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BOR - Papers in Press, published online ahead of print December 21, 2005.
Biol Reprod 2005, 10.1095/biolreprod.105.048157
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BIOLOGY OF REPRODUCTION 74, 788–797 (2006)
DOI: 10.1095/biolreprod.105.048157
© 2006 by the Society for the Study of Reproduction, Inc.

Research Article

Differential Impact of Intracellular Carboxyl Terminal Domains on Lipid Raft Localization of the Murine Gonadotropin-Releasing Hormone Receptor

Amy M. Navratil 2, Todd A. Farmerie 2, Jan Bogerd 3, Terry M. Nett 2, and Colin M. Clay 1 2

Department of Biomedical Sciences,2 Animal Reproduction and Biotechnology Laboratory, Colorado State University, Fort Collins, Colorado 80523 Department of Endocrinology,3 Utrecht University, 3584 Utrecht, The Netherlands

ABSTRACT

The mammalian type I GNRH receptor (GNRHR) is unique among G protein-coupled receptors (GPCRs) because of the absence of an intracellular C-terminus. Previously, we have found that the murine GNRHR is constitutively localized to low-density membrane microdomains termed lipid rafts. As such, association of the GNRHR with lipid rafts may reflect both a loss (C-terminus) and a gain (raft association address) of structural characteristics. To address this, we fused either the full-length C-terminus from the nonraft-associated LH receptor (LHCGR; GNRHR-LF) or a truncated (t631) LHCGR C-terminus to the GNRHR. These chimeric receptors are trafficked to the plasma membrane, bind ligand, and display increased agonist-induced receptor internalization, but they do not partition into lipid rafts. Thus, a heterologous C-terminus from a nonraft-associated GPCR redirects localization of the GNRHR to nonraft domains. In contrast to the murine GNRHR, the catfish GNRHR (cfGNRHR) possesses an intracellular C-terminus. We found that the cfGNRHR was localized to lipid rafts and that the cfGNRHR C-terminus did not alter raft localization of the mammalian receptor. Consistent with placement in different lipid microenvironments within the plasma membrane, fluorescence recovery after photobleaching revealed different lateral diffusion phenotypes of the raft-associated GNRHR and cfGNRHR versus the nonraft-associated GNRHR-LF fusion protein. We conclude that whereas an intracellular C-terminus is capable of redirecting the GNRHR to nonraft compartments, this is not a generalized feature of GPCR C-terminal tails. Thus, constitutive raft localization of the GNRHR is not simply a result of the loss of an intracellular C-terminus.

gonadotropin-releasing hormone receptor, mechanisms of hormone action, pituitary

INTRODUCTION

During the past decade, it has become evident that the plasma membrane is not a random sea of lipids but, rather, displays regions, typically less than 100 nm in diameter, that contain elevated levels of glycosphingolipid and cholesterol as compared to the bulk plasma membrane [1, 2]. The tight packing of the saturated acyl chains of the sphingolipids and the presence of cholesterol appears to result in a domain that is less fluid than the surrounding plasma membrane. Furthermore, this unique lipid composition appears to be responsible for two key characteristics of membrane microdomains: relative insolubility in nonionic detergents, and migration to low-density fractions in sucrose gradients. Thus, these lipid rafts appear to exist as distinct, liquid-ordered phase islands dispersed in the more disordered matrix of the lipid bilayer [2, 3].

It is thought that lipid rafts serve to colocalize membrane receptors and their cognate downstream signaling components in either preassembled complexes or in separate complexes that, on ligand activation, cosegregate to form a transient signaling platform [4–6]. We have shown that the mammalian type I GNRH receptor (GNRHR) and downstream signaling intermediates, including RAF1 (previously known as c-raf) and GNAQ (previously known as G{alpha}q), are detectable in low-density sucrose fractions prepared from the gonadotroph-derived {alpha}T3–1 cell line [7]. Furthermore, raft localization of the GNRHR appears to be independent of cell type, suggesting that segregation of the GNRHR into lipid rafts is intrinsic to the receptor. Unresolved, however, is the identity of the structural features of the GNRHR that dictate inclusion of this molecule in lipid microdomains.

Protein-targeting theory would predict that the segregation of raft-associated proteins into lipid microdomains is dependent on unique structural determinants found within the protein itself. Unfortunately, determination of these unique structural motifs remains elusive. At least in part, this likely is a consequence of the diversity of mechanisms that underlie protein targeting to lipid rafts [8–12]. For transmembrane proteins such as cell-surface receptors, critical amino acid residues have been identified in membrane-proximal extracellular domains and in membrane-spanning domains that function as molecular addresses for lipid rafts [10]. In regard to the latter, Anderson and Jacobson [9] have proposed that the lipid-binding properties of specific amino acid motifs located in membrane-spanning regions of raft-associated transmembrane proteins result in a lipid shell that directs the segregation of these proteins into lipid microdomains. Lipid or glycolipid modifications, such as glycosyl-phosphatidylinositol anchors, appear to be the essential targeting motif in several raft-associated proteins [13]. In this scenario, the acyl lipid modification (e.g., myristoylation, palmitoylation) may represent the anchor that directly interacts with membrane microdomains [8]. Finally, for some transmembrane proteins, critical amino acid residues in cytoplasmic domains function as molecular addresses [9]. In the case of G protein-coupled receptors (GPCRs), this could represent motifs that are present in one of three intracellular loops or the cytoplasmic C-terminus [14]. The latter is particularly intriguing, because cysteine residues located in the intracellular C-terminus are targets for palmitoylation in multiple GPCRs [15–17].

Although classified as a member of the rhodopsin class of GPCR, the mammalian GNRHR displays several unique structural features. Perhaps the most striking of these is an extremely short carboxyl terminal cytoplasmic domain of only one or two amino acids [18, 19]. In more prototypical GPCRs, this domain is quite extensive and contains phosphorylation sites for GPCR kinases, which are second messenger-regulated kinases, such as protein kinase A and casein kinases [20]. In many GPCRs, phosphorylation of the C-terminus appears to be requisite for subsequent interaction with ß-arrestin, which hinders further G-protein activation and targets the deactivated receptor for internalization [21, 22]. This is thought to be the case with GNRHRs found in nonmammalian vertebrates (e.g., catfish [23], goldfish [24], chicken [25], Xenopus sp. [26]), which possess C-terminal tails and appear to be phosphorylated by GPCR kinases, show rapid desensitization [27, 28] and undergo rapid agonist-induced internalization [29]; however, differences in arrestin dependence exist among these receptors [30]. The mammalian GNRHR shows a natural resistance to desensitization and internalization [31], which has been attributed to its lack of a C-terminal tail [32].

Significant insight regarding the role of C-terminal tails in signaling, trafficking, and internalization has been gained through the construction of chimeric type I GNRHRs [33]; however, to our knowledge, the potential impact of this structural motif on in-membrane localization has not been evaluated [28, 34]. We were intrigued with the possibility that raft localization of the mammalian type I GNRHR might reflect both the gain (raft localization) and the loss (C-terminal tail) of structural determinants. If correct, this loss-and-gain theory would predict that placement of a heterologous C-terminal domain on the type I (murine) GNRHR would redirect the localization of this receptor from a raft to a nonraft domain. To begin to address this, we fused the intracellular C-terminus from the rat LH receptor (LHCGR; previously known as LHR) onto the murine GNRHR. Unlike the GNRHR, the LHCGR is excluded from lipid rafts in the absence of hormone [35]. We also constructed a chimeric murine GNRHR that contained the intracellular C-terminal domain from the nonmammalian catfish GNRHR (cfGNRHR). Although not demonstrated directly, the ability of cholesterol depletion to attenuate internalization has been used as prima facie evidence for lipid raft localization of nonmammalian GNRHRs [36, 37]. Consistent with this interpretation, we find that the cfGNRHR, like its mammalian type I counterpart, is evident in detergent-resistant membrane fractions. Thus, we were positioned to evaluate the potential impact of intracellular C-terminal tails from GPCRs that either are (cfGNRHR) or are not (LHCGR) constitutively localized to lipid rafts.

MATERIALS AND METHODS

Materials

Antihemagglutinin (anti-HA) antibody was purchased from Roche. Secondary antibodies, as well as those against phosphorylated mitogen-activated protein kinase (phospho-MAPK; sold as "p-ERK (E-4)") and MAPK3 (sold as "ERK 1 (K23)"), were from Santa Cruz Biotechnology, Inc. D-Ala6-desGly10-GNRH-Pro9-ethylamine (D-Ala6-GNRH1), GNRH (GNRH1), and hCG were purchased from Sigma. Glass-bottom microwell dishes for confocal studies were obtained from Mat-Tek. Alexa 594-conjugated concanavalin A (ConA) was purchased from Molecular Probes.

Cell Culture

Chinese Hamster Ovary (CHO) cells from American Type Culture Collection were maintained in high-glucose Dulbecco modified Eagle medium (DMEM) containing 2 mM glutamine, 100 U/ml of penicillin, 100 µg/ml of streptomycin, and 1x nonessential amino acids (Mediatech) with 10% fetal bovine serum (Gemini Bioproducts). All cells were grown in 5% CO2 at 37°C in a humidified environment. All transfections were performed with Polyfect Reagent (Qiagen) following the manufacturer's instructions.

Plasmids

The pHA-GNRHR-GFP vector, which expresses a tagged murine GNRH receptor fused to green fluorescent protein (GFP), has been described previously [7]. The murine 3x-HA-GNRHR along with the parent 3x-HA vector (pKH3) were generous gifts from Dr. Mark Roberson (Cornell University, Ithaca, NY). For rat LHCGR or cfGNRHR C-terminal tail exchanges, PCR was performed on pHA-GNRHR-GFP using a cytomegalovirus-derived upstream primer and a downstream primer that inserted an EcoRV site beginning at position 2 of the codon for Gly323. The resulting product was cloned into pGEM-TEasy (Promega) and sequenced to confirm fidelity of PCR. A fragment was then excised with PpuMI (within GNRHR) and EcoRV (the resulting EcoRV half-site contains a single conservative substitution at position 3 of the Gly323 codon, stopping blunt after position 1 of codon Tyr324). Tail fragments were generated by PCR using rat LHCGR cDNA or cfGNRHR cDNA (provided by J. Bogerd, University of Utrecht, Utrecht, The Netherlands) with upstream primers at the fusion junction site, which completed an SspI half-site found beginning at position 2 of GNRHR codon Tyr324 (the added sequence being removed following subsequent SspI digestion) and included the codon for murine GNRHR Phe325, then switching to the corresponding LHCGR or cfGNRHR sequence, respectively. The products were cloned into pGEM-TEasy, sequenced, excised with SspI and BamHI, and ligated with a PpuMI/EcoRV-cut upstream fragment into pHA-GNRHR GFP, from which the C-terminal portion of the native receptor had been removed with PpuMI/BamHI (GNRHR-LF-GFP or GNRHR-CF-GFP). The truncated LHCGR-tailed GNRHR was constructed similarly, only a downstream tail primer inserted a BamHI site after codon Arg657 (GNRHR-LT-GFP). For the LHCGR with murine GNRHR C-terminus, a fusion tail segment was produced by PCR using an LHCGR primer upstream of a BglI site at codon Ser595 and one of two LHCGR/GNRHR fusion primers at the end of transmembrane domain 7, choosing for exchange points conserved residues at 3 (Phe627-LHR-GF) or 6 (Tyr630-LHR-GY) amino acids from the end of murine GNRHR and placing a BamHI site immediately downstream of the termination codon. The product was cloned into pGEM-TEasy, sequenced, isolated with BglI and BamHI, and relocated along with an EcoRI/BglI LHCGR fragment from pLHR-GFP [35] into pDsRed2-N1 (BD Clontech; the resulting vectors contained the DsRed cDNA, but being downstream of the termination codon, it was not part of the expressed receptor protein). The 3x-HA-cfGNRHR was constructed by digesting the cfGNRHR cDNA in pcDNA3 with XbaI (blunted with Klenow) and BamHI and placed into EcoRI (blunted)/BamHI-cut pKH3. A schematic of the chimeric receptors is illustrated in Figure 1. All the wild-type (wt) and chimeric GNRHRs were tagged with GFP at the C-terminus and an HA epitope at the N-terminus except for the 3x-HA-GNRHR and 3x-HA-cfGNRHR, which had only HA at the N-terminus.


Figure 1
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  		FIG. 1. Schematic diagram of the wt and chimeric GNRHR and LHCGR fusion proteins. Chimeric GNRHRs were constructed by the addition of either the full 70-amino-acid LHCGR C-terminus (GNRHR-LF), a 27-amino-acid truncated (t631) LHCGR C-terminus (GNRHR-LT), or the full 51-amino-acid cfGNRHR C-terminus (GNRHR-CF). The LHR-GF and LHR-GY represent chimeras in which the LHCGR C-terminus is replaced with either the final two or final five amino acids from the murine GNRHR. Exclamation marks represent potential palmitoylation sites; asterisks represent Ser phosphorylation sites.

Confocal Microscopy

The CHO cells were grown on glass-bottom microwell dishes and transfected with GNRHR-LF-GFP, GNRHR-LT-GFP, or GNRHR-CF-GFP. After 48 h, CHO cells were rinsed with PBS and labeled with 10 µg/ml of Alexa 594-conjugated ConA for 15 min at 4°C. Cells were washed twice with ice-cold PBS and fixed for 15 min with 4% paraformaldehyde. Confocal microscopy was conducted using the 488- and 543-nm laser lines of a Zeiss LSM510 confocal laser-scanning microscope (CLSM) under a 63x oil objective.

Fluorescence Recovery after Photobleaching Measurements

The CHO cells were grown on glass-bottom microwell dishes and transfected with the wt GNRHR-GFP, cfGNRHR-GFP, or GNRHR-LF-GFP for 48 h. Fluorescence recovery after photobleaching (FRAP) assays were conducted by CLSM at room temperature using a protocol established by Tanimura et al. [38]. First, cells were visualized using a 63x objective and a 488-nm argon laser line. Before photobleaching, we took a whole-cell scan at low laser power. Photobleaching was then carried out in a bleach region of interest (ROI) using 100% laser power and 100% transmission. Recovery was followed with low-power laser scans taken every 2 sec for 100 sec. All FRAP studies were completed within 15 min for a single dish to control for effects of room temperature. Following data collection, fluorescence intensities were analyzed in the selected bleach ROI (Ib) and a reference ROI (Ir) at each time point. We selected reference regions of identical size and fluorescence intensities similar to our bleach region for each cell (n = 10). Relative intensities (rI) at each time point were calculated by substracting bleach intensity from reference intensity (rI = Ib – Ir). Curves were plotted, and a second-order polynomial was derived for each curve using Microsoft Excel. Each of the resulting quadratics was solved for recovery at 60 sec.

Lipid Raft Preparation and Sucrose Gradients

Monolayer cultures of CHO cells (150-mm dish) were transfected for 48 h with specified vectors. Cells were harvested in PBS containing 5 mM EDTA and centrifuged for 3 min at 300 x g. The cell pellet was resuspended in 375 µl of MES (2-[N-morpholino]ethanesulfonic acid) buffer followed by the addition of 375 µl of a 2x TX-100 lysis buffer (0.2% TX-100 prepared in MES buffer). For the sample, the final lysis buffer concentration was 1x (TX-100 concentration, 0.1%), with a final volume of 750 µl. Cells were then homogenized by three passes through a 30-gauge needle. Next, 750 µl of 90% sucrose (in MES buffer) were added to the samples to yield a 45% sucrose fraction in a final volume of 1.5 ml. A discontinuous sucrose gradient was then prepared by layering 1.5 ml of 35% and 5% sucrose in a 5-ml ultracentrifuge tube. Isopycnic ultracentrifugation was then carried out at 46,000 rpm using a Beckman SW-55 rotor for 16–20 h at 4°C. Following ultracentrifugation, 250-µl samples were collected, representing a total of 18 fractions. Proteins that migrated to the interface of the 5% and 35% layers (approximately fractions 6 and 7) were considered to be raft-associated [7, 39].

Western Blot Analysis

Samples representing individual sucrose fractions were subjected to SDS-PAGE (acrylamide:bis-acrylamide ratio, 29:1) and electroblotted to nitrocellulose (Osmonics). Membranes were blocked in 5% nonfat dried milk in Tris-buffered saline (TBS). Anti-HA antibodies were used at a 1:1000 dilution with an incubation time of 1 h. Blots were washed and then incubated with a 1:10000 dilution of anti-mouse horseradish peroxidase (HRP) for 1 h at room temperature. All blots were washed for 60 min (six times for 10 min each time) with TBS after secondary antibody and then visualized by chemiluminescence using Pierce SuperSignal reagents.

MAPK (Formerly ERK) Activation Assays

A monolayer of CHO cells (2 x 105) in six-well tissue culture plates was transfected with the specified vectors. After 48 h, cells were washed twice with PBS and incubated in serum-free DMEM for 6 h. Then, either 0 or 100 nM GNRH1 was administered for a 15-min incubation. Cells were washed in ice-cold PBS and lysed in RIPA (radioimmuno-precipitation assay) buffer containing 20 mM Tris (pH 8.0), 137 mM NaCl, 10% glycerol, 1% NP-40, 0.1% SDS, 0.5% deoxycholate, and 0.2 mM PMSF. Next, 6x sample buffer (300 mM Tris-HCl [pH 6.8], 60% glycerol, 30 mM dithiothreitol, and 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 blot analysis. Nitrocellulose membranes were incubated for 2 h with a phospho-MAPK antibody (1:1000 dilution), followed by a 2-h incubation with a 1:2000 dilution of HRP-conjugated secondary antibody. Phospho-MAPK blots were then stripped at room temperature with 100 mM 2-mercaptoethanol, 2% SDS, and 62.5 mM Tris-HCl (pH 6.7) and 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, then reprobed with a 1:10000 dilution of an anti-MAPK-1 antibody that recognizes MAPK1 and MAPK3 (formerly ERK1 and ERK2) independent of phosphorylation state. After washing in TBS, blots were incubated with a 1:2000 dilution of anti-rabbit HRP, and immunoreactive bands were visualized by chemiluminescence.

Internalization Assay

The GNRHR internalization assays were performed 48 h after transfection of CHO cells with either wt GNRHR-GFP, GNRHR-LF-GFP, or GNRHR-LT-GFP vectors in 12-well plates. Radioiodinated D-Ala6-GNRH1 was prepared using a glucose-oxidase procedure and purified by chromatography in QAE Sephadex as described by Wagner et al [40]. Briefly, cells are incubated with 72 fmol (2 x 105 cpm) of [125I]D-Ala6-GNRH1 in the presence or absence of 72 pmol of unlabeled D-Ala6-GNRH1. Cells were incubated on ice for 4 h and then warmed to 37°C for 0, 5, 10, 15, 30, 60, or 90 min. Internalization was stopped by immediate cooling to 0°C and rapid washing with ice-cold PBS. Acid-sensitive radioligand binding (cell surface binding) was removed by addition of ice-cold acid solution (150 mM NaCl and 50 mM acetic acid; final pH 2.8) for 12 min. After removal of acid, cells are washed with ice-cold PBS and solubilized with 0.2 M NaOH and 1.0% SDS to measure acid-resistant (internalized) radioligand. Nonspecific binding (binding in the presence of 72 pmol of unlabeled D-Ala6-GNRH1) is subtracted at each time point. Surface binding is expressed as a percentage of initial values and internalized radioligand as a percentage of total cell-associated radioligand (internalized plus cell surface) at each time point. Differences in the percentage of each receptor internalized at 0, 5, 10, 15, 30, 60, and 90 min after the addition of ligand were compared using least-squares analysis of variance. Means between each receptor type at each specific time point were compared using the Duncan multiple-range test, which was protected by a significant (P < 0.05) F-value for receptor type, time after addition of ligand, and interaction between the two.

Hormone-Binding Assays

Approximately 10 million CHO cells were transfected in 150-mm dishes with selected vectors. After 48 h, cells were washed with PBS and centrifuged at 2 x g, and cell pellets were resuspended in assay buffer (10 mM Tris-HCl, 0.1% BSA, and 0.01 mM CaCl2) to a final concentration of 1 x 107 cells per 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-GNRH1 (61.4 pM) in 50 µl of assay buffer in the presence or absence of 50 µl of nonradioactive D-Ala6-GNRH1 (340 nM; 1000-fold excess). The total volume for each tube was adjusted to 250 µl by addition of ice-cold assay buffer. Following a 4-h incubation at 4°C, 3 ml of ice-cold assay buffer were added, and samples were immediately centrifuged for 15 min at 16000 x g. The supernatants were decanted, and radioactivity in the cell pellets was quantitated using an Apex automatic gamma counter (Micromedic Systems). Specific binding was determined by subtracting the cpm in samples containing [125I]D-Ala6-GNRH1 in the presence of unlabeled D-Ala6-GNRH1 from the cpm in samples containing only [125I]D-Ala6-GNRH1 samples. The same procedure was followed for the LHR-GY and LHR-GF constructs, only hCG was used as the [125I]-radiolabeled and nonlabeled ligand.

RESULTS

Chimeric GNRHRs Are Trafficked to the Plasma Membrane, Bind Ligand, and Capable of Signaling to MAPK

Appropriate trafficking and membrane localization of GNRHR-CF fusion proteins have been established [27, 33, 34]. To determine if the LHCGR C-terminus disrupts membrane trafficking of the GNRHR, CHO cells were transiently transfected with either wt GNRHR-GFP, GNRHR-LF-GFP, GNRHR-LT-GFP, or GNRHR-CF-GFP fusion proteins. At 48 h posttransfection, cells were stained with Alexa 594-conjugated ConA, a red fluorescent derivative of ConA that binds to plasma membrane carbohydrates. Cells were then fixed and imaged using the 488- and 543-nm laser lines of a Zeiss LSM 510 CLSM. The Alexa 594-conjugated ConA clearly delineated the plasma membrane of CHO cells. A similar membrane distribution of green fluorescence was seen for the wt GNRHR-GFP, GNRHR-LF-GFP, GNRHR-LT-GFP, and GNRHR-CF-GFP (Fig. 2). Colocalization of the red and green fluorophores is revealed as yellow in the overlay image (Fig. 2).


Figure 2
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  		FIG. 2. The GNRHR-LF, GNRHR-LT, and GNRH-CF colocalize with a plasma membrane marker in CHO cells. The CHO cells expressing either a wt GNRHR-GFP, GNRHR-LF-GFP, GNRHR-LT-GFP, or GNRHR-CF-GFP expression vectors were grown in glass-bottom microwell dishes and stained with an Alexa 594-conjugated ConA for 15 min at 4°C. Cells were then fixed with 4% paraformaldehyde for 10 min. The CLSM was conducted using the 63x objective and the 488- and 543-nm laser lines of a Zeiss LSM 510 confocal microscope.

The CLSM analysis revealed membrane localization of the chimeric GNRHRs. To determine if these receptors are capable of ligand binding, CHO cells were transiently transfected with wt GNRHR-GFP, GNRHR-LF-GFP, or GNRHR-LT-GFP or the LHCGR chimerics with the GNRHR "C-terminus" LHR-GF and LHR-GY for 48 h. Untransfected CHO cells, which do not express GNRHR, were used as a negative control. Cells were incubated with [125I]D-Ala6-GNRH1 in the presence or absence of cold D-Ala6-GNRH1 for 4 h at 4°C. This analysis revealed that the wt GNRHR-GFP, GNRHR-LF-GFP, and GNRHR-LT-GFP are all capable of binding [125I]D-Ala 6 GNRH1 (Fig. 3A). The CHO cells transfected with wt LHCGR displayed binding of [125I]hCG; however, neither the LHR-GF nor the LHR-GY fusion proteins displayed any significant binding of radioactive ligand (Fig. 3B). Thus, the extreme C-terminus of the GNRHR appears to be incapable of "rescuing" membrane trafficking of an LHCGR lacking the intracellular C-terminus. This is consistent with abrogation of membrane trafficking of LHCGR with simple truncation of the intracellular C-terminus [41].


Figure 3
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  		FIG. 3. The intracellular C-terminus from the LHCGR does not affect the binding [125I]D-Ala6-GnRH1 to GnRHR fusion proteins. A) The CHO cells were transiently transfected with expression vectors for the wt GnRHR-GFP, GnRHR-LF-GFP, or GnRHR-LT-GFP fusion proteins. Untransfected CHO cells were used as a negative control. At 48 h posttransfection, cells were harvested in ice-cold PBS, centrifuged, and placed in assay tubes with [125I]D-Ala6-GnRH1 in the presence or absence of unlabeled D-Ala6-GnRH1 (1 x 107 CHO cells per 50 µl). All samples were done in triplicate. Binding of [125I]D-Ala6-GnRH1 (61.4 pM) was determined as counts bound in the absence of unlabeled D-Ala6-GnRH1 subtracted from counts bound in the presence of unlabeled D-Ala6-GnRH1 (340 nM). B) CHO cells were transiently transfected for 48 h with LHCGR, LHCGR-GY, or LHCGR-GF, and binding was determined as in A except that [125I]hCG and hCG served as the radioactive and nonradioactive ligands, respectively. Bars represent the mean ± SEM for at least three replicates.

To confirm functional phenotype, we next assessed the ability of the chimeric GNRHRs to activate MAPK in response to GNRH1. The CHO cells were transiently transfected with the wt GNRHR-GFP, GNRHR-LF-GFP, GNRHR-LT-GFP, or GNRHR-CF-GFP for 48 h. Untransfected CHO cells were used as a negative control. Transfected cells were serum starved for 6 h and then incubated in the presence or absence of 100 nM GNRH1 for 15 min. Cells were lysed in RIPA buffer and subjected to probing by Western blot analysis for the dual-phosphorylated forms of MAPK (MAPK1 and MAPK3). Consistent with our previous studies, 15 min of exposure to GNRH1 increased MAPK phosphorylation in the wt GNRHR [7, 42]. A similar increase in MAPK phosphorylation was evident in RIPA lysates prepared from CHO cells transfected with the GNRHR-LF-GFP, GNRHR-LT-GFP, and GNRHR-CF-GFP fusion proteins (Fig. 4A).


Figure 4
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  		FIG. 4. The GNRHRs harboring heterologous intracellular C-terminal domains are capable of signaling to MAPK and display agonist-induced internalization. A) The CHO cells were transiently transfected with expression vectors for the wt GNRHR-GFP, GNRHR-LF-GFP, GNRHR-LT-GFP, or GNRHR-CF-GFP fusion proteins. Untransfected CHO cells were used a negative control. At 48 h posttransfection, cells were washed with PBS twice and incubated with serum-free medium alone for 6 h at 37°C. Cells were incubated with serum-free medium containing 0 or 100 nM GNRH1 for 15 min. Samples were then lysed in RIPA buffer, and lysates were examined by Western blot analysis using antibodies specific for phosphorylated MAPK (previously ERK1 and ERK2). After probing with anti-phospho-MAPK, blots were stripped and reprobed with an antibody that detects MAPK1 and MAPK3 independent of phosphorylation. B) The CHO cells were transiently transfected with expression vectors for the wt GNRHR-GFP, GNRHR-LF-GFP, or GNRHR-LT fusion proteins for 48 h. Cells were then incubated with [125I]D-Ala6-GNRH1 in the presence or absence of unlabeled D-Ala6-GNRH1 for 4 h at 4°C. Cells were warmed at 37°C for 0, 5, 10, 15, 30, 60, or 90 min. Surface binding is expressed as a percentage of initial values, and internalized radioligand is expressed as a percentage of total cell-associated radioligand (internalized + cell surface) at each time point. Percentage internalization at each time point represents the mean ± SEM.

Presence of an Intracellular C-Terminus Increases the Extent of GNRHR Internalization

The relatively blunted internalization kinetics of the mammalian type I GNRHR has been attributed to the absence of an intracellular C-terminus [32, 43]. Thus, the addition of heterologous C-terminal domains, including the cfGNRHR C-terminus, typically results in an increased rate and extent of receptor internalization [27]. To test this in our system, CHO cells were transiently transfected for 48 h with either the wt GNRHR-GFP, GNRHR-LF-GFP, or GNRHR-LT-GFP. Internalization of [125I]D-Ala6-GNRH1 was determined at 0, 5, 10, 15, 30, 60, and 90 min as described previously [44]. The GNRHR internalization reached a maximum of 32% at 90 min (Fig. 4B). At 60 and 90 min, the extent of internalization of the GNRHR harboring the full-length LHCGR C-terminus was significantly (P < 0.05) greater than that of the wt GNRHR (Fig. 4B). The presence of the truncated LHCGR C-terminus led to a further increase in the extent of internalization. It is interesting to note that the impact of the truncated LHCGR C-terminal domain exactly recapitulates what has been reported previously for the wt LHCGR. Specifically, the LHCGR C-terminal truncate displayed a higher rate and extent of internalization compared with the wt receptor [41].

Presence of an Intracellular C-Terminus Differentially Affects Lipid Raft Distribution of the Mammalian Type I GNRHR

In contrast to the constitutive residence of the type I GNRHR in lipid rafts [7], the LHCGR is detectable in lipid rafts only after ligand activation [35]. On confirming the ability of the tailed chimeric GNRHRs to recapitulate key functional attributes, including membrane trafficking, ligand binding, signaling, and internalization, we next asked if localization of the GNRHR in lipid rafts might reflect the loss of structural determinants in the intracellular C-terminal domains of more prototypical GPCRs. To address this issue, CHO cells were transfected with expression vectors for wt GNRHR, cfGNRHR, GNRHR-LF-GFP, GNRHR-LT-GFP, or GNRHR-CF-GFP. At 48 h posttransfection, raft preparations were prepared using a detergent-based method that has been described previously [7]. Raft fractions were separated using sucrose gradient centrifugation. As with our earlier studies, wt GNRHR was predominantly localized to low-density sucrose fractions [7]. In contrast, both the chimeric GNRHR-LF-GFP and GNRHR-LT-GFP were detectable only in high-density sucrose fractions (Fig. 5). Thus, the addition of the LHCGR intracellular C-terminus appears to fundamentally alter raft distribution of the murine GNRHR. Furthermore, this effect appears to be independent of C-terminal Ser phosphorylation sites. Interestingly, despite the presence of an extensive intracellular C-terminus, the cfGNRHR, like its nontailed mammalian counterpart, also localized to low-density sucrose fractions. Thus, raft trafficking may be a general feature of both mammalian (nontailed) and nonmammalian (tailed) GNRHRs. If correct, then in contrast to LHCGR, the C-terminal domain of the cfGNRHR would not, presumably, redirect raft trafficking of the mammalian type I receptor. Consistent with this, the C-terminal domain from the cfGNRHR did not alter raft distribution of the mammalian type I GNRHR (Fig. 5).


Figure 5
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  		FIG. 5. The intracellular C-termini from the LHCGR and cfGNRHR differentially affect raft localization of the murine GNRHR. The CHO cells (150-mm dish) were transfected with expression vectors for the indicated fusion proteins for 48 h. Raft samples were prepared using a TX-100 detergent buffer and separated by isopycnic ultracentrifugation through a nonlinear sucrose gradient (45%, 35%, and 5%). Fractions (250 µl) were collected from the top and separated by SDS-PAGE. Western blot analysis was conducted using an antibody directed against an HA epitope tag on the GNRHR, cfGNRHR, or GNRHR chimerics. Proteins that migrate to the 5%/35% interface (fractions 5–7) are considered to be raft associated [7].

FRAP Reveals Differences in Lateral Diffusion Properties of Tailed Mammalian Type I GNRHRs

Protein-protein interactions and the surrounding lipid environment affect the lateral motion of integral membrane proteins [45]. We have found that the major fraction of unoccupied GNRHRs in the plasma membrane are laterally mobile and display a relatively rapid rate of lateral movement [46]. Based on the ability of the LHCGR C-terminus to redirect the GNRHR to nonlipid raft domains, we reasoned that the in-membrane behavior of the GNRHR harboring the LHCGR C-terminus would display a different lateral diffusion phenotype. To address this, CHO cells were transfected with wt GNRHR-GFP, cfGNRHR-GFP, or GNRHR-LF-GFP expression vectors in glass-bottom microwell dishes, and FRAP analysis was carried out with CLSM [38]. Consistent with our earlier work, we found that the mobile fraction of wt GNRHRs is greater than 90% (Fig. 6A). The fraction of laterally mobile cfGNRHRs in the plasma membrane was similar to the wt mammalian type I receptor (Fig. 6B). In contrast to the in-membrane behavior of the raft-associated type I GNRHR and cfGNRHR, the percentage of laterally mobile GNRHR-LF-GFP fusion proteins was reduced by 45% ± 7% (Fig. 7). Thus, differential localization of the wt GNRHR, cfGNRHR and the chimeric GNRHR-LHCGR C-tail to lipid rafts is reflected as fundamental differences in the lateral diffusion properties of these molecules.


Figure 6
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  		FIG. 6. Fluorescence recovery after photobleaching reveals similar lateral diffusion characteristics of the mammalian GNRHR and cfGNRHR. The CHO cells were transiently transfected with expression vectors for wt GNRHR-GFP (A) or cfGNRHR-GFP (B) for 48 h. Cells were imaged using a 63x objective and the 488-nm argon laser line of a Zeiss LSM510 CLSM. A bleach ROI was used in the FRAP measurements. The ROI was photobleached using 100 % power of the 488-nm line of an argon laser. Fluorescence recovery was then obtained in the ROI using low-power laser scans at 2-sec intervals for 60 sec. Images and recovery curves from a representative experiments are shown. Fluorescence intensity and recovery was determined for both the bleach ROI and a reference ROI of the same size. The percentage recovery (%R) was determined as described in Materials and Methods


Figure 7
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  		FIG. 7. The intracellular C-terminus from the LHCGR alters the lateral diffusion phenotype of the mammalian GNRHR. The CHO cells were transiently transfected with an expression vector for the GNRHR-LF-GFP fusion protein for 48 h. The FRAP analysis was conducted as described in Figure 6. Images and the recovery curve from a representative experiment are shown. The percentage recovery (%R) in the bleach ROI was determined as described in Materials and Methods.

DISCUSSION

The construction of chimeric type I GNRHRs harboring heterologous C-terminal tails has yielded much insight regarding the role of these domains in signaling, trafficking, and internalization [33]; however, to our knowledge, the potential impact of this structural motif on lipid raft localization of the GNRHR has not been evaluated [28, 34]. To address this issue, we constructed chimeric murine GNRHRs that contained the intracellular C-terminal domain from GPCRs that either are (cfGNRHR) or are not (LHCGR) constitutively localized to lipid rafts [37]. Clearly, the utility of this approach is dependent on the degree to which the chimeric receptors display key functional attributes. Importantly, our data suggest that with the exception of the LHCGR harboring the C-terminus of the GNRHR, the chimeric receptors are trafficked to the plasma membrane and are capable of ligand binding and conveying an intracellular signal to the level of MAPK phosphorylation. Additionally, the internalization phenotype of the mammalian type I GNRHR is altered depending on the identity of the heterologous intracellular C-terminus such that the extent of internalization of GNRHRs harboring the t631 truncated LHCGR C-terminus is greater than either the GNRHR-LF fusion protein or wt GNRHR. Enhanced internalization of the type I GNRHR harboring the cfGNRHR C-terminal domain has been established [27].

We find that both the full-length and truncated forms of the intracellular C-terminal tail of the LHCGR redirects the GNRHR to nonraft domains. Interestingly, however, this is not simply the generic effect of adding a GPCR tail to the GNRHR. Specifically, when the intracellular C-terminus tail of the cfGNRHR is used to extend the type I GNRHR, this chimeric receptor, like the wt murine GNRHR, partitions into low-density fractions in sucrose gradients. Thus, the change in raft localization conferred by LHCGR is, presumably, caused by the presence of a specific structural feature within the C-terminal tail of LHCGR that overrides the intrinsic raft localization of the GNRHR. At issue, then, is the key difference between the cfGNRHR and LHCGR C-termini that account for the differential impact of these domains on raft trafficking of the mammalian type I GNRHR. At present, we cannot reach a definitive conclusion; however, several points can be made. First, the difference probably is not caused by phosphorylation or palmitoylation, because the C-terminal tails of both LHCGR and cfGNRHR likely are similarly modified [36, 47, 48]. Additionally, removal of the four distal Ser phosphorylation sites did not alter the impact of the LHCGR C-terminus on trafficking of the GNRHR to nonraft microdomains. One intriguing difference between these C-terminal domains is the dependence on ß-arrestin for internalization. Whereas the LHCGR displays an arrestin-dependent phenotype [49], internalization of the cfGNRHR or GNRHR-CF appears to be arrestin independent [50]. Although historically thought of as mediating desensitization and internalization of GPCRs [21, 22], more global roles for ß-arrestin family members, including scaffolding and trafficking, has emerged over the past several years [51, 52].

The lateral diffusion of proteins integrated into the plasma membrane is dependent on a number of factors, including interactions with other proteins, interactions with the cortical cytoskeleton, mode of membrane insertion, and surrounding lipid environment [53]. Consistent with our earlier studies, we find that the major fraction of unoccupied type I GNRHRs are laterally mobile in the plasma membrane and display a relatively rapid rate of lateral diffusion [46]. As such, it is interesting to note that the lateral diffusion characteristics of the cfGNRHR are equivalent to those of the type I receptor. Both proteins appear to be trafficked to low-density raft domains, so the in-membrane biophysical behavior of these receptors may reflect localization to a similar lipid microenvironment in the plasma membrane. In contrast, the addition of the LHCGR C-terminus not only redirects the type I GNRHR to nonraft domains but also fundamentally alters the lateral motions of the GNRHR, reflected as a significant reduction in the fraction of laterally mobile receptors on the cell surface. In fact, the lateral diffusion phenotype of the type I GNRHR harboring the LHCGR C-terminus is similar to what has been reported previously for the wt LHCGR expressed as a GFP fusion protein [54]. We should underscore that although limited lateral diffusion of membrane proteins has been used as a biophysical index of protein localization in confined plasma membrane microdomains [55], this relationship is far from clear. For example, in contrast to the view that raft-associated proteins will display more constrained lateral movements in the plasma membrane, Kenworthy et al. [45] found that raft proteins can display a high percentage mobile fraction, in some cases equivalent to or greater than that determined for nonraft proteins. As such, it likely is problematic to infer a mode of biophysical behavior of a raft-associated protein based on biochemical characterization. Thus, in our view, the key observation is that differential sorting of the GNRHR, cfGNRHR, and the chimeric GNRHR-LF to either low- or high-density sucrose fractions is reflected as a distinct difference in the lateral diffusion properties of these molecules. Finally, we recognize the caveats associated with assigning raft localization of a protein based on membrane homogenization and sucrose gradient separation [56]. In particular, Triton solubilization may artificially stabilize membrane domains [57]. Nevertheless, detergent resistance has served as one of the key functional, biochemical approaches for defining lipid raft association of membrane proteins. Certainly, we have used this approach to evaluate raft localization of the mammalian GNRHR; however, experimental results based on other methods, including nondetergent-based homogenization [7] and, more recently, colocalization with GM1 ganglioside (data not shown), are entirely consistent with placement of the mammalian type I GNRHR in lipid raft domains.

Given the role of fatty acyl modifications as a mechanism for raft association of a number of membrane proteins, including GPCRs [58], much of the work in this arena has focused on the intracellular C-terminal domain of GPCRs. Our data certainly support the notion that this domain not only contributes to raft targeting but also overrides an intrinsic raft "address" in the nontailed type I GNRHR. It is, however, also clear that the role(s) of the GPCR C-terminal tails in raft trafficking is not simple. Whereas the cfGNRHR, like the LHCGR, possesses an intracellular C-terminus, this receptor predominantly localizes to low-density fractions. Similarly, the cfGNRHR C-terminus does not alter raft distribution of the type I receptor. Thus, the field of study logically has focused on C-terminal acylation as a mechanism for raft localization of GPCRs, but it is important to underscore that other domains of these receptors are equally likely to be candidates for raft trafficking in both tailed and nontailed receptors.

In summary, we find that lipid raft localization of the mammalian type I GNRHR cannot be accounted for simply by the absence of an intracellular C-terminal domain. Thus, raft localization likely is not a default trafficking pathway resulting from the loss of an intracellular C-terminus. Whereas the LHCGR C-terminus is capable of directing the GNRHR from raft to nonraft domains, this is not the case for the intracellular C-terminus from the cfGNRHR. As such, it is tempting to speculate that lipid raft localization may be a generally conserved feature of both tailed and nontailed GNRHRs. Because raft placement appears to be critical for GNRHR coupling to GNAQ and, more recently, calmodulin in homologous cells [7, 59], elucidation of the motifs that lead to raft localization remains important to fully understand the structural organization of GNRHRs.

ACKNOWLEDGMENTS

The authors would like to thank Dr. Mark Roberson for the 3X-HA-GNRHR and pKH3 expression vectors and Dr. Clay Lents for assistance with statistical analyses.

FOOTNOTES

1 Correspondence: Colin M. Clay, Department of Biomedical Sciences-ARBL, 1683 Campus Delivery, Fort Collins, CO 80523. FAX: 970 491 3557; colin.clay{at}colostate.edu Back

Received: 3 October 2005.

First decision: 13 November 2005.

Accepted: 19 December 2005.

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