A Chicken Gonadotropin-releasing Hormone Receptor That Confers Agonist Activity to Mammalian Antagonists

Abstract

Mammalian receptors for gonadotropin-releasing hormone (GnRH) have over 85% sequence homology and similar ligand selectivity. Biological studies indicated that the chicken GnRH receptor has a distinct pharmacology, and certain antagonists of mammalian GnRH receptors function as agonists. To explore the structural determinants of this, we have cloned a chicken pituitary GnRH receptor and demonstrated that it has marked differences in primary amino acid sequence (59% homology) and in its interactions with GnRH analogs. The 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 contrast to the human receptor, it also had high affinity for chicken GnRH or D-isopropyl-Lys 6 moieties, functioned as pure antagonists in the human receptor but were full or partial agonists in the chicken receptor. This suggests that the Lys side chain interacts with functional groups of the chicken 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 extracellular 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.

Evolution of Constrained Gonadotropin-releasing Hormone Ligand Conformation and Receptor Selectivity

“…The overall cross-section of mammalian GnRH is larger due to the Arg 8 . Interestingly, this bulky side chain of Arg 8 is rather flexible, which supports data obtained from both the mouse (15) and the human (16) receptors that demonstrate Arg 8 is integral to binding via an acidic residue in extracellular loop 3 (15,16). The jump in cross-section in the earlier part of these dynamics measurements is expected, as the temperature of the low energy starting conformers is brought to thermal equilibration.…”

“…COS-7 and human embryonic kidney 293 cells were cultured as previously described for COS-1 cells (12,13) and transiently transfected with the human (14), chicken (15), and catfish (16) GnRH receptors as well as the recently cloned Ciona receptors (17) 3 using electroporation. These are Ciona A and Ciona B GnRH receptors previously referred to as CiGnRHR1 and Ci-GnRHR2.…”

Identification of new gonadotrophin-releasing hormone partial agonists

“…at the exoloop 2 or intraloop 2, which share 76 and 62% amino acid identity with the corresponding domains in the rGnRHR); in this setting, the particular structure of the rGnRHR antagonist might ultimately determine a preference for binding to the active receptor conformation of the hGnRHR and to both the inactive and active conformations of the rat counterpart, as would be expected for antagonist analogues. In fact, in a number of G protein-coupled receptors, including the GnRHR, mutations or species-dependent structural differences in either transmembrane domains (Strader et al 1989, Claude et al 1996, Noda et al 1996, Groblewski et al 1997, Han et al 1997, Spivak et al 1997, intraloops (Morin et al 1998, Wurch et al 1999 or exoloops (Sun et al 2001) may be limiting, to a variable extent, the level of maximal stimulation. It should be emphasized, however, that the effects of the compounds tested may be cell-specific and that depending on the relative levels of receptor, G proteins and other downstream effectors present in a particular tissue in vivo (i.e.…”

Evidence for Distinct Cation and Calcimimetic Compound (NPS 568) Recognition Domains in the Transmembrane Regions of the Human Ca2+ Receptor

“…Moreover, residues in the second extracellular loop of several GPCRs including the CCK5, adenosine, and gonadotropin receptors, have been shown to be important for high affinity agonist binding and activation of the receptors (25,26). Here we found that the mutant T903-RhocE/D5A receptor with Exo-loop2 Asp and Glu residues changed to Ala did not respond to Gd 3ϩ or poly-L-Arg in the absence or presence of NPS 568, and Ca 2ϩ elicited a significantly lower maximal IP response compared with the T903-Rhoc receptor.…”