Pro 7.33(303) of the human GnRH receptor regulates selective binding of mammalian GnRH

Abstract

Mammalian gonadotropin releasing hormone (GnRH) receptors have a conserved acidic residue (Glu 7.32(301) or Asp 7.32(302)) in extracellular loop (ECL) three that confers selectivity for mammalian GnRH, which has Arg 8. Comparison of mammalian and non-mammalian GnRH receptors suggested that the acidic residue is not the only determinant of ligand selectivity in mammalian receptors. The acidic residue is followed by a conserved Pro 7.33 in mammalian GnRH receptors, but not non-mammalian receptors. Unique structural constraints imposed by Pro residues suggested that Pro 7.33 determines selective binding of Arg 8-containing GnRH, by stabilising the conformation of the third extracellular loop of the receptor. Substituting Pro 7.33(303) or introducing Pro to position 7.31 decreased affinity for GnRH, but not analogs lacking Arg 8. Substituting Pro 7.33(303) changed the predicted α-helix content of the loop–helix interface. These results show that Pro 7.33(303) of the human GnRH receptor is required for selective high affinity binding of mammalian GnRH and supports the hypothesis that Pro 7.33(303) stabilises a loop conformation that is necessary for selective ligand binding.