One of the distinguishing features of the gonadotropin and thyrotropin hormone family is their heterodimeric structure, consisting of a common a subunit and a hormone-specific f8 subunit. Subunit assembly is vital to the function of these hormones: The conformation of the heterodimer is essential for controlling secretion, hormonespecific posttranslational modifications, and signal transduction. To address whether a and ,3 subunits can be synthesized as one chain and also maintain biological activity, a chimera composed of the human chorionic gonadotropin (hCG) ,3 subunit genetically fused to the a subunit was constructed. question of whether the common a subunit and a hormonespecific (3 subunit can be synthesized as a single chain to achieve the biological response. For structure-function studies, the ability to express a heterodimer as a single biologically active chain would likely avoid mutagenesis-induced defects in combination and secretion of individual subunits. In addition, since subunit dissociation will inactivate the in vivo activity of the heterodimer, single-chain analogs could have a longer biological half-life. A chimera composed of the C-terminal end of hCG(3 genetically fused to the N-terminal end of the a subunit was constructed because these regions can be modified without significant effects on the biological activity of the heterodimer (7,8). Expression of this chimeric gene in CHO cells produced a single polypeptide hCG molecule that was biologically active in vitro and in vivo. These results demonstrate that the a and hCG,B subunits encoded as single chain can fold into an appropriate conformation and that a noncovalent linkage of the a and 13 subunits is not required for biological activity. This approach can be used to further investigate structure-function relationships of hCG and related glycoprotein hormones that were previously not tractable because of the absolute dependence on subunit assembly for biological activity.

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.

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...

Disrupting disulfide loops in the human chorionic gonadotropin ␤ subunit (CG␤) inhibits combination with the ␣ subunit. Because the bioactivity requires a heterodimer, studies on the role of disulfide bonds on receptor binding/signal transduction have previously been precluded. To address this problem, we bypassed the assembly step and genetically fused CG␤ subunits bearing paired cysteine mutations to a wild-type ␣ (WT␣) subunit. The changes altered secretion of the single-chain mutants which parallel that seen for the CG␤ monomeric subunit. Despite conformational changes in CG disulfide bond mutants (assayed by gel electrophoresis and conformationally sensitive monoclonal antibodies), the variants bind to the lutropin/CG receptor and activated adenylate cyclase in vitro. The data show that the structural requirements for secretion and bioactivity are not the same. The results also suggest that the extensive native subunit interactions determined by the cystine bonds are not required for signal transduction.Moreover, these studies demonstrate that the singlechain model is an effective approach to structure-activity relationships of residues and structural domains associated with assembly of multisubunit ligands.Mutational and structural studies have revealed that small clusters of amino acids rather than large structural motifs often contribute to most of the energy involved in proteinprotein interactions such as hormone binding to its receptor (for review, see Refs. 1-3). However, it is not clear how the conformation of a peptide ligand contributes to the coupling of binding to signal transduction. This is especially an issue for the function of hormone-receptor complexes involving multisubunit ligands. A convenient model for studying the tertiary and quaternary determinants in signaling is the glycoprotein hormone family which include human chorionic gonadotropin (hCG), 1 lutropin (LH), follitropin (FSH), and thyrotropin. Each is a non-covalent heterodimer composed of a common ␣ and unique ␤ subunit which allows recognition of the corresponding G-protein-coupled receptor (for review, see Ref. 4).Recent crystallographic studies of hCG revealed a significant structural similarity to several growth factor families, e.g. transforming growth factor ␤, which contain a cystine knot motif composed of three pairs of bridged cysteine residues (5-8). Each hCG subunit contains a cystine knot configuring three disulfide-bonded loops which are the major structural motifs (7,8). Based on the crystallographic studies of hCG (7,8), the disulfide bonds in the CG␤ subunit are at positions 9 -57, 34 -88, 38 -90, 23-72, 93-100, and 26 -110 (Fig. 1A). The folding intermediates associated with the ordered formation of these bridges to acquire an assembly-competent form is well documented (9, 10). Current models of hCG action presume that the conformation of the dimer and the highly interactive contacts between the two subunits are critical for function (11)(12)(13)(14). One method to examine the functional role of the structural mo...

Incubation of cultured rat pituitary cells with gonadotropin-releasing hormone (GnRH, 1 nM) resulted in a rapid elevation of gonadotropin subunit steady-state mRNA levels(alpha, 2.2-fold, LH beta, 2.1-fold, and FSH beta 2.2-fold increases at 30 min). Addition of actinomycin D abolished the stimulatory effect of GnRH upon alpha and LH beta and reduced the effect upon FSH beta mRNA levels. The effect of GnRH is biphasic, where the early phase is being observed at 30-60 min, while the late phase is noticed between 12-24 h. A significant decrease in FSH beta mRNA levels was found after 6 h of incubation when using a stable GnRH analog. The unique profile of the time response enabled us to attempt to dissect the signal transduction cascade involved in the neurohormone action. Addition of the protein kinase C (PKC) activator, 12-O-tetradecanoylphorbol 13-acetate (TPA), or the Ca2+ ionophore, ionomycin, mimicked the profile of GnRH-induced alpha and LH beta mRNA elevation. The two phases of FSH beta mRNA elevation induced by GnRH could be mimicked by TPA, while the decrease at 6 h was mimicked by ionomycin. The rapid stimulatory effect of GnRH on gonadotropin subunit mRNA levels was abolished by the PKC inhibitors, staurosporine and GF 109203X. Similarly, the rapid stimulatory effect of GnRH on alpha and LH beta, but not FSH beta, was abolished in Ca(2+)-free medium. While additivity in LH release is obtained upon the combined addition of TPA and ionomycin for 30 min of incubation, LH beta and FSH beta gene expression is inhibited.(ABSTRACT TRUNCATED AT 250 WORDS)

Glycoprotein hormone receptors such as the lutropin/chorionic gonadotropin receptor (LHR) are characterized by a large N-terminal ectodomain (ECD), which is responsible for hormone-receptor interactions. For the closely related TSH receptor (TSHR), it has been proposed that the ECD also serves as a tethered inverse agonist. However, the exact role of the LHR-ECD for receptor activation remains elusive. Functional analysis of N-terminally truncated LHR mutants expressed in COS-7 cells revealed that the LHR-ECD does not act as an inverse agonist but facilitates active LHR conformations. This notion is supported by two observations: first, removal of the ECD tended to decrease basal LHR activity and secondly, mutationally induced constitutive receptor activity was diminished for most activating mutations in LHR lacking the ECD. In addition, swapping of the LHR-ECD for the ECD of the closely related TSHR was not sufficient to restore constitutive receptor activity induced by naturally occurring activating heptahelical LHR mutations. Thus, the ECD stabilizes an activation-competent conformation of the heptahelical region. While the full-length LHR fused to the cognate agonist, human chorionic gonadotropin (hCG), showed increased basal activity, fusion proteins between hCG and Nterminally truncated LHR did not yield constitutive receptor activity suggesting an important role of the ECD also for agonist-dependent LHR activity. Our experiments strengthen the concept of a major contribution of the LHR-ECD in the activation mechanism apart from hormone binding and provide evidence for a cooperative model with structural and functional interactions of the ECD and the transmembrane domain.

We have previously demonstrated a physical interaction between cytosolic phospholipase A 2 ␣ (cPLA 2 ) and the assembled NADPH oxidase on plasma membranes following neutrophil stimulation. The aim of the present study was to define the exact binding sites between these two enzymes. Here we show, based on blot overlay experiments, Förster resonance energy transfer analysis and studies in neutrophils from patients with chronic granulomatous disease deficient in p67 phox or p47 phox , that cPLA 2 specifically binds to p47 phox and that p47 phox is sufficient to anchor cPLA 2 to the assembled oxidase on the plasma membranes upon stimulation. Blot overlay and affinity binding experiments using subfragments of cPLA 2 and p47 phox demonstrated that the cPLA 2 -C2 domain and the p47 phox -PX domain interact to form a complex that is resistant to high salt. Computational docking was used to identify hydrophobic peptides within these two domains that inhibited the association between the two enzymes and NADPH oxidase activity in electro-permeabilized neutrophils. These results were used in new docking computations that produced an interaction model. Based on this model, cPLA 2 -C2 domain mutations were designed to explore its interaction p47 phox in neutrophil lysates. The triple mutant F35A/M38A/L39A of the cPLA 2 -C2 domain caused a slight inhibition of the affinity binding to p47 phox , whereas the single mutant I67A was highly effective. The double mutant M59A/H115A of the p47 phox -PX domain caused a significant inhibition of the affinity binding to cPLA 2 . Thus, Ile 67 of the cPLA 2 -C2 domain is identified as a critical, centrally positioned residue in a hydrophobic interaction in the p47 phox -PX domain.

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