From INSERM Unité 135, Hormones Gènes et
Reproduction, Institut Fédératif de Recherche 21, Hôpital Bicêtre, Assistance Publique Hopitaux de Paris,
94275 Le Kremlin Bicêtre, France and § Laboratoire de
Génétique et Physiologie du Développement,
Unité Mixte de Recherche 9943, Faculté des Sciences Luminy,
13288 Marseille, France
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ABSTRACT |
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The follicle-stimulating hormone receptor (FSHR) is physiologically localized in the basolateral compartment of the membrane of Sertoli cells. This localization is also observed when the receptor is experimentally expressed in Madin-Darby canine kidney cells. We thus used in vitro mutagenesis and transfection into these polarized cells to delineate the basolateral localization signal of the receptor.
The signal was localized in the C-terminal tail of the intracellular domain (amino acids 678-691) at a marked distance of the membrane. Mutation of individual amino acids highlighted the importance of Tyr684 and Leu689. The 14-amino acid sequence was grafted onto the p75 neurotrophin receptor and redirected this apical protein to the basolateral cell membrane compartment. Deletion of amino acids 677-695 did not modify the internalization of the FSHR, showing that the basolateral localization signal of the FSHR is not colinear with its internalization signal.
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INTRODUCTION |
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The FSH1 receptor (FSHR), along with the LH and TSH receptors, belongs to a subgroup of G protein-coupled receptors (reviewed in Refs. 1-5). These highly homologous proteins are unusual among G protein-coupled receptors in that they contain a very large extracellular hormone binding domain. FSH transduction pathways involve mainly Gs proteins coupling and the ensuing activation of adenylate cyclase (5).
The FSH receptor has a central role in reproduction through the control of gonadal development and gamete production (5). In males it is expressed in testicular Sertoli cells. These cells form an epithelial blood barrier and control the development of spermatogenesis (6). In females, the FSHR is expressed in the granulosa cells of the ovaries. It controls follicular growth and, in cooperation with the LH receptor, ovarian steroidogenesis (2).
Little is known of the intracellular trafficking of G protein-coupled receptors and, more specifically, of this subgroup of receptors. The pathways of internalization of the LH and TSH receptors have been studied at the ultrastructural level (7),2 but the molecular signals involved are still unknown. The LH receptor is also present in the vascular endothelium of the testes (8). This endothelial LH receptor is involved in receptor-mediated hormone transcytosis, leading to the accumulation of the hormone in close proximity to the target cells. FSHR has been detected in the vascular endothelial cells of the testes (9), but its role in hormone transcytosis has not been studied to date.
Another particularity of gonadotrophin and thyrostimulin receptors is their polarized cellular expression in several target tissues. Immunocytochemical studies with monoclonal antibodies have shown that the FSH receptor in Sertoli cells and the TSH receptor in thyroid follicular cells have a polarized basolateral expression (9, 10). By contrast, the LH receptor is expressed circumferentially in the ovarian granulosa and theca cells and in the testicular Leydig cells (11, 12). This difference in receptor distribution could result from either a difference in the structure of the receptors or from differential expression in polarized or nonpolarized cells. To distinguish between these possibilities, we have transfected the three receptors into polarized MDCK cells (13). We observed that all three receptors were directly delivered to the basolateral membrane of these cells, suggesting that they all contain a basolateral targeting signal.
The molecular signals involved in the polarized targeting of G protein-coupled receptors and more generally of hormone receptors in epithelial cells have still not been identified. We have thus undertaken to delineate this signal in the FSH receptor. In vitro mutagenesis was followed by establishment of permanent MDCK cell lines expressing mutated receptors. We report here the localization of the basolateral targeting signal, its structure, the functional importance of the individual amino acids, and the noncolinearity of this signal with the signal for receptor endocytosis.
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MATERIALS AND METHODS |
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Expression Vectors Encoding Deletion Mutants of FSHR--
The
vector encoding the wild-type FSH receptor cDNA (pSG5-FSHR) has
previously been described (9). Polymerase chain reaction-mediated oligonucleotide mutagnesis was used to introduce stop codons into the
pSG5-FSHR vector to produce vectors encoding FSHR657-695 and
FSHR
677-695.
Site-directed Mutagenesis of Receptor Basolateral Localization Signal-- Alanine scanning mutagenesis of the FSH receptor basolateral signal was performed using a series of synthetic oligonucleotides inserted between two restriction sites flanking the signal. The oligonucleotides were introduced either between the SapI (position 3075 of the pSG5 FSHR vector) and the KpnI sites or between the KpnI and the EcoRI (position 3145) sites.
The mutants Gly681Generation of the Truncated Neurotrophin Receptor (NTRt)/FSHR-(678-691) Hybrid Receptor-- The neurotrophin receptor (NTR) cDNA, cloned in the pCB6 vector, which lacks its intracellular domain (six residues downstream the transmembrane domain), has previously been described (14). The basolateral localization signal of the FSHR was introduced into the XbaI site. The stop codon TAG, located just before the basolateral signal, was then mutated to a GAG codon encoding a glutamine using the same strategy as described above, yielding the NTRt/FSHR-(678-691) hybrid receptor.
The control vector (NTRt) that was used contained a stop codon just before the first residue of the signal.Cell Culture and Expression of FSHR Mutants-- MDCK cells (type II) were seeded and grown on coverslips (Nunc) or filters (0.4-µm polycarbonate Transwell Costar Corp., Cambridge, MA) as described previously (13). The cells were cotransfected with expression vectors encoding either the wild-type or the mutated FSHR and with the plasmid pSV-neo, which confers resistance to the antibiotic G418. The clones were screened for receptor expression by immunocytochemistry (13) using the monoclonal anti-FSH receptor, FSHR323 (9). Sodium butyrate (10 mM) (Sigma) was added to increase the expression of the transfected cDNAs as described (15).
Control of Cell Polarity-- The transepithelial resistance of the clones was ~300 ohms/cm2. Indirect immunofluorescence studies were performed as described (13) using antibodies (a gift of A. Le Bivic) directed against a basolateral (BC11) or an apical (BB18) endogenous protein of MDCK cells. Polarized apical secretion of the endogenous glycoprotein complex gp80 (16) was verified as described previously (17).
Immunofluorescence and Confocal Microscopy-- MDCK cells were grown to confluence on coverslips as described (13). The cells were washed with PBS+ (phosphate-buffered saline (Dulbecco's formulation) with 0.1 mM CaCl2 and 1 mM MgCl2) and fixed for 15 min in 3% paraformaldehyde in PBS+. The cells were washed, and the aldehyde groups were quenched with 50 mM NH4Cl in PBS+ for 30 min. After 1 h of saturation with PBS+, 1% BSA (albumin, fraction V, Boehringer Mannheim), the cells were incubated for 2 h at room temperature in a humid chamber with the monoclonal anti-FSHR antibody FSHR323, (5 µg/ml in PBS+, 1% BSA). The cells where then washed with PBS+, 1% BSA, 0.1% Tween 20 (Sigma) and incubated for 1 h with a Cy3-labeled rabbit anti-mouse IgG (Sigma). After washing, the cells were mounted with fluorescent Montaing medium (DAKO) and observed with a Zeiss microscope (Axiovert 135M) in conjunction with a confocal laser scanning unit (Zeiss LSM 410). To open the tight junctions, MDCK cells were incubated for 1 h with Ca2+-free Dulbecco's modified Eagle's medium, washed twice with calcium and magnesium-free PBS, and then incubated with 10 mM EGTA in the same buffer (13). The cells were then fixed in 3% paraformaldehyde for 15 min and further processed as described above.
cAMP Assay-- cAMP was measured as described (9). The cells were incubated in the absence or in the presence of 50 nM FSH (Metrodine, Serono).
Surface Immunoprecipitation of Wild-type and Mutated FSH Receptors-- Polarized monolayers of MDCK cells were obtained after 2 days of culture on 24-mm filters. Receptors present on the cell surface were immunoprecipitated as described previously (13). Briefly, cells were pulse-labeled for 1 h with 1 mCi/ml Express 35S (NEN Life Science Products) and chased for 3 h in the same medium containing unlabeled amino acids and 1% BSA. Monoclonal antibody FSHR323 was added to the apical or to the basolateral compartment during the last hour of the chase period. Extraction and purification of receptor-antibody complex were performed as described (13). All experiments were performed at least twice with triplicate samples.
Surface Immunoprecipitation of the NTRt/FSHR-(678-691) Chimera-- MDCK cells grown on filters were incubated for 30 min in DMEM without cysteine and pulsed for 30 min in the same medium containing 1 mCi/ml [35S]cysteine (NEN Life Science Products) as described (18). After washing with DMEM, the cells were chased for 3 h in DMEM containing unlabeled amino acids.
Cell surface immunoprecipitation was performed as described (19). Briefly, the monoclonal antibody ME20.4 directed against the ectodomain of the human neurotrophin receptor was added either in the apical or in the basolateral compartment (5 µl of ascites fluid/ml) for 1 h at 4 °C. After the incubation, the filters were washed five times for 10 min with DMEM containing 0.5% BSA. Receptor-antibody complexes were extracted and purified as described (18).Electrophoresis and Autoradiography-- SDS-polyacrylamide gel electrophoresis was performed as described (13). The gels were fixed and processed for fluorography. Densitometric scanning was used for quantification.
Receptor-mediated FSH Internalization--
COS-7 cells were
transfected with wild-type FSHR or the truncated FSHR677-695 using
Superfect (Qiagen, Chatsworth, CA). Internalization from the cell
surface of wild-type FSHR or of the truncated FSHR
677-695 was
determined by using 125I-labeled FSH (NEN Life Science
Products, 130 µCi/µg, 26 µCi/ml). COS-7 cells plated on six-well
plates were quickly cooled to 4 °C using two washes with ice-cold
PBS, 1% BSA. The cells were incubated with 125I-FSH (7000 cpm/µl in PBS, 1% BSA) for 10 min at 37 °C. The cells were then
extensively washed at 4 °C to remove unbound ligand and were allowed
to internalize the ligand for various time periods at 37 °C. One set
of plates that were kept at 4 °C was used to measure the initial FSH
binding. The internalization was stopped by rapid cooling to 4 °C.
The ligand remaining on the cell surface was stripped with 50 mM glycine in PBS containing 1% BSA, pH 2, for 10 min at
4 °C. Finally, the cells were treated with 0.2 M NaCl,
and the total radioactivity was counted. Nonspecific binding was
determined in the presence of an excess of unlabeled FSH (Serono) and
with nontransfected COS-7 cells. Internalized ligand was expressed as
the percentage of the total radioactivity (corrected for nonspecific binding) initially bound to the cells.
Secondary Structure Predictions-- Secondary structure predictions were performed using computer modeling. Two methods were used: the protein sequence analysis method (20) and neural network prediction (21).
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RESULTS |
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The C-terminal Region of the Intracellular Domain of FHSR Is Involved in Basolateral Sorting-- To establish whether the intracellular domain of the FSH receptor contains basolateral sorting information, we constructed three mutants deleted for various parts of the C-terminal tail of the receptor (Fig. 2A). The truncated receptors were stably expressed in MDCK cell lines. For each mutant, several (at least five) independent clones exhibiting different levels of expression were analyzed with the anti-FSH receptor monoclonal antibodies by confocal microscopy (Fig. 1). Monoclonal antibodies BC11 (Fig. 1a) and BB28 (Fig. 1b) that recognize the ectodomain of endogenous antigens restricted to the basolateral and the apical compartments of MDCK cell membrane, respectively (13), were used as controls.
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The Truncated FSH Receptor 677-695 Is Directly Targeted to the
Apical Membrane of MDCK Cells--
The truncation of FSH receptor at
amino acid 677 might have provoked a change in receptor polarization by
several mechanisms: loss of a basolateral sorting signal, increased
transcytosis, or deletion of a basolateral anchoring sequence (reviewed
in Refs. 22 and 23).
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Individual Amino Acids Involved in the Basolateral Localization Signal-- To define precisely the amino acids involved in receptor basolateral targeting, we mutated each one of the 14 residues of the 678-691 sequence into an alanine (Table I). The polarized expression of the receptor mutants was analyzed by confocal microscopy and quantified by cell surface immunoprecipitation. As shown in Figs. 4 and 5, the main effects were observed with mutations of Tyr684 or Leu689. The corresponding receptor mutants exhibited a 55 and 45% delivery to the apical surface of MDCK cells, respectively (Fig. 5). More limited effects were observed when residues in the vicinity of Tyr684 and Leu689 were mutated; about 35% of Gly681, Ile685, and His691 mutant receptors were detected at the apical surface of MDCK cells (Fig. 5 and Table I). Basolateral localization was not altered by point mutations of serine and threonine into alanine, indicating that phosphorylation of these amino acids is not involved in the vectorial sorting of the FSHR.
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The FSHR Contains a Basolateral Targeting Signal That Is Autonomous and Transferable to a Heterologous Protein-- We have mapped within the C-terminal end of the intracellular tail of the FSHR a sequence corresponding to residues 678-691 that was very likely to correspond to a basolateral sorting signal. Indeed, the deletion or mutation of this sequence altered the basolateral expression of the receptor. However, it remained possible that this sequence was only a part of the signal and thus necessary but not sufficient to target the receptor to the basolateral membrane. To establish unequivocally that amino acids 678-691 corresponded to an autonomous basolateral signal sequence, it was necessary to demonstrate that this polypeptide could redirect a heterologous protein to the basolateral domain. We thus grafted this sequence onto an apically expressed protein: the p75 NTR truncated after the fifth amino acid of its cytoplasmic tail. This truncated receptor has been previously shown to be localized mainly (~80%) in the apical compartment of the MDCK cell membrane (19).
A hybrid receptor NTRt/FSHR-(678-691) was constructed, and the polarized expression of the hybrid receptor was studied by confocal microscopy and surface immunoprecipitation in parallel with that of the NTRt. Confocal microscopy confirmed the presence of NTRt at the apical surface of MDCK cells. In contrast, the chimera NTRt/FSHR-(678-691) was detected at the basolateral surface of the cells (Fig. 6). Quantitative surface immunoprecipitation confirmed this result (Fig. 7). While 90% of the NTRt was detected at the apical surface of the cells, about 90% of the hybrid molecules were observed on the basolateral membrane.
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The Basolateral Localization Signal of the FSHR Is Different from Its Endocytosis Signal and Is Not Involved in Gs Protein Coupling-- In several proteins, basolateral localization signals have been shown to coincide or overlap with internalization signals (reviews in Refs. 23 and 31). We thus compared the internalization of the wild-type FSHR with that of the receptor devoid of its basolateral localization signal in COS-7 cells. As shown in Fig. 8, both receptors were internalized at the same rate.
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DISCUSSION |
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We have previously shown that the FSHR displays a polarized basolateral expression in Sertoli cells of the testes (9). We went on to demonstrate that this location can also be observed in the MDCK model system (13). We have now used in vitro mutagenesis and expression in these cells to define the basolateral sorting signal of the FSHR. Deletion mutants allowed us to map the sorting sequence to the distal part of the intracellular domain at a distance of 47 amino acids from the membrane. Point mutations have allowed us to determine that this sequence comprises approximately 14 residues and is mainly dependent for its activity on Tyr684 and Leu689. This signal is autonomous and dominant; when transferred to an apically targeted heterologous membrane protein, the neurotrophin receptor, it redirects the chimeric construct to the basolateral domain of MDCK cells. Furthermore, we have shown that the FSHR basolateral targeting signal is independent from the endocytosis signal and is not linked to the Gs protein transduction pathway.
In receptor mutants with complete deletion of the basolateral targeting signal, the majority of receptor molecules were detected at the apical surface of MDCK cells at steady state. This reversion of receptor polarity suggests the existence of apical sorting information in the remaining part of the receptor. Glycosylation of the extracellular domain of the receptor may be involved (32).
The insulin receptor has been shown to be localized in the basolateral
compartment of rabbit kidney cells (33) and of transfected MDCK cells
(34). Among G protein-coupled receptors, the parathyroid hormone (35),
2-adrenergic (36, 37), melatonin (38), serotonergic (39,
40), calcium-sensing (41), and A1 adenosine (42) receptors also display
a polarized apical or basolateral localization. However, in none of
these cases has the targeting signal been identified. Recently,
in vitro mutagenesis experiments have been performed on the
2A-adrenergic receptor but have failed to identify its
targeting signal (43). The basolateral localization signal of the FSH
receptor is thus the first signal identified for a G protein-coupled
receptor and more generally for a hormone receptor.
Basolateral targeting signals have been described for a viral protein (vesicular stomatitis virus glycoprotein G) (27); for cell adhesion molecules (NCAM and CD44) (44, 45); for receptors involved in the transport of ligands (transferrin (46), polymeric immunoglobulin (15), FcIgG (47), low density lipoprotein (26), and asialoglycoprotein (25)); and for a growth factor receptor (epidermal growth factor receptor (48)). All of these signals are located in the vicinity of the membrane. The basolateral targeting signal of the FSHR is unusually localized at a distance (47 residues) from the membrane. In many cases, basolateral localization signals are colinear with internalization signals (reviewed in Ref. 31). However, it has been shown that the two sorting processes are differentially sensitive to various point mutations (28, 49, 50, 51). The basolateral localization signal of the epidermal growth factor (48), transferrin (46), and immunoglobulin (15) receptors; the distal signal of the low density lipoprotein receptor (26); and the signal of vesicular stomatitis virus glycoprotein G (27, 52) share with the FSH receptor the property of being independent of the internalization signal.
Comparison of sequences and point mutations of basolateral localization
signals has not allowed us to define a clear cut consensus sequence,
although in most cases either a tyrosine or a hydrophobic amino acid
doublet (Leu-Leu or Leu-Val) plays a major role in these signals
(reviewed in Ref. 23). Structure predictions have indicated in several
cases the existence of a -turn (53, 54). Only one basolateral
localization signal has been studied by NMR (29); in the isolated
polymeric immunoglobulin receptor signal, a
-turn was observed,
followed by a nascent helix. Mutations that compromised this structure
were shown to decrease the sorting fidelity of the protein.
In the case of the FSH receptor, both Tyr684 and
Leu689 were shown to play a major role. This is thus
different from the majority of known signals (see above). Only in the
case of the basolateral targeting signal of the rat lysosomal
glycoprotein 120 and of vesicular stomatitis virus glycoprotein G have
a Tyr and an Ile been shown to be of special importance for function
(49, 52). Structure predictions using several algorithms (20, 21) have indicated that the basolateral localization signal of FSHR contains a
-turn structure. Mutation of residues located within or at close
proximity to this putative structure have been shown to alter the
basolateral targeting of the receptor.
LH and TSH receptors also exhibit a basolateral localization in MDCK cells. These receptors are highly homologous to the FSH receptor. Mutagenesis experiments are now performed to compare their basolateral localization signals with that of the FSHR. These studies should shed light on the cellular trafficking mechanisms of G protein-coupled receptors. Furthermore, mutations of gonadotrophin and thyrostimulin receptors have been described in a variety of diseases (reviewed in Refs. 2 and 4). It is possible that in some cases such natural mutations may alter receptor cell trafficking.
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ACKNOWLEDGEMENTS |
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We thank Dr. P. Dessen for help in secondary structure predictions and P. Leclerc (Service Commun de Microscopie Confocale, Institut Fédératif de Recherche 21) for help in confocal microscopy analysis. We also thank M. Rodrigues for typing the manuscript.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
This work was supported by INSERM, the Faculté de Médecine Paris Sud, the Assistance Publique-Hôpitaux de Paris, the Association pour la Recherche sur le Cancer, and the Association de Recherches Scientifiques Paul Neumann.
¶ To whom correspondence should be addressed: Hôpital de Bicêtre, INSERM U.135, 3ème niveau, 78 Rue du Général Leclerc, 94275 Le Kremlin Bicêtre, France. Tel.: 33-1-45-21-33-29; Fax: 33-1-45-21-38-22.
1 The abbreviations used are: FSH, follicle-stimulating hormone; LH, luteinizing hormone; TSH, thyrotrophin; FSHR, follicle-stimulating hormone receptor; MDCK, Madin-Darby canine kidney; PBS, phosphate-buffered saline; BSA, bovine serum albumin; NTR, neurotrophin receptor; NTRt, truncated neurotrophin receptor; DMEM, Dulbecco's modified Eagle's medium.
2 C. Baratti-Elbaz, N. Ghinea, H. Loosfelt, C. Pichon, and E. Milgrom, unpublished results.
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REFERENCES |
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