Synthesis of multi-subunit domain gonadotropin complexes: A model for α/β heterodimer formation

The human glycoprotein hormones chorionic gonadotropin (CG), thyrotropin (TSH), lutropin (LH), and follitropin (FSH) are heterodimers, composed of a common α subunit assembled to a hormone-specific β subunit. The subunits combine noncovalently early in the secretory pathway and exist as heterodimers, but not as multimers. Little information is available regarding the steps associated with the assembly reaction. It is unclear if the initial αβ engagement results either in the formation of only mature heterodimer or if the nascent complex is reversible and can undergo an exchange of subunits or combine transiently with an additional subunit. This is relevant for the case of LH and FSH, because both are synthesized in the same cell (i.e., pituitary gonadotrophs) and several of the α subunit sequences required for association with either the LHβ or FSHβ subunits are different. Such features could favor the generation of short-lived, multi-subunit forms prior to completion of assembly. Previously, we showed that the CGβ or FSHβ subunit genes can be genetically fused to the α gene to produce biologically active single chains, CGβα and Fβα, respectively. Studies using monoclonal antibodies sensitive to the conformation of the hCG subunits suggested that in contrast to the highly compact heterodimer, the interactions between the β and α domains in the single chain are in a more relaxed configuration. That the tethered domains do not interact tightly predicts that they could combine with an additional subunit to firm triple domain complexes. We tested this point by cotransfecting CHO cells with the genes encoding Fβα and the CGβ subunit or the CGβα and FSHβ monomer. The CGβ subunit combined noncovalently with Fβα to form a Fβα/CGβ complex. Ternary complex formation was not restricted to a specific set of single chain/monomeric subunit, because a CGβα/FSHβ complex was also detected implying that triple domain intermediates could be transiently generated along the secretory pathway. Monoclonal antibodies specific for the CG heterodimer recognized the Fβα/CGβ complex, which suggests that the epitopes unique for dimeric CG were established. In addition, media containing Fβα/CGβ displayed high-affinity binding to both CG and FSH receptors. The presence of CG activity is presumptive for the existence of a functional Fβα/CGβ complex, because neither Fβα nor the uncombined CGβ subunit binds to CG receptor. These data show that the α subunit of the tether, although covalently linked to the FSHβ domain, can functionally interact with a different β subunit implying that the contacts in the nascent αβ dimer are reversible. The formation of a functional single chain/subunit complex was not restricted to the FSH single chain/CGβ subunit since CG single chain interacts with the monomeric FSHβ subunit and exhibits FSH activity. The presence of the triple domain configuration does not abolish bioactivity, suggesting that although the gonadotropins are heterodimers, the cognate receptor is capable of recognizing a larger ligand composed of three subunit domains.