Follicle-stimulating Hormone Interacts with Exoloop 3 of the Receptor

The human follicle-stimulating hormone (FSH) receptor consists of two distinct domains of ∼330 amino acids, the N-terminal extracellular exodomain and membrane-associated endodomain including three exoloops and seven transmembrane helices. The exodomain binds the hormone with high affinity, and the resulting hormone/exodomain complex modulates the endodomain where receptor activation occurs. It has been an enigma whether the hormone interacts with the endodomain. In a step to address the question, exoloop 3 of580KVPLITVSKAK590was examined by Ala scan, multiple substitution, assays for hormone binding, cAMP and inositol phosphate (IP) induction, and photoaffinity labeling. We present the evidence for the interaction of FSH and exoloop 3. A peptide mimic of exoloop 3 specifically and saturably photoaffinity-labels FSH α but not FSH β. This is in contrast to photoaffinity labeling of FSH β by the peptide mimic of the N-terminal region of the receptor. Leu583and Ile584are crucial for the interaction of FSH and exoloop 3. Substitutions of these two residues enhanced the hormone binding affinity. This is due to the loss of the original side chains but not the introduction of new side chains. The Leu583and Ile584side chains appear to project in opposite directions. Ile584appears to be so specific and to require flexibility and stereo specificity so that no other amino acids can fit into its place. Leu583is less specific. The improvement in hormone binding by substitutions was offset by the severe impairment of signal generation of cAMP and/or inositol phosphate. For example, the Phe or Tyr substitution of Leu583improved the hormone binding and cAMP induction but impaired IP induction. On the other hand, the substitutions for Ile584and Lys590abolished the cAMP and IP induction. Our results open a logical question whether Leu583, Ile584, and Lys590interact with the exodomain and/or the hormone. The answers will provide new insights into the mechanisms of hormone binding and signal generation.