Single-chain human chorionic gonadotropin analogs containing the determinant loop of the beta-subunit linked to the alpha-subunit

Abstract

Human chorionic gonadotropin (hCG) is a member of the family of glycoprotein hormones containing a common α-subunit and distinct β-subunits that confer hormonal specificity. hCG binds to the relatively large ectodomain of the human luteinizing hormone receptor (hLHR), a member of the G protein-coupled receptor superfamily, leading to increased intracellular production of cAMP. Using protein engineering, two miniaturized versions of hCGβ have been separately fused to the N-terminus of the α-subunit to give N-des[1-91]hCGβ-α-C and N-des[1-91,110-114]hCGβ-α-C, i.e. fusion proteins of the hCGβ determinant loop (extended to include the complete seat belt and carboxy-terminal peptide) coupled to the α-subunit. Bioactivity of these single-chain gonadotropin analogs was assessed in two systems following transient transfections into HEK 293 cells and subsequent cAMP measurements. In one, each mini-β-α cDNA was fused to that of hLHR and transfected into cells to create yoked miniaturized hCG-hLHR complexes; in the other, the cDNA of each single chain mini-β-α was co-transfected with that of hLHR in an effort to produce non-covalent miniaturized hCG-hLHR complexes. Using yoked hCG-hLHR and hLHR as positive and negative controls respectively, expression of each mini-hCG-hLHR complex was confirmed using antibody and ligand binding assays. The two mini-hCGs led to minimal activation of hLHR, suggesting weak intrinsic activity of the mini-β-α fusion proteins. These results suggest that potent agonists and antagonists will require the presence of other portions of hCGβ in addition to the determinant loop/seat belt.

The luteinizing hormone receptor: Insights into structure–function relationships and hormone-receptor-mediated changes in gene expression in ovarian cancer cells

“…Parallel studies using yoked hCG-FSHR and hCG-TSHR showed that each of the receptors could be activated by hCG, albeit to a small extent with FSHR (Schubert et al, 2003). This model system was also used with yoked α-LHR and yoked hCGβ-LHR to show that the individual subunits are incapable of activating LHR under conditions mimicking high concentrations (Narayan et al, 2002); likewise, yoked versions of minimized forms of hCGβ fused to α-LHR identified minimal responses at best (Schubert and Puett, 2003). This system has also been used to prepare and characterize soluble yoked hCG-LHR ECD C-terminal truncation complexes for circular dichroic spectroscopy in which it was demonstrated that secondary structure is lost as the hinge region is removed (Fralish et al, 2003).…”

Translational Fusion of Two β-Subunits of Human Chorionic Gonadotropin Results in Production of a Novel Antagonist of the Hormone

“…Several reports have shown that single-chain variants of glycoprotein hormones with C terminus of the ␤-subunit fused to the N terminus of the ␣-subunit display bioactivities comparable with those of the corresponding heterodimers (27)(28)(29)(30)(31). However, the relative position of the two subunit does not appear to be important for generation of a biologically active molecule (13).…”