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% ␣ helices contributed by the receptor, in agreement with the porcine ribonuclease inhibitor-based modelization.The LH/hCG 1 receptor, like the other members of the glycoprotein hormone receptor family (follicle-stimulating hormone and thyreo-stimulating hormone receptors), is composed of two domains of almost equal size. The N-terminal part, thought to form an extracellular domain (ECD), has been shown to bind ligands with high affinity (1-3) while being structurally related to the leucine-rich repeats (LRR) superfamily of proteins. The C-terminal moiety, including the seven transmembrane helices and their connecting loops, interacts with hormones and ECDs on one side, whereas it contacts the transducing G-proteins intracellularly. Dimeric glycoprotein hormones bound by these receptors, LH/hCG, follicle-stimulating hormone, and thyreostimulating hormone, share a common ␣ subunit non-covalently bound to an hormone-specific ␤ subunit; the 3D structure of hCG has been resolved by x-ray crystallography (4, 5).A number of structure-function studies (6 -9) identified several contacts between hormones and receptors. For receptor ECDs, models have been constructed based on the crystal structure of porcine ribonuclease inhibitor (pRI), the first structurally defined member of the LRR family (10, 11); according to pRI homology modeling, receptor ECDs would form a partial horseshoe-like structure generated by a succession of ␣ helices and ␤ sheets (12, 13). Although excluding the two N-and C-terminal cystein-rich clusters, the model allows hormone docking inside the horseshoe inner circumference formed by the parallel ␤ sheets (14).However, more than ten years after cloning of their cDNAs, little information on the precise topology of the glycoprotein hormone receptors is available. Expression have been reported in several recombinant systems, as membrane full-length, free ECDs or as membrane-anchored...

The LH/hCG receptor is a G protein-coupled receptor with an N-terminal extracellular domain involved in hormone-receptor interaction. The recombinant porcine receptor, stably expressed in Chinese hamster ovary (CHO) cells, has the same characteristics (Kd and cAMP production) as in Leydig cells. Six synthetic peptides derived from the receptor ectodomain and two polyclonal anti-peptide sera were tested in the homologous system porcine LH and porcine LH receptor. Their ability to inhibit hormone binding and signal transduction on CHO cells expressing the recombinant receptor was evaluated. Peptides 25-40 and 107-121 exhibited a high transduction inhibition as compared with hormone binding, peptides 21-36, 102-111, and 102-121 inhibited hormone binding more efficiently than signal transduction, and peptide 7-24 exhibited inhibition of both hormone binding and hormone-induced cAMP production. Immunoglobulins against peptides 21-36 and 102-111 inhibited both hormone binding and receptor activation suggesting that these sequences are located on the receptor surface. The data suggest that multiple, discontinuous regions of the extracellular domain of porcine LH receptor are involved in hormone binding and signal transduction. Two minimum critical sequences, 21-24 and 102-107, are involved in hormone binding and vicinal segments may be implicated in signal transduction.

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