A chicken gonadotropin-releasing hormone receptor that confers agonist activity to mammalian antagonists - Identification of D-Lys(6) in the ligand and extracellular loop two of the receptor as determinants

Mammalian receptors for gonadotropin-releasing hormone (GnRH) have over 85% sequence homology and similar ligand selectivity. Biological studies indic ated that the chicken GnRH receptor has a distinct pharmacology, and certai n antagonists of mammalian GnRH receptors function as agonists. To explore the structural determinants of this, we have cloned a chicken pituitary GnR H receptor and demonstrated that it has marked differences in primary amino acid sequence (59% homology) and in its interactions with GnRH analogs. Th e chicken GnRH receptor had high affinity for mammalian GnRH (K-i 4.1 +/- 1 .2 nM), similar to the human receptor (K-i 4.8 +/- 1.2 nM). But, in contras t to the human receptor, it also had high affinity for chicken GnRH ([Gln(8 )]GnRH) and GnRH II ([His(5),Trp(7),Tyr(8)]GnRH) (K-i 5.3 +/- 0.5 and 0.6 /- 0.01 nM). Three mammalian receptor antagonists were also pure antagonist s in the chicken GnRH receptor. Another three, characterized by D-Lys(6) or D-isopropyl-Lys(6) moieties, functioned as pure antagonists in the human r eceptor but were full or partial agonists in the chicken receptor. This sug gests that the Lys side chain interacts with functional groups of the chick en GnRH receptor to stabilize it in the active conformation and that these groups are not available in the activated human GnRH receptor. Substitution of the human receptor extracellular loop two with the chicken extracellula r loop two identified this domain as capable of conferring agonist activity to mammalian antagonists. Although functioning of antagonists as agonists has been shown to be species-dependent for several GPCRs, the dependence of this on an extracellular domain has not been described.