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Molecular Endocrinology, doi:10.1210/me.2006-0289
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Molecular Endocrinology 21 (2): 538-549
Copyright © 2007 by The Endocrine Society

Signaling Complexes Associated with the Type I Gonadotropin-Releasing Hormone (GnRH) Receptor: Colocalization of Extracellularly Regulated Kinase 2 and GnRH Receptor within Membrane Rafts

Stuart P. Bliss, Amy M. Navratil, Matthew Breed, Donal C. Skinner, Colin M. Clay and Mark S. Roberson

Department of Biomedical Sciences (S.P.B., M.B., M.S.R.), Cornell University, Ithaca, New York 14853; Department of Biomedical Sciences (A.M.N., C.M.C.), Colorado State University, Fort Collins, Colorado 80523; and Department of Zoology and Physiology (D.C.S.), University of Wyoming, Laramie, Wyoming 82071

Address all correspondence and requests for reprints to: Mark S. Roberson, Ph.D., T3-004d Veterinary Research Tower, Department of Biomedical Sciences, Cornell University, Ithaca, New York 14853. E-mail: msr14{at}cornell.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Our previous work demonstrated that the type I GnRH receptor (GnRHR) resides exclusively and constitutively within membrane rafts in {alpha}T3-1 gonadotropes and that this association was necessary for the ability of the receptor to couple to the ERK signaling pathway. G{alpha}q, c-raf, and calmodulin have also been shown to reside in this compartment, implicating a raft-associated multiprotein signaling complex as a functional link between the GnRHR and ERK signaling. In the studies reported here, we used subcellular fractionation and coimmunoprecipitation to analyze the behavior of ERKs with respect to this putative signaling platform. ERK 2 associated partially and constitutively with low-density membranes both in {alpha}T3-1 cells and in whole mouse pituitary. Cholesterol depletion of {alpha}T3-1 cells reversibly blocked the association of both the GnRHR and ERKs with low-density membranes and uncoupled the ability of GnRH to activate ERK. Analysis of the kinetics of recovery of ERK inducibility after cholesterol normalization supported the conclusion that reestablishment of the association of the GnRHR and ERKs with the membrane raft compartment was not sufficient for reconstitution of signaling activity. In {alpha}T3-1 cells, the GnRHR and ERK2 coimmunoprecipitated from low-density membrane fractions prepared either in the presence or absence of detergent. The GnRHR also partitioned into low-density, detergent-resistant membrane fractions in mouse pituitary and coimmunoprecipitated with ERK2 from these fractions. Collectively, these data support a model in which coupling of the GnRHR to the ERK pathway in gonadotropes involves the assembly of a multiprotein signaling complex in association with specialized microdomains of the plasma membrane.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
THE COMPLEX SIGNALING events initiated by engagement of the mammalian type I GnRH receptor (GnRHR) have been the subject of much investigation. In gonadotropes, the GnRHR couples to G{alpha}q/11, leading to activation of phospholipase Cß, and subsequent elaboration of the second messengers, phosphatidylinositol-3-phosphate and diacylglycerol (1). Diacylglycerol-dependent activation of protein kinase C isozymes contribute to a sharp rise in intracellular calcium concentration, which derives from both phosphatidylinositol-3-phosphate-mediated release of intracellular calcium stores, as well as influx of extracellular calcium through L-type voltage-gated channels (2, 3). GnRH also induces rapid activation of the MAPK pathways, of which the ERK pathway has been most thoroughly characterized. In gonadotropes, activation of the ERK pathway has been linked to the expression of a complement of immediate early genes such as c-fos (4, 5), ATF-3 (6), and Per-1 (7), as well as several genes essential for gonadotrope function, including the glycoprotein hormone {alpha}-subunit (8), the LHß subunit (5, 9), and a regulatory MAPK phosphatase (MKP2) (10). Several requirements for ERK activation by GnRH have been defined, including influx of extracellular calcium through L-type voltage-gated calcium channels (11), activation of protein kinase C isozymes (12, 13), and calmodulin (14). Nevertheless, the precise mechanism by which the GnRHR couples to the ERK pathway remains unclear.

An important mechanism for regulation of signaling activity that has received widespread attention in recent years involves compartmentalization of signaling molecules through association with specialized low-density membrane microdomains, termed "membrane rafts" (15, 16, 17). These domains are cholesterol- and sphingolipid-rich regions of membrane that appear in many cell types to function as platforms for the assembly of multiprotein signaling complexes (18). Rafts are enriched in lipid species with highly saturated acyl chains, particularly glycosphingolipids and sphingomyelin (19). Interactions between sphingolipids and cholesterol contribute to the formation of a liquid-ordered (lo) phase. The lo phase is described as a localized lipid environment in which the acyl chains of individual lipid molecules assume an extended, closely packed, and highly ordered conformation akin to a gel phase, but in which the lipids retain high lateral mobility within the plane of the membrane, as in a fluid phase (20). Liquid-ordered domains present a unique membrane environment for which integral and peripheral membrane proteins may show highly different affinities. These domains thus provide a mechanism for regulation of protein-protein interactions through the partitioning of individual proteins into, or out of, lo regions.

We have previously reported that the GnRHR resides constitutively and exclusively within a detergent-resistant, low-density membrane environment in {alpha}T3-1 gonadotrope cells (21). Cholesterol depletion shifted the receptor into a detergent-soluble nonraft environment and led to a reversible blockade of ERK activation by GnRH. The GTP-binding protein G{alpha}q/11, as well as c-raf, the putative upstream activator of the ERK pathway in these cells, colocalized with the receptor in low-density membrane fractions, suggesting that these membrane domains may be of functional significance in coupling the GnRHR to components of the ERK pathway (21). To further explore this model, we now report the use of subcellular fractionation techniques to examine the behavior of the ERK proteins with respect to association with low-density membrane domains. We find that ERKs 1 and 2 associate with low-density domains of the plasma membrane in conjunction with the GnRHR in a constitutive and cholesterol-dependent manner, both in {alpha}T3-1 gonadotropes and in whole mouse pituitary. Membrane-associated ERKs are activated rapidly after exposure of cells to a GnRH agonist. In addition, the GnRHR and ERKs coimmunoprecipitate from low-density membrane preparations, lending further support for our model of a raft-associated multiprotein signaling complex as a functional link between the GnRHR and the ERK signaling module.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
GnRHR Colocalizes with the Ganglioside GM1 in {alpha}T3-1 Cell Plasma Membrane
Low-density membrane microdomains (membrane rafts) are enriched in the ganglioside GM1. The B subunit of cholera toxin (CtxB) binds specifically to plasma membrane GM1 and has been used as a marker for GM1-enriched plasma-membrane subdomains in living cells (22). We have reported biochemical data indicating that the GnRHR resides constitutively within low-density membrane domains in a cholesterol-dependent manner (21). To further characterize the tendency of the GnRHR to partition into subdomains of the plasma membrane, we probed {alpha}T3-1 cells that stably express a GnRHR-green fluorescent protein (GFP) fusion protein with Alexa-594-conjugated CtxB and examined the cells by laser confocal scanning microscopy (Fig. 1Go). GnRHR-GFP localized exclusively to the plasma membrane and colocalized with GM1-positive domains. As a control, endogenously expressed transferrin receptor did not colocalize with CtxB-Alexa-594/GM1 -positive membrane domains, suggesting that GnRHR colocalization with GM1 was specific in this assay. Consistent with our previous biochemical studies, these data indicate that the GnRHR resides constitutively within regions of the plasma membrane marked by GM1.


Figure 1
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  		Fig. 1. GnRHR Colocalizes with GM1-Positive Membrane Domains in {alpha}T3-1 Cells

A, {alpha}T3-1 cells transiently expressing a GnRHR-GFP fusion protein were stained with Alexa 594-conjugated CtxB and fixed. Confocal images of the separated fluorescence channels show membrane localization of the GnRHR-GFP (green channel) and Alexa 594 GM1 (red channel). Merged images show the overlay of the two flurophores. {alpha}T3-1 cells were stained for GM1 using Alexa 594 CtxB. Cells were then fixed and immunostained for transferrin receptor (TfR) using an Alexa 488-conjugated second antibody. Confocal images of the separated fluorescence channels show membrane localization of the TfR (green channel) and Alexa 594 GM1 (red channel). Merged images show the overlay images of the two flurophores. B, Using quantitative colocalization analysis, the merged GnRHR-GFP and Alexa 594 GM1 image was computed and expressed as a scatter diagram. Colocalization of fluorophores produces a clean diagonal line running from the bottom left to the top right indicating that GnRHR and GM1 are colocalized on the plasma membrane. In contrast, differences between fluorescence localization causes an irregular distribution in the scatter diagram indicating that TfR and GM1 display little colocalization.

 
Anti-GnRHR Immune Serum Binds Specifically to Its Cognate Epitope in Both Immunoblotting and Immunohistochemical Applications
The anti-GnRHR antibody used in these studies was raised against a conserved region of the second extracellular loop of the mammalian type I GnRHR. To validate the performance of this antibody in the studies reported here, we treated {alpha}T3-1 cells with GnRHa (the GnRH agonist buserelin) for various time periods and examined whole-cell lysates for GnRHR protein expression by immunoblot analysis. As shown in Fig. 2AGo, the GnRHR antiserum generated a predominant band of immunoreactivity of approximately 50 kDa. Consistent with previous reports of GnRH-induced up-regulation of GnRHR in gonadotropes (23), the target of this antibody showed clear up-regulation in response to GnRHa. When used for immunohistochemical examination of sections of mouse pituitary, the same antiserum discretely marked a subpopulation of cells within the anterior lobe; costaining with an antibody against LHß subunit indicated nearly complete colocalization of GnRHR and LHß immunofluorescence (Fig. 2BGo). These validation studies provide evidence that this antibody recognizes both denatured and nondenatured conformations of its cognate epitope on the GnRHR with high specificity.


Figure 2
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  		Fig. 2. Validation of GnRHR Antiserum

A, {alpha}T3-1 cells were treated with GnRHa for the indicated times. Whole-cell lysates were analyzed by SDS-PAGE and immunoblotting using anti-GnRHR immune serum. ß-Actin was used as a loading control. B, Histological sections of mouse pituitary were probed simultaneously with anti-GnRHR immune serum and an antibody against LH. Sections were then incubated in fluorochrome-conjugated secondary antibodies, mounted, and examined by fluorescence microscopy. AP, Anterior pituitary; FITC, fluorescein isothiocyanate; PP, posterior pituitary.

 
ERK 2 Associates Constitutively with Detergent-Resistant, Low-Density Membrane Domains in {alpha}T3-1 Cells
We previously reported that the GnRHR, G{alpha}q/11, and c-raf, the putative upstream activator of the ERK signaling pathway in gonadotropes, associate constitutively with low-density, detergent-resistant membrane domains (DRMs) in {alpha}T3-1 cells (21). Based on these data, we proposed that raft domains may play a key role in GnRH-induced ERK activation by serving as a platform for the association of key intermediates of this signaling pathway. To further explore this model, we fractionated {alpha}T3-1 cells in the presence of 0.1% Triton X-100 and analyzed low-density fractions for the presence of the ERK proteins. A small proportion of total cellular ERK 2 partitioned constitutively into low-density fractions (Fig. 3AGo). When the microsomal suspensions in the low-density fractions were rehomogenized and repelleted by high-speed centrifugation, ERK immunoreactivity appeared only in the membrane pellet, indicating that the ERK protein recovered in association with DRMs is indeed membrane-associated (data not shown). Phosphorylated ERK was detected in the low-density fractions within 5 min of GnRHa administration, and the kinetics of activation of membrane-associated ERK appeared to parallel the kinetics of non-membrane-associated ERK. (Fig. 3BGo). Consistent with our previous results, the GnRHR, as well as the G{alpha}q/11 heterotrimeric G-protein subunit and the lipid raft marker flotillin-1, associated preferentially with the low-density fractions (Fig. 3AGo). As a further control, the membrane-associated {alpha}1-subunit of the L-type voltage-gated calcium channel was not present in low-density fractions. This latter observation supports the specificity of the fractionation method and provides a useful negative control for analysis of membrane rafts in gonadotropes.


Figure 3
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  		Fig. 3. ERK2 Associates with Detergent-Resistant, Low-Density Membrane Fractions in {alpha}T3-1 Cells

A, {alpha}T3-1 cells were homogenized in the presence of 0.1% Triton X-100 and subjected to discontinuous sucrose density gradient centrifugation. Fractions were collected from the top of the gradient and analyzed by SDS-PAGE and immunoblotting using antibodies against the GnRHR, G{alpha}q/11, and ERK2, as well as the lipid raft marker flotillin-1, and the non-raft-associated Cavpan {alpha}-1 calcium channel subunit. B, Cells were treated with GnRHa or vehicle for the indicated times and fractionated as in panel A. Fractions were collected and aliquots of low- and high-density fractions were analyzed by SDS-PAGE and immunoblotting using antibodies against phosphorylated ERK (p-ERK) and ERK2.

 
The DRMs analyzed in Fig. 3Go were isolated after homogenization of cells in the presence of 0.1% Triton X-100. Under these conditions, the association of ERKs with low-density membranes was unaffected by administration of GnRHa (data not shown). In contrast, homogenization of unstimulated cells in the presence of 1.0% Triton X-100 interrupted the association of ERKs with DRMs (Fig. 4AGo). However, after 15 min of exposure to GnRHa, the association of ERKs with DRMs was rendered refractory to the effects of high-detergent concentration (Fig. 4AGo). Phosphorylated ERK was also detected in the low-density fractions of GnRHa-treated cells after homogenization in the presence of the higher detergent concentration (Fig. 4BGo). This observation is consistent with the behavior of c-raf under similar experimental conditions and suggests that the signaling activity induced by GnRHR occupancy is capable of stabilizing interactions among the participants of a putative raft-associated signaling complex.


Figure 4
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  		Fig. 4. GnRHa Stabilizes the Association of ERK2 with Detergent-Resistant, Low-Density Membrane Domains in {alpha}T3-1 Cells

Cells were treated with GnRHa or vehicle for 15 min and then homogenized in the presence of 1.0% Triton X-100. Homogenates were fractionated by discontinuous sucrose density gradient centrifugation, and fractions were collected from the top of the gradient. Fractions were analyzed by immunoblot using an antibody against ERK2 (A) or phosphorylated ERK (p-ERK, panel B).

 
ERK2 and the GnRHR Associate Preferentially with Low-Density Membranes Isolated in the Absence of Detergent
Resistance to detergent solubilization is an important operational characteristic of membrane rafts (24). Indeed, sucrose density gradient centrifugation of cell homogenates prepared in the presence of low concentrations of nonionic detergents remains the most common method for isolation of these membrane domains. However, controversy remains regarding the effects of detergents on cellular membranes, and some evidence suggests that detergent solubilization may itself enhance the ability of lipid bilayers to adopt a lo conformation (25). To further assess the validity of our observations regarding the association of ERKs with DRMs, we adapted a technique based on sucrose density gradient centrifugation of postnuclear supernatant for fractionation of subcellular membranes in the absence of detergent (specific protocol found at http://www.i-med.ac.at/cellbio/downloads/preparative_gradients.RTF). Using this method, Huber and colleagues (30) describe clear separation of low-, intermediate-, and high-density subcellular membrane fractions enriched in late endosomes/lysosomes, early endosomes, and plasma membrane, respectively. We reasoned that this method might simultaneously result in separation of low- and high-density subdomains of plasma membrane, thereby providing a complementary method for detergent-free isolation of membrane rafts. Initially, we used cell surface biotinylation to track the plasma membrane proteins during the fractionation. Consistent with previous reports (27), in the absence of cell surface biotinylation, streptavidin overlay assays revealed the presence of a small number of endogenously biotinylated proteins in {alpha}T3-1 whole cell lysates (Fig. 5AGo). After cell surface biotinylation and separation of cell homogenates into cytosolic and total membrane fractions, streptavidin overlay assays revealed the majority of biotinylation within the membrane fraction, verifying the cell impermeability of the biotinylation reagent and validating this assay for tracking of bulk plasma membrane during fractionation (Fig. 5AGo). Discontinuous sucrose density gradient fractionation of {alpha}T3-1 cell postnuclear supernatants after cell surface biotinylation revealed that the majority of plasma membrane localized to the high-density fraction (35–40% sucrose interface, Fig. 5BGo). Microsomal suspensions retrieved from the middle fraction of the sucrose gradient (25–35% sucrose interface) were enriched in the early endosomal marker EEA-1 (Fig. 5CGo). However, both the GnRHR and the lipid raft marker flotillin-1 partitioned exclusively into this middle fraction, indicating that this technique allows effective separation of low-density subdomains of plasma membrane. Both the ERKs as well as G{alpha}q/11 associated with the lower-density middle fraction in a manner unaffected by GnRHa treatment (Fig. 5CGo). After GnRHa treatment, phosphorylated ERK was similarly detected in the lower-density middle fraction. Immunoreactivity for the proteins examined was undetectable in the low-density upper fraction (data not shown).


Figure 5
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  		Fig. 5. In {alpha}T3-1 Cells, GnRHR and ERK2 Associate with Low-Density Membrane Domains Isolated in the Absence of Detergent

A, {alpha}T3-1 cells were surface biotinylated and separated into whole-cell lysate (WCL), cytosolic (CYT), and crude membrane (CM) fractions. Fractions along with nonbiotinylated whole-cell lysate were subjected to SDS-PAGE and analyzed by streptavidin overlay. Molecular masses (MW) are indicated in kilodaltons. B and C, Cells were homogenized in a detergent-free buffer, and the postnuclear supernatant was subjected to discontinuous sucrose density gradient centrifugation (40.6, 35, 25, and 5%). Microsomal suspensions were collected from the upper (U), middle (M), and lower (L) interfaces of the gradient, and the membranes were repelleted by centrifugation at 100,000 x g. Membrane pellets were electrophoresed and analyzed by streptavidin overlay (B) and immunoblotting using antibodies against the indicated proteins (C).

 
Both the Association of ERK2 with Low-Density Membrane Domains as Well as ERK Inducibility by GnRHa Are Susceptible to Perturbation of Cellular Cholesterol Levels
Analysis of lipid behavior in model membranes indicates that lo domain formation is highly dependent upon cholesterol (28). Cholesterol is also understood to be critically important in the formation of membrane raft domains in living cells (29). We previously reported that, in {alpha}T3-1 cells, depletion of cellular cholesterol led to a dissociation of the GnRHR from a detergent-resistant, low-density membrane environment as well as a reversible uncoupling of the ability of GnRH to activate the ERK pathway (21). To further define the importance of cholesterol-dependent membrane organization for the function of this signaling pathway, we used a depletion/repletion strategy to define the kinetics of recovery of GnRH-induced ERK activation after cholesterol perturbation. Exposure of cells to methyl-ß-cyclodextrin (CD) for 40 min resulted in a complete loss of both the GnRHR and the ERKs from DRMs (Fig. 6AGo). However, the association of the membrane raft marker flotillin-1 with DRMs was unaffected, indicating that cholesterol depletion did not promote complete solubilization of this membrane compartment (Fig. 6AGo). Consistent with our previous results, CD exposure resulted in a significant decrease in cellular cholesterol to approximately 50% of control levels and led to a complete block of ERK activation by GnRHa (Fig. 6BGo). Repletion of cholesterol by exposure of cells to CD preloaded with cholesterol (CDChol) for 60 min normalized cellular cholesterol levels and led to reassociation of both the GnRHR and ERKs with DRMs (Fig. 6AGo). However, despite normalization or even mild elevation of total cellular cholesterol, ERK inducibility by GnRH was not restored for up to 3 h (Fig. 6BGo). Moreover, exposure of {alpha}T3-1 cells to CDChol alone led to an approximately 3-fold increase in cellular cholesterol levels and caused a block in ERK activation by GnRH that developed with highly similar kinetics (data not shown). These results suggest that cholesterol homeostasis is critical for normal signaling through the GnRHR and implicate a complex process of dynamic membrane (re-)organization as an important regulatory feature in the functional reestablishment of the ERK pathway after perturbation of cholesterol homeostasis in cell membranes.


Figure 6
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  		Fig. 6. Normalization of Cellular Cholesterol Restores the Association of ERK2 with Low-Density Membrane Domains in {alpha}T3-1 Cells after Cholesterol Depletion, but This Is Not Sufficient to Reconstitute Cholesterol-Dependent ERK Inducibility by GnRH

A, Cells were cultured in the absence (Control) or presence (Depletion) of CD for 40 min. Some cells were then incubated in media containing 0.5 mg/ml of cholesterol in the form of CDChol for 60 min (Depletion + repletion). Cells were homogenized in the presence of 0.1% Triton X-100 and fractionated by discontinuous sucrose density gradient centrifugation. Fractions were analyzed by immunoblot using anti-GnRHR immune serum, or antibodies against ERK2 and the lipid raft marker Flotillin-1. B, Cells were subjected to cholesterol depletion and repletion as in panel A. Some samples were then incubated for the indicated times in cholesterol-free media (Media). Whole-cell lysates were analyzed by immunoblot for phosphorylated and total ERK proteins. Total cellular cholesterol measurements were normalized to total protein concentrations and are expressed as mean ± SEM for four separate experiments. Asterisks indicate significant differences from the control group (P ≤ 0.01)

 
GnRHR and ERK2 Coimmunoprecipitate from Low-Density Membrane Domains in {alpha}T3-1 Cells
Our experimental results to date suggest the possibility that ERK activation after GnRHR occupancy may be initiated through the interaction of several signaling intermediates (G{alpha}q/11, c-raf, ERKs) that are compartmentalized in proximity to the GnRHR through association with low-density plasma membrane microdomains. However, our data do not rule out the possibility that cofractionation of multiple signaling proteins in this pathway represents the isolation of a complex, mixed population of subcellular membrane compartments. For example, many components of the ERK pathway have been shown in other systems to be assembled in association with endosomal membranes through interactions with specific scaffolds and adaptors (30, 31). To further determine whether the GnRHR and ERKs associate with a common membrane environment within the raft compartment, we immunoprecipitated the GnRHR from low-density membrane suspensions and examined the immunoprecipitates for the presence of ERKs. ERKs coimmunoprecipitated with the GnRHR from DRMs (Fig. 7Go, A and B), as well as from microsomal suspensions prepared in the absence of detergent (Fig. 7CGo). The reciprocal immunoprecipitations targeting ERK2 followed by immunoblot analysis for the GnRHR were uninformative due to a high level of nonspecific signal arising from the heavy chain of the anti-ERK2 primary antibody.


Figure 7
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  		Fig. 7. GnRHR and ERK2 Coimmunoprecipitate from Low-Density Membrane Fractions in {alpha}T3-1 Cells

A, Cells were homogenized in the presence of 0.1% Triton X-100 and fractionated through a discontinuous sucrose gradient. Fractions were collected and aliquots were analyzed by immunoblot using an antibody against ERK2. B, GnRHR was immunoprecipitated from fraction 3 of the experiment in panel A (lanes 3–5) using anti-GnRHR immune serum (IS), nonimmune serum (NRS), or beads alone (Beads). Control immunoprecipitations were performed in sucrose alone (lanes 6–8). Whole-cell lysate (WCL, 1 µg lane 1), 5% of the input for each input (IP, lane 2), and the immunoprecipitates were analyzed by immunoblot using an anti-ERK2 antibody. C, Cells were homogenized in the absence of detergent and fractionated by discontinuous sucrose density gradient centrifugation (40.6, 35, 25, and 8%). GnRHR was immunoprecipitated from microsomal suspensions at the 25–35% sucrose interface using anti-GnRHR immune serum (IS), nonimmune serum (NRS), or beads alone (Beads). Whole-cell lysate (WCL, 1 µg, lane 1), 5% of the input for each input (IP, lane 2), and the immunoprecipitates (lanes 3–5) were analyzed by immunoblot using an anti-ERK2 antibody.

 
GnRHR Associates with Low-Density Membrane Domains in Whole Mouse Pituitary and Coimmunoprecipitates with ERK2 in Mouse Pituitary Homogenates
Our data from {alpha}T3-1 cells strongly implicate low-density membrane microdomains in the regulation of ERK activation by GnRH. To determine whether these domains may play a similar role in the organization of this signaling pathway in differentiated gonadotropes in vivo, we examined DRMs from homogenates of whole mouse pituitaries. The GnRHR partitioned into low-density DRMs in a manner similar to that observed in {alpha}T3-1 cells (Fig. 8AGo). Flotillin-1 was present exclusively in the low-density fractions, whereas transferrin receptor was detected only in the high-density solubilized fractions (data not shown). ERKs were also detectable in low-density fractions from pituitary homogenates (Fig. 8AGo). Whole pituitary represents a diversity of cell types in which ERKs are ubiquitously expressed; thus the presence of ERKs within low-density fractions of pituitary homogenates does not provide evidence for cell type specificity regarding the association of ERKs with low-density membranes. To further determine whether ERKs associate with a low-density membrane compartment in gonadotropes, we immunoprecipitated the GnRHR from DRMs of pituitary homogenates and examined the precipitates for the presence of ERKs. Consistent with our observations in {alpha}T3-1 cells, ERKs coimmunprecipitated with the GnRHR, indicating that these proteins associate with a common membrane environment in vivo (Fig. 8BGo).


Figure 8
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  		Fig. 8. GnRHR and ERK2 Coimmunoprecipitate from Detergent-Resistant, Low-Density Membranes in Whole Mouse Pituitaries

A, Whole mouse pituitaries were homogenized in the presence of 0.1% Triton X-100 and subjected to discontinuous sucrose density gradient centrifugation. Fractions were collected from the top of the gradient, and aliquots of each fraction were analyzed by immunoblot using anti-GnRHR immune serum and anti-ERK2 antibody. B, GnRHR was immunoprecipitated from fraction 3 of the same experiment using anti-GnRHR immune serum (IS), nonimmune serum (NRS), or beads alone (Beads). Whole-cell lysate (WCL, 1 µg, lane 1), 5% of the input for each IP (Input, lane 2), and the immunoprecipitates (lanes 3–5) were analyzed by immunoblot using an anti-ERK2 antibody.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Subcellular compartmentalization of signaling proteins is now recognized as a mechanism by which signaling activity may be temporally and spatially regulated within the cell, or by which the activation of a given signaling pathway may be linked to a diversity of biological outcomes (32, 33, 34). This has become particularly evident in the area of G protein-coupled receptor (GPCR) signaling. Whereas early studies of GPCR function focused on the role of activated G protein {alpha}-subunits in the generation of diffusible second messengers and modulation of ion conductances, current models emphasize the spatial segregation and trafficking of cell surface receptors among discrete subdomains of the plasma membrane, as well as the ability of internalized GPCRs to couple to diverse intracellular signaling cascades, including the ERK module of the MAPK system (35). Consistent with these themes, we previously demonstrated that, unlike other GPCRs, the GnRHR partitions constitutively and exclusively into membrane rafts, and that this raft association was required for the ability of this GPCR to couple to the ERK pathway in the {alpha}T3-1 gonadotrope cell line (21). The results described here expand upon these earlier findings and show that the GnRHR partitions into low-density membrane domains in whole mouse pituitary as well in {alpha}T3-1 cells. These results further support the use of this cell line as a model of GnRHR behavior in pituitary gonadotropes and suggest that compartmentalization of the GnRHR into low-density membrane microdomains is of functional significance in vivo.

After ligand activation, most GPCRs are rapidly phosphorylated, desensitized, and targeted for clathrin-mediated endocytosis through association with ß-arrestins (36). Arrestins have also been shown to provide an important link between many GPCRs and the ERK pathway by nucleating specific signaling complexes on the surface of receptor-laden early endosomes (37, 38, 39). However, the GnRHR is not phosphorylated after ligand binding and does not associate with arrestins (40). Furthermore, the delayed kinetics of GnRHR internalization suggest that GnRH-induced ERK activation occurs independently of receptor internalization (41). Previously, we proposed a model in which coupling of the GnRHR to the ERK pathway involves, at least in part, the assembly of a raft-associated signaling complex consisting minimally of the GnRHR, G{alpha}q, and c-raf (21). Our present confocal imaging data show that the vast majority of the GnRHR population localizes to GM1-enriched regions of the plasma membrane in {alpha}T3-1 cells. Furthermore, we show that the GnRHR and ERKs coimmunoprecipitate from low-density membrane fractions both in {alpha}T3-1 cells as well as whole mouse pituitary. These data demonstrate a close physical proximity between the GnRHR and ERKs at the level of the plasma membrane and provide further support for our model of a specific plasma membrane-associated signaling complex as a link between the GnRHR and the ERK pathway in gonadotropes.

In these studies, the behavior of the ERKs with respect to their association with low-density membrane domains was similar to our previous observations of c-raf (21). Both c-raf and the ERKs partitioned constitutively and partially into low-density membrane fractions, both in {alpha}T3-1 cells and in whole mouse pituitary. In addition, these kinases were rapidly activated within this membrane compartment after GnRHa stimulation (21). The small proportion of total cellular ERKs that partitioned into the low-density membrane compartment may reflect the overall small size of this compartment, or may indicate a relative paucity of ERK docking sites within this compartment, dictated perhaps by the specific architecture of raft-associated complexes. Also, because signaling complexes are characterized by low-affinity interactions, it is possible that dissociation and loss of some of these complexes from low-density membranes during homogenization and fractionation led to an underestimation of the proportion of ERKs that reside within this compartment in intact cells. Like c-raf, the association of ERKs with membrane rafts in resting cells was completely disrupted by homogenization of cells in higher concentrations of the nonionic detergent Triton X-100. Importantly, however, stimulation with GnRHa rescued the raft-association of ERKs after exposure of cells to higher detergent concentrations. The biophysical basis for this increased resistance to detergent solubilization is unclear; however, the apparent increase in affinity of both c-raf and ERKs for the low-density membrane compartment that is induced by GnRH argues that modulation of this membrane affinity is a specific biological effect of GnRHR activation. Finally, unlike c-raf, association of ERKs with low-density membranes was susceptible to cholesterol depletion and was restored after cholesterol replenishment, in a manner that paralleled the GnRHR. Interestingly, ERKs do not possess a defined lipid-binding domain, and, to our knowledge, have not been shown to interact directly with membranes (42). Loss of ERKs from the low-density membrane compartment under conditions of cholesterol depletion likely reflects the cholesterol dependence of an intermediate, membrane-associated adapter protein(s). Our analysis of the kinetics of recovery of ERK inducibility after cholesterol depletion and repletion indicated that normalization of total cellular cholesterol levels and restoration of the raft association of the GnRHR and ERKs are not sufficient for reconstitution of signaling. This temporal lag in recovery of signaling is consistent with a requirement for reorganization of a raft-associated multiprotein signaling complex.

Although numerous methods for the study of membrane rafts have been reported, the most commonly used method for their isolation remains extraction of cells with low concentrations of a nonionic detergent, followed by fractionation of the cell homogenate by density gradient centrifugation. However, studies of model membranes have raised concerns regarding the ability of nonionic detergents to promote the formation of a cholesterol-dependent lo phase (24, 43). To address these concerns, our previous work analyzed DRMs, as well as membrane fractions prepared using a detergent-free, carbonate-based method, to demonstrate association of the GnRHR with low-density membranes (21). Here we report the use of an additional complementary technique for fractionation of subcellular membranes in the absence of detergent. This method was originally used for isolation of microsomal suspensions enriched in early endosomes. Huber and colleagues (30) have shown that early endosomes are largely separable from plasma membrane on the basis of differential buoyant density alone. Because membrane rafts are distinguishable from bulk plasma membrane by their lower buoyant density, as well as their detergent resistance, we reasoned that this technique might allow separation of membrane rafts from bulk plasma membrane in the absence of detergent. Indeed, our results with this method show that both the GnRHR and the membrane raft marker flotillin-1 partitioned into a low-density microsomal fraction that is enriched in early endosomes. Using cell-surface biotinylation to track the plasma membrane during the fractionation, our data indicate that the majority of plasma membrane partitions into a higher density fraction, consistent with the notion that the membrane raft domains occupied by GnRHR constitute a small proportion of the total plasma membrane in these cells. Interestingly, membrane-associated ERKs showed a preference for the lower-density fraction. Because this low-density fraction represents a mixed population of endosomal and plasma membrane elements, we cannot exclude the possibility that a significant proportion of the ERKs that partitioned into this fraction were associated with endosomes. Indeed, assembly of signaling complexes on endosomes is a prominent feature of the organization of the ERK pathway (30, 44). However, our ability to coimmunoprecipitate the GnRHR and ERKs from this fraction indicates that at least some of the ERKs within this compartment are associated specifically with GnRHR-enriched plasma membrane rafts. The appearance of phosphorylated ERK within this fraction after GnRHa treatment further supports this conclusion. Overall, these data corroborate our analyses of DRMs and lend further support to our model of a raft-based GnRHR signaling platform.

Whereas our data provide evidence for the association of ERKs with plasma membrane rafts in gonadotropes, the functions and substrates of this membrane-associated ERK are unknown. In most cell types, including gonadotropes, a substantial portion of activated ERK undergoes nuclear translocation after activation; indeed, phosphorylation of nuclear substrates involved in transcriptional regulation is often considered the predominant endpoint of pathway activation. However, ERKs have also been shown to target a variety of extranuclear substrates including cytoskeletal regulatory proteins and cytoskeletal elements, proteins involved in intracellular membrane trafficking, proteosomal subunits, and translational regulators (45, 46, 47). Although the functional link between the ERK pathway and the activity of many of these substrates has not been thoroughly defined, a recent report indicated that the ERKs play a key role in the GnRH-induced up-regulation of LHß mRNA translation in LßT2 cells through phosphorylation of specific elongation initiation factors (48). As a further example of the complex and multifunctional organization of this pathway, it has been shown that mitogen-induced activation and nuclear translocation of the ERK substrate p90RSK1 are preceded by its transient translocation to the plasma membrane via a mechanism that involves ERK interaction but is independent of ERK phosphotransferase activity (49). This raises the interesting possibility that, in addition to their prominent role as kinases, activated ERK proteins may serve adaptor roles under certain circumstances. Given the small proportion of total cellular ERK that resides within the raft compartment in our studies, it seems unlikely that the totality of ERK activation that occurs within these cells takes place within the context of the raft-associated GnRHR signaling platform proposed in our model. However, we are intrigued by the possibility that raft-associated ERKs play a role in the cellular response to GnRH by targeting a specific subcellular complement of substrates. Determination of the functional importance of raft-associated ERKs in gonadotropes will require identification of the molecular determinants required for association of these proteins with membrane rafts, as well as the composition of the protein complexes that form within this compartment.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Cells, Antibodies, and Chemicals
{alpha}T3-1 cells, an immortalized mouse gonadotrope cell line (generously provided by Dr. Pamela Mellon, University of California, San Diego), were cultured as described previously (21). Anti-ERK2 (sc-154-G), anti-G{alpha}q/11 (sc-352), and horseradish peroxidase (HRP)-conjugated secondary antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Secondary antibodies and fluorescein isothiocyanate-streptavidin used for immunohistochemistry were from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA). Anti-phospho-ERK1/2 antibody was from Sigma Chemical Co. (St. Louis, MO). Anti-Flotillin-1 antibody was from BD Biosciences (San Jose, CA). Anti-EEA-1 antibody was from Upstate Biotechnology (Lake Placid, NY). Anti-Ca pan {alpha}-1 antibody was from Alomone Laboratories (Jerusalem, Israel). Anti-LH antibody was from A. C. Parlow and the National Hormone and Peptide Program sponsored by National Institute of Diabetes and Digestive and Kidney Diseases. Antitransferrin receptor antibody was purchased from Zymed Laboratories (South San Francisco, CA). Tissue-Tech OCT compound was from Miles, Inc. (Elkhart, IN). HRP-conjugated streptavidin was from Vector Laboratories (Burlingame, CA). The GnRHR antibody was raised in a rabbit against 20 amino acids of the second extracellular loop (amino acids 193–212) of the ovine GnRHR. This sequence shows no overlap with any other receptor or peptide. Buserelin (des-GLY10 [D-Ser(t-But)6]-LH-RH Ethylamide; referred to as GnRHa), CD, and CDChol, and all other chemicals were obtained from Sigma. In all experiments, GnRHa was used at 10 nM. Alexa Fluor 594 membrane raft labeling kit (CtxB) was obtained through Molecular Probes (Eugene, OR). Glass-bottom microwell dishes for confocal studies were obtained from Mat-Tek (Ashland, MA). Superfect transfection reagent was purchased from QIAGEN (Valencia, CA).

Confocal Microscopy
{alpha}T3-1 cells grown on glass-bottom microwell dishes were transiently transfected with a GnRHR construct with a GFP fluorophore linked to the C terminus for 48 h. After 48 h of transfection, GM1 domains were labeled with Alexa Fluor 594 CtxB at 4 C. Anti-CtxB antibody was then used to cross-link the CtxB-labeled domains. Cells were fixed in 4% paraformaldehyde for 10 min at room temperature and then imaged. Imaging was done utilizing the 63x oil objective and the 488-nm and 543-nm laser lines of a Zeiss LSM 510 Meta confocal microscope (Carl Zeiss, Inc., Thornwood, NY). The Alexa594 and GFP signals were acquired using multitrack mode, and no cross talk between Alexa594 and GFP was observed. Transferrin receptor colocalization with GM1 was assessed in {alpha}T3-1 cells using the same Alexa Fluor 594 CtxB staining approach described above. After GM1 labeling, {alpha}T3-1 cells were fixed in 4% paraformaldehyde for 10 min at room temperature. Cells were blocked in PBS containing 3% BSA and then incubated with antitransferrin antibody overnight at 4 C. The following day, cells were washed and labeled with a Alexa Fluor 488-conjugated antimouse secondary antibody for 1 h. Cells were then imaged by confocal laser scanning microscopy as above. Quantitative analysis of fluorophore colocalization is expressed as a scatter diagram (50). Identical images (colocalization) produce a clean diagonal line running from the bottom left to the top right. Differences between the images cause an irregular distribution in the scatter diagram.

Tissue Preparation and Immunohistochemistry
Male mice were anesthetized with ketamine and transcardially perfused with 100 ml 1% sodium nitrite in 0.9% NaCl, followed by 200 ml of fixative (4% paraformaldehyde; 15% saturated picric acid in 0.1 M phosphate buffer; pH 7.4) and then 100 ml cryoprotectant (20% sucrose in 0.1 M phosphate buffer). Pituitaries were removed and stored in cryoprotectant overnight at 4 C. Tissue was embedded in Tissue-Tech OCT compound and frozen by immersion in liquid nitrogen-cooled isopentane. Sections (20 µm) were mounted on Silane-coated slides and stored at –80 C.

Sections were washed (all washes 3 x 3 min in 0.01 M PBS) and incubated (48 h) in a solution containing a rabbit anti-GnRHR antibody (1:2000; 10% goat serum, 0.3% Triton in PBS) as well as a horse antiovine LH antibody (1:5000). Sections were washed and immersed in biotinylated goat antirabbit IgG and goat antihorse IgG linked to Texas Red (90 min; both at 1:200). After additional washing, sections were immersed in fluorescein isothiocyanate-streptavidin (90 min; 1:200), washed, mounted, and examined by conventional immunofluorescence microscopy.

Cell Surface Biotinylation
Cell surface biotinylation was performed using a commercial kit (Molecular Probes/Invitrogen, Carlsbad, CA) with minor modifications. Biotin-XX-sulfosuccinimidyl ester was dissolved in dimethylsulfoxide at a concentration of 0.2 mg/ml and then diluted to a final concentration of 0.5 µg/ml in PBS. {alpha}T3-1 cells were serum starved in DMEM for 2 h and then placed on ice. Cells were washed twice with cold PBS and then incubated on ice for 15 min in 1 ml of Biotin-XX-sulfosuccinimidyl ester in PBS per 12 cm2 of culture dish surface area. Cells were then washed twice in cold PBS before collection.

Cell Fractionations
To assess the specificity of cell-surface biotinylation, {alpha}T3-1 cells were grown to 80% confluence in 10-cm2 dishes and surface biotinylated as described above. Cells were scraped into PBS and pelleted by centrifugation. Cell pellets were resuspended in 500 µl of homogenization buffer (HB) containing 250 mM sucrose, 3 mM imidazole (pH 7.4), 2 mM EDTA, and protease inhibitors. A 50-µl aliquot of the whole cell suspension (representing 10% of the total input) was reserved. The remaining suspension was incubated on ice for 10 min, homogenized in a glass Dounce, and centrifuged for 10 min at 3000 rpm. The postnuclear supernatant was collected and centrifuged at 100,000 x g at 4 C for 30 min. The resulting supernatant and pellet (representing cytosolic and total membrane fractions, respectively), as well as the input were boiled in sodium dodecyl sulfate (SDS) load buffer.

Detergent-resistant, low-density membrane fractions were prepared essentially as described previously (21). Briefly, {alpha}T3-1 cells were grown to 70–80% confluence in 15-cm2 dishes. After the treatments indicated, cells (~1.5 x 108 per dish) were washed twice in cold PBS and scraped into PBS with protease inhibitors. Cells were pelleted by centrifugation and then resuspended in 2-[N-morpholino]ethanesulfonic acid buffer (MBS) containing 25 mM MES (pH 6.5), 130 mM NaCl, and protease inhibitors to a final volume of 400 µl. The samples were adjusted to the indicated concentration of Triton X-100 and incubated on ice for 10 min. After Dounce homogenization (20 strokes), the samples were mixed with an equal volume of 90% sucrose, placed in a 5-ml ultracentrifuge tube, and overlaid with a discontinuous gradient of sucrose in MBS consisting of 35% (3.7 ml) and 5% (500 µl) layers. The gradients were centrifuged at 116,000 x g in a SW50.1 rotor for 20 h at 4 C. Low-density detergent resistant membranes were visible as a band of flocculent material at the 35–5% interface. Fractions (500 µl) were collected starting from the top of the gradient.

Detergent-free fractionations were performed as described previously (26) with minor modifications. {alpha}T3-1 cells (~4 x 108 cells) were washed and scraped into PBS as described above. Cells were pelleted by centrifugation and washed once in 5 ml cold HB. Cell pellets were resuspended in 2.2 ml HB containing 50 µg/ml cycloheximide, and gently homogenized in a glass Dounce (eight strokes). Nuclei and unbroken cells were pelleted by centrifugation, and 2 ml of the postnuclear supernatant was collected and mixed with 2.4 ml of HB-sucrose containing 62% sucrose, 3 mM imidazole (pH 7.4), 2 mM EDTA, and protease inhibitors to achieve a final sucrose concentration in the sample of 40.6%. The homogenate was transferred to a 13-ml ultracentrifuge tube and overlaid with a discontinuous gradient of HB-sucrose consisting of 35% (4 ml), 25% (3 ml), and HB (600 µl). Samples were centrifuged in a SW41 rotor at 35,000 rpm, 4 C for 1 h. Translucent bands of material visible at each interface were collected, diluted 4-fold in cold PBS, and repelleted by centrifugation at 100,000 x g for 30 min at 4 C. Pellets were resuspended in SDS loading buffer and boiled.

Female B6/129 mice (8–20 wk of age) were euthanized by CO2 asphyxiation. Pituitaries were collected and placed in DMEM containing 10% fetal bovine serum on ice. Pituitaries were washed once in cold PBS and once in cold MBS and homogenized as described above. Animal use and experimental protocols for these studies were approved by the Cornell University Institutional Animal Care and Use Committee. For whole pituitary fractionations, pituitaries (n = 20) were resuspended in 400 µl cold MBS containing 0.1% Triton X-100, homogenized in a glass Dounce (20 strokes), and subjected to discontinuous sucrose density centrifugation as described above for preparation of detergent-resistant low-density membranes.

Preparation of Cell Lysates, Immunoblotting, and Streptavidin Overlay Assays
After the indicated treatments, cells were washed twice in cold PBS and scraped into cold PBS containing 5 mM sodium vanadate, 0.2 mM phenylmethylsulfonyl fluoride, and 5 mM benzamidine. Cells were pelleted by centrifugation, and pellets were resuspended in a radioimmunoprecipitation assay buffer containing 20 mM Tris-HCl (pH 8.0), 137 mM NaCl, 10% glycerol, 1% Nonidet P-40, 0.1% SDS, 0.5% deoxycholate, 2 mM EDTA, 5 mM sodium vanadate, 0.2 mM phenylmethylsulfonyl fluoride, 5 mM benzamidine. Lysates were cleared by centrifugation and protein concentrations of the lysates were determined by Bradford assay. Protein samples were boiled for 5 min in SDS loading buffer, resolved by SDS-PAGE, and transferred to polyvinylidene difluoride membranes by electroblotting. Membranes were blocked with 5% nonfat dry milk in TBST (10 mM Tris-HCl, pH 7.5; 150 mM NaCl; 0.05% Tween 20) and then incubated with primary and HRP-conjugated secondary antibodies. For streptavidin overlay assays, blots were blocked as described and then incubated at room temperature for 1 h in a 1:10,000 dilution of streptavidin-HRP in TBST. Protein bands were visualized using enhanced chemiluminescence according to the manufacturer’s instructions (PerkinElmer, Boston, MA).

Cholesterol Depletion and Repletion
{alpha}T3-1 cells were cultured in 6-cm2 dishes to 70–80% confluence. After serum starvation for 2 h, cells were incubated in DMEM or DMEM containing 2% CD for the indicated times. For cholesterol repletion, CD-containing media were exchanged for DMEM containing 0.5 mg/ml cholesterol in the form of cholesterol-loaded CD for the indicated times. Total cellular cholesterol was measured from 2-µl aliquots of clarified radioimmunoprecipitation assay lysate using a commercial kit (Amplex Red, Molecular Probes/Invitrogen) according to the manufacturer’s instructions.

Immunoprecipitation
Aliquots (400 µl) of fractions containing suspensions of low-density membranes, or sucrose alone, were diluted in an equal volume of PBS and adjusted to 0.01% Triton X-100. Anti-GnRHR immune serum (IS, 25 µl), nonimmune rabbit serum (NRS, 25 µl), or vehicle (PBS, 25 µl), were added, and the samples were rocked for 4 h at 4 C. Protein A/G agarose beads (30 µl) were added, and the samples were rocked an additional hour at 4 C. Beads were then washed three times in PBS with 0.01% Triton X-100, resuspended in 60 µl of SDS loading buffer, and boiled.

Statistical Analysis
Data on cholesterol concentration were analyzed using a one-way ANOVA with a Bonferroni post hoc multiple comparisons test. Significance for each pair-wise comparison was determined at P ≤ 0.01.


   FOOTNOTES
 
Disclosure Statement: The authors have nothing to disclose.

First Published Online October 26, 2006

Abbreviations: CD, Methyl-ß-cyclodextrin; CDChol, CD preloaded with cholesterol; CtxB, B subunit of cholera toxin; DRM, detergent-resistant membrane domain; GFP, green fluorescent protein; GnRHR, GnRH receptor; GPCR, G protein-coupled receptor; HB, homogenization buffer; HRP, horseradish peroxidase; MBS, 2-[N-morpholino]ethanesulfonic acid buffer; SDS, sodium dodecyl sulfate.

Received for publication July 13, 2006. Accepted for publication October 17, 2006.


   REFERENCES
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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

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A Preformed Signaling Complex Mediates GnRH-Activated ERK Phosphorylation of Paxillin and FAK at Focal Adhesions in L{beta}T2 Gonadotrope Cells
Mol. Endocrinol., November 1, 2009; 23(11): 1850 - 1864.
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E Robin, J Cognie, F Foulon-Gauze, J Fontaine, and X Cayla
Disruption of Lipid Rafts Induces Gonadotropin Release in Ovine Pituitary and LbetaT2 Gonadotroph Cells
Biol Reprod, July 1, 2008; 79(1): 17 - 25.
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