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Neuroendocrinology; |
Wadsworth Center,6 David Axelrod Institute for Public Health, New York State Department of Health, and Department of Biomedical Sciences, State University of New York at Albany, Albany, New York 12208
Research Unit in Reproductive Medicine,4 Hospital de Ginecobstetricia "Luis Castelazo Ayala," Instituto Mexicano del Seguro Social, México 10101 D.F., México
Depto de Fisicoquímica,5 Facultad de Química, Universidad Nacional Autónoma de México (UNAM), México 04510 D.F., México
ABSTRACT
The carboxyl-terminal segment of G protein-coupled receptors has one or more conserved cysteine residues that are potential sites for palmitoylation. This posttranslational modification contributes to membrane association, internalization, and membrane targeting of proteins. In contrast to other members of the glycoprotein hormone receptor family (the LH and thyroid-stimulating hormone receptors), it is not known whether the follicle-stimulating hormone receptor (FSHR) is palmitoylated and what are the effects of abolishing its potential palmitoylation sites. In the present study, a functional analysis of the FSHR carboxyl-terminal segment cysteine residues was carried out. We constructed a series of mutant FSHRs by substituting cysteine residues with alanine, serine, or threonine individually and together at positions 629 and 655 (conserved cysteines) and 627 (nonconserved). The results showed that all three cysteine residues are palmitoylated but that only modification at Cys629 is functionally relevant. The lack of palmitoylation does not appear to greatly impair coupling to Gs but, when absent at position 629, does significantly impair cell surface membrane expression of the partially palmitoylated receptor. All FSHR Cys mutants were capable of binding agonist with the same affinity as the wild-type receptor and internalizing on agonist stimulation. Molecular dynamics simulations at a time scale of
100 nsec revealed that replacement of Cys629 resulted in structures that differed significantly from that of the wild-type receptor. Thus, deviations from wild-type conformation may potentially contribute to the severe impairment in plasma membrane expression and the modest effects on signaling exhibited by the receptors modified in this particular position.
biosynthesis, expression, follicle-stimulating hormone, follicle-stimulating hormone receptor, palmitoylation
The follicle-stimulating hormone (FSH), LH, and thyroid-stimulating hormone (TSH) receptors are glycoproteins that belong to the superfamily of G protein-coupled receptors (GPCR), specifically the family of rhodopsin-like receptors [1]. These receptors consist of a single polypeptide chain of variable length that threads back and forth across the lipid bilayer seven times forming characteristic 
-helical transmembrane domains (TM) connected by alternating extracellular and intracellular (i) loops (L), with an extracellular amino-terminal domain and an intracellular carboxyl-terminal segment (Ctail) [1, 2]. Glycoprotein hormone receptors are related to each other by the presence of a large extracellular domain containing several leucine-rich repeats as well as by the homologous structure of their corresponding ligands, which are noncovalently bound heterodimeric glycoproteins [3]. The human (h) FSH receptor (R) (FSHR) consists of 695 amino acids (the first 17 amino acids encoding the signal sequence) [4–6]. On agonist binding, the activated receptor stimulates a number of intracellular signaling pathways. In the classical, linear signaling cascade, occupancy of the FSHR causes activation of the heterotrimeric Gs protein and stimulation of the effector adenylyl cyclase with the consequent increase in the synthesis of the second messenger cAMP, activation of protein kinase A, phosphorylation of cAMP response element-binding protein, and activation of transcription [7]. Nonetheless, increasing evidence indicates that in addition to the adenylyl cyclase/cAMP/protein kinase-A signaling pathway, activation of the FSH receptor by its cognate ligand also triggers activation of other intracellular signaling cascades, including the MAPK and phosphoinositol-3-kinase/Akt pathways [8–12].
Site-directed mutagenesis and chimeric studies of several GPCRs belonging to the family of rhodopsin-like receptors indicate that several cytoplasmic domains of these receptors, particularly the iL2, the juxtamembrane portions of the iL3, and the Ctail, are involved in several functions, including signal transduction, plasma membrane expression, receptor internalization and turnover, and interaction of the receptor with adapter proteins [1, 13]. In the FSHR, several structural and functional features characterize the Ctail domain: 1) It is rich in serine and threonine residues (which are potential sites for phosphorylation [14]) and plays an important role in determining nonvisual arrestin binding capacity and the fate of the internalized receptor [14, 15]; 2) it contains a minimal BBXXB motif reversed in its juxtamembrane region (residues 614–618), which is involved in receptor expression and to a lesser extent in Gs protein activation [16–18]; the last two residues of this motif (Arg617 and Arg618) and the preceding Phe616 are part of the NH2-end of the highly conserved F(X)6LL motif required for receptor transport to the cell membrane [19]; and 3) this domain exhibits two conserved cysteine residues (at positions 629 and 655) and one nonconserved Cys residue (at position 627) (Fig. 1) whose function is unknown but that may be the target for S-acylation with palmitic acid. This contrasts with the TSHR and LHCGR, in which palmitoylation of their conserved Ctail Cys residues has been well documented [20–23]; further, in these receptors, mutation of their corresponding palmitoylation sites results in abnormal intracellular transport and/or altered turnover of the mutant receptors [21–25].
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In the present study, a functional analysis of the Cys residues present in the Ctail of the hFSHR was carried out. For this purpose, a series of mutant hFSHRs were constructed in which Cys residues at positions 627, 629, and 655 were substituted by Ala, Ser, or Thr both individually and together. Mutant hFSHRs were transiently expressed in human embryonic kidney-293 (HEK-293) cells, and their function and ability to incorporate [3H]-labeled palmitic acid was analyzed. In addition, to analyze how the mutations may affect the structural and dynamic properties of the hFSHR Ctail, long molecular dynamics simulations for a model of the wild-type (Wt) receptor and eight Cys hFSHR mutants were performed. One-hundred-nanosecond-long trajectories were reached employing a novel approach that optimizes the conformational sampling of protein regions with low computational cost.
Construction of the hFSHR mutants was performed employing the full-length Wt hFSHR cDNA [26] (GenBank accession no. S59900) cloned into the mammalian expression vector pSG-5 (Stratagene). Site-directed mutagenesis was performed using the QuikChange site-directed mutagenesis kit (Stratagene), following the manufacturer instructions. Each cysteine residue (at positions Cys627, Cys629, and Cys655) present in the Ctail (residues 614–678) of the receptor (Fig. 1A) were individually replaced by Ala, Ser, or Thr. Double and triple mutants in which Cys residues were replaced with Ala (hFSHR-C627/629A, -C629/655A, and -C627/629/655A) were also constructed employing the same mutagenesis procedure (Table 1). Forward and reverse mutagenic oligonucleotide primers (Life Technologies) were designed according to the cDNA sequence reported for the testicular hFSHR [26] (see Supplemental Table 1 available online at www.biolreprod.org). A chimeric hFSHR/rat (r) Lhcgr-Ctail cDNA was constructed employing the following procedure. The expression vector pcDNA3.1-rLhcgr (GenBank accession no. NM_012978] was used as template and the phosphorylated oligonucleotides 5'-ATCTTCACGAAGGCGTTTCAGAG-3' and 5'-ACCACCGAGAGCGTTAACTCACTAG-3' as primers. The 219-bp fragment corresponding to the carboxyl-terminal segment of the rLHCGR (residues 604 to 674) was amplified by PCR and ligated into the expression vector pcDNA3.1 (Invitrogen) at the Eco RV restriction site (pcDNA3.1-rLhcgr-Ctail construct). The rLhcgr-Ctail cloned started with ATC allowing the recovery of the EcoRV site. An 1890-bp hFSHR fragment (residues 1–611) was amplified from the expression vector pSG5-hFSHR by PCR employing the phosphorylated oligonucleotides 5'-CTAGCCACCATGGCCCTGCTCCTGGTCTC-3' and 5'-GTGCCAACCCCTTCCTCTATGCC-3' as primers. Pfu DNA polymerase (Invitrogen) was used in all PCR reactions. The amplified product was then ligated into the pcDNA3.1-rLhcgr-Ctail construct at the new Eco RV restriction site. Finally, the chimeric hFSHR/rLhcgr-Ctail fragment (2109 bp) was subcloned into the pSG5 vector at the Eco RI restriction site to obtain the pSG5-hFSHR(1–611)/rLhcgr-Ctail (604–674) expression plasmid. The identity of all mutant hFSHR constructs and the correctness of the PCR-derived sequences were verified by DNA sequencing using an automated sequencer 377 (Applied Biosystems). For transfection, large-scale plasmid DNAs were prepared using an Endofree maxiprep kit (Qiagen).
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Cell Culture and Transfection of Wt and Mutant hFSHR cDNAs
Human embryonic kidney 293 cells (HEK-293 cell line was kindly provided by Dr. Aaron J.W. Hsueh, Stanford University, Stanford CA) were maintained in an humidified atmosphere of 5% CO2 at 37°C in low-glucose Dulbeco modified Eagle medium (DMEM) (Life Technologies), supplemented with 5% fetal calf serum (FCS), 5 µg/ml geneticin, and antibiotic-antimycotic reagent (Life Technologies). Cells were grown to 70%–80% in 75-cm2 flasks (Costar) and then replated at an initial density of 3.75 to 7.5 x 104 cells per well in 24-well or 48-well cell culture plates for 24 h at 37°C. Cells were then washed with unsupplemented DMEM and transfected with 0.4 µg DNA per well by liposome-mediated endocytosis in OPTIMEM (Life Technologies).
In Vitro Bioassay and Measurement of cAMP Production
Forty-eight hours after the start of transfection, the medium was removed and the cells were washed twice with DMEM-5% FCS and then stimulated with increasing doses (0.7–200 ng/ml) of human recombinant FSH (Serono de México S.A. de C.V.) in DMEM-5% FCS supplemented with 0.125 mM 3-isobutyl-methyl-xantine (Sigma Chemical Co.). At the end of the incubation period (18 h), the medium was removed, and total (extracellular plus intracellular) cAMP accumulation was measured in acetylated samples by radioimmunoassay, as described previously [27].
Human pituitary FSH (specific activity 24 µCi/µg protein) was radiolabeled as described previously [28]. HEK-293 cells (75 000 cells/well in 24-well dishes) were transfected with Wt or mutant hFSHR cDNAs as described previously. Forty-eight hours after transfection, the medium was removed, replaced with fresh medium, and allowed to continue incubation at 37°C for 1 h. After the preincubation period, the medium was removed, and serum-free DMEM containing 20 ng/ml [125I]-FSH was added to each well in the presence or absence of 1 µg/ml unlabeled recombinant single-chain FSH to assess for nonspecific binding [29]. Hormone was allowed to bind for 1 h at 37°C before the cultures were placed on ice and washed twice with ice-cold PBS. Cell surface radiolabeled FSH was eluted with ice-cold 50 mM glycine/100 mM NaCl, pH 3.0, for 10 min on ice, and the eluate was removed to a glass tube and counted with a gamma counter (Wallac 1470 Wizard).
Internalization of hFSHR Species under Equilibrium and Nonequilibrium Conditions
Equilibrium-binding internalization assays were performed by incubating HEK-293 cells with 20 ng/ml [125I]-FSH in serum-free media in the presence or absence of 1 µg/ml unlabeled single-chain recombinant FSH [29] for 1 h at 37°C. Unbound radiolabeled hormone was removed, fresh buffer was replaced, and the cells were incubated at 37°C. At each time point (0–90 min), cells were removed from the incubator, placed on ice, washed with 1x PBS, and incubated on ice in elution buffer (50 mM glycine/100 mM NaCl, pH 3.0) for 20 min to elute cell surface FSH. After the eluate was removed for counting, the cells were washed with PBS and then solubilized in 2 M NaOH for 1 h at room temperature to allow measurement of cell-associated counts per minute.
Internalization under nonequilibrium binding conditions was measured by binding assays performed as described previously but with [125I]-FSH removed at the various times (0, 5, 10, 15, 30, 45, 60, and 90 min). Cell surface radiolabeled FSH was eluted as described previously. After the eluate was removed for counting, the cells were washed with 1x PBS and solubilized in 2 M NaOH for 1 h at room temperature to allow measurement of cell-associated counts per minute. Data were analyzed using a spreadsheet designed in our laboratory. The endocytotic rate constant (Ke) was calculated from the slope generated by least squares regression of the line resulting from graphing the cell-associated counts per minute against the integral of the cell surface binding; this calculation includes time and specifically bound counts per minute. The half-time (t
) for internalization was calculated using the formula 0.692/Ke = t.
The binding affinity of Wt and mutant hFSHRs was determined in an equilibrium displacement binding isotherm assay using [125I]-FSH as previously described [30, 31]. HEK-293 cells were transfected as described previously and cultured for 48 h at 37°C. Cell numbers were adjusted according to binding assays to give comparable cpm of [125I]-FSH binding in order to accurately compare Kd values between Wt and mutant receptors. Increasing amounts (0 to 650 ng/ml) of unlabeled pituitary FSH competed with [125I]-FSH for binding to the FSHR. After the incubation period (18 h at room temperature), 2 ml cold assay buffer (50mM Tris pH 7.5, 25 mM MgCl2, 0.3% BSA) were added to each tube and the cells were pelleted at 3000 rpm for 30 min. Pellets were counted in a gamma counter, and the data were analyzed using an in-house-built computer program.
SDS-PAGE and Western Blot Analysis
Human embryonic kidney 293 cells were transfected with 0.4 µg of hFSHR Wt or mutant plasmid DNA. After 48 h of the start of transfection, cells were lysed with Passive Lysis Buffer (Promega), containing 1% Igepal, 0.4% DOC, 10 mM Tris pH 7.5, and 6.6 mM EDTA, and protein extracts (15 µg/lane) were resolved by 7.5% SDS-PAGE. Following electrophoreses, proteins were transferred to Immobilon-P membranes, probed with primary antibody (5 µg mAb 106.105), and then incubated with secondary anti-mouse IgG horseradish peroxidase conjugate (Biosource International), as previously described [31, 32]. Signal was developed using the Pierce ECL Western Blotting detection kit. Equal protein loading was confirmed in a stripped, washed, and reprobed membrane with a 1:2000 anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (Sigma) and 1:10 000 goat-anti-mouse IgG conjugated with horseradish peroxidase (Biosource).
Metabolic Labeling of hFSHR with [35S]-Methionine/Cysteine and [3H]-Palmitic Acid
Cells transiently expressing Wt or mutant FSHR were labeled with [3H]-palmitic acid following the procedure described by Zhu et al. [22] with some modifications. Briefly, transfected cells in 60-mm plates were preincubated for 20 min with serum-free DMEM containing 10 mM HEPES. After preincubation, media were replaced with serum-free medium containing 285 µCi/ml 3H palmitic acid (PerkinElmer) and incubated at 37°C for 2 h under 5% CO2. At the end of the incubation period, the medium was removed, and the cells were washed twice with PBS and allowed to continue incubation at 37°C for 4 h in fresh, serum-free DMEM. Cells were harvested after labeling and lysed in lysis buffer (1% Igepal, 0.4% DOC, 10 mM Tris pH 7.5, 6.6 mM EDTA) containing protease inhibitors. Cell lysates were then precleared with protein A agarose and centrifuged at 14 000 x g for 20 min, and the resultant supernatants were used for the immunoaffinity extraction (at 4°C for 16 h) employing mAb 106.105 antibody conjugated to agarose beads. After extensive washes, the proteins were eluted from the beads by incubation at room temperature for 15 min with SDS sample buffer containing 5% β-mercaptoethanol. Labeled proteins were then separated by SDS-PAGE (7.5%). Gels were dried at 50°C for 2 h and exposed to Kodak X-Omat AR film at –70°C for 18 days. The level of palmitoylation of Wt and mutant hFSHR is expressed as the ratio of total [3H]-palmitate incorporation (determined by densitometric analysis) relative to overall receptor expression levels (i.e., receptor number determined in binding assays). To verify palmitate attachment to cysteine residues, immunoprecipitates were incubated with 100 µl 1 M hydroxylamine or Tris buffer, pH 7.0, during 4 h at 20°C, at which time the beads were washed, and the bound material was eluted and electrophoresed as described previously.
Metabolic labeling of hFSHR species with [35S]-methionine/cysteine and immuno-extraction of labeled receptors were performed as described previously using cysteine-free DMEM containing 10 mM HEPES before labeling. At the end of the preincubation period, the medium was removed, replaced with cysteine-free medium containing 200 µCi/ml [35S]-methionine/cysteine (Amersham Biosciences) and allowed to continue incubation at 37°C for 2 h under 5% CO2. Labeled proteins were processed as described previously.
Computational Modeling and Molecular Dynamics Simulations of Wt and Ctail Mutant hFSHRs
To analyze the conformational changes provoked by mutations at positions 627, 629, and/or 655 of the hFSHR Ctail, a rough model of the whole hFSHR was developed. Intensive refinement of the Ctail and the intracellular loops of the hFSHR was performed by molecular dynamics simulations. Specifically, the fragment of the hFSH receptor between Gly344 and Pro650 was modeled using the MODELLER server [33] with the structure of rhodopsin (Protein Data Bank ID 1U19A) as a template. The homology score between both receptors was 15%. Since this study was focused on the intracellular loops and the Ctail, the first 343 residues were ignored. No homologous sequences with well-defined secondary structure were found for the last 28 residues of the Ctail by using the PROF program [34, 35] together with the Predict Protein server [36]. These programs suggest a disordered conformation for the last segment of the hFSHR Ctail with a low probability of short -helices or β-sheets formation at certain residues. Thus, the last 28 residues of the Ctail were simply attached as a random coil to the previously obtained model by imposing the corresponding peptidic bond between Pro650 and Arg651. A simulated annealing optimization in vacuum was performed as a first step to refine the structure. In order to preserve the helices of the TMs, their corresponding backbone atoms were kept at fixed positions during this stage. All atoms from the side chains of the helices as well as the whole intracellular loops and the Ctail residues were allowed to move. The low homology score and the apparent lack of secondary structure in the last 28 amino acids of the FSHR made convenient a further refinement. With this aim, molecular dynamics (MD) simulations together with a partial solvation (PS) approach that optimize the conformational sampling of local protein regions with low computational cost were employed. Briefly, the PS approach consists of solvating the region of interest only, leaving free all the space of the simulation box that is not occupied by the protein itself or by these few solvent molecules. Then an MD simulation at constant volume is performed, freezing the atoms of the protein residues that are not hydrated to center the analysis on the intracellular domains of the receptor. The PS approach outlined was employed initially to refine the intracellular loops and the Ctail of the model as follows: 1) Three thousand water molecules were used to partially solvate the Ctail and the intracellular loops of the Wt hFSHR model, 2) eight chloride ions were also added to neutralize the total positive charge of the receptor at biological pH, and 3) the resulting system was placed in a 30-nm cubic box and a 6.5-nsec trajectory at 277 K was generated to optimize the protein-water interactions as well as the conformation of the intracellular region of the receptor. In this low-temperature simulation, the positions of those atoms forming the backbone of the TM region were kept fixed. Under such conditions, most of the water molecules kept in contact with the flexible region of the receptor (Fig. 2 and Supplemental Movie available online at www.biolreprod.org), creating a convenient local environment. The conformation so obtained was taken as the mother structure for the subsequent studies on both the Wt receptor and the Cys hFSHR mutants.
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Starting from the Wt hFSHR model described previously, eight Cys
Ala or CysThr/Ser hFSHR mutants were generated (Table 2). Using the PS approach described previously, 100-nsec-long MD simulations at 310 K were performed for the Wt hFSHR and the single and double cysteine mutant receptor models; because of the high instability of the hFSHR C627/629/655A, the corresponding simulation was carried out at 298 K during 45 nsec. These long simulations would allow detection of evident mutation-induced differences in the intracellular region of the receptor that may affect the protein structure or dynamics in the absence of palmitoylation. The PS approach is of limited accuracy because only the atoms of the intracellular loops and the Ctail are allowed to move, and because of the particular distribution of the solvent molecules, no lipids are employed, and only 3000 water molecules are located in the intracellular domain. Nevertheless, omission of the lipid membrane allowed to perform long simulation times, which were essential for the purpose of the modeling. Although the PS approach is of limited accuracy because of the lack of involvement of lipid membranes, it is worth mentioning that the dielectric permittivity of vacuum (the means surrounding the TM helices in the employed PS approach) is comparable to that of the hydrophobic lipid tails in an actual membrane [37]. Further, the thickness of the water layer imposed around the intracellular domains (>4 Å) should suffice for keeping its behavior as biological water [38]. The last assumption on the PS approach employed is that the movement of the residues that are inside the cell can be separated from the movement of the helices. This is reasonable since RMSF values of TM helices obtained from MD simulations of other GPCRs at the time scale of tens of nanoseconds are lower than RMSF values of loops or the Ctail [39].
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The GROMACS package version 3.3.1 [40] with the GROMOS96 (53a6) force field [41] and the simple point charge water model [42] were used. This force field treats aliphatic hydrogen atoms as united atoms, together with the carbon atom to which they are attached [43]. Periodic boundary conditions with a cubic box as the basic unit cell in the canonical ensemble were used. The receptor, ions, and water molecules were coupled separately to a temperature bath using a coupling constant of 0.1 psec [44]. Nonbonded interactions were evaluated using a twin range cutoff of 0.8 and 1.4 nm, the interactions within the shorter and longer cutoffs being updated every step and every five steps, respectively. Beyond the 1.4-nm cutoff, a reaction field correction with a dielectric constant of 62.0 was used. Although the time step was 2 fsec, in the case of the triple mutant it was reduced to 1 fsec in an attempt to make more stable the simulation. The bond lengths and angle in water were constrained using the SETTLE algorithm [45], whereas the LINCS algorithm [46] was used to constrain bond lengths within the peptide. The equations of motion were integrated using the leapfrog method [47]. The trajectories were first analyzed by visual inspection using the viewers VMD [48] and PyMOL [49]. Root mean square positional deviations (RMSD) of distinct receptor segments were calculated as a function of time throughout all trajectories. Analyses of the secondary structure were performed using the definitions of the dictionary of protein secondary structure [50].
Differences between the responses analyzed were calculated employing one-way ANOVA followed by unpaired t-tests. Maximal responses (Rmax) and ED50 were calculated from dose-response curves using the software Origin 7.0 (OriginLab Co.) fitted to a sigmoidal dose-response curve. Probabilities <0.05 were considered statistically significant. Results are presented as means ± SEM from three or more experiments or from a representative experiment.
Metabolic Labeling of the Wt and Mutant hFSHR Species with [35S]-Cysteine/Methionine and [3H]-Palmitic Acid
To determine whether the hFSHR is palmitoylated and to identify the specific cysteine residue(s) at which this posttranslational modification occurs, cells transfected with the Wt and Cys mutant hFSHR cDNAs were first incubated with [35S]-cysteine/methionine to visualize the expression levels of mature receptors after a brief 2-h incubation period and immunoprecipitation with the anti-hFSHR 106.105 monoclonal antibody. Since it has been demonstrated that the rat LHCGR is palmitoylated at cysteine residues 620 and 621 [21, 22], we included cells transiently expressing the chimeric hFSHR/rLhcgr-Ctail construct as a positive control for these experiments. This chimeric receptor is highly expressed at the plasma membrane, efficiently binds agonist, and promotes cAMP accumulation on exposure to agonist (Fig. 3A and Table 1). In agreement with the results from immunoblotting analyses (see the following discussion), labeling with [35S]-cysteine/methionine for 2 h of cells expressing the Wt, chimeric, and Cys mutant hFSHRs but not the mock transfected cells allowed identification of the mature 80-kD hFSHRs at intensities that varied depending on the particular hFSHR species transfected. The intensity of this band was higher in cells expressing the Wt, C627A, and hFSHR/rLHCGR-Ctail receptors than in those transfected with the remaining hFSHR Cys mutants (Fig. 3, B–D).
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Metabolic labeling with [3H]-palmitic acid revealed that both the hFSHR Wt and the hFSHR/rLHCGR-Ctail chimera are efficiently palmitoylated. Incubation of immunoprecipitates from [3H]-palmitic acid or [35S]-cysteine/methionine-labeled Wt and chimeric hFSHRs with hydroxylamine resulted in the complete loss of 3H- but not 35S-signal from both receptors (Fig. 3B, inset). To map the residues at which this particular esterification occurs in the hFSHR, we mutated individually or simultaneously into alanine the three potential acylation sites located in the Ctail of the receptor and evaluated the effects of these mutations on [3H]-palmitic acid incorporation. Cells bearing these Cys mutants were labeled with [3H]-palmitic acid, immunoprecipitated, and analyzed by SDS-PAGE. Incorporation of [3H]-labeled palmitate was present in the mature forms of all single and double mutant receptors tested but not in the triple C627/629/655A hFSHR mutant (Fig. 3, B–E), indicating that all Cys residues present in the Ctail of the hFSHR are the target for palmitoylation. Overexposure of [3H]-palmitic acid- or [35S]-cysteine/methionine-labeled blots to radiographic films revealed the presence of high- (>118 kDa) and low- (<80 kDa) molecular-weight bands that presumably represent dimers or oligomers of the hFSHR [51] and immature precursors of the glycoprotein [31], respectively. These bands, however, were detected only in extracts from cells transfected with the Wt and C627A hFSHR cDNA but not from those expressing C629A mutants, consistent with their lower expression/decreased stability (Fig. 3F).
cAMP Production by HEK-293 Cells Expressing Cys Mutant hFSHRs
The ability of palmitoylation deficient mutant hFSHRs to evoke signal transduction was established by assaying HEK-293 cells expressing mutant FSH receptor for cAMP production in response to increasing doses of recombinant FSH (Fig. 4 and Table 1). Maximal agonist-stimulated cAMP production was virtually unaltered by the expression of the hFSHR-C627A mutant, whereas Ala substitution at C629 or C655 decreased maximally stimulated cAMP production by 15%–19%; replacement of C655 by Ser or Thr yielded similar results (Fig. 4, A and B). A further (10%) reduction in signal transduction ability was detected for the double hFSHR C627/629A mutant but not for the C629/655A hFSHR. Alanine substitution at all three Cys residues resulted in a mutant hFSHR species with markedly reduced (to 60%) efficacy to evoke cAMP production in response to agonist (Fig. 4C). When differences in the expression levels of different constructs (see the following discussion) were reduced by adjusting the concentration of Wt hFSHR cDNA transfected to yield similar levels of cell surface expression (Fig. 4H), the efficacy of the palmitoylation-deficient mutant FSHRs to elicit maximal cAMP accumulation in response to agonist still remained reduced (by 8%–20%) as compared to that achieved by the Wt receptor (Fig. 4, D–F, and Table 1).
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Cell Surface Expression and [125I]-Labeled FSH Binding of Cys Mutant hFSHRs
Plasma membrane expression of wild-type and Cys mutant hFSHRs was examined by Western blot analysis. A 80-kDa band representing the mature, fully glycosylated FSHR was clearly identified in HEK-cells transiently expressing the Wt and the C627A hFSHRs. The intensity of this band was decreased in the C629A, C627/C629A, C655A/S/T, and C629/655A mutants and barely detected in the triple C627/629/655A hFSHR mutant (Fig. 4G). The level of plasma membrane expression detected by Western blotting correlated with the quantity of functional receptors capable of binding ligand (Table 1 and Fig. 5A). The capability of cells transfected with the hFSHRs to maximally bind [125I]-FSH was particularly reduced for those expressing the C629A, C627/629A, C629/655A, and the triple C627/629/655A receptor mutants; the latter mutant hFSHRs maximally bound [125I]-labeled FSH at levels <30% from that exhibited by the Wt hFSHR counterpart (Fig. 5A). None of the mutations in Cys residues altered the affinity of the hFSHR to bind agonist (Fig. 5B); in fact, the dissociation constants of the altered receptors were similar to that exhibited by the Wt receptor (Table 1). These results indicate that the decreased capability of the mutant hFSHRs to maximally bind agonist was due to the reduced plasma membrane expression resulting from replacing the cysteine residues located at the Ctail, particularly at position 629.
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Internalization of Mutant hFSHR
Given that the Ctail of the rat FSHR is involved in the internalization dynamics of the receptor [15] and that mutations in palmitoylation sites of the human and rat LHCGR enhanced the rate of ligand-induced receptor internalization [21, 52, 53], we investigated whether replacement of the cysteine residues present in the hFSHR Ctail modified agonist-induced receptor internalization. For this purpose we measured the rate of disappearance of labeled ligand (and presumably receptor as well) from the cell surface in equilibrium and nonequilibrium conditions. Employing the first approach and in agreement with the data shown in Figure 5A, mutation of Cys residues present in the Ctail of the hFSHR caused a decrease in the total hormone bound; all Cys mutant receptors exhibited similar internalization rates over time (up to 90 min) than that followed by the Wt hFSHR (Fig. 6). Nevertheless, when tested under nonequilibrium conditions, internalization of Cys655 hFSHR mutants but not C627A, C629A, C627/629A, C629/655A, and C627/629/655A mutants was significantly decreased (Table 1). Figure 7A shows the line generated by graphing the cell-associated hormone (i.e., hormone resistant to elution from cells at acid pH) against the integral of the cell surface hormone at each time point from a representative internalization experiment with the Cys655 mutants; the slope of each line represents the endocytotic rate constant (Ke) of each hFSHR species. Accordingly, the Ke was measured to be 0.0110 ± 0.0008 (Wt), 0.0075 ± 0.0006 (C655A), 0.0068 ± 0.0005 (C655S), and 0.0066 ± 0.0003 (C655T)/min. The Ke measured for the remaining mutants were 0.0099 ± 0.0008 (C627A), 0.0080 ± 0.0013 (C629A), 0.0086 ± 0.0006 (C627/629A), 0.0083 ± 0.00013 (C629/655A), and 0.0091 ± 0.00009 (C627/629/655A)/min.
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Computational Modeling and Molecular Dynamics Simulations of Wt and Ctail-Cys Mutant hFSHRs
Employing the partial solvation approach proposed, nearly 2 nsec of MD simulations per day and per processor could be produced. This represents an important save of computational time (approximately by factor of 10) when compared to typical membrane protein simulations, which consider hundreds of lipids and thousands of water molecules in addition to ions and protein atoms [54, 55]. Even though a large volume of the simulation boxes is bared in the initial conformations, most of the water molecules employed to partially hydrate the receptors as well as the ions added to compensate for the total charge of the system keep close to the unconstrained residues; this occurred even at long time scales due to favorable protein-water, ion-water, and water-water interactions. As expected, a significant amount of water molecules evaporate to form a vapor phase (Supplemental Movie). Differences in structure among the intracellular regions of the Wt receptor and the Cys mutants were determined by calculating the RMSD of the last 65 residues together with the three intracellular loops (black dashed curves in Supplemental Figure 1). In all the trajectories, the reference structure was the final Wt hFSHR conformation obtained after 100 nsec of simulation. The C655A, C655T, and C655S mutants presented the lowest RMSD values followed by the double C627/629A mutant. The highest RMSDs corresponded to the C629A and C629/655A mutants (Table 2 and Supplemental Figure 1). Two representative examples of the final conformations obtained after 100 nsec of the simulation are shown in Figure 8. The figure shows the final conformation attained by the C655A and C629A hFSHR mutants (yellow) superposed on the final structure of the Wt receptor (red). To avoid artifacts from the TM helices (which were not considered in the calculations and the simulations), only the intracellular loops and Ctail atoms were taken into account to fit the structures. As expected from the RMSD values shown in Table 2, the conformation of the Ctail bearing the C655A mutation was more closed to that exhibited the Wt receptor than the conformation reached by the C629A mutant; differences in the relative orientation of the helices were also more evident in this latter mutant than in the C655A modified receptor. It is worth being reminded that the initial conformation was the same for all sequences simulated. RMSDs for the F(X)6LL motif were also calculated with respect to the same motif in the final structure of the Wt receptor (red solid curves in Supplemental Figure 1). Again, the lowest RMSD values were produced by two of the mutants involving the position 655 (C655T and C655S), while the largest positional deviations were found in mutants involving positions 627 and 629, which was probably due to the relatively short distance between the replaced residue and the F(X)6LL motif. As previously suggested by the PROF predictor [34, 36], the general results from the dynamic simulations indicate that the Ctail is essentially unstructured in both the Wt receptor and the Cys mutants. Overall, the simulations proved to be sensitive for specific mutations; mutants involving Cys655 yielded conformations more closed to the Wt receptor than any other mutant, whereas mutants involving the position 629 yielded the highest RMSDs and the most unstable structures, with the triple mutant structure resulting highly unstable even at 298 K.
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The present study was specifically designed to determine the functional significance of the palmitoylation of cysteine residues present in the Ctail of the hFSHR, an issue that had not been previously explored. Cysteine residues in the Ctail of several GPCR, belonging mainly to family A, have been shown to be the target for S-acylation with palmitic acid [56]. Palmitoylation may potentially influence the local conformation of the Ctail and thus regulate several functions, including G protein coupling, desensitization, intracellular trafficking, and targeting of the receptors to the plasma membrane [56–58]. In some GPCRs, such as the β2-adrenergic receptor, mutation of the palmitoylated cysteine leads to greatly decreased interaction of the mutant receptor with the Gs protein [59], whereas in others it has little or no effect in receptor function [60, 61]. As with the TSHR and LHCGR, the hFSHR exhibits in the proximal region of its Ctail a conserved cysteine residue with a neighboring basic residue (Lys680, Lys619, and Lys626 in the hTSHR, hLHCGR, and hFSHR, respectively) that appears to be important for palmitoylation [62]. In the present study, we found that metabolic labeling with [3H]-palmitic acid revealed attachment of this fatty acid to the hFSHR. Further, our data are consistent with a model where the hFSHR Ctail Cys residues are targets for palmitoylation regardless of their location in the carboxyl-terminus of the receptor. Although palmitoylation of GPCRs usually occurs at one or more Cys residues proximal to the TM-7 [56, 57], palmitoylation of cysteines positioned close to the carboxyl-terminal end of the receptor has also been detected, as in the case of the 5-hydroxytryptamine 4(a) receptor [63].
Employing site-directed mutagenesis, we here demonstrate for the first time that Cys residues in positions 629 and 655 but not at 627 of the hFSHR Ctail are important for receptor function. This is in agreement with the multiple alignments of FSHR sequences from other mammalian species (Fig. 1C) in which Cys629 and Cys655 appear as highly conserved residues, whereas Cys627 in the sequence of the hFSHR is replaced mostly with the hydrophobic phenylalanine and thus is not palmitoylated. The functional behavior of the hFSHR C627A mutant was, in fact, very similar to that exhibited by the Wt receptor. By contrast, replacement of Cys655 (a conserved Cys residue distal to the NH2-terminal end of the Ctail) by alanine, serine, or threonine modestly decreased maximally stimulated cAMP production and plasma membrane expression and slowed receptor internalization. Alanine substitution at Cys629 or simultaneously at Cys627/629, Cys629/655, or Cys627/629/655 decreased even more maximal FSH-stimulated cAMP accumulation, and significantly impaired receptor plasma membrane expression as disclosed by immunoblotting and radioligand-binding experiments. Even when Cys629 was absent, the modified receptors exhibited ED50s for cAMP production similar to that showed by the Wt receptor, indicating that cysteine residues at positions 629 and 655 play a minor role, if any, on the signaling efficiency of the FSHR to couple the Gs protein. Similarity of maximal cAMP production, in the face of differing amounts of expression, is reminiscent of the spare receptor concept, where activation of only a fraction of receptors is necessary and sufficient for a full cAMP response [64].
Even though Cys627 and Cys655 appear to be a target for palmitoylation, cell surface levels of the hFSHR C627A mutant and, to a lesser extent, of the C655A mutant were only slightly reduced compared to those showed by their Wt counterpart, suggesting that S-acylation in these positions is not essential for efficient hFSHR cell surface membrane targeting. This is in contrast to what was observed for all C629A mutants, whose plasma membrane expression was markedly affected. Previous studies in other GPCRs have shown that posttranslational palmitoylation is often required for efficient delivery of the protein to the cell surface membrane [22–24, 58, 65–67]; in fact, in stable transfected cells, some palmitoylation-deficient GPCR mutants are retained in an intracellular compartment, primarily in the endoplasmic reticulum (ER) [22, 58], whereas others (e.g., the TSHR) exhibit only delayed cell surface expression without detectable intracellular trapping [24]. Moreover, in the case of rhodopsin, it has been suggested that the palmitoylated groups on intracellular Cys residues are involved in stabilizing the dimeric structure of the receptor [68]. In the present study, analysis of the expression of C629A mutant hFSHRs by immunoblotting and [35S]-cysteine/methionine labeling allowed detection of only the mature, cell surface membrane-inserted (molecular weight 80 kDa) species of the receptor [31] but not of the bands corresponding to immature (molecular weight <80 kDa) or high-molecular-weight (
175,000 kDa) hFSHR forms that presumably represent, respectively, precursors and oligomers of the mature receptor [51]. Failure to detect such forms of the mutant receptors by immunological methods may reflect the occurrence of rapid degradation of the newly synthesized palmitoylation-deficient C629A receptors into fragments that cannot be recognized by the anti-hFSHR antibody employed or alternatively to our inability to detect this bands because of their overall reduced biosynthesis resulting from increased proteolysis. In this vein, it has been shown that palmitoylation-deficient human A1 adenosine receptors are more prone to form proteolytic fragments and to degrade than are wild-type receptors [69], suggesting that S-acylation may protect nascent receptors from rapid degradation. The finding that a fraction of the double and triple Cys629 mutants were still detected as mature FSHR species is not necessarily in contradiction with the latter possibility considering that overexpression of the mutant receptors may have overwhelmed the quality control mechanisms of the cell allowing some palmitoylation-deficient receptor molecules to evade proteolytic processing. In this scenario, the hFSHR might be palmitoylated at position Cys629 (whose presence appears critical for cell surface membrane expression) posttranslationally at the ER and/or Golgi compartment [57, 58], while palmitoylation of the receptor at positions Cys627 and Cys655 may occur either in these compartments or locally after insertion of the mature receptor in the plasma membrane, as shown for other GPCRs [57].
The spectrum of structural requirements that determine export trafficking or targeting for proteasomal degradation of GPCRs remains poorly defined. Although in some receptors palmitoylation plays a significant role in receptor export from the ER [65–67], in others single mutations at locations not involving potential palmitoylation sites or disulfide bond formation may lead to misfolded/unstable structures that are eventually targeted for degradation [70]. Such mutations may alter motifs important for receptor ER export (e.g., the highly conserved F(X)6LL motif at the NH2-terminal end of the Ctail) or allow exposure of ER retention motifs [19]. In the present study, the stability of the receptor on mutation of one or several Cys residues at positions 627, 627, and/or 655 was analyzed by MD simulations. The PS approach employed for this purpose was expected to detect reliable structural mutation-induced changes because the surrounding of the simulated region mimics well the actual environment of those residues. The limited amount of water employed should not represent a poor approximation since it has been proved both experimentally [71, 72] and by MD simulations [73–75] that a few-water-molecule layer is sufficient to reproduce the dynamic behavior of proteins. Such a water layer has been estimated to be nearly 4 Å wide and named biological water [38]. Besides, the region of the simulation box that is not hydrated is bare and then can be considered as a hydrophobic means. This includes the immediate vicinity of the TM helices that in fact should be immersed in the hydrophobic tails of the lipids forming the membrane. Significant mutation-dependent structural changes in the intracellular region were found by using this approach. Replacement of Cys629 produced the most unstable structures, which may, in part, explain the reduced membrane expression of the mutants. In this regard, it is known that failure of the ER quality control system to stabilize unstable conformations results in destruction of the incorrectly manufactured protein [70]. On the contrary, single mutations at position 655 yielded structures much more similar to the Wt receptor, a finding that correlates with the experiments showing efficient plasma membrane expression (80% of that shown by the Wt receptor) of the Cys655 mutant FSHRs. Although the results from the MD simulations do not allow to reach definitive conclusions on the role of palmitoylation in receptor function, it is worth noting that, except for the case of the C627A mutant, all RMSD values correlated well with the experimental estimates of plasma membrane expression (Tables 1 and 2). In the case of the C627A mutant, the discrepancies between experimental and computational data may be explained by the following: 1) the simulation time scale employed, which could be still insufficient to allow reaching equilibrium of the structure in this particular system; 2) the possibility that the intracellular residues of the mutant may have been trapped in an ensemble of metastable conformations that eventually prevented its structure optimization; or 3) that the final conformation simulated may actually be compatible with ER export. Alternatively, it is also possible that the presence of palmitoylation at Cys629 may have counteracted the effects of misfolding provoked by the mutation and thereby contribute to the capacity of the C627A receptor to fully express at the cell surface membrane.
It has been proposed that palmitoylation of conserved cysteine residues in the Ctail of GPCRs may provide an additional site for anchoring of the receptor to the plasma membrane, creating a fourth intracellular loop [56, 57]. This palmitic acid-mediated membrane anchoring may potentially decrease agonist-induced receptor internalization thereby extending the residence time of the ligand-bound receptor in the cell surface. Mutation of the rat and human LHCGR palmitoylation sites leads to increased rate of receptor internalization through a mechanism involving hyperphosphorylation of the depalmitoylated receptor and, presumably, increased nonvisual arrestin recruitment [21, 52, 53]. Further, prevention of hLHCGR palmitoylation by site-directed mutagenesis reduces recycling of the depalmitoylated, internalized receptor back to the cell surface [76]. In the rat FSHR, phosphorylation occurs on the iL1 and iL3 as well as on a short Ser/Thr-rich sequence located in the middle of the Ctail (residues 638–644) [14, 77]; mutation of this sequence into alanine residues delays internalization of the receptor [14]. Whereas association of the FSHR with arrestin-3 occurs in a phosphorylation-independent fashion and is modulated by a short sequence located near the carboxyl-terminal end of the Ctail [15, 78], phosphorylation of the Ser/Thr-rich Ctail cluster is apparently important for recruitment of arrestin-1 and -2 [14]. Although phosphorylation of the hFSHR Ctail has not been documented, the high homology (84%) between the rat and human FSHR Ctail (see Fig. 1C) predicts that agonist-provoked phosphorylation of the latter may, in fact, occur at the same Ser/Thr-rich motif (residues 639–644). In this scenario, it would be expected that abrogation of palmitoylation of the hFSHR would enhance the rate of receptor internalization, as documented for the LHCGR [25]. In the present study, the dynamics of hFSHR internalization under equilibrium conditions was not altered by the replacement of the Ctail Cys residues, and all mutants internalized at similar rates. However, under nonequilibrium binding conditions (which does not allow for saturation of the recycling/degradative pathways), the internalization kinetics of the Cys655 mutants was significantly delayed. This finding suggests that palmitoylation at Cys655 may contribute to hFSHR receptor internalization through regulating the receptor-nonvisual arrestins interaction as demonstrated for the thyrotropin-releasing hormone receptor [78]. Alternatively, the possibility also exists that the slight conformational changes provoked by the replacement at this particular position may by its self lower the affinity and/or accessibility of the hFSHR Ctail for nonvisual arrestins.
Our previous work has established that epitope tags placed on the Ctail of the FSHR are cleaved prior to FSH insertion into the plasma membrane [51]. The present work establishes that the clip site occurs beyond cysteine 655. As discussed previously, it is intriguing that failure to palmitoylate the receptor leads to decreased plasma membrane expression. We anticipate that epitope tagged FSHR once palmitoylated has its Ctail clipped beyond Cys655 and then the receptor is allowed to proceed to the plasma membrane. Studies to address this hypothesis are currently under way.
It was interesting to find that the hFSHR/rLHCGR-Ctail chimera consistently showed higher levels of cell surface membrane expression and FSH-stimulated cAMP accumulation than the Wt hFSHR species. Although the reasons for these differences are not readily apparent, a plausible explanation may be that insertion of the rLHCGR-Ctail, which is palmitoylated in two contiguous cysteine residues (Cys621 and Cys622) [21, 22], conferred the hFSHR a higher efficiency for intracellular transport and cell surface targeting.
Taken together, the results of this study provide the first evidence that cysteine residues present in the Ctail of the hFSHR play an important role in receptor function. The functional effects of the Cys residues at this domain are apparently mediated by palmitoylation mainly at its conserved 629 and 655 cysteines. In addition to its importance in receptor palmitoylation, the presence of a Cys residue at position 629 seems critical to maintain the conformation of the Ctail. The lack of palmitoylation does not appear to greatly impair coupling to Gs but when absent at position 629 significantly impairs net biosynthesis and cell surface membrane expression probably because of rapid degradation of the palmitoylation-deficient receptor. Palmitoylation of the conserved Cys655 apparently increases the efficiency of the hFSHR to internalize on agonist stimulation and influences uncoupling of the FSH response.
ACKNOWLEDGMENTS
The authors are grateful to the Dirección General de Servicios de Cómputo Académico (DGSCA) of the Universidad Nacional Autónoma de México (UNAM) for computer time and excellent service. The CV-27 cAMP antiserum was obtained from Dr. A.F. Parlow, National Hormone and Peptide Program.
FOOTNOTES
1Supported by grants 2005/1/I/002 from the FOFOI-IMSS, México (to A.U.-A.); grants 45991 (to A.U.-A.) and J-49811Q (to Á.P.) from CONACyT, México; and grant HD18407 from the NIH, Bethesda, MD (to J.A.D.). A.U.-A. is recipient of a Research Career Development Award from the Fundación IMSS, México. Presented in part at the 88th Annual Meeting of the Endocrine Society, Boston, Massachusetts, June 2006, Abstract P3–280. 
Correspondence: 2Alfredo Ulloa-Aguirre, Research Unit in Reproductive Medicine, IMSS, Apdo. Postal 99–065, Unidad Independencia, México 10101 D.F., México. FAX: 52 55 5616 2278; e-mail: aulloaa{at}servidor.unam.mx
Correspondence: 3These authors contributed equally to this work.
Received: 3 July 2007.
First decision: 16 August 2007.
Accepted: 8 January 2008.
REFERENCES
2-adrenergic receptors based upon characteristics in primary structure. J Biol Chem 1992; 267:8342–8346.S protein coupling and receptor expression. Mol Cell Endocrinol 2007; 260:153–162.[CrossRef][Medline]
opioid receptor. Protein Sci 2004; 13:1997–2008.[CrossRef][Medline] opioid receptor. J Biol Chem 2006; 281:15780–15789.2A-adrenergic receptor that eliminate detectable palmitoylation do not perturb receptor-G-protein coupling. J Biol Chem 1993; 268:8003–8011.
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P < 0.001 vs. Wt hFSHR, hFSHR/hLHCGR-Ctail, and C655A, C655S, C655T, and C627A hFSHR mutants, and P < 0.01 vs. C629A, C627/629A, and C629/655A hFSHR mutants. B) Displacement of [125I]-FSH by increasing concentrations of unlabeled FSH in cells transiently expressing the Wt and the C627A, C629A, C627/629A, and C655A/S/T hFSHRs in the radioreceptor binding assay. Inset: Radioreceptor assay of the C629/655A and the C627/629/655A hFSHR mutants and the hFSHR/rLHCGR-Ctail chimera. Results are the mean values from three independent assays.
