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PNAS 109 (31): 12491-12496

Copyright © 2012 by the National Academy of Sciences.

Structure of follicle-stimulating hormone in complex with the entire ectodomain of its receptor

Xuliang Jianga, Heli Liub, Xiaoyan Chenb, Po-Han Chenb, David Fischera, Venkataraman Sriramana, Henry N. Yua, Steve Arkinstalla, and Xiaolin Heb,1

a EMD Serono Research Institute, Billerica, MA 01821; and bDepartment of Molecular Pharmacology and Biological Chemistry, Northwestern University Feinberg School of Medicine, Chicago, IL 60611


Figure 01
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Fig. 1. Structure of human FSH in complex with human FSHRED. (A) Ribbon model of human FSH bound to FSHRED: FSH α-subunit is shown in green, FSH β-subunit is shown in purple, and FSHRED is shown in magenta. The side chain of sulfated Y335 is depicted as sticks for the tyrosine stem and colored balls for the sulfate group. The carbohydrates are depicted as balls. The disordered residues in the receptors are marked as dashed lines. (B) Top view of the trimer observed in the asymmetrical unit.

 

Figure 02
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Fig. 2. Structure of FSHRED. (A) Schematic diagram of the topology of the FSHR structure. The regular secondary structure elements of LRRs are shown as arrows for the β-strands and as a cylinder for the α-helix. Disulfide bonds are shown as yellow lines linking cysteines, the N-linked glycosylation sites are shown as "Y," and the exon boundaries in the FSHR gene are shown as black triangles ({blacktriangledown}). (B) Ribbon representation of FSHRED. The hormone-binding subdomain is shown in yellow, and the signal specificity subdomain (hinge region) is shown in magenta. (Inset) In the close-up view of the signal specificity subdomain, motif CF3 is colored green, the hormone-binding region is colored yellow, and the rhodopsin-like extracellular sequence is colored magenta. The disulfide bonds are shown as brown sticks, sulfated Y335 is shown as sticks for the phenyl ring and balls for the sulfate group, and the side chain of S273 is shown as balls. As a reference, FSH is included as a gray surface.

 

Figure 03
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Fig. 3. Hormone-receptor interactions outside the primary binding site. (A) sTyr site, with close-up views of the interactions within the FSH–FSHRED complex monomer (Upper Inset) and the electrostatic potential of the sulfated tyrosine-binding pocket (Lower Inset). (B) Potential exosite related to trimerization, with a close-up view (Inset) of the interactions originating from the FSH–FSHRED complex oligomerizations. The receptor trimer is shown as a magenta surface. FSH is shown as ribbons: green ribbons for the α-chain and light purple ribbons for the β-chain. The FSH N52α glycan is shown as yellow balls.

 

Figure 04
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Fig. 4. Formation of the sulfate-binding pocket in FSH at the sTyr site. (A and B) Comparison of the L2β loop of FSH in free form (red wire) and FSHRED-bound form (brown wire). FSH α-subunit is shown as green ribbons, and the rest of the β-subunit is shown as blue ribbons. Sulfated FSHR Y335 is shown as balls as a reference. The orientation of FSH in A and B is related by a rotation of 60°. Residues around A43β in the L2β loop of FSH swing ~10 Å from the free form to the FSHRED-bound form to form the sulfate-binding pocket. (B, Inset) Newly formed FSH pocket is structurally supported by the hydrophobic interactions between the left wall of the pocket and the FSHR residues around V221 and I222. (C and D) Surface representations show the rearrangement of the FSH L2β loop on receptor binding. FSH α- and β-subunits are colored green and blue, respectively.

 

Figure 05
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Fig. 5. Two-step FSH/FSHR recognition and functional validations of the sTyr site and the exosite. (A) Schematic diagram of the two-step recognition. The GPHR’s extracellular LRRs, in a putative orientation relative to the seven-transmembrane (7TM) domain, are shown as magenta blocks with the flexible loop as a hairpin, and the 7TM domain is shown as cylinders with the inactivated state colored gray and the activated state colored green. The hormone-binding subdomain is labeled as HBSD, and signal specificity subdomain is labeled as SSSD. Sulfated Y335 is shown as yellow balls, residue S271 is shown as green stars, and disulfide bonds are shown as brown sticks. Heterotrimeric Gs or β-arrestin protein is shown as a green ellipsoid. GPH heterodimer is represented in purple, whereas carbohydrates at N52α are shown as a yellow Y-shaped stick. Shown here is only one of the possible orientations of the extracellular domain relative to the 7TM domain. (B) Importance of electrostatic potential of FSH in lifting the Y335-harboring FSHR hairpin for receptor activation. Negative potential is colored red, and positive potential is colored blue. FSHRED is represented as magenta ribbons, with the side chain of sulfated Y335 shown as sticks. (C) Validation of the roles of the sTyr site and the exosite in FSHR activation by FSH mutagenesis. (Left) Relative amount of β-arrestin recruited for binding to the activated FSHR inside the CHO cell on stimulation by FSH or its mutants. The amount of recruited β-arrestin is normalized to 100% for the maximum response of FSH. Data represent experiments performed with quadruplicate samples. (Right) Relative amount of estradiol production inside primary granulosa cells from immature rats on stimulation by FSH or its mutants. The amount of estradiol production is normalized to 100% for the maximum response of FSH. Data represent experiments performed with triplicate samples.

 


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