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• W2009566252 abstract "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. 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. The FSH 1The 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. receptor (FSHR), along with the LH and TSH receptors, belongs to a subgroup of G protein-coupled receptors (reviewed in Refs. 1Segaloff D.L. Ascoli M. Endocr. Rev. 1993; 14: 324-347PubMed Google Scholar, 2Misrahi M. Beau I. Meduri G. Bouvattier C. Atger M. Loosfelt H. Ghinea N. Vu Hai M.T. Bougneres P. Milgrom E. Baillieres Clin. Endocrinol. Metab. 1998; (in press)PubMed Google Scholar, 3Misrahi M. Milgrom E. Weetman A.P. Grossman A. Pharmacotherapeutics of the Thyroid Gland. 128. Springer-Verlag, Berlin1997: 33-73Google Scholar, 4Vassart G. Parma J. Van Sande J. Dumont J.E. Endocr. Rev. 1994; 3: 77-80Google Scholar, 5Simoni M. Gromoll J. Nieschlag E. Endocr. Rev. 1997; 18: 739-773Crossref PubMed Scopus (720) Google Scholar). 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 (5Simoni M. Gromoll J. Nieschlag E. Endocr. Rev. 1997; 18: 739-773Crossref PubMed Scopus (720) Google Scholar). The FSH receptor has a central role in reproduction through the control of gonadal development and gamete production (5Simoni M. Gromoll J. Nieschlag E. Endocr. Rev. 1997; 18: 739-773Crossref PubMed Scopus (720) Google Scholar). In males it is expressed in testicular Sertoli cells. These cells form an epithelial blood barrier and control the development of spermatogenesis (6Dym M. Fawcett B.W. Biol. Reprod. 1970; 3: 308-321Crossref PubMed Scopus (886) Google Scholar). 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 (2Misrahi M. Beau I. Meduri G. Bouvattier C. Atger M. Loosfelt H. Ghinea N. Vu Hai M.T. Bougneres P. Milgrom E. Baillieres Clin. Endocrinol. Metab. 1998; (in press)PubMed Google Scholar). 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 (7Ghinea N. Vu Hai M.T. Groyer-Picard M.T. Houllier A. Schoëvaërt D. Milgrom E. J. Cell Biol. 1992; 118: 1347-1358Crossref PubMed Scopus (87) Google Scholar), 2C. Baratti-Elbaz, N. Ghinea, H. Loosfelt, C. Pichon, and E. Milgrom, unpublished results. but the molecular signals involved are still unknown. The LH receptor is also present in the vascular endothelium of the testes (8Ghinea N. Vu Hai M.T. Groyer-Picard M.T. Milgrom E. J. Cell Biol. 1994; 125: 87-97Crossref PubMed Scopus (63) Google Scholar). 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 (9Vannier B. Loosfelt H. Meduri G. Pichon C. Milgrom E. Biochemistry. 1996; 35: 1358-1366Crossref PubMed Scopus (88) Google Scholar), 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 (9Vannier B. Loosfelt H. Meduri G. Pichon C. Milgrom E. Biochemistry. 1996; 35: 1358-1366Crossref PubMed Scopus (88) Google Scholar, 10Loosfelt H. Pichon C. Jolivet A. Misrahi M. Caillou B. Jamous M. Vannier B. Milgrom E. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3765-3769Crossref PubMed Scopus (176) Google Scholar). By contrast, the LH receptor is expressed circumferentially in the ovarian granulosa and theca cells and in the testicular Leydig cells (11Meduri G. Vu Hai M.T. Jolivet A. Milgrom E. Endocrinology. 1992; 131: 366-373Crossref PubMed Scopus (42) Google Scholar, 12Meduri G. Vu Hai M.T. Jolivet A. Takemori S. Kominami S. Driancourt M.A. Milgrom E. J. Endocrinol. 1996; 148: 435-446Crossref PubMed Scopus (41) Google Scholar). 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 (13Beau I. Misrahi M. Gross B. Vannier B. Loosfelt H. Vu Hai M.T. Pichon C. Milgrom E. J. Biol. Chem. 1997; 272: 5241-5248Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). 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. The vector encoding the wild-type FSH receptor cDNA (pSG5-FSHR) has previously been described (9Vannier B. Loosfelt H. Meduri G. Pichon C. Milgrom E. Biochemistry. 1996; 35: 1358-1366Crossref PubMed Scopus (88) Google Scholar). Polymerase chain reaction-mediated oligonucleotide mutagnesis was used to introduce stop codons into the pSG5-FSHR vector to produce vectors encoding FSHRΔ657–695 and FSHRΔ677–695. In the case of the FSHRΔ657–695, a 821-base pair fragment (positions 1244–2065 of the cDNA sequence, +1 being the first base of the initiation codon) containing a stop codon at residue 657 was constructed. After digestion with PflMI, the purified fragment was subcloned into the pSG5-FSHR vector previously digested with the same enzyme. In the case of the FSHRΔ677–695, the same strategy was used. A 486-base pair fragment (positions 1793–2279 of the cDNA sequence) containing a stop codon at residue 677 was digested withSapI and EcoRI. It was cloned into the pSG5-FSHR vector previously digested with SapI andEcoRI. The FSHRΔ692–695 mutant was prepared by generating a KpnI site at position 3098 of pSG5-FSHR using the same strategy as described above for the FSHRΔ677–695 mutant. This mutation yields a conservative substitution of threonine 682 for a serine. This substitution does not alter the polarity or the functional properties of the receptor. A synthetic double strand mutated oligonucleotide was then inserted between the KpnI and the EcoRI sites. All of the constructs were verified by double-stranded DNA sequencing. 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 theSapI (position 3075 of the pSG5 FSHR vector) and theKpnI sites or between the KpnI and theEcoRI (position 3145) sites. The mutants Gly681 → Ala and Ser682 → Ala were constructed using polymerase chain reaction-mediated oligonucleotide mutagenesis. A 486-base pair fragment (positions 1793–2279 of the cDNA sequence) containing the Gly681→ Ala or the Ser682 → Ala substitution was digested with SapI and EcoRI, purified, and cloned into the SapI–EcoRI-digested pSG5-FSHR. 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 (14Hempstead B.L. Patil N. Thiel B. Chao M.V. J. Biol. Chem. 1990; 265: 9595-9598Abstract Full Text PDF PubMed Google Scholar). 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. 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 (13Beau I. Misrahi M. Gross B. Vannier B. Loosfelt H. Vu Hai M.T. Pichon C. Milgrom E. J. Biol. Chem. 1997; 272: 5241-5248Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). 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 (13Beau I. Misrahi M. Gross B. Vannier B. Loosfelt H. Vu Hai M.T. Pichon C. Milgrom E. J. Biol. Chem. 1997; 272: 5241-5248Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar) using the monoclonal anti-FSH receptor, FSHR323 (9Vannier B. Loosfelt H. Meduri G. Pichon C. Milgrom E. Biochemistry. 1996; 35: 1358-1366Crossref PubMed Scopus (88) Google Scholar). Sodium butyrate (10 mm) (Sigma) was added to increase the expression of the transfected cDNAs as described (15Casanova J.E. Apodaca G. Mostov K.E. Cell. 1991; 66: 65-75Abstract Full Text PDF PubMed Scopus (226) Google Scholar). The transepithelial resistance of the clones was ∼300 ohms/cm2. Indirect immunofluorescence studies were performed as described (13Beau I. Misrahi M. Gross B. Vannier B. Loosfelt H. Vu Hai M.T. Pichon C. Milgrom E. J. Biol. Chem. 1997; 272: 5241-5248Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar) 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 (16Urban J. Parczyk K. Leutz A. Kayne M. Kondor-Kock C. J. Cell Biol. 1987; 150: 2735-2743Crossref Scopus (146) Google Scholar) was verified as described previously (17Breitfeld P.P. Casanova J.E. McKinnon W.C. Mostov K.E. J. Biol. Chem. 1990; 265: 13750-13757Abstract Full Text PDF PubMed Google Scholar). MDCK cells were grown to confluence on coverslips as described (13Beau I. Misrahi M. Gross B. Vannier B. Loosfelt H. Vu Hai M.T. Pichon C. Milgrom E. J. Biol. Chem. 1997; 272: 5241-5248Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). 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 (13Beau I. Misrahi M. Gross B. Vannier B. Loosfelt H. Vu Hai M.T. Pichon C. Milgrom E. J. Biol. Chem. 1997; 272: 5241-5248Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). The cells were then fixed in 3% paraformaldehyde for 15 min and further processed as described above. cAMP was measured as described (9Vannier B. Loosfelt H. Meduri G. Pichon C. Milgrom E. Biochemistry. 1996; 35: 1358-1366Crossref PubMed Scopus (88) Google Scholar). The cells were incubated in the absence or in the presence of 50 nmFSH (Metrodine, Serono). 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 (13Beau I. Misrahi M. Gross B. Vannier B. Loosfelt H. Vu Hai M.T. Pichon C. Milgrom E. J. Biol. Chem. 1997; 272: 5241-5248Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Briefly, cells were pulse-labeled for 1 h with 1 mCi/ml Express35S (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 (13Beau I. Misrahi M. Gross B. Vannier B. Loosfelt H. Vu Hai M.T. Pichon C. Milgrom E. J. Biol. Chem. 1997; 272: 5241-5248Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). All experiments were performed at least twice with triplicate samples. 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 (18Le Bivic A. Real F.X. Rodriguez-Boulan E. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 9313-9317Crossref PubMed Scopus (150) Google Scholar). After washing with DMEM, the cells were chased for 3 h in DMEM containing unlabeled amino acids. Cell surface immunoprecipitation was performed as described (19Le Bivic A. Sambuy Y. Patzak A. Patil N. Chao M. Rodriguez-Boulan E. J. Cell Biol. 1991; 115: 607-618Crossref PubMed Scopus (116) Google Scholar). 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 (18Le Bivic A. Real F.X. Rodriguez-Boulan E. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 9313-9317Crossref PubMed Scopus (150) Google Scholar). SDS-polyacrylamide gel electrophoresis was performed as described (13Beau I. Misrahi M. Gross B. Vannier B. Loosfelt H. Vu Hai M.T. Pichon C. Milgrom E. J. Biol. Chem. 1997; 272: 5241-5248Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). The gels were fixed and processed for fluorography. Densitometric scanning was used for quantification. COS-7 cells were transfected with wild-type FSHR or the truncated FSHRΔ677–695 using Superfect (Qiagen, Chatsworth, CA). Internalization from the cell surface of wild-type FSHR or of the truncated FSHRΔ677–695 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. Internalization was also studied in MDCK cells permanently expressing the wild-type FSHR or the truncated FSHRΔ677–695. The cells were seeded on filters and incubated 48 h later with125I-FSH added to the basolateral compartment (10 min at 37 °C). The MDCK cells were then processed as described above for the COS-7 cells. Secondary structure predictions were performed using computer modeling. Two methods were used: the protein sequence analysis method (20Stultz C.M. White J.V. Smith T.F. Protein Sci. 1993; 2: 305-314Crossref PubMed Scopus (156) Google Scholar) and neural network prediction (21McGregor M.J. Flores T.P. Sternberg M.J.E. Protein Eng. 1989; 2: 521-526Crossref PubMed Scopus (96) Google Scholar). 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. 2 A). 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. 1 a) and BB28 (Fig. 1 b) that recognize the ectodomain of endogenous antigens restricted to the basolateral and the apical compartments of MDCK cell membrane, respectively (13Beau I. Misrahi M. Gross B. Vannier B. Loosfelt H. Vu Hai M.T. Pichon C. Milgrom E. J. Biol. Chem. 1997; 272: 5241-5248Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar), were used as controls.Figure 1Localization of the basolateral sorting signal. Confocal microscopic examination of the distribution of FSHR deletion mutants in polarized MDCK cells is shown. Polarized monolayers of cells were grown to confluence on coverslips. They were treated with EGTA. The cells were processed for indirect immunofluorescence microscopy as described under “Materials and Methods.” Primary antibodies corresponded to monoclonal antibodies BC11 (a) and BB18 (b) recognizing, respectively, a basolateral and an apical endogenous antigen of MDCK cells. Monoclonal antibody FSHR 323 was also used to analyze the wild type (c) and the Δ657–695 (d), Δ677–695 (e), and Δ692–695 (f) receptor mutants. In each case, the upper panel shows xyhorizontal focal sections (parallel through MDCK cells) taken in the plane of the apical (a, c, and f) or lateral (b, d, and e) membrane. In each panel the lower part represents the corresponding xz section taken in 0.1-μm steps through the en face view represented above (at 90° to the xy sections). When the immunolabeling was detected in one compartment, no labeling or very faint labeling was observed in the opposite compartment of the cell membrane (not shown). Bar, 10 μm.View Large Image Figure ViewerDownload (PPT) In the first mutant (FSHRΔ657–695), the major part of the intracellular domain of the receptor was deleted. The cell lines expressing mutant or wild-type receptors were incubated with the anti-FSHR ectodomain antibody in the presence of EGTA (Fig. 1). This compound opens the tight junctions (13Beau I. Misrahi M. Gross B. Vannier B. Loosfelt H. Vu Hai M.T. Pichon C. Milgrom E. J. Biol. Chem. 1997; 272: 5241-5248Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar) and thus allows antibodies to reach the totality of the cell surface. In cells expressing the wild-type receptor, a strong labeling of the basolateral membrane was observed with a reticular pattern characteristic of basolateral antigens (see in Fig. 1 c a confocal horizontal section close to the basal pole of the cell). In cells expressing the truncated FSHRΔ657–695 receptor, there was a strong apical labeling (see in Fig. 1 d a confocal horizontal section close to the apical pole of the cell). Confocal vertical sections confirmed that the wild-type FSHR was predominantly expressed in the basolateral domain (Fig. 1 c) and the FSHRΔ657–695 in the apical domain of cell membranes (Fig. 1 d). To narrow down the sequences of the cytoplasmic tail of the FSHR necessary for basolateral targeting, we next analyzed by confocal microscopy two additional deletion mutants: FSHRΔ677–695 and FSHRΔ692–695. While the first mutant exhibited mainly an apical localization very similar to that of the FSHRΔ657–695 mutant (Fig. 1 e), the second mutant, FSHRΔ692–695, had a predominant basolateral polarized expression indistinguishable from the wild-type receptor (Fig. 1 f). Thus, the sequence located between residues 678 and 691 is very likely to contain basolateral sorting information. To quantify the relative proportion of receptor targeted to each membrane domain at steady state, we performed surface immunoprecipitation experiments. MDCK cells expressing wild-type or mutated receptors were pulse-labeled with [35S]methionine and cysteine for 1 h and chased for 3 h with a medium containing unlabeled amino acids (see “Materials and Methods”). Antireceptor antibodies were added either to the basal or to the apical compartment of MDCK cells during the last hour of the chase period. The receptor-antibody complexes were purified and analyzed by gel electrophoresis. For the wild-type FSHR, ∼85% of the receptor molecules were immunoprecipitated from the basolateral surface (Fig. 2 B). The same result was obtained for the FSHRΔ692–695 truncated mutant, while for clones expressing the Δ677–695 or Δ657–695 truncated mutants only ∼30% of the receptor molecules were found on the basolateral surface of MDCK cells, the majority of receptors being detected on the apical domain. These results thus confirm the immunocytochemical studies localizing the signal between residues 678 and 691 of the FSHR. 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. 22Mostov K.E. Apodaca G. Aroeti B. Okamoto C. J. Cell Biol. 1992; 116: 577-583Crossref PubMed Scopus (191) Google Scholar and 23Matter K. Mellman I. Curr. Opin. Cell Biol. 1994; 6: 545-554Crossref PubMed Scopus (392) Google Scholar). Analysis of the kinetics of appearance of the newly synthesized protein on the basolateral and apical cell surfaces allows us to distinguish between these mechanisms (15Casanova J.E. Apodaca G. Mostov K.E. Cell. 1991; 66: 65-75Abstract Full Text PDF PubMed Scopus (226) Google Scholar, 19Le Bivic A. Sambuy Y. Patzak A. Patil N. Chao M. Rodriguez-Boulan E. J. Cell Biol. 1991; 115: 607-618Crossref PubMed Scopus (116) Google Scholar, 24Monlauzeur L. Rajasekaran A. Chao M. Rodriguez-Boulan E. Le Bivic A. J. Biol. Chem. 1995; 270: 12219-12225Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 25Geffen I. Fuhrer C. Leitinger B. Weiss M. Huggel K. Griffiths G. Speiss M. J. Biol. Chem. 1993; 268: 20772-20777Abstract Full Text PDF PubMed Google Scholar, 26Matter K. Hunziker W. Mellman I. Cell. 1992; 71: 741-753Abstract Full Text PDF PubMed Scopus (304) Google Scholar). Such an analysis was performed by metabolic labeling of the cells followed by selective surface immunoprecipitation at various time intervals. As shown in Fig. 3, after 30 min of chase, the newly synthesized FSH receptor Δ677–695 was already detected on the apical surface of MDCK cells. A minority of receptor molecules (∼25%) were also delivered to the basolateral surface of MDCK cells at this time. This was in marked contrast to what has been previously observed for the wild-type receptor, which was mainly detected on the basolateral membrane of MDCK cells in the same conditions (13Beau I. Misrahi M. Gross B. Vannier B. Loosfelt H. Vu Hai M.T. Pichon C. Milgrom E. J. Biol. Chem. 1997; 272: 5241-5248Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar). Throughout the chase period, delivery of the mutant receptor to each surface remained constant at each time period. Approximately 60–70% of the labeled receptor molecules were found on the apical surface. There was no indication that the truncated receptor presented an increased transcytosis or decreased anchorage at the basolateral surface of the cells. This experiment thus suggested that a basolateral localization signal has been inactivated in the truncated receptor. Furthermore, the fact that the majority of receptor molecules were delivered to the apical cell surface indicates that apical sorting signal(s) may be present elsewhere in the FSHR protein and be revealed by the deletion of basolateral targeting sequences. 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 (TableI). 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.Table IPoint mutations in the basolateral targeting signal of the FSH receptorReceptorSequenceApical receptor%FSHR wild typeVTSGS¯TYILVPLSH20FSHR V678AA————-20FSHR T679A-A————20FSHR S680A–A———–20FSHR G681A—A———-35FSHR S682A—-A———25FSHR T683A—–A——–20FSHR Y684A——A——-55FSHR I685A——-A——35FSHR L686A——–A—–20FSHR V687A———A—-20FSHR P688A———-A—25FSHR L689A———–A–45FSHR S690A————A-20FSHR H691A————-A35A series of point alanine substitutions were generated in the 678–691 sequence of the FSHR as described under “Materials and Methods.” The substitutions performed are indicated on the left. Dashes represent unchanged residues. Expression of the mutants was analyzed in stably transfected MDCK cells by cell surface immunoprecipitation. Densitometric analysis of the gels was performed, and the percentage of total FSHR present on the basolateral and on the apical surfaces of the cells was determined. The percentage of apical receptor is indicated on the right and corresponds to the average of at least two experiments. The bar above the wild-type sequence indicates the residues are predicted to form a β-turn. Open table in a new tab Figure 5Point mutants in the basolateral signal of FSHR. Analysis by cell surface immunoprecipitation of the FSH receptor is shown. Cell surface immunoprecipitation was performed using antibody FSHR 323 as indicated in the legend of Fig. 1. Representative gels corresponding to the FSHR mutants Tyr684 → Ala, Leu689 → Ala, Gly681 → Ala, Ile685 → Ala, and His691 → Ala are shown.View Large Image Figure ViewerDownload (PPT) A series of point alanine substitutions were generated in the 678–691 sequence of the FSHR as described under “Materials and Methods.” The substitutions performed are indicated on the" @default.
• W2009566252 created "2016-06-24" @default.
• W2009566252 creator A5004044904 @default.
• W2009566252 creator A5004646984 @default.
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• W2009566252 date "1998-07-01" @default.
• W2009566252 modified "2023-10-05" @default.
• W2009566252 title "The Basolateral Localization Signal of the Follicle-stimulating Hormone Receptor" @default.
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