed to dry for 24 h and stored in glass at room temperature. A CLCD working solution was prepared by adding 50 mg of the CLCD crystals to 1 ml of serum-free DMEM, warming to 37 °C, and vortexing briefly.

Detergent-free Preparation of Lipid Rafts—{alpha}T3-1 cells, a generous gift from Dr. Pam Mellon, or CHO cells were grown to confluence in 150-mm tissue culture plates. Cells were harvested in phosphate-buffered saline (PBS) and centrifuged for 3 min at 300 x g. Cells were resuspended in PBS to a final volume of 1 ml and administered either vehicle or 100 nM GnRH for 15 min at 37 °C followed by centrifugation for 3 min at 300 x g. Detergent-free lipid raft preparations were conducted according to Song et al. (34). The cell pellet was resuspended in 2 ml of 500 mM sodium carbonate buffer (pH 11). Cells were then homogenized using a Wheaton loose fitting glass dounce homogenizer (10 strokes) followed by sonication (three 20-s bursts) on ice using a Branson 250 sonicator. Two ml of 90% sucrose prepared in MES buffer (20% glycerol, 150 mM NaCl, 2 mM EDTA, 25 mM MES, pH 6.5) was added to the homogenized samples yielding a final concentration of 45% sucrose in a total volume of 4 ml. A discontinuous sucrose gradient was then layered on the surface of the 45% fraction (4 ml of 35% sucrose, 4 ml of 5% sucrose in MES containing 250 mM sodium carbonate). Isopycnic ultracentrifugation was then carried out at 38,000 rpm using a SW 41 rotor for 16–20 h at 4 °C. Following ultracentrifugation, 645-µl samples were collected representing a total of 18 fractions. Proteins that migrated to the interface of the 5 and 35% gradients (approximately fractions 6 and 7) were considered to be raft-associated (34).

CD treated samples were prepared as above with the exception that {alpha}T3-1 cells in monolayer were incubated in 12 ml of serum-free medium containing 2% CD for1hat37 °C followed by2hof serum-free medium alone. Control cells were incubated in serum-free medium without CD over the same time period. For the cholesterol repletion studies, {alpha}T3-1 cells were treated with CD as described above. Following CD treatment, cells were washed with PBS and incubated in 12 ml of serum-free medium containing 1 mg/ml of CLCD for 2 h. Raft samples were then prepared as described above.

Triton X-100 Preparation of Lipid Rafts—{alpha}T3-1 or CHO cells were grown to confluence in monolayer cultures. Cells were harvested and treated with hormone as in the detergent-free raft method. Following centrifugation, the cell pellet was resuspended to a final volume of 500 µl in PBS. 500 µl of 2x Triton X-100 lysis buffer (0.2% Triton X-100 prepared in MES buffer) was then added to yield a final 1x lysis buffer concentration (Triton X-100 concentration = 0.1%). Cells were then homogenized either by 3 passes through a 30-gauge needle or douncing. PBS was added to adjust the final volume to 1 ml. 1 ml of 80% sucrose (in MES buffer) was added to the samples to yield a 40% sucrose fraction in a final volume of 2 ml. A discontinuous sucrose gradient was then prepared by layering 2 ml of each sucrose fraction (80, 60, 40%-containing sample, 30, 20, and 10%) in a 12-ml ultracentrifuge tube (35) (Figs. 1D and 2B) or layering 4 ml of 45, 35, and 5% sucrose in a 12-ml ultracentrifuge tube (Fig. 6). Samples were subjected to isopycnic ultracentrifugation in an SW 41 rotor for 20 h at 37,000 rpm. Following ultracentrifugation, 645-µl samples were collected representing a total of 18 fractions and Western analyses were conducted. In Fig. 7, whole mouse pituitaries were obtained following euthanasia, minced, rinsed free of blood in PBS, and lysed in a 0.1% Triton lysis buffer (in MES) by douncing. Whole pituitary lysates were adjusted to 45% sucrose and layered within a sucrose gradient (45 (sample), 35, and 5% sucrose) in a total volume of ~3.5 ml to reduce a potential dilution effect of the larger gradients described for {alpha}T3-1 and CHO cells. These samples were centrifuged as described above in an SW50.1 rotor. Following centrifugation, gradients were harvested in 10 equal fractions.



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  		FIG. 1.
Endogenous GnRHR in {alpha}T3-1 cells localizes to low density membrane microdomains. A, raft samples were prepared using a detergent free carbonate buffer and separated by isopycnic ultracentrifugation through a non-linear sucrose gradient (45, 35, 5%). Sucrose fractions were electrophoresed in PAGE and then silver-stained to determine the efficiency of membrane separation. B, {alpha}T3-1 cells were incubated in the presence or absence of 100 nM GnRH for 15 min at 37 °C. Raft samples were then prepared using a detergent-free carbonate buffer and separated by isopycnic ultracentrifugation through a non-linear sucrose gradient (45, 35, 5%). Fractions were collected from the top and separated by SDS-PAGE. Western blots were conducted using an antibody directed against the C terminus of the GnRHR, tubulin, or flotillin-1. C, whole cell RIPA lysates were prepared from either {alpha}T3-1 or CHO cells. Western blots were conducted using a pancaveolin antibody that detects caveolin-1, caveolin-2, and caveolin-3. D, {alpha}T3-1 cells were incubated in the absence or presence of 100 nM GnRH for 15 min at 37 °C. Samples were then prepared using a 0.1% Triton X-100-based raft preparation and separated by isopycnic ultracentrifugation through a non-linear sucrose gradient (80, 60, 40, 30, 20, 10%). Fractions were collected, and electrophoresis and immunoblotting were performed as above.

 


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  		FIG. 2.
Stably transfected GnRHR localizes to low density membrane microdomains in CHO cells. A, CHO cells stably transfected with HA-GnRHR-GFP were administered either control vehicle or 100 nM GnRH for 15 min at 37 °C. Detergent-free raft samples were prepared as in Fig. 1A. Fractions were collected and separated by SDS-PAGE. Western blots were conducted using antibodies directed against an HA epitope tag on the GnRHR, tubulin, or caveolin-1. B, using a 0.1% Triton X-100 raft preparation as in Fig. 1C, CHO cells were incubated either in the absence of hormone or in the presence of 100 nM GnRH, 10 nM superagonist ([D-Ala6]GnRH), or 10 nM antagonist (antide). Western blots were conducted using an antibody against an HA tag on the GnRHR, tubulin, and caveolin-1.

 


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  		FIG. 6.
GnRH stabilizes the association of c-raf kinase with low density membrane microdomains. A, {alpha}T3-1 cells were incubated in the presence or absence of 100 nM GnRH for 15 min at 37 °C and then solubilized using a 0.1% Triton X-100 detergent raft preparation. Western blots were probed with an antibody specific for c-raf kinase. B, {alpha}T3-1 cells were incubated with 100 nM GnRH for 15 min and solubilized using a 1.0% Triton X-100 raft preparation. Western blots were probed for an antibody specific for c-raf kinase.

 


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  		FIG. 7.
c-raf kinase localizes to low density membrane microdomains in mouse pituitary lysates. A pooled sample representing 10 whole mouse pituitaries was prepared using a 0.1% Triton X-100 raft procedure and then separated by sucrose gradient centrifugation. Ten fractions were collected, and Western blotting was carried out using antibodies specific for c-raf kinase and caveolin-1.

 

Silver Staining—Sucrose gradient fractions from {alpha}T3-1 cells prepared with carbonate lysis were resolved by SDS-PAGE. The resolved proteins within the gel were visualized by silver staining using methods as described by Blum et al. (36). Identical results were obtained using gradient fractions from {alpha}T3-1 lysates generated from 0.1% Triton X-100 lysis buffer (data not shown).

Western Blots—Samples representing individual fractions were subjected to SDS-polyacrylamide gel electrophoresis (acrylamide:bis-acrylamide ratio of 29:1) and electroblotted to nitrocellulose or polyvinylidene difluoride membranes (PerkinElmer Life Sciences). In Fig. 5B, SDS-PAGE was carried out using a 37.5:1 acrylamide:bis-acrylamide ratio to favor the separation of phosphorylated and non-phosphorylated c-raf kinase. Membranes were blocked in 5% nonfat dried milk in Tris-buffered saline (TBS) or TBS containing 0.1% Tween-20 (TBST). Anti-GnRHR antibody (1:500 dilution in 5% milk) was incubated for 8 h at 4 °C on an orbital shaker. Blots were washed for 30 min (3 washes x 10 min) with TBS and then incubated with anti-goat HRP (1:5,000) for 2 h. Anti-G{alpha}q/11 antibody was used at a 1:500 dilution for 2 h at room temperature followed by washing with TBS and incubation with anti-rabbit HRP (1:5,000) for 2 h. Anti-HA and anti-flotillin antibodies were used at a 1:1,000 dilution with an incubation time of 1 h. Blots were washed and then incubated with a 1:10,000 dilution of anti-mouse HRP for 1 h at room temperature. Anti-c-raf, b-raf, and H+-ATPase antibodies were used at a dilution of 1:1,000 and incubations were overnight at 4 °C. Blots were washed in TBST and then incubated with a 1:5,000 dilution of anti-rabbit HRP for 2 h at room temperature. Anti-caveolin-1, anti-tubulin, and anti-pan caveolin were used at a 1:2,000 dilution with a 1:10,000 dilution of the appropriate secondary antibody for 1 h. All blots were washed for 60 min (6 x 10 min) with TBS or TBST after secondary antibody and then visualized by chemiluminescence using either Pierce SuperSignal or PerkinElmer Western Lightening reagents.



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  		FIG. 5.
c-raf kinase localizes to lipid rafts in {alpha}T3-1 cells. A, {alpha}T3-1cells were treated with PBS or 100 nM GnRH for 15 min at 37 °C. Following detergent-free raft preparation and density gradient centrifugation, Western blots were probed for antibodies specific for c-raf kinase, b-raf-kinase and anti-H+-ATPase. B, low density fractions 6 and 7 were isolated from the sucrose gradient and subjected to electrophoresis in a low cross-linking gel to determine the effects of GnRH treatment on c-raf kinase.

 

ERK and c-Fos Activation Assays—Monolayers of {alpha}T3-1 cells in 6-well tissue culture plates were subjected to 1 of 3 treatments. First, control cells were washed with PBS and then incubated with 1 ml of serum-free medium for 3 h. Second, for cholesterol depletion, cells were washed with PBS and incubated with 1 ml of serum-free medium containing 2% CD for 1 h. Cells were then washed with PBS and incubated for2hin serum-free medium. Third, for cholesterol repletion, cells were washed with PBS and administered 1 ml of 2% CD in serum-free medium for 1 h. Cells were then washed with PBS and incubated for 2 h in serum-free medium containing 1 mg/ml CLCD. Following the 3-h treatment protocols, cells were washed with PBS and serum-free medium was replaced containing either 0 or 100 nM GnRH. After a 30- or 60-min incubation, cells were washed in ice cold PBS and lysed in RIPA buffer containing 20 mM Tris (pH 8.0), 137 mM NaCl, 10% glycerol, 1% Nonidet P-40, 0.1% SDS, 0.5% deoxycholate, and 0.2 mM phenylmethylsulfonyl fluoride. 6x sample buffer (300 mM Tris-HCl, pH 6.8, 60% glycerol, 30 mM dithiothreitol, 6% SDS) was added to yield a final concentration of 1x. Aliquots (15 µl) of each lysate were heated to 95 °C for 5 min and subjected to SDS-PAGE and Western analysis. Nitrocellulose membranes were incubated for 2 h with either a phospho-ERK or c-Fos antibody (both at 1:1,000 dilutions) followed by a 2-h incubation with a 1:2,000 dilution of HRP conjugated secondary antibody. Phospho-ERK blots were then stripped at room temperature with 100 mM 2-mercaptoethanol, 2% SDS, 62.5 mM Tris-HCl (pH 6.7) heated to 50 °C for 30 min. After stripping, membranes were washed twice for 15 min with TBS and blocked with 5% milk for 1 h. Blots were then re-probed with a 1:10,000 dilution of an anti-ERK antibody that recognizes ERK-1 and ERK-2 independent of phosphorylation state. Following washing in TBS, blots were incubated with a 1:2,000 dilution of anti-rabbit HRP, and immunoreactive bands were visualized by chemiluminescence.

Cholesterol Assays—{alpha}T3-1 cells were either incubated in serum-free medium alone for 3 h, cholesterol-depleted or cholesterol-depleted followed by incubation with CLCD as described under "ERK and c-Fos Activation Assays" above. At the end of the 3-h treatment period, all cells were washed and harvested in PBS. Cells were pelleted by centrifugation and resuspended to a final volume of 500 µl in PBS. An equal volume of 2x lysis buffer containing 0.2% Triton X-100 in MES buffer cooled to 4 °C was then added to solubilize the plasma membrane. Cholesterol content was determined using Infinity cholesterol reagent (Sigma). Briefly, lysates were diluted 1:5 with cholesterol reagent and samples were incubated for 10 min at 37 °C followed by spectrophotometric analysis (absorption at 500 nm). Cholesterol concentration was determined using a 6-point standard curve generated with known concentrations of cholesterol. To standardize cholesterol content, protein concentrations were determined using a BCA protein assay (Pierce). Data are expressed as mg of cholesterol per mg of protein.

Transmission Electron Microscopy (TEM)—CHO and {alpha}T3-1 cells were cultured to ~60–70% confluence then scraped from dishes in ice-cold Hepes-buffered saline (HBS; pH 7.4) and washed once in HBS. The cell pellets were fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4 for 30 min at room temperature followed by 1.5hat4 °C. The cells were then washed three times 10 min each in 0.1 M sodium cacodylate buffer (pH 7.4). The cells were postfixed in 2% osmium tetroxide for 1 h at room temperature, followed by washes in sodium cacodylate buffer as described above. Cells were then dehydrated through a standard graded ethanol series followed by an acetone rinse. Gradual infiltration of epon araldite resin followed, after which the cells were placed into Beem capsules and cured in a 60 °C oven. The resin blocks were cut on a Reichert OmU2 ultramicrotome and ~70-nm sections were stained with uranyl acetate and lead citrate. The grids were viewed in a Tecnai 12 Biotwin transmission electron microscope (FEI Corp). For quantitation of each cell type, 100 cells were visualized and the number of putative caveolae was obtained. Digital images were captured with a Gatan Model 791 Multiscan Camera.

125I-[D-Ala6]GnRH Binding Assay—[D-Ala6] GnRH was radioiodinated using a glucose-oxidase procedure and purified by chromatography in QAE-Sephadex as described by Wagner et al. (37). Following a 1-h incubation in either serum-free medium alone or serum-free medium containing 2% CD, {alpha}T3-1 cells were washed with PBS and incubated for 2 h in serum-free medium alone. Cells were harvested in ice-cold PBS. Following centrifugation, cell pellets were resuspended in assay buffer (10 mM Tris-HCl, 0.1% bovine serum albumin, .01 mM CaCl2) to a final concentration of 1 x 107 cells/50 µl. Triplicate 12 x 75 mm assay tubes were prepared containing 50-µl aliquots of cell suspension and 5 x 104 cpm of 125I-[D-Ala6]GnRH (61.4 pM) in 50-µl assay buffer in the presence or absence of 50 µl of non-radioactive [D-Ala6]GnRH (340 nM). The total volume for each tube was adjusted to 250 µl by addition of ice-cold assay buffer. Following a 2-h incubation at 4 °C, 3 ml of ice-cold assay buffer was added, and samples were immediately centrifuged for 15 min at 16,000 x g. The supernatants were decanted and radioactivity in the cell pellets was quantitated using an Apex automatic gamma counter (Micromedic systems, Horsham, PA). Specific binding was determined by subtracting the cpm in samples containing 125I-[D-Ala6]GnRH in the presence of unlabeled [D-Ala6]GnRH from the cpm in samples containing only 125I-[D-Ala6]GnRH samples. To standardize binding for protein concentration, a BCA protein assay (Pierce) was performed. The binding activity presented in Fig. 3A (inset) was adjusted for protein concentration.



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  		FIG. 3.
Plasma membrane topologies differ between aT3-1 and CHO cells using TEM. A, 70-nm sections of CHO and {alpha}T3-1 cells were fixed, stained with uranyl acetate and lead citrate, and viewed by transmission electron microscopy. B, to quantify the relative amounts of putative caveolae, the number of membrane invaginations per cell section was counted and the mean was calculated for 100 cell sections.

 

[3H]Inositol Assays—Phospholipase C activity was assessed by quantifying cellular accumulation of phosphorylated inositol using previously described methods (38). Briefly, {alpha}T3-1 cells were plated overnight in 24-well culture plates. The DMEM culture medium was washed from the cells with serum-free M199 culture medium (Mediatech; Herndon, VA) supplemented to 0.37% (w/v) sodium bicarbonate, 20 mM HEPES buffer, 100 units of penicillin/ml, and 100 µg streptomycin/ml. After washing, cells were incubated at 37 °C for 5 h in 0.3 ml of serum-free M199 containing 2 µCi of myo-[2-3H]inositol. The labeled cells were then washed with serum-free DMEM containing 5 mM LiCl and incubated for an additional hour at 37 °C in either 1 ml of serum-free DMEM with 5 mM LiCl or 1 ml of serum-free DMEM containing 5 mM LiCl and 2% CD. After 1 h of cholesterol depletion, this medium was removed and then control and cholesterol-depleted cells remained untreated or were challenged with 100 nM GnRH in 1 ml serum-free DMEM containing 5 mM LiCl. These treatment conditions were maintained at 37 °C for 1 h, after which the medium was removed, and cells were immediately lysed by addition of 0.05 ml of RIPA buffer (described above) and 1 ml of water heated to 95 °C. The cells were then frozen overnight and thawed at room temperature. Cell lysates from each individual well were collected and loaded separately onto Dowex 1-X8, 200–400 mesh, formate-form columns with an approximate bed volume of 0.4 ml. Free, unphosphorylated inositol was eluted from the lysate by the addition of 10 bed volumes of water. After collection of the eluent containing the free inositol, total inositol phosphates were collected by the addition of 10 bed volumes of 1 M ammonium formate in 0.1 M formic acid. The amounts of radioactivity in both the free and phosphorylated inositol eluents were quantitated using a Beckman LS-5000CE liquid scintillation counter. Data are presented as phosphorylated inositol expressed as a percentage of the total [3H]inositol.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
The GnRHR Is a Resident Protein in Low Density Membrane Microdomains—Silver staining of non-detergent fractions from {alpha}T3-1 cells was conducted to assess the efficacy of protein separation using sucrose gradients (Fig. 1A). This analysis revealed enrichment of distinct populations of proteins associated with either low density (fractions 5 and 6) or high density fractions (fractions >10) suggestive of effective separation of raft-associated proteins. To assess membrane sublocalization of the GnRHR, {alpha}T3-1 cells were administered either vehicle or 100 nM GnRH for 15 min. Cells were then sonicated in a detergent-free sodium carbonate buffer and fractions separated in a discontinuous sucrose gradient. Fractions were isolated and subjected to SDS-PAGE followed by Western blotting using an anti-GnRHR, anti-tubulin, or anti-flotillin antibody. Immunodetectable GnRHR localized to low density fractions within the gradient independent of GnRH treatment (Fig. 1A, lanes 6 and 7). Lanes 6 and 7 represent the interface of the 5 and 35% sucrose fractions where raft-associated proteins would migrate (34). The absence of caveolin expression in {alpha}T3-1 cells (Fig. 1C) precluded the utility of this protein as a marker protein for low density membrane microdomains, thus, we used an antibody directed against flotillin, another raft-associated protein (39, 40). Using the same detergent free raft isolation method in {alpha}T3-1 cells, flotillin appropriately localized to low density fractions within the sucrose gradient (Fig. 1B). Not considered a raft-associated protein (41), tubulin did not partition into membrane rafts in these conditions. To confirm GnRHR localization to membrane microdomains, an independent approach based on resistance to detergent solubilization was utilized to prepare samples prior to isopycnic centrifugation. This technique takes advantage of the relative insolubility of lipid rafts in non-ionic detergents at 4 °C. As with the detergent-free raft preparations, the GnRHR localized to the low density fractions regardless of hormone treatment. Tubulin was again localized to high density fractions (Fig. 1D).

To assess whether GnRHR localization to low density membrane microdomains was specific to a gonadotrope-derived, non-caveolin expressing cell line, we next studied an HA-tagged GnRHR-GFP fusion protein stably expressed in CHO cells (33). Using the detergent-free raft preparation, we found that the GnRHR migrated to low density fractions 6 and 7 (the 5/35% interface). As in {alpha}T3-1 cells, the migration of the GnRHR in the sucrose gradient was unaffected by hormone treatment (Fig. 2A). Similar results were evident when cells were prepared using non-ionic detergent (Fig. 2B). Specifically, the GnRHR localized to the low density fractions, and the migration of the GnRHR in the sucrose gradient was unaffected by treatment with agonist (GnRH), super-agonist ([D-Ala6]GnRH), or antagonist (antide). With both the detergent and non-detergent preparations, tubulin failed to migrate out of the high density fractions of the gradient. Finally, unlike {alpha}T3-1 cells, CHO cells express the caveolar raft marker protein caveolin-1 that appropriately localized to low density fractions. Thus, GnRHR localization to membrane microdomains or lipid rafts appears to be independent of cell type or the presence of caveolin-1.

Transmission Electron Microscopy Reveals Topological Differences in the Plasma Membrane of {alpha}T3-1 and CHO Cells— The absence of caveolin expression in {alpha}T3-1 cells suggests that the GnRHR localizes to non-caveolar rafts in homologous cells, consistent with expression of flotillin-1. We wanted to examine the potential topological differences that could possibly exist in the plasma membrane of {alpha}T3-1 and CHO cells. The topological differences should be expressed as the presence of flask-shaped invaginations with the plasma membrane of CHO cells but not {alpha}T3-1 cells. For quantitation, 100 cells were visualized, and the number of flask-shaped invaginations in the plasma membrane of each cell type was determined (Fig. 3A). Consistent with non-detectable levels of caveolin-1, {alpha}T3-1 cells contain 20-fold fewer morphologically distinct membrane invaginations relative to CHO cells (Fig. 3B). These ultrastructural studies support the notion that GnRHR most likely partitions to noncaveolar lipid rafts.

GnRH Activation of ERK and c-Fos Expression Is Lost with Cholesterol Depletion—The microenvironment necessary for raft formation is sensitive to cholesterol depletion (42). Thus, to assess the functional relevance of GnRHR localization in lipid rafts we sought to disrupt raft organization utilizing the cholesterol-sequestering agent, methyl-{beta}-cyclodextrin (CD) (5). {alpha}T3-1 cells were incubated for 1 h in serum-free medium with or without 2.0% CD. 1 h exposure to 2.0% CD effectively reduced cholesterol content by ~55% (Fig. 4A). To address the functional consequence of cholesterol depletion and, presumably, raft disruption we next examined the effects of CD treatment on GnRH activation of ERK. Consistent with our previous studies (22, 43), 30 min exposure to GnRH increased the dual phosphorylated forms of ERK (ERK1 and ERK2) (Fig. 4B). In contrast, phosphorylated ERK was not detectable in cells that had been exposed to 2.0% CD. GnRH activation of c-fos gene expression proceeds through an ERK-dependent mechanism (44, 45). Consistent with this notion, the effects of cholesterol depletion were also evident as a loss of GnRH activation of c-fos expression (Fig. 4C). Finally, it seemed possible that the loss of GnRH activation of ERK and c-fos gene expression in CD-treated cells might simply reflect a significant attenuation in the number of cell surface GnRH receptors available for binding. This would not, however, appear to be the case as cholesterol depleted {alpha}T3-1 cells retained the ability to bind 125I-[D-Ala6]GnRH at levels ~72% that of control cells (Fig. 4A, inset).



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  		FIG. 4.
Cholesterol depletion leads to a loss of ERK and c-Fos activation by GnRH in aT3-1 cells. A, {alpha}T3-1 cells were incubated in serum-free medium containing 2.0% CD for 1 h while control cells received serum-free medium alone. Samples were lysed in MES buffer containing 0.1% Triton X-100. Cholesterol content was assayed as described under "Experimental Procedures" and adjusted for protein concentration. Inset, binding of 125I-[D-Ala6]GnRH (61.4 pM) to {alpha}T3-1 cells ± CD treatment was determined as counts bound in the absence of unlabeled [D-Ala6]GnRH subtracted from counts bound in the presence of unlabeled [D-Ala6]GnRH (340 nM). Binding was then adjusted for protein concentration. B, {alpha}T3-1 cells were treated for 1 h with serum-free medium containing 2% CD while control cells received serum-free medium alone. Cells were then washed with PBS and incubated with serum-free medium containing 0 or 100 nM GnRH for 30 min at 37 °C. Samples were then lysed in RIPA buffer and lysates analyzed by Western blotting using antibodies specific for phosphorylated ERK. After probing with anti-phospho-ERK, blots were stripped and reprobed with an antibody that detects ERK 1 and 2 independent of phosphorylation. C, treatment of {alpha}T3-1 cells was identical to B except that RIPA lysates were prepared following 1 h of GnRH treatment and analyzed in Western blots using a c-Fos-specific antibody.

 

c-raf Kinase Localizes to Low Density Membrane Microdomains in {alpha}T3-1 Cells—GnRH receptor activation of the ERK cascade and c-fos gene expression is thought to proceed through a c-raf kinase-dependent mechanism (22). Thus, we were intrigued with the possibility that the loss of ERK and c-Fos activation associated with cholesterol depletion may reflect disruption of membrane microdomains containing both the GnRHR and c-raf kinase. Consistent with this possibility, we find that c-raf kinase is also localized to low density microdomains in {alpha}T3-1 cells prepared using the non-detergent based approach (Fig. 5A). Partitioning of c-raf kinase to the low density sucrose fractions did not appear to be affected by hormone treatment. Thus, c-raf kinase was constitutively but not exclusively present in lipid rafts. In contrast to c-raf kinase, b-raf-kinase, a closely related member of the raf kinase family, was only evident in high density fractions. Thus, partitioning into lipid rafts appeared to be specific to the c-raf isoform of raf kinase. Also supportive of the specificity of fractionation, an integral membrane protein H+-ATPase migrated to high density sucrose fractions. Finally, to test if raft-associated c-raf kinase is a target for GnRH-mediated phosphorylation, fractions 6 and 7 were isolated from the sucrose gradient and subjected to electrophoresis in a low cross-linking gel. Consistent with GnRH-induced phosphorylation, the electrophoretic mobility of raft-associated c-raf kinase was reduced with GnRH treatment (Fig. 5B). Previous studies have demonstrated that the retarded electrophoretic mobility of c-raf kinase correlates with GnRH-induced catalytic activation of this enzyme (22).

GnRH Binding Enhances the Association of c-raf Kinase with Lipid Microdomains—We next sought to confirm raft localization of c-raf kinase using the detergent-based preparation. As with the non-detergent approach, immunoblots of lysates prepared using a 0.1% Triton X-100 lysis buffer revealed constitutive but not exclusive localization of c-raf kinase low density fractions independent of hormone treatment (Fig. 6A). It is interesting to note, however, that increasing the concentration of Triton X-100 from 0.1 to 1.0% resulted in a distinctly different pattern of segregation such that c-raf kinase was evident in the low density fractions only under conditions of GnRH binding (Fig. 6B). Thus, in the unbound state, the association of c-raf kinase in lipid rafts was more susceptible to disruption by non-ionic detergent suggesting that GnRH treatment may act to stabilize or enhance the association of c-raf kinase in lipid microdomains.

c-raf Kinase Is Present in Low Density Membrane Microdomains in Mouse Pituitary Cells—To address whether c-raf kinase localization to lipid rafts was a unique property of the {alpha}T3-1 cell line, detergent-free raft preparations were prepared from CHO, JEG3 (choriocarcinoma), and NIH 3T3 cells and subjected to sucrose density gradient centrifugation. As with {alpha}T3-1 cells, a raft-associated population of c-raf kinase was detectable in all 3 cell lines (data not shown). Thus, c-raf kinase is present in low density membrane microdomains independent of cell type and expression of caveolin-1. Most importantly, c-raf kinase was also evident in low density fractions prepared from whole pituitary lysates from adult female mice (Fig. 7). Pituitary expression of caveolin-1 allowed us to use this protein as a marker for low density microdomains. Clearly, we do not suggest that these results reflect a unique contribution of GnRHR expressing cells (gonadotropes) as there are multiple cell lineages present in the adult pituitary. These data do, however, demonstrate that raft localization of c-raf kinase is evident in non-transformed, GnRHR expressing tissues and is not simply an aberrant feature of the {alpha}T3-1 cell line. Unfortunately, we were unable to detect the GnRHR in mouse pituitaries due to the low number of gonadotropes and insufficient protein concentrations.

Disruption of Raft Localization of GnRHR by CD Treatment of {alpha}T3-1 Cells Is Reconstituted by Cholesterol Repletion—In Fig. 4, we demonstrate that GnRH activation of ERK and c-Fos expression is lost in cholesterol-depleted {alpha}T3-1 cells. Presumably, the loss of cholesterol is associated with raft disruption. If correct, then the effects of CD should be revealed as a loss of both GnRHR and potentially c-raf kinase in low density sucrose fractions. Furthermore, if the effects of CD are specific to cholesterol depletion then reconstitution of cholesterol content should accordingly reconstitute raft microdomains. To directly test these possibilities, {alpha}T3-1 cells were incubated in the presence or absence of 2.0% CD for 1 h. Cholesterol repletion was accomplished by incubation of cholesterol-depleted cells with 1 mg/ml CLCD for 2 h. Cellular lysates were then prepared in sodium carbonate buffer and subjected to sucrose density gradient centrifugation. As in the previous study (Fig. 4), 1 h exposure to CD resulted in an approximate 55% reduction in cholesterol in CD-treated cells as compared with control cells (Fig. 8A). Subsequent incubation of cholesterol-depleted cells with CLCD was effective in restoring cholesterol content to ~85% of control levels (Fig. 8A). Consistent with raft disruption, cholesterol depletion was associated with a loss of GnRHR from the low density fractions 6 and 7 (Fig. 8B). Importantly, however, cholesterol repletion was effective in reconstituting the localization of GnRHR to low density fractions in the sucrose gradient (Fig. 8B). In contrast, partitioning of c-raf kinase into lipid rafts was not remarkably affected by cholesterol depletion (Fig. 8C) suggesting that fundamental differences exist in the mechanism(s) underlying the partitioning of c-raf kinase and GnRHR into low density compartments.



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  		FIG. 8.
Disruption of GnRHR raft localization can be rescued by cholesterol repletion. A, {alpha}T3-1 cells were incubated for 1 h in serum-free medium containing 2.0% CD at 37 °C. Cells were then incubated with either 1 mg/ml of CLCD or serum-free medium alone for 2 h. Control cells received 3 h of serum-free medium alone. Cholesterol content was assayed as described under "Experimental Procedures" and then adjusted for protein concentrations. B, {alpha}T3-1 cells were incubated for 1 h in serum-free medium containing 2.0% CD at 37 °C followed by treatment with 1 mg/ml of CLCD or serum-free medium alone for 2 h. Western blots were probed with an antibody that detects GnRHR. C, {alpha}T3-1 cells were incubated for 1 h in serum-free medium containing 2.0% CD at 37 °C. Cells were then incubated with serum free media alone for 2 h. Control cells received 3 h of serum-free medium alone. Western blots were probed with an antibody that detects c-raf kinase.

 

Cholesterol Repletion Reconstitutes GnRH Activation of ERK and c-Fos Expression in CD-treated {alpha}T3-1 Cells—Based on the data in the previous section, incubation of cholesterol depleted {alpha}T3-1 cells with CLCD effectively restored cholesterol content and raft localization of GnRHR. Next, we sought to determine if this is sufficient to reconstitute GnRH signaling to ERK and c-Fos. Accordingly, GnRH activation of ERK and c-Fos expression was assessed utilizing the same cholesterol depletion/repletion paradigm. As in Fig. 4, GnRH-induced phosphorylation of ERK and c-Fos expression was lost upon incubation of {alpha}T3-1 cells for 1 h in 2.0% CD (Fig. 9). Partial reconstitution of both parameters was, however, evident after incubation of cholesterol-depleted cells with CLCD (Fig. 9).



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  		FIG. 9.
Repletion of membrane cholesterol in {alpha}T3-1 cells restores the ability of GnRHR to signal to ERK and c-Fos. {alpha}T3-1 cells were incubated for 1 h in serum-free medium containing 2.0% CD at 37 °C. Following the CD treatment, cells were incubated with either 1 mg/ml of CLCD or serum-free medium alone for 2 h. Control cells received serum-free medium alone for 3 h. After treatments, media was removed and replaced with serum-free medium containing 100 nM GnRH for 1 h. Separate Western blots were then probed with antibodies specific for the phosphorylated forms of ERK and c-Fos. Phospho-ERK blots were subsequently stripped and reprobed with an antibody that detects ERK independent of phosphorylation.

 

Cholesterol Depletion Uncouples GnRHR- but Not Phorbol Ester-mediated Activation of ERK—To begin to localize the lesion in GnRH signaling to ERK we next asked if ERK activation in response to phorbol ester (PMA) treatment was retained in cholesterol-depleted cells. The use of PMA in these studies would, presumably, directly activate PKC isozymes thus effectively bypassing the GnRHR, G{alpha}q/11, and phospholipase C. Consistent with earlier studies (43–46), both GnRH and PMA induced ERK phosphorylation in control cells (Fig. 10A). As expected, cholesterol depletion resulted in a loss of GnRH-induced ERK phosphorylation. Importantly, however, CD treatment did not visibly compromise ERK activation in response to PMA. Thus, cholesterol depletion and the resulting raft disruption appears to uncouple the GnRHR from signaling intermediates that lie upstream of PKC isozymes and may, in fact, reflect uncoupling of the GnRHR from its cognate heterotrimeric G-protein complex. Consistent with this possibility, we find that, like the GnRHR, cholesterol depletion of {alpha}T3-1 cells leads to an attenuation in the amounts of immunodetectable G{alpha}q/ll localized to low density fractions (Fig. 10B) suggesting that raft organization may be critical for GnRH coupling to G{alpha}q. If correct, disruption of raft organization should be revealed as a loss of GnRH signaling to phospholipase C and, as a consequence, attenuation in the ability of GnRH to liberate IP3 from membrane phospholipid. In accordance with this we find that CD treatment virtually eliminates the GnRH-induced IP3 response in {alpha}T3-1 cells (Fig. 10C).



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  		FIG. 10.
Cholesterol depletion uncouples GnRH but not phorbol ester-mediated activation of ERK. A, {alpha}T3-1 cells were incubated for 1 h in serum-free medium containing 2.0% CD at 37 °C. CD media was removed and replaced with serum-free medium for 2 h. Control cells received serum-free medium alone for 3 h. After 2 h of serum-free medium, samples were incubated with 100 nM GnRH or 100 nM PMA for either 30 or 60 min. Western blots were then probed with antibodies specific for the phosphorylated forms of ERK. Phospho-ERK blots were subsequently stripped and reprobed with an antibody that detects ERK independent of phosphorylation. B, {alpha}T3-1 cells were incubated in the presence or absence of 2.0% CD for 1 h at 37 °C followed by detergent-free raft preparation and density gradient centrifugation as described under "Experimental Procedures." Western blots were probed using an antibody specific for G{alpha}q/11. C, {alpha}T3-1 cells were preloaded with [3H]inositol for 5 h and then incubated with or without 2.0% CD for 1 h followed by treatment with either 0 or 100 nM GnRH for an additional hour. Lysates were prepared and subjected to ion exchange chromatography to separate free and phosphorylated inositol. Phosphorylated inositol is expressed as a percentage of total [3H]inositol.

 


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The organization of cell surface receptors and their cognate signaling proteins into pre-formed signaling platforms contributes to maintaining the specificity and integrity of ligand-induced intracellular signaling cascades (47). Furthermore, it appears that these signaling platforms are not uniformly distributed throughout the plasma membrane but rather are often localized to discrete microdomains characterized by an enriched population of sphingolipids and cholesterol (1). The relative buoyancy of these microdomains in sucrose gradients accounts for the evolution of the term "lipid raft" as a general descriptor of these regions of the plasma membrane (1). Over the past 3–5 years, a wide array of signaling proteins, scaffolding proteins, and receptors has been shown to associate with lipid rafts (6). Typically, this association is characterized as either static or dynamic. For example, caveolin is constitutively associated with and, in fact, defines a unique subpopulation of microdomains termed caveolae (3). Dynamic partitioning from the bulk plasma membrane to lipid rafts characterizes ligand activation of a number of cell-surface receptors including members of the GPCR superfamily such as the muscarinic acetylcholine receptor and B2 bradykinin receptor (11, 13). In contrast to this dynamic behavior, we find that, independent of ligand activation, the mammalian GnRHR is constitutively localized to non-caveolar lipid rafts in the gonadotrope-derived {alpha}T3-1 cell line. Raft localization of the GnRHR was equally evident in heterologous cells (CHO) suggesting that this constitutive segregation into lipid rafts is not unique to {alpha}T3-1 cells but rather is intrinsic to the GnRHR itself.

As yet we do not know what structural features of the GnRHR account for raft localization. However, perhaps the most unique structural feature of this GPCR is the virtual absence of an intracellular C terminus (24). In more prototypical GPCRs, this region is quite extensive and important for coupling to G-proteins, agonist-induced receptor internalization, and phosphorylation-mediated desensitization (48). Phosphorylation of the C terminus is typically thought of as requisite for subsequent interaction with {beta}-arrestin, which hinders further G-protein activation and targets the deactivated receptors for internalization (26, 49). Due to the absence of an intracellular C terminus, the mammalian GnRHR is neither phosphorylated nor internalized via a {beta}-arrestin-dependent mechanism (29, 31, 50). Finally, in several GPCRs, cysteine residues located in the intracellular C terminus are targets for reversible palmitoylation (51–53), a particularly intriguing observation as palmitoylation has been implicated in increasing protein affinity with lipid rafts and caveolae (54, 55). In short, given this fundamental difference in the structural and functional properties of the GnRHR vis a vis prototypical GPCRs, it is tempting to speculate that the unusual behavior of the GnRHR in the plasma membrane may partially reflect the absence of an intracellular C terminus. In this regard, it should be of particular interest to examine the inmembrane behavior of non-mammalian and Type II GnRH receptors, which possess intracellular C-terminal tails (56–58).

Regardless of the structural features of the GnRHR that direct raft association it appears that localization of the GnRHR to lipid rafts is functionally significant. In support of this notion, raft disruption resulting from the removal of cholesterol leads to a loss of GnRH activation of ERK and c-fos gene expression. Importantly, this loss of signaling was not due to a loss of cell-surface binding and was reversible by cholesterol replenishment and reconstitution of lipid rafts. It is also important to underscore that the lesion in GnRH signaling resulting from cholesterol depletion was not reflected as a generalized loss of signaling. Specifically, while GnRH activation of ERK was lost in CD-treated cells, this same paradigm had little effect on the efficacy of PMA induced ERK phosphorylation. Thus, the ERK signaling cascade in cholesterol-depleted {alpha}T3-1 cells is sufficiently intact to transduce a PMA signal but not a GnRH signal. As such, the primary lesion in the GnRH response likely lies upstream of PKC. Based on several lines of evidence we suggest that this lesion may partially reflect uncoupling of the GnRHR from its cognate heterotrimeric G-protein complex. First, consistent with others (68), we find that the presence of G{alpha}q in low density membrane fractions is susceptible to disruption by cholesterol depletion. Second, cholesterol depletion significantly attenuates the ability of GnRH to increase intracellular levels of IP3.

GnRH activation of ERK proceeds through c-raf kinase (22, 59). Consistent with this, we find that, like the GnRHR, c-raf kinase also segregates into low density sucrose fractions prepared from {alpha}T3-1 cells, whole mouse pituitaries, and several clonal cell lines. Thus, the localization of at least a portion of c-raf kinase to low density membrane compartments is not cell type specific. It is also clear, however, that the localization of c-raf to low density microdomains is not exclusive. The limited presence of c-raf kinase in lipid rafts relative to the larger pool of c-raf kinase associated with cytosol or other membrane compartments (higher density fractions) may reflect the rate-limiting presence of a putative platform or scaffolding protein(s) reminiscent of the yeast protein Ste5p that is known to bind multiple members of the ERK signaling cascade (60). Similarly, several mammalian scaffold proteins have also been characterized including 14-3-3 proteins and multiple isoforms of JNK inhibitory protein (JIP) (7, 8, 61). If correct, then compartmentalization of such a scaffold or platform into lipid rafts associated with GnRHR would reflect a key mechanism for the initiation and organization of GnRH signaling in gonadotropes. Finally, we should point out that caveolin itself has been implicated as a scaffolding protein (7); however, the absence of expression of caveolin in {alpha}T3-1 cells would suggest that this protein is not requisite for organizing the GnRHR and c-raf kinase into a signaling platform or scaffold.

The constitutive presence of c-raf kinase within lipid rafts is consistent with studies of the insulin signaling cascade and, more recently, the use of artificial rafts to examine the interaction of c-raf kinase with lipid microdomains. In regard to the latter, U. Rapp and co-workers (62) demonstrate c-raf kinase preferentially associated with artificial lipid rafts with high affinity. In the absence of conditions that promote lipid raft formation, raf kinase displayed moderate binding affinity directly with cholesterol; however, the binding affinity increased markedly in the context of lipid rafts. Similar observations with c-raf kinase were demonstrated for binding of ceramides within lipid rafts. In bulk plasma membrane not associated with rafts, c-raf kinase associated with phospholipids such as phosphotidylserine and phosphatidic acid, a product of phospholipase D catalytic activity. In addition to these studies, c-raf kinase has been shown to associate with lipid rafts in the context of insulin signaling via recruitment into internalized endosomes (63, 64). In this system, c-raf kinase appears to be recruited to membrane rafts by an interaction with raft-associated phosphatidic acid generated by insulin activation of phospholipase D. This interaction appears to be necessary for activation of the ERK cascade in a ras-dependent manner. This mechanism stands in contrast to the present studies in which c-raf kinase is constitutively present in low density fractions independent of any recruitment by signaling intermediates downstream of GnRHR activation.

In the insulin signaling system, cholesterol depletion and, presumably, raft disruption blocks the recruitment of ras to lipid rafts and ERK activation but does not appear to affect c-raf kinase membrane association or activation (63). In the case of {alpha}T3-1 cells, cholesterol depletion to levels ~50% of control cells was sufficient to disrupt GnRHR association with lipid rafts. These same conditions, however, did not affect the association of c-raf kinase with lipid rafts. Thus, it is possible that the association of c-raf kinase with lipid rafts in {alpha}T3-1 cells may be independent of cholesterol content within the membrane. Alternatively, if raft association of c-raf kinase in {alpha}T3-1 cells is cholesterol dependent then it is possible that the cholesterol depletion was not sufficient to disrupt this association. Unfortunately, a marked reduction in {alpha}T3-1 cell viability precludes more extensive cholesterol depletion. Finally, it is interesting to note that the association of c-raf kinase within membrane rafts was sensitive to increasing levels of non-ionic detergent. In relatively low detergent conditions, c-raf kinase was constitutively localized to membrane rafts independent of GnRH administration. However, while increasing detergent concentrations effectively reduced c-raf localization to membrane rafts in unstimulated {alpha}T3-1 cells, GnRH receptor activation restored c-raf kinase to this low density membrane compartment. This increased resistance to detergent solubilization raises the possibility that GnRH action resulting in modification of c-raf kinase (such as phosphorylation) may increase the stability of the association of c-raf kinase with lipid microdomains.

Since the isolation of the first cDNA encoding the mammalian GnRHR much progress has been made in identifying structure-function relationships in this unique GPCR (23, 24). Additionally, the use of epitope- or fluorophore-tagged GnRH receptors has allowed direct observations of the "in-membrane" behavior of unoccupied, agonist occupied, and antagonist-occupied receptors. For example, we and others (65–67) have utilized resonance energy transfer methods to demonstrate that agonist but not antagonist leads to self-association of GnRH receptors in the plasma membrane. Based on the present studies, we suggest that this self-association occurs in the context of discrete lipid microdomains that contain not only the GnRHR but also downstream signaling components, such as G{alpha}q and c-raf kinase, that serve to transduce a GnRH signal to the level of ERK activation. As such, disrupting these microdomains effectively lesions the ability of GnRH to activate ERK and increase c-fos gene expression. In this model then, the GnRHR exists as a resident protein in a pre-formed signaling platform that is poised to both receive and efficiently transmit the GnRH signal. Confirmation of this model awaits definition of the complement of proteins organized within this platform including identification of potential scaffolding proteins and definition of the structural features of the GnRHR that direct its association with lipid microdomains.


   FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

To whom correspondence should be addressed. Tel.: 970-491-7571; Fax: 970-491-3557; E-mail: colin.clay{at}colostate.edu.

1 The abbreviations used are: GPCR, G-protein-coupled receptor; GnRH, gonadotropin-releasing hormone; DMEM, Dulbecco's modified Eagle's medium; MES, 4-morpholineethanesulfonic acid; HA, hemagglutinin; HRP, horseradish peroxidase; PBS, phosphate-buffered saline; RIPA, radioimmune precipitation assay buffer; ERK, extracellular signal-regulated kinase; TEM, transmission electron microscopy; PKC, protein kinase C; CHO, chinese hamster ovary; CD, methyl-{beta}-cyclodextrin; IP3, inositol 1,4,5-trisphosphate; MAPK, mitogen-activated protein kinase. Back



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 ABSTRACT
 INTRODUCTION
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 DISCUSSION
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