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Endocrinology, doi:10.1210/en.2005-1046
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Endocrinology Vol. 147, No. 4 1789-1795
Copyright © 2006 by The Endocrine Society

Luteinizing Hormone Receptors Translocate to Plasma Membrane Microdomains after Binding of Human Chorionic Gonadotropin

Steven M. L. Smith, Ying Lei, Jingjing Liu, Mary E. Cahill, Guy M. Hagen, B. George Barisas and Deborah A. Roess

Department of Biomedical Sciences (S.M.L.S., M.E.C., D.A.R.), Cell and Molecular Biology Program (Y.L., J.L.), and Department of Chemistry (G.M.H., B.G.B.), Colorado State University, Fort Collins, Colorado 80523

Address all correspondence and requests for reprints to: Dr. Deborah A. Roess, Department of Biomedical Sciences, Colorado State University, Fort Collins, Colorado 80523. E-mail: deborah.roess{at}colostate.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Receptor-mediated signal transduction by G protein-coupled receptors can involve redistribution of plasma membrane receptors into membrane structures that are characterized by insolubility in Triton X-100 and low buoyant density in sucrose gradients. Here we describe the translocation of wild-type (wt) rat LH receptors (LHR-wt) from the bulk membrane into membrane microdomains (rafts) after the binding of human chorionic gonadotropin (hCG). In sucrose gradient ultracentrifugation of plasma membranes from cells stably expressing FLAG-tagged LHR-wt, receptors were located in high-density membrane fractions before binding of hormone and in low-density fractions after hCG treatment. Receptor translocation to low-density sucrose fractions did not occur when cells were pretreated with 1% methyl-ß-cyclodextrin, which reduces membrane cholesterol and disrupts rafts. Single-particle tracking of individual FLAG-LHR-wt receptors showed that hCG-treated receptors become confined in small compartments with a diameter of 86 ± 36 nm, significantly smaller than 230 ± 79 nm diameter regions accessed by the untreated receptor. Receptors were no longer confined in these small compartments after disruption of rafts by methyl-ß-cyclodextrin, a treatment that also decreased levels of cAMP in response to hCG. Finally, translocation of LHR into rafts required a functional hormone-receptor complex but did not occur after extensive receptor cross-linking that elevated cAMP levels. Thus, retention of LHR in rafts or small membrane compartments is a characteristic of functional, hormone-occupied LHR-wt. Although raft translocation was not essential for cAMP production, it may be necessary for optimizing hormone-mediated signaling.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
SEVERAL LINES OF evidence suggest that functional LH receptors (LHR) are clustered within high-molecular-weight structures after the binding of hormone. Electron micrographs of LHR on rat granulosa cells show large clusters of receptors that form only after binding of hormone (1) as does immunofluorescent labeling of rat receptors in granulosa cells (2). Wild-type (wt) rat LHR (LHR-wt) tagged with green fluorescent protein (LHR-GFP) aggregated within minutes after binding of either LH or human chorionic gonadotropin (hCG) to receptors on viable cells (3). This aggregation was accompanied by close interactions between individual receptors, as suggested previously by other strategies for evaluating fluorescence resonance energy transfer between LHR (4), and indicated by comparatively high values for fluorescence energy transfer efficiency between receptors tagged with variants of green fluorescent protein (5). The presence of receptors in physically large structures has also been suggested by lateral diffusion studies of the LHR in luteal cells from sheep (6) and rat (7) in which most LHR were laterally immobile.

Because clustering of the rat LHR occurs within minutes and, upon microscopic inspection, involves the movement of diffusely distributed receptors into discrete membrane sites (5), one question is whether receptors cluster at arbitrary sites on the plasma membrane or become confined in membrane microdomains. Membrane microdomains include so-called rafts that, because of their high cholesterol and sphingolipid content, float in sucrose gradients. Rafts are enriched not only with sphingolipids and cholesterol (8) but also with glycosylphosphatidylinositol (GPI)-anchored proteins (9). The lateral diffusion of specific membrane proteins within these microdomains is also reduced (10). Membrane domains, which on some cells may comprise a substantial fraction of the plasma membrane (11, 12), can contain membrane proteins necessary for cell signaling such as G proteins (13) and adenylate cyclase (14). There is also evidence for transient protein targeting to membrane rafts including, as an example, regulators of G protein signaling (15). Together, these observations have led to suggestions that rafts might serve as signaling platforms for G protein-coupled receptors and that this may by accomplished by targeting receptors to environments that favor receptor-mediated signaling.

An additional question raised by these observations is whether LHR function is dependent on receptor translocation into rafts. There are physical differences between functional hormone-receptor complexes, i.e. hCG- and LH-occupied LHR-wt, and those complexes that do not activate adenylate cyclase. As examples, binding of the hCG antagonist deglycosylated hCG (16) to rat LHR does not produce receptor self-association (17) or slow receptor rotational diffusion to the same extent observed for hCG-occupied LHR (18). These results indicate that larger receptor-containing structures do not form after receptor binding of deglycosylated hCG. Similarly, there is no self-association of rat LHR containing a substitution of arginine for lysine at amino acid 583 (LHR-K583R) that either partially (19) or fully (20) eliminates the cAMP response to hCG. Thus, if rafts serve as signaling platforms for rat LHR, nonfunctional receptors may be excluded in some manner from these structures or, alternatively, lack some critical receptor feature needed to direct the hormone-occupied receptor to rafts.

To address these questions, we have isolated membrane fragments from hCG- or deglycosylated hCG-treated Chinese hamster ovary (CHO) cells expressing LHR-wt tagged at their N terminus with the FLAG sequence and from hCG-treated CHO cells expressing a FLAG-tagged LHR containing a lysine-to-arginine mutation at position 583 (LHR-K583R). To further demonstrate the localization of functional LHR within rafts, we have treated cells with methyl ß-cyclodextrin (MßCD), which can efficiently remove cholesterol from the plasma membranes of live cells (21, 22) and thus disrupt raft structure. To independently examine the effects of hCG on LHR motions within the plasma membrane, we have used single-particle tracking methods to evaluate the size of membrane compartments accessed by individual receptors as visualized microscopically on viable cells (23). Finally, we examined whether disruption of membrane rafts is accompanied by altered signaling in CHO cells in response to hCG (17).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
DMEM containing 4.5 g/liter glucose was purchased from VWR (Denver, CO). Geneticin was purchased from Invitrogen (Carlsbad, CA). HEPES and nonessential amino acids were purchased from Sigma-Aldrich (St. Louis, MO) as was MßCD. Fetal bovine serum was purchased from Gemini Bio Products (Woodland, CA). hCG was purchased from Research Diagnostics Inc. (Flanders, NJ). Cellular cAMP was measured using a Direct Cyclic AMP Correlate-EIA kit (Assay Designs, Ann Arbor, MI) as per the manufacturer’s instructions. Colloidal gold (40 nm) was purchased from Ted Pella, Inc. (Redding, CA).

Cell lines
To test whether the rat LHR-wt becomes associated with membrane rafts after binding of ligand, we generated a stable cell line expressing the FLAG-tagged receptor. Dr. K. J. Menon from the University of Michigan kindly provided us with N-terminal FLAG-tagged LHR subcloned into the pFLAG vector (Sigma Chemical Co., St. Louis, MO). Using the FLAG-LHR vector, we made a mutation of lysine 583 to arginine (FLAG-LHR-K583R). CHO cells were stably transfected with 5 µg of the FLAG-LHR or K583R-FLAG-LHR vector using Lipofectamine-Plus (Life Technologies, Inc.-BRL, Gaithersburg, MD) as per the manufacturer’s instructions. Selection of stable clones expressing the FLAG-tagged receptors was based on the acquisition of geneticin (G418) resistance. CHO cell lines were maintained in cell medium that included DMEM supplemented with 4.5 g/ml glucose and containing 10% fetal bovine serum, 100 U/ml penicillin, 100 µg/ml streptomycin, and 1x MEM nonessential amino acids (Sigma) that was supplemented with 400 µg/ml G418.

Isolation of plasma membrane rafts
Cells were incubated with either 100 nM hCG or 100 nM deglycosylated hCG (16), the kind gift of Dr. Henry Keutman, or PBS for 1 h at 37 C before cell lysis. To isolate membrane rafts from LHR-wt and LHR-K583R cells, 5 x 107 cells were washed two times with PBS, pH 7.2, and lysed for 5–10 min on ice in 1 ml of a buffer containing 25 mM 2-[N-morpholino]ethanesulfonic acid, 150 mM NaCl, 2 mM EDTA, 20% glycerol, 0.25% Triton X-100, and Complete Mini protease inhibitor cocktail (Roche, Indianapolis, IN). A low-speed (300 x g) spin was used to remove cell nuclei and large cell debris, and 1 ml of the supernatant from this spin, which contained plasma membrane fragments, was then combined with 1 ml of 80% sucrose containing 0.25% Triton X-100 and protease inhibitors to produce a 40% sucrose solution. A discontinuous sucrose gradient from 10–80% was created with the sample in 40% sucrose layered within this gradient. The gradient was loaded into a Beckman SW-41 swinging bucket rotor and spun at 175,000 x g for 20 h at 4 C. After the spin, 18 650-µl fractions were carefully collected from the top of the gradient downward. A 50-µl aliquot from each fraction was diluted 1:1 with 95% SDS and 5% ß-mercaptoethanol. After separation of proteins from each fraction using SDS-PAGE and transfer of proteins to nitrocellulose, the LHR was identified using 30 µg of an anti-FLAG M2 monoclonal antibody (Sigma). The amount of receptor in each fraction was measured using a Bio-Rad (Hercules, CA) GS-800 calibrated densitometer. The sucrose concentration in each fraction was determined using a Bausch and Lomb refractometer. In some experiments, cells were pretreated for 1 h at 37 C with 1% MßCD in serum-free DMEM containing high glucose before incubation with hCG or PBS or with 100 nM anti-FLAG antibody for 45 min at room temperature followed by a second 45-min incubation with 1 µM antimouse IgG (Sigma). MßCD neither binds nor inserts in the plasma membrane of cells but rather extracts cholesterol by including it in a central nonpolar cavity of cyclic oligomers of glucopyranoside in {alpha}-1,4-glycosidic linkages (9). At this concentration, it was nontoxic to cells and did not compromise cell integrity (data not shown) (24).

Single-particle tracking of FLAG-LHR-wt receptors on individual cells
Lateral dynamics and the size of domains accessed by individual FLAG-LHR-wt were evaluated using single-particle tracking methods as described by Kusumi and co-workers (23). The 40-nm nanogold particles were conjugated with the lowest possible concentration of anti-FLAG monoclonal antibody needed to stabilize the gold solution and were then incubated with CHO cells expressing FLAG-LHR-wt receptors. The anti-FLAG-gold concentration, typically 15 µg/ml, was then further reduced by addition of 1% BSA in PBS until there were approximately one to four gold particles per cell. This binding was specific for FLAG-tagged receptors; when cells were preincubated with a 10-fold excess of anti-FLAG antibody, no anti-FLAG-gold particles were detected on cells. In some experiments, cells were treated with 100 nM hCG for 1 h after labeling of receptors with anti-FLAG monoclonal antibody or were pretreated with 1% MßCD for 1 h before labeling with anti-FLAG antibody.

Individual nanoparticles were imaged by differential interference contrast with a 1.4 numerical aperture x63 objective in a Zeiss Axiovert 135 microscope. Images were acquired using a Dage IFG-300 camera and were recorded for 2 min (3600 frames) at approximately 30 nm/pixel under the control of Metamorph software from Universal Imaging. The trajectories for individual gold particles were segmented into domains by calculation of statistical variance in particle position over times using a procedure similar to that developed by a number of investigators (25, 26, 27). The variance of a particle’s position can be calculated within windows of varying duration. These windows are translated along the particle trajectory, producing a variance plot that exhibits peaks that indicate interdomain boundaries. These results can be analyzed to yield the domain size and residence time for each particle. Effective macroscopic diffusion constants were calculated as the square of the domain diagonal divided by four times the residence time in the domain as previously described (27).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Functional, but not nonfunctional, LHR appear in membrane rafts after hCG binding
After isopycnic centrifugation of plasma membrane fractions from CHO cells expressing FLAG-LHR-wt, unoccupied LHR were found in sucrose fractions with relatively high densities (Fig. 1Go). Immunoblots of FLAG-LHR-wt receptors identified by anti-FLAG antibody were developed as previously shown (28), and the relative amount of FLAG-LHR-wt in each sucrose fraction was quantified using densitometry. Over 90% of FLAG-LHR-wt receptors were localized in sucrose fractions 10–15 where sucrose concentrations ranged from approximately 36–56%. After treatment of cells with 100 nM hCG, there was a marked change in the distribution of receptors to lower-density sucrose fractions. Over 80% of hCG-treated FLAG-LHR-wt receptors consistently appeared in fractions 3–7, which, on average, contained 14–26% sucrose, and over 90% of the receptors appeared in less dense sucrose fractions than did untreated receptors.


Figure 1
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  		FIG. 1. Before exposure to hCG, LHR-wt appear in high-density fractions of the sucrose gradient. After treatment of CHO cells with 100 nM hCG, receptors appear in lower-density fractions. Membrane fractions were separated as described in Materials and Methods, and densitometry was used to estimate the relative amount of LHR contained in each fraction. Results shown are the mean and SEM for at least five individual experiments. The sucrose concentration in each fraction was evaluated in five separate experiments using a Bausch and Lomb refractometer together with a standard curve. Because, for any given fraction, the sucrose concentration did not vary appreciably from experiment to experiment, the average sucrose concentration for five representative experiments is shown.

 
The translocation of LHR from high- to low-density membrane fractions required functional hormone-receptor complexes; LHR from CHO cells expressing FLAG-LHR-K583R treated with hCG or FLAG-LHR-wt cells treated with deglycosylated hCG remained associated with higher-density sucrose fractions (Fig. 2Go) despite the presence of bound ligand. Cross-linking of FLAG-LHR-wt receptors using an anti-FLAG antibody either alone or together with an excess of a secondary polyclonal antimouse antibody (Fig. 2Go) also had no significant effect on the distribution of FLAG-LHR-wt within the sucrose gradient.


Figure 2
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  		FIG. 2. Translocation of LHR into buoyant membrane fractions required functional LHR. LHR-wt treated with 100 nM deglycosylated hCG (Degly-hCG), LHR-K583R treated with hCG or antibody treated (anti-FLAG antibody alone), or extensively cross-linked LHR-wt (anti-FLAG antibody followed by excess antimouse IgG) were not found in plasma membrane rafts.

 
Depleting membrane cholesterol disrupts membrane rafts and decreases cAMP signals in response to hCG treatment
Preincubation of cells for 30 min with 1% MßCD disrupted membrane rafts containing the LHR. Over 93% of hCG-treated FLAG-LHR-wt were found in fractions 10–12 after exposure of cells to 1% MßCD (Fig. 3Go), which was essentially unchanged from the distribution of unoccupied FLAG-LHR-wt receptors on CHO cells treated with MßCD (data not shown) and from the distribution of unoccupied receptors shown in Fig. 1Go. Interestingly, disruption of LHR-containing rafts with MßCD did not affect caveolae. Western blots prepared in tandem with those used to identify FLAG-LHR-wt were probed with an anticaveolin antibody from Santa Cruz Biotechnology (Santa Cruz, CA). Caveolin remained broadly distributed in lower-density sucrose fractions under all experimental conditions (Fig. 3Go), suggesting that LHR are associated with membrane microdomains that are distinct from caveolin-containing membrane regions.


Figure 3
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  		FIG. 3. Disruption of plasma membrane rafts by extraction of cholesterol from the plasma membrane eliminated translocation of FLAG-LHR-wt into low-density sucrose fractions. Results shown are the mean and SEM for two separate experiments. In addition, anti-caveolin-1 antibody was used to identify caveolin in sucrose fractions. The distribution of caveolin in fractions 1–10 remained relatively constant in untreated cell samples and in samples exposed to hCG or MßCD.

 
Disruption of membrane rafts also reduced cell signaling in response to hormone. It has been suggested that plasma membrane rafts may serve as signaling platforms, effectively concentrating receptors and other plasma membrane molecules necessary for initiating downstream signaling events. If this were correct, disruption of membrane rafts should reduce or eliminate selective signaling events dependent on this membrane microenvironment. We examined accumulation of cAMP in response to hCG in cells pretreated with 1% MßCD. Although, in our hands, changes in cAMP levels in CHO cells were modest, typically 2- to 7-fold over basal levels, pretreatment of cells with MßCD nonetheless reduced hCG-mediated increases in intracellular cAMP to basal levels (Fig. 4Go). Interestingly, extensive cross-linking of LHR-wt caused a significant increase in cAMP production to levels comparable to those of hCG-treated CHO cells or cells treated with forskolin even though this antibody-induced form of receptor self-association was not sufficient to drive receptor translocation from the bulk membrane to rafts (Fig. 2).


Figure 4
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  		FIG. 4. cAMP levels were assayed in CHO cells using a colorimetric cAMP kit from Assay Designs. There was an approximately 3-fold increase in cAMP in response to cell treatment with either 100 nM hCG or forskolin, and 1% MßCD reduced cAMP levels in hCG-treated samples to basal levels. Although exposure of FLAG-LHR-wt to monoclonal anti-FLAG antibody (Ab) had no effect on cAMP levels, cross-linking of the receptor with an excess of antimouse IgG elevated cAMP levels to values comparable to those of hCG-treated cells. Results are the mean and SEM for at least five experiments performed in triplicate.

 
Single-particle tracking of hCG-occupied FLAG-LHR-wt receptors demonstrates trapping of receptors in small membrane compartments
To independently assess the localization of hCG-treated FLAG-LHR-wt in membrane microdomains, single-particle tracking methods were used. This technique identifies individual LHR on the surface of viable cells and tracks their motions over approximately 2 min. The centroid for a 40-nm gold particle attached to an individual receptor can be identified visually on video obtained from each experiment, and its motions can be quantitatively described. Figure 5Go, which shows a representative particle track for an untreated LHR and for an hCG-treated receptor, demonstrates that a gold particle bound to FLAG-LHR-wt exhibited distinctive motions in the presence and absence of hCG. hCG treatment reduced the size of compartments containing FLAG-LHR-wt from 230 ± 79 to 86 ± 36 nm (Table 1Go). Although the average residence time for receptors within microdomains and the number of microdomains, 6 ± 2 and 5 ± 2, accessed by the receptor in 2 min did not differ significantly for untreated and hCG-treated cells, respectively, an individual receptor’s rate of diffusion D within each domain was reduced by a factor of 10 after hCG treatment. Figure 6Go illustrates these results for untreated and hCG-treated cells and shows that as receptors access progressively larger membrane microdomains, their rate of diffusion within those microdomains increases.


Figure 5
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  		FIG. 5. Representative trajectories for a single FLAG-LHR-wt receptor on an untreated cell or a cell treated with hCG. Each track represents data obtained during a single experiment of approximately 2 min. The representative track for untreated FLAG-LHR-wt cells shows receptor confinement within five compartments, whereas the representative track for hCG-treated cells shows confinement within three compartments. Individual compartments within a given particle trajectory were identified as described in Materials and Methods.

 

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  		TABLE 1. Tracking of individual anti-FLAG-gold particles bound to FLAG-LHR-wt on CHO cells

 

Figure 6
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  		FIG. 6. Single-particle tracking of individual FLAG-LHR-wt receptors labeled with gold-conjugated anti-FLAG antibody. The compartment size and diffusion coefficient for the LHR within that compartment were calculated as described in Materials and Methods. Results shown are from individual cells that were untreated ({triangleup}), treated with hCG after labeling of receptors with gold-conjugated anti-FLAG antibody ({square}), or pretreated with 1% MßCD before labeling with anti-FLAG antibody and treatment with hCG ({blacktriangledown}). Data presented in this figure for each condition are from cells examined in separate experiments on three different days.

 
Because MßCD pretreatment largely reversed the effects of hCG on compartment size and receptor lateral diffusion, it seems plausible that compartments retaining hCG-treated receptors are rafts. As shown in Table 1Go and illustrated in Fig. 5Go, MßCD pretreatment results in a significant increase in the size of membrane compartments containing LHR-wt. The size of these compartments does not differ significantly from those accessed by untreated FLAG-LHR-wt receptors and, together with results from sucrose density gradient centrifugation experiments, suggests that these unbound receptors are residing in the bulk membrane rather than rafts.

Finally, we evaluated single-particle tracks for nonfunctional hormone-receptor complexes. As shown in Fig. 6Go, FLAG-LHR-wt receptors treated with deglycosylated hCG as well as FLAG-LHR-K583R receptors treated with hCG remained in large membrane compartments where they exhibited comparatively fast lateral motions. These results were consistent with previous studies of LHR rotational dynamics that suggest that receptors within nonfunctional hormone-receptor complexes exhibit comparatively short rotational correlation times, suggesting that they are located within smaller complexes (18).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our results demonstrate that LHR-wt, if occupied by a functional ligand, are capable of moving from the bulk membrane into small nanometer-diameter plasma membrane compartments with characteristics of so-called rafts. The translocation of LHR to low-density membrane regions appears to be characteristic of the functional hormone-receptor complex and is not observed either when the LHR is nonfunctional or when a hormone antagonist has bound to the wild-type receptor. Moreover, the regions within which hormone-treated LHR-wt receptors are confined are small. Translocation of receptors into rafts is also implicated in receptor-mediated signaling as demonstrated by the decrease in hCG-mediated cellular levels of cAMP when rafts were disrupted. The appearance of receptors within low-density, detergent-insoluble membrane fractions occurs upon binding of hormone agonist to LHR. This is similar to results of Bramley and Ryan (29) who separated membrane fragments from homogenated ovaries of superovulated ewes on continuous sucrose gradients. They isolated two distinct membrane fractions that displayed hCG binding and, as a result, suggested that there might be two populations of LHR present on granulosa cells. They also characterized selected proteins in heavy and light membrane fractions containing LHR including adenylate cyclase which, interestingly, associated with the heavy receptor population.

The mechanism involved in targeting of LHR to rafts is not clear. Proteins found in plasma membrane rafts include transmembrane proteins with attached lipid groups including, most commonly, GPI or palmitate (30). There is evidence for palmitoylation of the LHR at two cysteines located in the receptor’s intracellular carboxy terminus (31). Although mutations to the palmitoylated cysteines did not affect cAMP production, the mutated receptors were internalized more quickly than wild-type receptors, apparently through an arrestin-mediated pathway (32). Point mutations to palmitoylation sites on the LHR C terminus also eliminates LHR-wt translocation into rafts (33). However, it is not known whether there is reversible palmitoylation of the LHR-wt or whether the binding of ligand alters equilibrium between palmitoylated and nonpalmitoylated LHR. This appears to be the case for ß-adrenergic receptors, G protein-coupled receptors structurally related to LHR, which undergo palmitoylation/depalmitoylation cycling with binding of agonist favoring receptor depalmitoylation (34). If palmitoylation were necessary for targeting of a ligand-activated G protein-coupled receptor to rafts, depalmitoylation would be predicted to reduce the likelihood of finding, for example, ß-adrenergic receptors in rafts. However, the role for palmitoylation in receptor-mediated signaling (35) and raft localization is generally unclear and may prove ultimately to be receptor specific.

Receptor aggregation, either together with receptor palmitoylation or independently, may increase the affinity of the LHR for rafts. Signaling by multichain receptors such as T-cell and B-cell antigen receptors are present in the bulk membrane before cross-linking by multivalent antigens (36, 37). Cross-linking may shift the equilibrium distribution for membrane proteins within the lipid bilayer and favor interactions between proteins and more ordered lipid microdomains such as rafts as has been demonstrated for lipid-anchored proteins within artificial lipid monolayers (38). In our hands, LHR self-association (17) as well as raft localization are characteristic of functional hormone-receptor complexes. One could hypothesize that agonist-induced associations between LHR causes translocation of receptors to rafts. Interestingly however, presumably random interactions between LHR receptors after antibody cross-linking are not sufficient to drive receptor translocation to rafts but are sufficient for activation of adenylate cyclase in the absence of hCG. Although targeting of proteins to rafts can be initiated by antibody cross-linking of, for example, T-cell and B-cell antigen receptors (36, 37) and the Type I Fc{epsilon} receptor on rat basophilic leukemia cells (39), this was not the case for antibody-cross-linked LHR, suggesting that LHR required binding of a hormone agonist and potentially specific receptor conformations for targeting of activated receptors to rafts.

It appears that membrane rafts may serve as signaling platforms for the LHR, although the specific signaling event(s) are uncertain. Spatial coordination of key signaling proteins in lipid rafts may provide a rapid, efficient, and specific mechanism for promoting signal transduction from extracellular to intracellular mechanisms while also preventing cross-talk between pathways (40, 41). As an example, type I Fc{epsilon} receptors form, at a minimum, receptor dimers that are tyrosine phosphorylated by Lyn and then colocalize with Lyn in membrane rafts (39). Other downstream signaling molecules also appear within rafts. Oh and Schnitzer (41) have shown that Gi and Gs are targeted to, and concentrated in, lipid rafts, and some isoforms of adenylate cyclase are associated with these membrane structures (42, 43, 44). If rafts serve to concentrate these proteins, cholesterol depletion and raft disruption may disperse raft-associated proteins and reduce the likelihood of protein-protein interactions necessary for cell signaling. Indeed, MßCD treatment reduced fluorescence resonance energy transfer between GPI-anchored proteins (45) and signal transduction by these proteins (46). Other studies have shown that cholesterol is important for lipid raft formation and that its depletion decreases signal transduction efficiency (24, 47).

Single-particle tracking studies provide some insight into the nature of LHR-containing structures. Upon binding ligand, the LHR-wt becomes largely confined within small compartments with an average diameter of 86 nm. For the most part, the receptor remains within these regions for comparatively long times and appears to diffuse pseudo-randomly before being captured within another compartment of similar size. Similar behavior has been described and analyzed by Kusumi and co-workers (25) for selected phospholipids and for transferrin receptor (48) and by Daumas et al. (26) for the µ-opioid receptor, a G protein-coupled receptor involved in pain responses. Daumas argues that µ-opioid receptor motions reflect its diffusion within the bulk membrane followed by confinement within a domain that itself diffuses slowly and suggests that this confinement is a result of interactions with the confining molecules. Alternatively, Ritchie et al. (49) suggest that these interactions may be with proteins forming a continuous barrier (fences) or discontinuous protein barrier (pickets). Fences or pickets can confine and limit receptor diffusion within small membrane regions while still permitting intermittent escape from a compartment zone followed by faster diffusion in the bulk membrane. Our previous studies of LHR lateral diffusion using fluorescence photobleaching recovery methods suggest that actin microfilaments may provide fences or organizing structures for pickets that restrict the lateral motions of the receptor (50). Nonetheless, one caveat in interpreting these results is that the compartments occupied by hCG-occupied LHR should not be equated a priori with structures identified in biochemical studies such as plasma membrane rafts. The relationship of biochemically identified rafts to membrane compartments visualized via single-particle tracking remains a topic of active debate.

Finally, disruption of the membrane rafts reduced, but did not completely eliminate, LH signaling. This result is reasonable if hormone-mediated signaling is most efficient within rafts where higher concentrations of downstream signaling molecules exist. Nonetheless, some signaling proteins remain available within the bulk membrane, albeit at reduced concentrations, where they are capable of relaying a productive signal. The questions that remain are what role receptor aggregation plays in signaling and raft localization, whether small membrane compartments accessed by hCG-occupied receptors are the same structures isolated in low-density sucrose fractions, and whether rafts are essential for LHR function. These questions seem likely to be resolved only if it can be demonstrated that forcing an LHR, either with or without ligand, into the raft environment, can produce a downstream signal and if some, or all, of the components involved in LH-receptor-mediated signaling can be localized with LHR in the same membrane compartments. Alternatively, raft localization may be a convenient, but not essential, method for concentrating membrane proteins involved in signal transduction.


   Footnotes
 
This project was supported in part by National Institutes of Health Grant HD23236 (to D.A.R.) and National Science Foundation DBI-0138322 (to B.G.B.).

First Published Online January 12, 2006

Abbreviations: CHO, Chinese hamster ovary; GPI, glycosylphosphatidylinositol; hCG, human chorionic gonadotropin; LHR, LH receptor; MßCD, methyl-ß-cyclodextrin; wt, wild type.

Received August 16, 2005.

Accepted for publication January 5, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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