Analysis of glycoprotein hormone receptor extracellular domain interactions using a solid-phase capture assay.
The receptors for the glycoprotein hormones are unique in having a large extracellular domain that is responsible for mediating ligand binding. We describe the characterization, validation, and application of a solid-phase radioligand binding assay that can be used to assess the interaction of peptides and small molecules at the extracellular domain (ECD) of the follicle-stimulating hormone receptor (FSHR). The assay utilizes a C-terminal tag on the FSHR-ECD, which is used to capture the ECD and position it in a sterically favorable orientation on a solid-phase platform. Competition experiments with the cognate ligand, FSH, indicated that the interaction at the FSHR-ECD using the solid-phase assay was comparable to the full-length receptor assayed using a standard filtration assay. The utility of the assay was evaluated by competing several peptides and a small molecule for both the full-length FSHR and the FSHR-ECD. The solid-phase capture format allowed for the establishment of an assay to specifically evaluate compounds that interact at the ECD or require the full-length receptor, thereby facilitating structure-activity studies. This assay format should be applicable to the other receptors of this family.
Stevis
Panayiotis E
PE
Contraception and Reproductive Endocrinology Group, Women's Health and Bone, Wyeth Research, 500 Arcola Road, Collegeville, PA 19426, USA. stevisp@wyeth.com
Deecher
Darlene C
DC
D001667:Binding, Competitive; D004353:Drug Evaluation, Preclinical / Q000379:methods*; D043373:Follicle Stimulating Hormone, Human / Q000737:chemistry*; D006023:Glycoproteins / Q000737:chemistry; D006801:Humans; D008024:Ligands; D010455:Peptides / Q000737:chemistry; D017434:Protein Structure, Tertiary; D011869:Radioligand Assay; D011962:Receptors, FSH / Q000737:chemistry*
D043373:Follicle Stimulating Hormone, Human; D006023:Glycoproteins; D008024:Ligands; D010455:Peptides; D011962:Receptors, FSH