The position of the alpha and beta subunits in a single chain variant of human chorionic gonadotropin affects the heterodimeric interaction of the subunits and receptor-binding epitopes

The glycoprotein hormone family represents a class of heterodimers, which i nclude the placental hormone human chorionic gonadotropin (CG) and the ante rior pituitary hormones follitropin, lutropin, and thyrotropin. They are co mposed of common alpha subunit and a hormone-specific beta subunit. Based o n the CG crystal structure, it was suggested that the quaternary subunit in teractions are crucial for biological activity. However, recent observation s using single chain glycoprotein hormone analogs, where the beta and alpha subunits are linked (NH2-CG beta-alpha; CG beta alpha orientation), implie d that the heterodimeric-like quaternary configuration is not a prerequisit e for receptor binding/signal transduction. To study the heterodimeric alig nment of the two subunit domains in a single chain and its role in the intr acellular behavior and biological action of the hormone, a single chain CG variant was constructed in which the carboxyl terminus of a was fused to th e CG beta amino terminus (NH2-alpha -CG beta; alpha CG beta orientation). T he secretion rate of alpha CG beta from transfected Chinese hamster ovary c ells was less than that seen for CG beta alpha. The alpha CG beta tether wa s not recognized by dimer-specific monoclonal antibodies and did not bind t o lutropin/CG receptor. To define if one or both subunit domains were modif ied in alpha CG beta, it was co-transfected with a monomeric a or CGP gene. In each case, alpha CG beta/alpha and alpha CG beta /CG beta complexes wer e formed indicating that CG dimer-specific epitopes were established. The a lpha CG beta/alpha complex bound to receptor indicating that the beta domai n in the alpha CG/beta tether was still functional. In contrast, no signifi cant receptor binding of alpha CG beta /CG beta was observed indicating a m ajor perturbation in the alpha domain. These results suggest that although dimeric-like determinants are present in both alpha CG beta/alpha and alpha CG beta /CG beta complexes, the receptor binding determinants in the alpha domain of the tether are absent. These results show that generating hetero dimeric determinants do not necessarily result in a bioactive molecule. Our data also indicate that the determinants for biological activity are disti nct from those associated with intracellular behavior.