The Position of the α and β 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, Irving Boime

Research output: Contribution to journalArticle

16 Citations (Scopus)

Abstract

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 α subunit and a hormone-specific β 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 β and α subunits are linked (NH 2 -CGβ-α; CGβα 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 α was fused to the CGβ amino terminus (NH 2 -α -CGβ; αCGβ orientation). The secretion rate of αCGβ from transfected Chinese hamster ovary cells was less than that seen for CGβα. The αCGβ 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 αCGβ, it was co-transfected with a monomeric α or CGβ gene. In each case, αCGβ/α and αCGβ/CGβ complexes were formed indicating that CG dimer-specific epitopes were established. The αCGβ/α complex bound to receptor indicating that the β domain in the αCGβ tether was still functional. In contrast, no significant receptor binding of αCGβ/CGβ was observed indicating a major perturbation in the α domain. These results suggest that although dimeric-like determinants are present in both αCGβ /α and αCGβ/CGβ complexes, the receptor binding determinants in the α 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.

Original languageEnglish (US)
Pages (from-to)29871-29879
Number of pages9
JournalJournal of Biological Chemistry
Volume276
Issue number32
DOIs
StatePublished - Aug 10 2001

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Chorionic Gonadotropin
Epitopes
LH Receptors
Hormones
Luteinizing Hormone
Bioactivity
Dimers
Glycoproteins
Placental Hormones
Anterior Pituitary Hormones

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

The Position of the α and β Subunits in a Single Chain Variant of Human Chorionic Gonadotropin Affects the Heterodimeric Interaction of the Subunits and Receptor-binding Epitopes. / Ben-Menahem, David; Jablonka-Shariff, Albina; Hyde, Ricia K; Pixley, Mary R.; Srivastava, Shivaji; Berger, Peter; Boime, Irving.

In: Journal of Biological Chemistry, Vol. 276, No. 32, 10.08.2001, p. 29871-29879.

Research output: Contribution to journalArticle

Ben-Menahem, David ; Jablonka-Shariff, Albina ; Hyde, Ricia K ; Pixley, Mary R. ; Srivastava, Shivaji ; Berger, Peter ; Boime, Irving. / The Position of the α and β Subunits in a Single Chain Variant of Human Chorionic Gonadotropin Affects the Heterodimeric Interaction of the Subunits and Receptor-binding Epitopes. In: Journal of Biological Chemistry. 2001 ; Vol. 276, No. 32. pp. 29871-29879.
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abstract = "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 α subunit and a hormone-specific β 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 β and α subunits are linked (NH 2 -CGβ-α; CGβα 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 α was fused to the CGβ amino terminus (NH 2 -α -CGβ; αCGβ orientation). The secretion rate of αCGβ from transfected Chinese hamster ovary cells was less than that seen for CGβα. The αCGβ 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 αCGβ, it was co-transfected with a monomeric α or CGβ gene. In each case, αCGβ/α and αCGβ/CGβ complexes were formed indicating that CG dimer-specific epitopes were established. The αCGβ/α complex bound to receptor indicating that the β domain in the αCGβ tether was still functional. In contrast, no significant receptor binding of αCGβ/CGβ was observed indicating a major perturbation in the α domain. These results suggest that although dimeric-like determinants are present in both αCGβ /α and αCGβ/CGβ complexes, the receptor binding determinants in the α 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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