Ligand Binding to Mammalian and Non-Mammalian GnRH Receptors Apparently Involves Similar Receptor Contact Sites in Different Spatial Arrangements

K Pfleger; T Ott; R Millar

OC27

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Endocrine Abstracts (2001) 2 OC27

SFE2001 Oral Communications Reproduction (8 abstracts)

Ligand Binding to Mammalian and Non-Mammalian GnRH Receptors Apparently Involves Similar Receptor Contact Sites in Different Spatial Arrangements

KDG Pfleger , TR Ott & RP Millar

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MRC Human Reproductive Sciences Unit, Edinburgh, UK.

Ligands have different binding affinities for mammalian and non-mammalian GnRH receptors. We have investigated whether this is due to differences in ligand contact sites and/or configuration of the receptors.

The majority of residues that are contact sites in the mammalian receptor are conserved across species and are located in the extracellular loops (ECL's). The different binding affinities of ligands to non-mammalian receptors may be due to different contact sites or to changes in the spatial arrangement of conserved contact sites. Chimeric receptors of human GnRH receptor (hGnRH-R) containing ECL's of catfish GnRH receptor (cfGnRH-R) in single, double and triple combinations were produced. Membrane binding assays using transfected COS-7 cells established binding affinities of GnRH, [D-Trp⁶]GnRH, GnRH-II, [D-Lys⁶]GnRH-II and Antagonist 135-18.

The hGnRH-R had higher binding affinities for GnRH, [D-Trp⁶]GnRH and Antagonist 135-18 than the cfGnRH-R (7, 10 and 47-fold respectively). The triple chimera had decreased binding affinities for these ligands of 4, 8 and 2-fold respectively relative to hGnRH-R. The hGnRH-R had lower binding affinities for GnRH-II and [D-Lys⁶]GnRH-II than the cfGnRH-R (104 and 27-fold respectively). The triple chimera had increased binding affinities for these ligands of 4 and 2-fold respectively relative to hGnRH-R.

The lower binding affinity of cfGnRH-R for GnRH and [D-Trp⁶]GnRH is predominately due to the catfish ECL's. The binding affinities of the chimeras for GnRH-II, [D-Lys⁶]GnRH-II and Antagonist 135-18 is not substantially different from that of wild type hGnRH-R. This may be due to:

1) The presence of different contact sites in the transmembrane helices

2) Non-mammalian receptors having a different conformation of the transmembrane helices compared with mammalian receptors. This gives rise to a different spatial arrangement of conserved ECL contact sites. This is particularly likely because GnRH-II is known to interact with the conserved Asp98 and Asn102 residues in ECL1 of hGnRH-R.