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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 alpha subunit and a hormone-specific beta 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 beta and alpha subunits are linked (NH(2)-CGbeta-alpha; CGbetaalpha 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 alpha was fused to the CGbeta amino terminus (NH(2)-alpha-CGbeta; alphaCGbeta orientation). The secretion rate of alphaCGbeta from transfected Chinese hamster ovary cells was less than that seen for CGbetaalpha. The alphaCGbeta 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 alphaCGbeta, it was co-transfected with a monomeric alpha or CGbeta gene. In each case, alphaCGbeta/alpha and alphaCGbeta/CGbeta complexes were formed indicating that CG dimer-specific epitopes were established. The alphaCGbeta/alpha complex bound to receptor indicating that the beta domain in the alphaCGbeta tether was still functional. In contrast, no significant receptor binding of alphaCGbeta/CGbeta was observed indicating a major perturbation in the alpha domain. These results suggest that although dimeric-like determinants are present in both alphaCGbeta/alpha and alphaCGbeta/CGbeta complexes, the receptor binding determinants in the alpha 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.
Highlights
The glycoprotein hormones lutropin (LH),1 follitropin (FSH), thyrotropin, and choriogonadotropin (CG) are heterodimers, which consist of a common ␣ subunit and a unique  subunit that confer the receptor specificity of the ligand
To examine if the two subunit domains in ␣CG could form intrachain heterodimeric-like interactions, media samples were electrophoresed under nonreduced conditions without heating, and the blots were probed with polyclonal ␣ antiserum and a panel of CG dimer-specific monoclonal antibodies (mAbs) (Fig. 3)
The data described above demonstrated that the presence of the carboxyl-terminal peptide (CTP) as a linker in ␣cCG⌬T partially restores activity of the ␣CG. We examined if this linker would reconstitute binding determinants in the tethered subunit domains and enhance the binding affinity when co-transfected with the monomeric ␣, CG, or CG⌬T subunits (Fig. 9). (As expected, ␣cCG⌬T/␣ was immunoreactive with dimer-specific mAbs.) The affinity of the ␣cCG⌬T/CG complex was the same as ␣cCG⌬T
Summary
The glycoprotein hormones lutropin (LH),1 follitropin (FSH), thyrotropin, and choriogonadotropin (CG) are heterodimers, which consist of a common ␣ subunit and a unique  subunit that confer the receptor specificity of the ligand. 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.
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