No abstract

These results suggest that hCG, through its binding to the LH/CG receptor, may augment proliferation and inhibit apoptosis in choriocarcinoma JAr cells, and that the introduction of an antisense gene may be a potential approach to the inhibition of choriocarcinoma cell growth.

The common alpha subunit of glycoprotein hormones contains five disulfide bonds. Based on the published crystal structure, the assignments are 7-31, 59-87, 10-60, 28-82, and 32-84; the last three comprise the cystine knot, a structure also seen in a variety of growth factors. Previously, we demonstrated that the efficiency of secretion and the ability to form heterodimers by alpha subunits bearing single cysteine residue mutants in the cystine knot were significantly reduced. These results suggested that the cystine knot is critical for the intracellular integrity of the subunit. To assess if the presence of the free thiol affected the secretion kinetics, we constructed paired cysteine mutants of each disulfide bond of the alpha subunit. The secretion rate for these monomers was comparable with wild type except for the alpha-10-60 mutant, which was 40% lower. The recovery of the alpha7-31 and alpha59-87 mutants was greater than 95%, whereas for the cystine knot mutants, it was 20-40%. Co-expression of the wild-type chorionic gonadotropin beta subunit with double cysteine mutants did not enhance the recovery of alpha mutants in the media. Moreover, compared with wild-type, the efficiency of heterodimer formation of the alpha10-60 or alpha32-84 mutants was less than 5%. Because subunit assembly is required for biological activity, studies on the role of these disulfide bonds in signal transduction were not possible. To bypass the assembly step, we exploited the single chain model, where the alpha and beta subunits are genetically fused. The recovery of secreted tethered gonadotropins bearing mutations in the cystine knot was increased significantly. Although dimer-specific monoclonal antibodies discriminated the conformation of single chain alpha10-60 and alpha32-84 mutants from the native heterodimer, these mutants were nevertheless biologically active. Thus, individual bonds of cystine knot are important for secretion and heterodimer formation but not for in vitro bioactivity. Moreover, the data suggest that the native heterodimer configuration is not a prerequisite for receptor binding or signal transduction.

No abstract

The gonadotropin/thyrotropin hormone family is characterized by a heterodimeric structure composed of a common ␣ subunit noncovalently linked to a hormonespecific ␤ subunit. The conformation of the heterodimer is essential for controlling secretion, hormone-specific post-translational modifications, and signal transduction. Structure-function studies of follicle-stimulating hormone (FSH) and the other glycoprotein hormones are often hampered by mutagenesis-induced defects in subunit combination. Thus, the ability to overcome the limitation of subunit assembly would expand the range of structure-activity relationships that can be performed on these hormones. Here we converted the FSH heterodimer to a single chain by genetically fusing the carboxyl end of the FSH ␤ subunit to the amino end of the ␣ subunit in the presence or absence of a linker sequence. In the absence of the CTP linker, the secretion rate was decreased over 3-fold. Unexpectedly, however, receptor binding/signal transduction was unaffected by the absence of the linker. These data show that the single-chain FSH was secreted efficiently and is biologically active and that the conformation determinants required for secretion and biologic activity are not the same.One of the hallmarks of the gonadotropin and thyrotropin hormone family is their heterodimeric structure, consisting of a common ␣ subunit and a unique ␤ subunit (1). Subunit assembly is vital to the function of these hormones: (i) only the dimers are bioactive, (ii) maturation of the hormone-specific oligosaccharides is dependent on the formation of the heterodimer complex, and (iii) the secretion efficiency of the dimer is determined by the ␤ subunit. Previously, we constructed a chimera composed of the human chorionic gonadotropin (hCG) 1 ␤ subunit genetically fused to the ␣ subunit, and the resulting single polypeptide chain was efficiently secreted and was biologically active (2). Because subunit dissociation would lead to inactivation of the heterodimer, a single-chain form could have higher biological activity.Although the gonadotropin dimers have similar structural features, they are nevertheless unique. The ␣ subunit in the dimers has a different conformation which is manifested by distinct immunological and spectral characteristics (3-6). In addition, the carbohydrates on the ␣ subunits are not the same in all the gonadotropin dimers (7-9) and the receptor contact sites on the ␣ subunit differ among the hormones (10, 39, 40). Thus, a priori one cannot predict based on the CG model that other members of the glycoprotein hormone family can be converted to single chains.Tethering a variety of multisubunit complexes into single chains has been performed by several laboratories to increase protein stability or activity (11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22). In such studies, a linker sequence was designed to give optimal alignment of determinants. In the case of the glycoprotein hormones, we presumed that a linker would be required for successful expression of the correspondin...