High Affinity Hormone Binding to the Extracellular N-terminal Exodomain of the Follicle-stimulating Hormone Receptor Is Critically Modulated by Exoloop 3

Abstract

The human follicle-stimulating hormone receptor (FSH-R) consists of two distinct domains of >330 amino acids, the N-terminal extracellular exodomain and membrane-associated endodomain. The exodomain alone binds hormone with high affinity, whereas the endodomain is the site of receptor activation. Coordination of these two domains is essential for successful hormone action but little is known about their functional and structural relationship. In this communication, we report that exoloop 3 of FSH-R constrains follicle-stimulating hormone binding to the exodomain. When the FSH-R exodomain was prepared by truncating its endodomain, the hormone binding affinity of the exodomain was slightly improved, compared with the wild type receptor. The binding affinity was further improved by >3-fold when the exodomain was attached to the membrane-associated domain of CD8. These results suggest that the FSH-R endodomain attenuates hormone binding at the exodomain. As a first step to test this hypothesis, the 11 amino acids except Ala 589 of exoloop 3 were individually substituted with Ala. Ala substitution for Leu 583 or Ile 584 improved the hormone binding affinity by 4 -6-fold while totally abolishing cAMP induction, indicating an inverse relationship. The Ala substitution for Lys 580 or Pro 582 had a similar trend but to a lesser extent. This significant improvement in the binding affinity suggests that the four residues at the N-terminal region of exoloop 3 interact with the exodomain and constrain the hormone binding in the wild type receptor. This effect is specific since substitutions for other than the 4 residues did not improve the hormone binding affinity. Computer modeling shows that the 4 residues can be positioned on one side of exoloop 3. This result and the apparent inverse relationship of hormone binding and cAMP induction suggest that these two essential functions may work against each other. Therefore, hormone binding might be compromised to preserve cAMP inducibility while maintaining a reasonably high, but below maximum, binding affinity. The FSH1 receptor and other glycoprotein hormone (LH/CG and TSH) receptors belong to a structurally unique subfamily of G protein-coupled receptors (1). Unlike other receptor subfamilies, they comprise two equal halves, an extracellular Nterminal half (exodomain) and a membrane-associated C-terminal half (endodomain) (2-5) as shown in Fig. 1. The exodomain is ϳ350 amino acids long, which alone is capable of high affinity hormone binding (6 -9) with hormone selectivity (10 -12) but without hormone action (8, 13). Receptor activation occurs in the endodomain (14), which is structurally equivalent to the entire molecule of many other G protein-coupled receptors (15). Existing evidence suggests that glycoprotein hormones initially bind to the exodomain, and then the resulting hormone-exodomain complex interacts with the endodomain (1). This secondary interaction is responsible for signal generation (1, 14). Despite the importance of this secondary contact and the relationship...

Follicle-stimulating Hormone Interacts with Exoloop 3 of the Receptor

“…In a step toward this goal, we examined exoloop 3 of FSHR for its involvement in the activation of the two effectors and interaction with FSH. It is the shortest of the three exoloops, consisting of 11 amino acids, and has been implicated in the cAMP signal generation (15,20,21). Our observations show, for the first time, the interaction of exoloop 3 with FSH, in particular the FSH ␣ subunit, the mode of this interaction, and its role in the AC and PLC␤ activation.…”

“…P582A produced a detectable level of IP 1 but not IP 2 and IP 3 . These results raise the question of whether the non-responding mutant receptors were expressed on the cell surface in this study, although they were in the previous study (15). Therefore, the cells stably transfected with the mutants were assayed for 125 I-FSH binding as well as FSH-dependent cAMP production.…”

“…The sequence is also conserved among the glycoprotein hormone receptor family except 587 SKA 589 near the C terminus. The previous Ala scan has demonstrated that exoloop 3 constrains the hormone binding at the exodomain and plays a crucial role in cAMP induction (15). However, little is known about the mechanism or its role in IP induction.…”

“…However, there is little information on the subject, particularly concerning the FSH receptor. Since the exodomain lacking the endodomain is capable of high affinity hormone binding (5)(6)(7)(8)15), the high affinity hormone binding appears to be independent of the endodomain. Contrary to this view, it has been reported that FSH and human chorionic gonadotropin binding to their cognate receptors is regulated by certain residues of exoloops 2 and 3 of the endodomain (15,16).…”

“…Since the exodomain lacking the endodomain is capable of high affinity hormone binding (5)(6)(7)(8)15), the high affinity hormone binding appears to be independent of the endodomain. Contrary to this view, it has been reported that FSH and human chorionic gonadotropin binding to their cognate receptors is regulated by certain residues of exoloops 2 and 3 of the endodomain (15,16). Furthermore, the hinge region of the exodomain interacts with exoloop 2 and modulates cAMP induction (17)(18)(19).…”