FSH and TSH binding to their respective receptors: similarities, differences and implication for glycoprotein hormone specificity R Nu ´n ˜ ez Miguel 1,2 , J Sanders 1 , D Y Chirgadze 2 , T L Blundell 2 , J Furmaniak 1 and B Rees Smith 1 1 FIRS Laboratories, RSR Ltd, Parc Ty Glas, Llanishen, Cardiff CF14 5DU, UK 2 Department of Biochemistry, University of Cambridge, 80 Tennis Court Road, Cambridge CB2 1GA, UK (Correspondence should be addressed to B Rees Smith; Email: firs@rsrltd.eclipse.co.uk) Abstract The crystal structures of the leucine-rich repeat domain (LRD) of the FSH receptor (FSHR) in complex with FSH and the TSH receptor (TSHR) LRD in complex with the thyroid-stimulating autoantibody (M22) provide opportunities to assess the molecular basis of the specificity of glycoprotein hormone–receptor binding. A comparative model of the TSH–TSHR complex was built using the two solved crystal structures and verified using studies on receptor affinity and activation. Analysis of the FSH–FSHR and TSH–TSHR complexes allowed identification of receptor residues that may be important in hormone-binding specificity. These residues are in leucine-rich repeats at the two ends of the FSHR and the TSHR LRD structures but not in their central repeats. Interactions in the interfaces are consistent with a higher FSH-binding affinity for the FSHR compared with the binding affinity of TSH for the TSHR. The higher binding affinity of porcine (p)TSH and bovine (b)TSH for human (h)TSHR compared with hTSH appears not to be dependent on interactions with the TSHR LRD as none of the residues that differ among hTSH, pTSH or bTSH interact with the LRD. This suggests that TSHs are likely to interact with other parts of the receptors in addition to the LRD with these non-LRD interactions being responsible for affinity differences. Analysis of interactions in the FSH–FSHR and TSH–TSHR complexes suggests that the a-chains of both hormones tend to be involved in the receptor activation process while the b-chains are more involved in defining binding specificity. Journal of Molecular Endocrinology (2008) 41, 145–164 Introduction In mammals, the glycoprotein hormone (GPH) family comprises three gonadotrophins: lutrophin (lutein- ising hormone, LH), follitrophin (follicle-stimulating hormone, FSH) and choriogonadotrophin (CG) and one thyrotrophin (thyroid-stimulating hormone, TSH). There are, however, only three high affinity GPH receptors as LH and CG bind the LH receptor (LHR). GPHs control several major physiological processes, with TSH regulating metabolism through the production of thyroid hormones and the gonado- trophins regulating reproductive functions (Pierce & Parsons 1981, Combarnous 1992). The GPHs are heterodimers composed of non- covalently linked a- and b-subunits. The N-linked oligosaccharides of these hormones are necessary for proper folding, assembly, secretion, metabolic clearance and biological activity (Baenziger & Green 1988). Whereas b-subunits are hormone specific, a-subunits are identical within a species, although there may be differences in the oligosaccharides. The structure of each subunit comprises a cystine-knot motif with five disulphide bonds in the a-subunit and six in the b-subunit (McDonald & Hendrickson 1993). The core of the cystine-knot motif involves three disulphide bridges arranged so that two disulphides link adjacent antiparallel strands of the peptide chain and form a ring penetrated by the third disulphide. On one side of the knot, there is a loop of double-stranded b-sheet-like structure; on the other, there are two hairpin loops lying in almost parallel planes (Wu et al. 1994, Fox et al. 2001). The receptors of the GPHs belong to the rhodopsin- like class of G protein-coupled receptors (GPCRs) and are w45% identical at the amino acid level (Dias 1992). In contrast to other receptors in their class, GPH receptors are characterised by a large extracellular region consisting of an N-terminal tail, a leucine-rich repeat domain (LRD) and a cystine-rich domain (CD). The structure is completed with a transmembrane domain (TMD) consisting of seven membrane spanning helices and a C-terminal tail (Szkudlinski et al. 2002, Farid & Szkudlinski 2004, Nu ´n ˜ez Miguel et al. 2004). In both the TSH receptor (TSHR) and the FSH receptor (FSHR), the N-terminal tail contains four half-cystines (i.e. one of the two disulphide-bonded cysteines) and is 145 Journal of Molecular Endocrinology (2008) 41, 145–164 DOI: 10.1677/JME-08-0040 0952–5041/08/041–145 q 2008 Society for Endocrinology Printed in Great Britain Online version via http://www.endocrinology-journals.org Downloaded from Bioscientifica.com at 06/17/2023 11:41:20AM via free access