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

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 ␣ subunit and a hormone-specific ␤ 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 ␤ and ␣ subunits are linked (NH 2 -CG␤-␣; CG␤␣ 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 ␣ was fused to the CG␤ amino terminus (NH 2 -␣-CG␤; ␣CG␤ orientation). The secretion rate of ␣CG␤ from transfected Chinese hamster ovary cells was less than that seen for CG␤␣. The ␣CG␤ 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 ␣CG␤, it was co-transfected with a monomeric ␣ or CG␤ gene. In each case, ␣CG␤/␣ and ␣CG␤/CG␤ complexes were formed indicating that CG dimer-specific epitopes were established. The ␣CG␤/␣ complex bound to receptor indicating that the ␤ domain in the ␣CG␤ tether was still functional. In contrast, no significant receptor binding of ␣CG␤/CG␤ was observed indicating a major perturbation in the ␣ domain. These results suggest that although dimeric-like determinants are present in both ␣CG␤/␣ and ␣CG␤/CG␤ complexes, the receptor binding determinants in the ␣ 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.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. The subunits combine non-covalently early in the secretory pathway, and formation of the heterodimer is crucial for binding to the gonadal and thyroid receptors. Although it is well established that both subunits contain residues critical for bioactivity, recent evidence indicates that the quaternary interactions between the two subunits are essential for intracellular behavior but not for in vitro bioactivity (1-3). This conclusion was based on the single chain gonadotropin model where the amino end of the common ␣ subunit was genetically fused to the carboxyl end of CG␤ subunit (designated CG␤␣; Fig. 1) (4). This analog was secreted efficiently and was active in in vitro and in vivo bioassays. The CG␤␣ ori...

Modulation of the steroidogenic related activity according to the design of single-chain bovine FSH analogs

“…The generation of variants in the presence or absence of a linker sequence to space the tethered subunit domains, as well as switching their relative position, enabled to generate SC gonadotropin analogs with differences in the conformation (Argos, 1990;Ben-Menahem et al, 2001Garcia-Campayo and Boime, 2001;Grinberg et al, 2008;Sugahara et al, 1995Sugahara et al, , 1996b. Interestingly, gonadotropin analogs, mostly SC variants but also mutated heterodimers and nonpeptide mimetics, with a conformation that is different from that of the wild-type heterodimer, bound to the cognate receptor and stimulated cAMP formation, which is a key signaling mediator for steroidogenesis (Ben-Menahem et al, 1997;Fralish et al, 2003;Garcia-Campayo and Boime, 2001;Grossmann et al, 1997;Heikoop et al, 1997;Jackson et al, 1999;Maclean et al, 2004;Moyle et al, 1987;Perlman et al, 2003;Sato et al, 1997;Sugahara et al, 1995;van Straten et al, 2002;Weenen et al, 2004;Xing et al, 2001).…”

Consequences of Single-Chain Translation on the Structures of Two Chorionic Gonadotropin Yoked Analogs in α-β and β-α Configurations

“…Our laboratory and others have also reversed the order of subunit tether to N-␣-CTP-␤-C (YhCG3; Refs. [19][20][21]. This analog bound LHR poorly, but activated the receptor efficiently, thus uncoupling the two activities (19).…”

“…Using monoclonal antibodies, single-chain hCG (N-␤-␣-C) was shown to retain biological activity when some of the native quaternary interactions at the interface between the subunits were disrupted by mutation (26). However, complete disruption of subunit interactions, as in the single chain N-␣-␤-C (without linker), causes nearly complete loss of activity (21). From these elegant studies, it is apparent that native tertiary and quaternary structure are not a prerequisite to biological activity.…”

“…From these elegant studies, it is apparent that native tertiary and quaternary structure are not a prerequisite to biological activity. Indeed, these structural characteristics have ascribed more importance to intracellular behavior (21)(22)(23)26).…”

Single-chain human chorionic gonadotropin analogs containing the determinant loop of the beta-subunit linked to the alpha-subunit

“…We and others have shown that biologically active fusion proteins of hCG could be constructed in the manner N-hCG --C (Narayan et al 1995, Sugahara et al 1995, Heikoop et al 1997, Schubert et al 2003 and N--CTP-hCG -C (Narayan et al 2000a, Sen Gupta & Dighe 2000, Ben-Menahem et al 2001. These studies were expanded to include yoked hCG-rat LHR complexes (YHR) of the form, N-hCG --CTP-LHR-C (Wu et al 1996) and N--hCG -LHR-C (Narayan et al 2000a), resulting in ligand-mediated constitutive receptor activation.…”