GLUTAMATE-301 OF THE MOUSE GONADOTROPIN-RELEASING-HORMONE RECEPTOR CONFERS SPECIFICITY FOR ARGININE-8 OF MAMMALIAN GONADOTROPIN-RELEASING-HORMONE

The Arg residue at position 8 of mammalian GnRH is necessary for high affinity binding to mammalian GnRH receptors. This requirement has bee n postulated to derive from an electrostatic interaction of Arg(8) wit h a negatively charged receptor residue. In order to identify such a r esidue, 8 conserved acidic residues of the mouse GnRH receptor were mu tated to isosteric Asn or Gin. Mutant receptors were tested for decrea sed preference for Arg(8)-containing Ligands by ligand binding and ino sitol phosphate production. One of the mutants, in which the Glu(301) residue was mutated to Gin, exhibited a 56-fold decrease in apparent a ffinity for mammalian GnRH. The mutant receptor also exhibited decreas ed affinity for [Lys(8)]GnRH, but its affinity for [Gln(8)]GnRH was un changed compared with the wild type receptor. The apparent affinity of the mutant receptor for the acidic analogue, [Glu(8)]GnRH, was increa sed more than 10-fold. The mutant receptor did not, therefore, disting uish mammalian GnRH from analogues with amino acid substitutions at po sition 8 as effectively as the wild type receptor. This loss of discri mination was specific for the residue at position 8, because the mutan t receptor did distinguish mammalian GnRH from analogues with favorabl e substitutions at positions 5, 6, and 7. These findings show that Glu (301) Of the GnRH receptor plays a role in receptor recognition of Arg (8) in the ligand and are consistent with an electrostatic interaction between these 2 residues.