Purification and structural analysis of a soluble human chorionogonadotropin hormone-receptor complex.
Receptors for the luteotropin/human chorionogonadotropin hormone belong to the G-protein-coupled receptor family by their membrane-anchoring domains. They also possess a large extracellular domain (ECD) responsible for most of the hormone-receptor interactions. Structure-function studies identified several contacts between hormone and receptor ECD, but the precise topology of the complex is still unknown because of the lack of suitable heterologous expression means. Receptor ECDs exhibit leucine repeats and have been modelized on the basis of the three-dimensional structure of the porcine ribonuclease inhibitor, the first structurally known leucine-rich repeats protein. Here we report overexpression (up to 20 mg per liter) and purification to homogeneity of a soluble human chorionogonadotropin-ECD receptor complex secreted by stably cotransfected Chinese hamster ovary cells. Biochemical analysis and surface plasmon resonance data were in favor of a unique dimer with a 1:1 ligand-receptor stoichiometry. Immunopurified complex was submitted to circular dichroism characterization; CD spectra deconvolution indicated more than 25% alpha helices contributed by the receptor, in agreement with the porcine ribonuclease inhibitor-based modelization.
Remy
J J
JJ
Biologie Cellulaire et Moleculaire, Institut National de la Recherche Agronomique, 78352 Jouy-en-Josas, France. remy@biotec.jouy.inra.fr
Nespoulous
C
C
Grosclaude
J
J
Grébert
D
D
Couture
L
L
Pajot
E
E
Salesse
R
R
D001483:Base Sequence; D006063:Chorionic Gonadotropin / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003432:Cross-Linking Reagents; D017931:DNA Primers; D006801:Humans; D011487:Protein Conformation; D011974:Receptors, LH / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
D006063:Chorionic Gonadotropin; D003432:Cross-Linking Reagents; D017931:DNA Primers; D011974:Receptors, LH