HIGH-AFFINITY HORMONE-BINDING TO THE EXTRACELLULAR N-TERMINAL EXODOMAIN OF THE FOLLICLE-STIMULATING-HORMONE RECEPTOR IS CRITICALLY MODULATED BY EXOLOOP-3

The human follicle-stimulating hormone receptor (FSH-R) consists of tw o distinct domains of >330 amino acids, the N-terminal extracellular e xodomain and membrane-associated endodomain. The exodomain alone binds hormone with high affinity, whereas the endodomain is the site of rec eptor activation. Coordination of these two domains is essential for s uccessful hormone action but little is known about their functional an d structural relationship, In this communication, we report that exolo op 3 of FSH-R constrains follicle-stimulating hormone binding to the e xodomain, When the FSH-R exodomain was prepared by truncating its endo domain, the hormone binding affinity of the exodomain was slightly imp roved, compared with the wild type receptor. The binding affinity was further improved by >3-fold when the exodomain was attached to the mem brane-associated domain of CD8, These results suggest that the FSH-R e ndodomain attenuates hormone binding at the exodomain, As a first step to test this hypothesis, the II amino acids except Ala(589) of exoloo p 3 were individually substituted with Ala, Ala substitution for Leu(5 83) or Ile(584) improved the hormone binding affinity by 4-6-fold whil e totally abolishing cAMP induction, indicating an inverse relationshi p. The Ala substitution for Lys(580) or Pro(582) had a similar trend b ut to a lesser extent. This significant improvement in the binding aff inity suggests that the four residues at the N-terminal region of exol oop 3 interact with the exodomain and constrain the hormone binding in the wild type receptor. This effect is specific since substitutions f or other than the 4 residues did not improve the hormone binding affin ity. Computer modeling shows that the 4 residues can be positioned on one side of exoloop 3, This result and the apparent inverse relationsh ip of hormone binding and cAMP induction suggest that these two essent ial 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.