Assembly and structural characterization of an authentic complex between human follicle stimulating hormone and a hormone-binding ectodomain of its receptor

Abstract

Follicle stimulating hormone (FSH) is secreted from the pituitary gland to regulate reproduction in vertebrates. FSH signals through a G-protein coupled receptor (FSHR) on the target cell surface. We describe here the strategy to produce a soluble FSH-FSHR complex that involves the co-secretion of a truncated FSHR ectodomain (FSHR(HB)) and a covalently linked FSHalphabeta heterodimer from baculovirus-infected insect cells. FSH binds to FSHR(HB) with a high affinity comparable to that for the full-length receptor. The crystal structure of the FSH-FSHR(HB) complex provides explanations for the high affinity and specificity of FSH interaction with FSHR, and it shows an unexpected dimerization of these complexes. Here we also compare the crystal structure with theoretical models of the FSH-FSHR-binding mode. We conclude that the FSH-FSHR(HB) structure gives an authentic representation of FSH binding to intact FSHR.

Fig. 1

(A) Schematic diagram of the baculovirus expression constructs for human FSHR_HB and the covalently linked human FSHα and FSHβ subunits. (B) Western blot of the culture medium of insect cells that were infected with the recombinant virus for FSHR_HB and single-chain FSH. Culture samples were concentrated 20-fold and run on a 4-15% SDS gel in the presence of 100 mM dithiothreitol (DTT). The blot was developed using the M2 anti-FLAG antibody as the primary antibody.

Fig. 2

(A) Gel filtration column chromatography of the FSH-FSHR_HB complex. Samples of the indicated peak fraction were assayed on (B) 8–25% SDS gel and (C) 8–25% native gel. Samples of purified FSH-FSHR_HB (5 μg) and free hormone ligands were also analyzed by 8–25% native gel electrophoresis for possible additional interactions upon mixture: (D) for 5 μg of free CG, and (E) for 7.5 μg of free FSH.

Fig. 3

Fully glycosylated (control) and partially deglycosylated FSH-FSHR_HB complex were assayed on (A) 8–25% SDS gel and (B) 8–25% native gel. (C) A crystal of fully glycosylated FSH-FSHR_HB complex grown at 4°C from 16% tert-butanol and 0.1 M sodium citrate, pH 6.0. (D) Thin plate crystals of partially deglycosylated FSH-FSHR_HB complex grown at 20°C from 10% PEG 3350 and 0.1 M Li₂SO₄.

Fig. 4

Ribbon diagram of the crystal structure of human FSH bound to FSHR_HB. FSH α- and β-chains are in green and cyan, respectively. FSHR_HB is in red. The observed N-linked carbohydrates at Asn52, Asn78 of FSHα , Asn7 and Asn24 of FSHβ , and Asn191 of FSHR_HB are in yellow. Disulfide bonds are in black. Adapted with permission from Nature (Fan and Hendrickson, 2005a).

Fig. 5

Dimeric associations. (A–C) Ribbon diagrams of quasi-symmetric crystallographic dimers of FSH-FSHR_HB complexes, colored as in Fig. 4. The view is parallel to the symmetry axis, as if looking at the membrane. (D–E) Schematic illustrations of dimeric receptors on the cell membrane. Projected outlines from the complexes shown above and from rhodopsin are shown for the hormone (cyan), hormone-binding domain (orange), linker and 7TM transmembrane domain (black dotted trace). The view is perpendicular to figures directly above, parallel with the membrane plane. (A) Principal crystallographic dimer. (B) Principal dimer with outline of an offset 7TM dimer disposed for interaction with one of the bound FSH molecules. (C) Alternative crystallographic dimer. (D) Symmetric principal dimer with both 7TM domains engaged. (E) Asymmetric principal dimer with only one 7TM domain engaged. Note that dimer axes for the two components are not aligned. (F) Symmetric alternative dimer with both 7TM domains engaged.

Fig. 6

Ribbon diagrams comparing the predicted hCG-LHR_HB interaction model (hCG in green; LHR_HB model in gray) with the crystal structure of human FSH-FSHR_HB complex (FSH in blue; FSHR_HB in red). The structure of hCG in the predicted model was superimposed onto the structure of receptor-bound FSH. (A) Top view. (B) Side view.