TY - JOUR
T1 - 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
AU - Ben-Menahem, David
AU - Jablonka-Shariff, Albina
AU - Hyde, Ricia K.
AU - Pixley, Mary R.
AU - Srivastava, Shivaji
AU - Berger, Peter
AU - Boime, Irving
PY - 2001/8/10
Y1 - 2001/8/10
N2 - 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 (NH2-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 (NH2-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.
AB - 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 (NH2-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 (NH2-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.
UR - http://www.scopus.com/inward/record.url?scp=0035839426&partnerID=8YFLogxK
U2 - 10.1074/jbc.M104687200
DO - 10.1074/jbc.M104687200
M3 - Article
AN - SCOPUS:0035839426
SN - 0021-9258
VL - 276
SP - 29871
EP - 29879
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 32
ER -