Hormone-induced Conformational Change of the Purified Soluble Hormone Binding Domain of Follitropin Receptor Complexed with Single Chain Follitropin
2001; Elsevier BV; Volume: 276; Issue: 26 Linguagem: Inglês
10.1074/jbc.m100057200
ISSN1083-351X
AutoresAnja Schmidt, Robert MacColl, Barbara Lindau‐Shepard, David R. Buckler, James A. Dias,
Tópico(s)Hormonal and reproductive studies
ResumoHuman follicle-stimulating hormone receptor (hFSHR) belongs to family I of G protein-coupled receptors. FSHR extracellular domain (ECD) is predicted to have 8–9 αβ or leucine-rich repeat motif elements. The objective of this study was to identify elements of the FSHR ECD involved in ligand binding. Preincubation of recombinant hFSHR ECD with rabbit antisera raised against synthetic peptides of hFSHR ECD primary sequence abolished follitropin binding primarily in the region of amino acids 150–254. Accessibility of hFSHR ECD after hormone binding, captured by monoclonal antibodies against either ECD or FSH, was decreased for the region of amino acids 150–220 but additionally for amino acids 15–100. Thus, when hFSH bound first, accessibility of antibody binding was decreased to a much larger extent than if antibody was bound first. This suggestion of a conformational change upon binding was examined further. Circular dichroism spectra were recorded for purified single chain hFSH, hFSHR ECD, and hFSHR ECD-single chain hFSH complex. A spectral change indicated a small but consistent conformational change in the ECD·FSH complex after hormone binding. Taken together, these data demonstrate that FSH binding requires elements within the leucine-rich repeat motifs that form a central region of hFSHR ECD, and a conformational change occurs upon hormone binding. Human follicle-stimulating hormone receptor (hFSHR) belongs to family I of G protein-coupled receptors. FSHR extracellular domain (ECD) is predicted to have 8–9 αβ or leucine-rich repeat motif elements. The objective of this study was to identify elements of the FSHR ECD involved in ligand binding. Preincubation of recombinant hFSHR ECD with rabbit antisera raised against synthetic peptides of hFSHR ECD primary sequence abolished follitropin binding primarily in the region of amino acids 150–254. Accessibility of hFSHR ECD after hormone binding, captured by monoclonal antibodies against either ECD or FSH, was decreased for the region of amino acids 150–220 but additionally for amino acids 15–100. Thus, when hFSH bound first, accessibility of antibody binding was decreased to a much larger extent than if antibody was bound first. This suggestion of a conformational change upon binding was examined further. Circular dichroism spectra were recorded for purified single chain hFSH, hFSHR ECD, and hFSHR ECD-single chain hFSH complex. A spectral change indicated a small but consistent conformational change in the ECD·FSH complex after hormone binding. Taken together, these data demonstrate that FSH binding requires elements within the leucine-rich repeat motifs that form a central region of hFSHR ECD, and a conformational change occurs upon hormone binding. follicle stimulating hormone circular dichroism extracellular domain FSH receptor glycoprotein hormone GPH, receptor human luteinizing hormone LH receptor leucine-rich repeat monoclonal antibody enzyme-linked immunosorbent assay polymerase chain reaction Tris-buffered saline with Tween ribonuclease inhibitor single chain thyroid-stimulating hormone amino acid(s) Follicle stimulating hormone (FSH),1 together with luteinizing hormone (LH), thyroid stimulating hormone, and chorionic gonadotropin, belongs to the group of pituitary or placental glycoprotein hormones (GPH). Members of this group belong to the cysteine knot fold family. A common α-subunit and a hormone-specific β-subunit, each sharing the same fold, form the functional heterodimer. FSH plays a central role in the regulation of mammalian reproduction. In females, it is essential for ovarian and follicular development and maturation, whereas in males it regulates spermatogenesis (1Simoni M. Gromoll J. Nieschlag E. Endocr. Rev. 1997; 18: 739-773Crossref PubMed Scopus (700) Google Scholar, 2Chappel S.C. Howles C. Hum. Reprod. 1991; 6: 1206-1212Crossref PubMed Scopus (279) Google Scholar).FSH binds specifically to follicle-stimulating hormone receptors (FSHR) on granulosa cells in the ovary or Sertoli cells in the testis. Binding of FSH to its receptor induces signal transduction by stimulation of adenylate cyclase, which leads to a specific cell response through protein kinase A-dependent pathways (3Reichert Jr., L.E. Dattatreyamurty B. Biol. Reprod. 1989; 40: 13-26Crossref PubMed Scopus (58) Google Scholar).The FSHR belongs to the family of G protein-coupled receptors and consists of an uncharacteristically large extracellular domain (ECD) and a seven-pass helical transmembrane domain (reviewed by Simoniet al. (1Simoni M. Gromoll J. Nieschlag E. Endocr. Rev. 1997; 18: 739-773Crossref PubMed Scopus (700) Google Scholar)). Sequence analysis suggests that the ECD of the glycoprotein hormone receptors (GPHR) contains 8–9 imperfect leucine-rich repeats (LRR) like the porcine ribonuclease inhibitor (RI), a member of the LRR family. Crystal structure analysis showed that RI consists of 15 LRR, which have a β-α hairpin unit structure, where the units are aligned parallel to a common axis (4Kobe B. Deisenhofer J. Nature. 1993; 366: 751-756Crossref PubMed Scopus (540) Google Scholar). The resulting structure resembles a horseshoe with β-strands forming the inner circumference, whereas the α-helices form the outer circumference. This kind of structure shows high flexibility in structure, and binding of RNase A results in a conformational change of the RI (4Kobe B. Deisenhofer J. Nature. 1993; 366: 751-756Crossref PubMed Scopus (540) Google Scholar, 8Kobe B. Deisenhofer J. Nature. 1995; 374: 183-186Crossref PubMed Scopus (569) Google Scholar, 9Kobe B. Deisenhofer J. Curr. Opin. Struct. Biol. 1995; 5: 409-416Crossref PubMed Scopus (322) Google Scholar).Based on this structure, theoretical models have been proposed for the GPHR, where the hormone presumably mainly interacts with the inner circumference of the receptor LRR motifs (5Jiang X. Dreano M. Buckler D.R. Cheng S. Ythier A. Wu H. Hendrickson W.A. Tayar N.E. Structure ( Lond. ). 1995; 15: 1341-1353Abstract Full Text Full Text PDF Scopus (175) Google Scholar, 6Moyle W.R. Campbell R.K. Rao S.N. Ayad N.G. Bernard M.P. Han Y. Wang Y. J. Biol. Chem. 1995; 270: 20020-20031Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 7Kajava A.V. Vassart G. Wodak S.J. Structure ( Lond. ). 1995; 3: 867-877Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar). Using charge inversion mutagenesis, Bhowmick et al. (10Bhowmick N. Huang J. Puett D. Isaacs N.W. Lapthorn A.J. Mol. Endocrinol. 1996; 10: 1147-1159PubMed Google Scholar, 11Bhowmick N. Narayan P. Puett D. Endocrinology. 1999; 140: 4558-4563Crossref PubMed Scopus (38) Google Scholar) could show the involvement of several residues between aa 40 and 206 in hormone binding, located in the concave surface of the LRR model of the LHR. More recently, mutational analysis of the LRR of the FSHR and LHR indicated that several regions within this domain of the receptor are involved in binding (12Song Y.S. Ji I. Beauchamp J. Isaacs N.W. Ji T.H. J. Biol. Chem. 2000; 276: 3426-3435Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 13Song Y.S. Ji I. Beauchamp J. Isaacs N.W. Ji T.H. J. Biol. Chem. 2000; 276: 3436-3442Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Furthermore, antibody inhibition combined with peptide binding studies gave rise to evidence that the most N-terminal part of the FSHR, aa 9–30, is also important in the interaction of receptor and hormone (14Dattatreyamurty B. Reichert Jr., L.E. Endocrinology. 1993; 133: 1593-1601Crossref PubMed Scopus (35) Google Scholar, 15Dattatreyamurty B. Reichert Jr., L.E. Mol. Cell. Endocrinol. 1992; 87: 9-17Crossref PubMed Scopus (39) Google Scholar, 16Nechamen C.A. Dias J.A. Mol. Cell. Endocrinol. 2000; 166: 101-110Crossref PubMed Scopus (28) Google Scholar).The specificity of binding, however, seems to be located differently in the LHR and FSHR. Fragments of the LHR ECD as short as 206 aa are able to bind LH with little reduction in affinity, and chimera studies demonstrate the importance of aa 1–164 for the specificity of the LHR, whereas aa 1–257 are needed for specific FSH binding, with the main part of the specificity being located after LRR5, beyond aa 165 (17Braun T. Schofield P.R. Sprengel R. EMBO J. 1991; 10: 1885-1890Crossref PubMed Scopus (306) Google Scholar,18Thomas D. Rozell T.G. Liu X. Segaloff D.L. Mol. Endocrinol. 1996; 10: 760-768PubMed Google Scholar). Altogether, these data indicate that the binding domain of FSHR is composed of multiple regions of the ECD, but exactly which elements of the receptor interact and whether the FSHR exhibits the same flexibility as the RI and changes upon ligand binding has not yet been determined.It has been difficult to address this issue because the full-length receptor is located within the membrane and, thus, might be sterically restricted and less accessible to antibodies or peptides, and no biophysical data have been provided yet for the receptor-hormone interaction. We reasoned that using solely the ECD might provide a suitable solution for this problem. Over the past several years there have been several attempts to express the ECD of the GPCR to analyze issues like glycosylation (19Thomas D.M. Segaloff D.L. Endocrinology. 1994; 135: 1902-1912Crossref PubMed Scopus (40) Google Scholar, 20Zhang R. Cai H. Fatima N. Buczko E. Dufau M.L. J. Biol. Chem. 1995; 270: 21722-21728Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar) or trafficking (21Peterson A.J. Lindau-Shepard B. Brumberg H.A. Dias J.A. Mol. Cell. Endocrinol. 2000; 160: 203-217Crossref PubMed Scopus (12) Google Scholar). However, expression in Escherichia coli or mammalian or insect cells yielded ECD trapped in the cell, with none or just a little secretion of the recombinant protein (19Thomas D.M. Segaloff D.L. Endocrinology. 1994; 135: 1902-1912Crossref PubMed Scopus (40) Google Scholar, 21Peterson A.J. Lindau-Shepard B. Brumberg H.A. Dias J.A. Mol. Cell. Endocrinol. 2000; 160: 203-217Crossref PubMed Scopus (12) Google Scholar, 22Stevis P.E. Deecher D.C. Lopez F.J. Frail D.E. Endocrine. 1999; 10: 153-160Crossref PubMed Scopus (17) Google Scholar, 23Chen W. Bahl O.P. Mol. Cell. Endocrinol. 1993; 91: 35-41Crossref PubMed Scopus (26) Google Scholar, 24Chazenbalk G.D. Rapoport B. J. Biol. Chem. 1995; 270: 1543-1549Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar).This study used a recombinant form of the hFSHR ECD, secreted from insect cells, that was able to bind FSH with high affinity. This form of the hFSHR ECD allowed analysis of hormone binding independently of a cell system, providing a highly accessible protein. The objective of this study was to utilize this model system to locate the specific elements of the FSHR interacting with the hormone and to collect biophysical data of the interaction.EXPERIMENTAL PROCEDURESMaterialsIodination grade human pituitary LH and human pituitary TSH were from the National Pituitary Hormone Program. Chemicals were purchased from Sigma, Pierce, and Bio-Rad.Expression of hFSHR ECD in Hi 5 Insect CellsThe expression vector used was kindly provided by Ares Advanced Technologies (Randolph, MA). Briefly, plasmid pACgp67-FSHR-ECD contains the hFSHR ECD cDNA (aa 1–349) under the control of the polyhedrin promoter. The secretion of the ECD is facilitated by the signal sequence of the baculovirus gp67 envelope surface glycoprotein fromAutographa californica (25Murphy C.I. McIntire J.R. Davis D.R. Hodgdon H. Seals J.R. Young E. Protein Expression Purif. 1993; 4: 349-357Crossref PubMed Scopus (43) Google Scholar). This creates a fusion protein with three additional amino acids beyond the consensus cleavage site at the N-terminal end, resulting in a secreted protein with the N terminus ADQCHHR. Recombinant baculovirus was amplified in Sf9 cells maintained in TMN-FH medium and titered by a plaque assay (26King L.A. Possee R.D. The Baculovirus Expression System: A Laboratory Guide. Chapman & Hall, London1992Crossref Google Scholar). For large scale production of hFSHR ECD, 24 h after seeding in roller bottles 2 × 108 Hi 5 cells (Invitrogen) were infected with recombinant virus at a multiplicity of infection of 2–4 × 108 for 3 days. The media was collected, cell debris was spun out for 30 min at 10,000 rpm, and the media was filtered through a 0.2-μm filter. After the addition of 0.001 mprotease inhibitor phenylmethylsulfonyl fluoride and 0.02% sodium azide, the media was either stored at 4 °C or frozen at −20 °C until further use.hFSHR ECD PurificationSecreted hFSHR ECD was purified from insect cell media using affinity chromatography. The protein A-purified monoclonal antibody (mAb) 106.105, which is directed against aa 300–315 of the hFSHR (27Lindau-Shepard B.A. Brumberg H.A. Peterson A.J. Dias J.A. J. Reprod. Immunol. 2000; 49: 1-19Crossref Scopus (37) Google Scholar), was coupled to CNBr-activated Sepharose (Amersham Pharmacia Biotech) and then blocked and washed according to the manufacturer's protocol. The column was loaded with secreted hFSHR ECD-containing media until saturated, as determined by enzyme-linked immunosorbent assay (ELISA). After the column was extensively washed with 1% Triton X-100 in wash buffer (0.1 m phosphate, 0.3 m NaCl, pH 7.2) followed by wash buffer, a short wash in 1% octoglucoside in wash buffer, and an extensive wash with wash buffer overnight, ECD was eluted in 0.1 m glycine, 1% acetic acid, pH 2.2. The addition of 30% glycerol to the elution buffer decreased the pH-related loss of biological activity of the purified hFSHR ECD.Expression and Purification of Single Chain (sc) hFSHPreparation of the schFSH expression plasmid was performed as follows. The hFSHβ cDNA was inserted into the mutagenesis vector pKR5.2 as previously described (28Roth K.E. Dias J.A. Mol. Cell. Endocrinol. 1995; 109: 143-149Crossref PubMed Scopus (21) Google Scholar). A mutagenesis oligo to the β-subunit 3′-end was designed to replace the stop codon with the amino acid alanine. The hFSH α-subunit with 5′-spacer was produced by PCR; the 5′-α-PCR primer was designed with a SalI site followed by a Ser-Gly-Ser-Gly-Ser spacer linked to nucleotide 121 of the α-subunit, thus excluding the signaling sequence of the α-subunit. The 3′-α-PCR primer was designed with aPstI-cloning site at the 3′-end and a silent mutation at Cys-84 to delete an undesired PstI site. The PCR product was then digested with restriction enzymes SalI andPstI and cloned into theSalI-PstI-site of pKR5.2. The construct was sequenced to ensure that the tandem FSH subunits were in frame and that no PCR error was introduced. The single chain FSH cDNA was then cloned into the EcoRI-PstI sites of the baculovirus vector pVL 1393. Single chain FSH virus was amplified in Sf9 cells, and Hi 5 cells were infected for expression using a titer of 5–10 multiplicity of infection (see above). Purification of expressed schFSH from media was performed by affinity chromatography using a 46.3H6.B7 column as previously described (29Dias J.A. Zhang Y. Liu X. J. Biol. Chem. 1994; 269: 25289-25294Abstract Full Text PDF PubMed Google Scholar). Purified schFSH was dialyzed against 0.01 m phosphate buffer, pH 7.2, and stored either frozen or lyophilized.Polyacrylamide Gel Electrophoresis and Western Blot AnalysisTo analyze the expression of hFSHR ECD, media was collected as described above, and aliquots were boiled in Laemmli sample buffer. The samples were separated on a 10% SDS-polyacrylamide gel. After electrophoresis, proteins were stained either by Coomassie blue or silver staining (Bio-Rad) or electroblotted onto Immobilon-P membranes (Milipore, Bedford, MA) using a Transblot semi-dry electrophoresis transfer apparatus (Bio-Rad) and 48 mm Tris, 39 mm glycine, 20% methanol, 1.3 mm SDS, pH 9.2, transfer buffer. The membranes were blocked overnight in TBST (10 mm Tris, 145 mm NaCl, pH 7.2, 0.5% Tween 20) containing 5% nonfat milk at 4 °C. The following day, blots were probed with 5 μg of protein A-purified anti-FSHR mAb 106.105 (27Lindau-Shepard B.A. Brumberg H.A. Peterson A.J. Dias J.A. J. Reprod. Immunol. 2000; 49: 1-19Crossref Scopus (37) Google Scholar) diluted in TBST containing 5% nonfat milk for 2 h at room temperature. Blots were washed (4× for 10 min) in TBST and incubated with goat anti-mouse IgG conjugated to horseradish peroxidase (BIOSOURCE International, Camarillo, CA) diluted 1:10,000 in TBST + 5% nonfat milk for 1 h at room temperature. Blots were washed (4× for 10 min), and signals were developed using ECL Western blot detection kit (Amersham Pharmacia Biotech) or Pierce Super Signal kit.Polyclonal Anti-peptide AntibodiesSome of the antibodies used in this study have been described in other studies (30Liu X. Depasquale J.A. Griswold M.D. Dias J.A. Endocrinology. 1994; 135: 682-691Crossref PubMed Scopus (43) Google Scholar). Briefly, rabbits were immunized with synthetic peptides encompassing nearly the entire FSHR ECD primary sequence. Antisera were tested in Western blot and in ELISA assays for their specificity of reaction with both immunizing peptides and hFSHR ECD. Antisera testing positive (see Table I) were precipitated with ammonium sulfate according to the protocol of Harlow and Lane (31Harlow E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1988: 298-299Google Scholar) and dialyzed against PBS with 0.02% sodium azide.Table ISynthetic peptide and resulting specific polyclonal antibodiesResidue number in hFSHRAntiserum (name)1–15X80315–44W971, W96345–7255872–100513150–183W953, W970183–220562221–254X180265–296W955, X179 Open table in a new tab Solid-phase Radio Receptor AssayTo analyze the binding characteristics of secreted hFSHR ECD, a new solid-phase radio receptor assay format was developed. Nunc® tubes (Nalge Nunc International, Rochester, NY) were coated with 1 μg of mAb 106.105 in 200 μl of coating buffer (50 mm Tris, pH 9.5) at 4 °C overnight. The tubes were washed once with 1 ml of wash buffer (phosphate-buffered saline with 0.02% azide and 0.05% Tween 20; all the following washes were done in this wash buffer) and blocked with 500 μl of 10% nonfat milk, 2% bovine serum albumin in coating buffer for 3 h at 37 °C. After the tubes were washed (3× 1 ml of wash buffer), 200 μl of insect media containing hFSHR ECD diluted in binding buffer (phosphate-buffered saline, 0.127 mm EDTA, 0.25% bovine serum albumin, 0.05% Tween 20) was added and incubated for 2 h at 37 °C.To analyze the binding affinity of hFSHR ECD, the tubes were washed three times, and radiolabeled purified pituitary hFSH (125I-FSH, 150,000 cpm) as well as unlabeled pituitary hFSH in increasing concentrations was added and incubated for 2 h at 37 °C. The specificity of binding was analyzed in competitive displacement assays utilizing unlabeled purified pituitary LH, TSH, and FSH. Counts bound were measured in a γ-counter and plotted using the PRISM3 program. Affinity constants were calculated based on the ED50 from data sets with limiting amounts of captured hFSHR ECD using National Institutes of Health radioimmunoassay software.To assess the effect of binding of the anti-peptide antibodies on125I-FSH binding to ECD, captured hFSHR ECD was incubated with different dilutions of antibodies (dilutions ranging from 1:3 to 1:100) for 2 h at 37 °C followed by washing and incubating with125I-FSH as described above. Radioactivity counts were compared with the addition of normal rabbit serum.ELISAThe ELISA format was used not only to determine the relative concentration of hFSHR ECD in media but also to examine the effects of FSH-hFSHR·ECD complex formation on the accessibility of hFSHR ECD epitopes for the anti-peptide antibodies. Two complementary approaches were used for protein footprinting in which either hFSHR ECD (see the next paragraph, “hFSHR ECD-capture ELISA”) or hormone (see “FSH-capture ELISA,” below) was captured by specific monoclonal antibodies.hFSHR ECD-capture ELISAImmulon 4HBX 96-well plates (DYNEX Technologies Inc., Alexandria, VA) were coated with 500 ng of mAb 106.105 in 100 μl of coating buffer at 4 °C overnight. Plates were washed thoroughly (4–6 times) with wash buffer (phosphate-buffered saline with 0.02% azide and 0.05% Tween 20; all the following washes are done with this buffer) between all of the following steps. Plates were blocked for 2 h at 37 °C with 200 μl of 5% nonfat milk in coating buffer, and hFSHR ECD from 100 μl of insect media was captured on the antibody for 1 h at 37 °C (Hi 5 cell-conditioned media was used as control). To detect hFSHR ECD captured, plates were incubated with polyclonal anti-peptide antibody W970 (1:1000 in 100 μl of binding buffer; anti-hFSHR 150–183) for 1 h at 37 °C followed by a 1-h incubation with alkaline phosphatase-labeled goat anti-rabbit IgG (1:4000 in 100 μl of binding buffer, BIOSOURCE International, Camarillo, CA). For detection, 100 μl of development substrate (p-nitrophenyl phosphate in diethylamine buffer, Bio-Rad) was added, and absorbance at 410 nm was measured after 30–60 min at room temperature using as Dynatech MR700 plate reader.The effect of FSH binding on epitope accessibility was assessed by protein footprinting. Excess purified schFSH (1 μg/well) or binding buffer as control was added to the captured ECD and incubated for 1–2 h at 37 °C. After being washed extensively, the captured ECD was challenged with different polyclonal anti-peptide antibodies (1:25–1:100 in binding buffer) for 1 h at 37 °C. Antibody binding was detected with goat anti-rabbit IgG coupled to alkaline phosphatase followed by substrate incubation as described above.FSH-capture ELISATo confirm the footprinting results achieved with the ECD-capture ELISA, a complementary approach was developed. Immulon 4HBX 96-well plates were coated with 500 ng of βFSH-specific mAb 46.3H6.B7 in 100 μl of coating buffer at 4 °C overnight. The plate was blocked and washed (see above), and 500 ng of purified schFSH was bound to the antibody for 1 h at 37 °C. After washing extensively, the plate was incubated with 100 μl of either hFSHR ECD or conditioned insect cell media (control) for 1 h at 37 °C. To determine the accessibility of hFSHR ECD bound to captured schFSH, the plate was then challenged with each polyclonal anti-peptide antibody (1:25–1:100 in binding buffer) for 1 h at 37 °C. Antibody binding was detected with goat anti-rabbit IgG coupled to alkaline phosphatase followed by substrate incubation as described above.Circular Dichroism StudiesExperiments were carried out on a JASCO J-720 spectropolarimeter at 20 or 6 °C using a cell with a path length of 0.05 cm. Proteins were dialyzed against 0.01 m phosphate buffer, pH 7.2, overnight before the measurements. Spectra were measured with the following settings: 1.0 nm bandwidth, 20-nm/min speed, 0.2-nm resolution, wavelength between 260 and 180 nm with 3–9 accumulations per measurement. Protein concentrations were determined by amino acid analysis. Spectra were determined for the schFSH (3.4–5.8 μm in 0.01 m phosphate buffer) and hFSHR ECD (0.5–0.8 μm in 0.01 m phosphate buffer) individually as well as for a mixture containing 3.4–5.8 μm schFSH and 0.5–0.8 μm hFSHR ECD, thereby containing exactly the same concentration of each protein as in the individually measured samples. schFSH was thus used in a 4–12-fold molar excess to ECD in separate experiments. Before obtaining the spectra, the individual proteins as well as a mixture of both were incubated at room temperature or 37 °C for 0.5–2 h.Individual spectra were analyzed using the JASCO software to subtract the background and smooth the spectra. The computer program SELCON 3 was used to calculate the secondary structure estimates for schFSH and hFSHR ECD utilizing two data bases containing 37 and 42 proteins, respectively, enabling an analysis in the wavelength range from 185 to 260 nm (32Sreerama N. Woody R.W. Anal. Biochem. 1993; 209: 32-44Crossref PubMed Scopus (942) Google Scholar, 33Sreerama N. Woody R.W. J. Mol. Biol. 1994; 242: 497-507PubMed Google Scholar, 34Sreerama N. Venyaminov S.Y. Woody R.W. Protein Sci. 1999; 8: 370-380Crossref PubMed Scopus (637) Google Scholar, 35Johnson W.C. Proteins. 1999; 35: 307-312Crossref PubMed Scopus (618) Google Scholar). For comparison of the spectra (in molar ellipticity) of the hFSHR ECD·schFSH complex versus theoretically calculated ECD + FSH spectra, the individual spectra of FSH and ECD were added after background subtraction and smoothed, and the molar ellipticity (mean residue ellipticity [Θ]) was calculated. This sum of the individual spectra, representing a mixture of noninteracting schFSH and hFSHR ECD, was compared with the spectra experimentally determined for the hFSHR ECD·schFSH complex. Follicle stimulating hormone (FSH),1 together with luteinizing hormone (LH), thyroid stimulating hormone, and chorionic gonadotropin, belongs to the group of pituitary or placental glycoprotein hormones (GPH). Members of this group belong to the cysteine knot fold family. A common α-subunit and a hormone-specific β-subunit, each sharing the same fold, form the functional heterodimer. FSH plays a central role in the regulation of mammalian reproduction. In females, it is essential for ovarian and follicular development and maturation, whereas in males it regulates spermatogenesis (1Simoni M. Gromoll J. Nieschlag E. Endocr. Rev. 1997; 18: 739-773Crossref PubMed Scopus (700) Google Scholar, 2Chappel S.C. Howles C. Hum. Reprod. 1991; 6: 1206-1212Crossref PubMed Scopus (279) Google Scholar). FSH binds specifically to follicle-stimulating hormone receptors (FSHR) on granulosa cells in the ovary or Sertoli cells in the testis. Binding of FSH to its receptor induces signal transduction by stimulation of adenylate cyclase, which leads to a specific cell response through protein kinase A-dependent pathways (3Reichert Jr., L.E. Dattatreyamurty B. Biol. Reprod. 1989; 40: 13-26Crossref PubMed Scopus (58) Google Scholar). The FSHR belongs to the family of G protein-coupled receptors and consists of an uncharacteristically large extracellular domain (ECD) and a seven-pass helical transmembrane domain (reviewed by Simoniet al. (1Simoni M. Gromoll J. Nieschlag E. Endocr. Rev. 1997; 18: 739-773Crossref PubMed Scopus (700) Google Scholar)). Sequence analysis suggests that the ECD of the glycoprotein hormone receptors (GPHR) contains 8–9 imperfect leucine-rich repeats (LRR) like the porcine ribonuclease inhibitor (RI), a member of the LRR family. Crystal structure analysis showed that RI consists of 15 LRR, which have a β-α hairpin unit structure, where the units are aligned parallel to a common axis (4Kobe B. Deisenhofer J. Nature. 1993; 366: 751-756Crossref PubMed Scopus (540) Google Scholar). The resulting structure resembles a horseshoe with β-strands forming the inner circumference, whereas the α-helices form the outer circumference. This kind of structure shows high flexibility in structure, and binding of RNase A results in a conformational change of the RI (4Kobe B. Deisenhofer J. Nature. 1993; 366: 751-756Crossref PubMed Scopus (540) Google Scholar, 8Kobe B. Deisenhofer J. Nature. 1995; 374: 183-186Crossref PubMed Scopus (569) Google Scholar, 9Kobe B. Deisenhofer J. Curr. Opin. Struct. Biol. 1995; 5: 409-416Crossref PubMed Scopus (322) Google Scholar). Based on this structure, theoretical models have been proposed for the GPHR, where the hormone presumably mainly interacts with the inner circumference of the receptor LRR motifs (5Jiang X. Dreano M. Buckler D.R. Cheng S. Ythier A. Wu H. Hendrickson W.A. Tayar N.E. Structure ( Lond. ). 1995; 15: 1341-1353Abstract Full Text Full Text PDF Scopus (175) Google Scholar, 6Moyle W.R. Campbell R.K. Rao S.N. Ayad N.G. Bernard M.P. Han Y. Wang Y. J. Biol. Chem. 1995; 270: 20020-20031Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 7Kajava A.V. Vassart G. Wodak S.J. Structure ( Lond. ). 1995; 3: 867-877Abstract Full Text Full Text PDF PubMed Scopus (213) Google Scholar). Using charge inversion mutagenesis, Bhowmick et al. (10Bhowmick N. Huang J. Puett D. Isaacs N.W. Lapthorn A.J. Mol. Endocrinol. 1996; 10: 1147-1159PubMed Google Scholar, 11Bhowmick N. Narayan P. Puett D. Endocrinology. 1999; 140: 4558-4563Crossref PubMed Scopus (38) Google Scholar) could show the involvement of several residues between aa 40 and 206 in hormone binding, located in the concave surface of the LRR model of the LHR. More recently, mutational analysis of the LRR of the FSHR and LHR indicated that several regions within this domain of the receptor are involved in binding (12Song Y.S. Ji I. Beauchamp J. Isaacs N.W. Ji T.H. J. Biol. Chem. 2000; 276: 3426-3435Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar, 13Song Y.S. Ji I. Beauchamp J. Isaacs N.W. Ji T.H. J. Biol. Chem. 2000; 276: 3436-3442Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Furthermore, antibody inhibition combined with peptide binding studies gave rise to evidence that the most N-terminal part of the FSHR, aa 9–30, is also important in the interaction of receptor and hormone (14Dattatreyamurty B. Reichert Jr., L.E. Endocrinology. 1993; 133: 1593-1601Crossref PubMed Scopus (35) Google Scholar, 15Dattatreyamurty B. Reichert Jr., L.E. Mol. Cell. Endocrinol. 1992; 87: 9-17Crossref PubMed Scopus (39) Google Scholar, 16Nechamen C.A. Dias J.A. Mol. Cell. Endocrinol. 2000; 166: 101-110Crossref PubMed Scopus (28) Google Scholar). The specificity of binding, however, seems to be located differently in the LHR and FSHR. Fragments of the LHR ECD as short as 206 aa are able to bind LH with little reduction in affinity, and chimera studies demonstrate the importance of aa 1–164 for the specificity of the LHR, whereas aa 1–257 are needed for specific FSH binding, with the main
Referência(s)