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J. Biol. Chem., Vol. 276, Issue 32, 29871-29879, August 10, 2001

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The Position of the  and  Subunits in a Single Chain Variant of Human Chorionic Gonadotropin Affects the Heterodimeric Interaction of the Subunits and Receptor-binding Epitopes^*

David Ben-Menahem, Albina Jablonka-Shariff^§, Ricia K. Hyde, Mary R. Pixley, Shivaji Srivastava, Peter Berger^¶, and Irving Boime

From the Department of Molecular Biology and Pharmacology, Washington University School of Medicine, St. Louis, Missouri 63110 and the ^¶ Institute for Biomedical Aging Research, Austrian Academy of Science, A-6020 Innsbruck, Austria

The glycoprotein hormone family represents a class of heterodimers, which include the placental hormone human chorionic gonadotropin (CG) and the anterior pituitary hormones follitropin, lutropin, and thyrotropin. They are composed of common  subunit and a hormone-specific  subunit. Based on the CG crystal structure, it was suggested that the quaternary subunit interactions are crucial for biological activity. However, recent observations using single chain glycoprotein hormone analogs, where the  and  subunits are linked (NH₂-CG-; CG orientation), implied that the heterodimeric-like quaternary configuration is not a prerequisite for receptor binding/signal transduction. To study the heterodimeric alignment of the two subunit domains in a single chain and its role in the intracellular behavior and biological action of the hormone, a single chain CG variant was constructed in which the carboxyl terminus of  was fused to the CG amino terminus (NH₂--CG; CG orientation). The secretion rate of CG from transfected Chinese hamster ovary cells was less than that seen for CG. The CG tether was not recognized by dimer-specific monoclonal antibodies and did not bind to lutropin/CG receptor. To define if one or both subunit domains were modified in CG, it was co-transfected with a monomeric  or CG gene. In each case, CG/ and CG/CG complexes were formed indicating that CG dimer-specific epitopes were established. The CG/ complex bound to receptor indicating that the  domain in the CG tether was still functional. In contrast, no significant receptor binding of CG/CG was observed indicating a major perturbation in the  domain. These results suggest that although dimeric-like determinants are present in both CG/ and CG/CG complexes, the receptor binding determinants in the  domain of the tether are absent. These results show that generating heterodimeric determinants do not necessarily result in a bioactive molecule. Our data also indicate that the determinants for biological activity are distinct from those associated with intracellular behavior.

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