Alternatively folded choriogonadotropin analogs. Implications for hormone folding and biological activity.

Most heterodimeric proteins are stabilized by intersubunit contacts or disulfide bonds. In contrast, human chorionic gonadotropin (hCG) and other glycoprotein hormones are secured by a strand of their beta-subunits that is wrapped around alpha-subunit loop 2 "like a seatbelt." During studies of hCG synthesis in COS-7 cells, we found that, when the seatbelt was prevented from forming the disulfide that normally "latches" it to the beta-subunit, its carboxyl-terminal end can "scan" the surface of the heterodimer and become latched by a disulfide to cysteines substituted for residues in the alpha-subunit. Analogs in which the seatbelt was latched to residues 35, 37, 41-43, and 56 of alpha-subunit loop 2 had similar lutropin activities to those of hCG; that in which it was latched to residue 92 at the carboxyl terminus had 10-20% the activity of hCG. Attachment of the seatbelt to alpha-subunit residues 45-51, 86, 88, 90, and 91 reduced lutropin activity substantially. These findings show that the heterodimer can form before the beta-subunit has folded completely and support the notions that the carboxyl-terminal end of the seatbelt, portions of alpha-subunit loop 2, and the end of the alpha-subunit carboxyl terminus do not participate in lutropin receptor interactions. They suggest also that several different architectures could have been sampled without disrupting hormone activity as the glycoprotein hormones diverged from other cysteine knot proteins.

The surface of alpha-subunit loop 1 distant from the subunit interface is exposed in the hCG lutropin receptor complex.

Interactions of the placental glycoprotein hormone human choriogonadotropin (hCG) with lutropin receptors (LHR) are required for maintenance of early pregnancy. Knowledge of how hCG interacts with LHR is useful for understanding the mechanism of receptor function, an issue of considerable debate. A large surface of hCG remains exposed after the hormone binds the LHR and can be readily detected with monoclonal antibodies. Here we show that the surface of hCG alpha-subunit loop 1 furthest from the beta-subunit interface can also be recognized by a monoclonal antibody when hCG is bound to the LHR. This extends the area of hCG known to be exposed in the hormone receptor complex, an observation that further restricts models of hCG-LHR interaction.

Deglycosylation of a bifunctional lutropin-follitropin agonist reduced its follitropin activity more than its lutropin activity.

OBJECTIVE: To design a drug that blocks the gonadal actions of lutropins and follitropins. DESIGN: Controlled in vitro study. SETTING: Academic laboratory. PATIENT(S): None. INTERVENTION(S): We removed three glycosylation signals from an hCG-hFSH chimera known to have high affinity for LH and FSH receptors, expecting this would create a bifunctional antagonist (dgCFC). To offset the inhibition of subunit combination caused by deglycosylation of alpha-subunit loop 2, we prepared dgCFC as a single-chain fusion protein containing the alpha-subunit downstream of the chimeric beta-subunit. MAIN OUTCOME MEASURE(S): Receptor binding, cyclic adenosine monophosphate accumulation. RESULT(S): dgCFC bound LH or FSH receptors similar to hCG or hFSH. It was a partial agonist and had one tenth the efficacy of hFSH and two thirds the efficacy of hCG. CONCLUSION(S): The surprising high residual lutropin activity of dgCFC indicated that its FSH residues offset the effects of deglycosylation, suggesting this approach to preparing a bifunctional antagonist is unlikely to lead to a useful drug. The increased lutropin efficacy of dgCFC relative to deglycosylated hCG supports the idea that oligosaccharides modulate glycoprotein hormone efficacy through an influence on hormone conformation.

Desensitization and resensitization of lutropin receptors expressed in transfected Y-1 adrenal cells.

Stimulation of gonadal cells by lutropins such as human chorionic gonadotropin (hCG) is often transient and followed by down-regulation and/or desensitization of lutropin receptors (LHR). Here we describe desensitization/resensitization of LHR in Y-1 adrenal cell lines (termed Y-1L) expressing a rat cDNA lacking most 5' and 3' LHR untranslated regions under the control of a metallothionein promoter. Using a simple morphological assay in which stimulated cells are round and unstimulated cells are flat, we identified clones that rounded and remained round and others that became insensitive to lutropin stimulation and reverted to their flat appearance within 2-4 h. Flattened cells were insensitive to further hormonal stimulation but rounded after treatments with cholera toxin, forskolin, or cyclic AMP, showing that loss of responsiveness was associated with an early step in signal transduction, not loss of rounding potential. Removing the lutropin stimulus for at least 90-120 min reversed hormone insensitivity, even in the presence of the protein synthesis inhibitor puromycin. The number of surface bound receptors did not change during a cycle of rounding/flattening and hCG bound to rounded or flattened cells was replaced equally by radioiodinated hCG during incubations at 4 degrees C. Thus, desensitization/resensitization of LHR in Y-1L cells occurred in the absence of new receptor synthesis, receptor degradation, or receptor recycling. These observations suggest that LHR desensitization/resensitization in Y-1L cells was closely coupled to receptor occupancy and that this cell line may be useful for identifying factors that modulate the activities of occupied receptors.

Addition of an N-terminal dimerization domain promotes assembly of hCG analogs: implications for subunit combination and structure-function analysis.

Human chorionic gonadotropin (hCG) is a heterodimeric placental glycoprotein hormone that acts through ovarian lutropin receptors (LHR) to maintain early pregnancy. Its ability to distinguish LHR and follitropin receptors (FSHR) is controlled by 20 beta-subunit 'seatbelt' residues that surround alpha-subunit loop 2. Positively charged amino acids between residues 93-100, a small loop within the seatbelt, have been postulated to make essential LH receptor contacts. Previous studies showed that analogs containing negatively charged amino acids in this small loop had 5-10% the activity of hCG and 1-10% the lutropin activities of hCG/hFSH chimeric analogs capable of binding LHR and FSHR. These effects might be due to the influence of these residues on receptor contacts or on hormone conformation. During efforts to distinguish these possibilities, we increased and decreased the number of residues in this loop, mutations we anticipated would distort its conformation. Consistent with this supposition, these changes inhibited dimer formation, precluding assessment of these mutations on hormone activity. Addition of Fos and Jun dimerization domains to the N-termini of hCGalpha- and hCG/hFSHbeta-subunit chimeras overcame the effects of the seatbelt mutations on subunit combination and enabled preparation of heterodimers containing six, seven, or nine residues in their seatbelt loops. These had 0.1-10% the lutropin and 3-60% the follitropin activities of bifunctional chimeras containing 8 residues derived from hCG in the seatbelt loop. The abilities of N-terminal dimerization domains to promote subunit combination may permit structure/function analysis of other residues that influence heterodimer formation.

Lutropins appear to contact two independent sites in the extracellular domain of their receptors.

Human chorionic gonadotropin (hCG) and bovine lutropin (bLH), a hormone chemically more similar to most mammalian lutropins than hCG, interact with the extracellular domains of their gonadal lutropin receptors (LHRs). These portions of the rat and human LHRs are 85% identical and both receptors bind hCG with high, albeit not identical, affinity. However, at least 1000-fold more bLH is required to inhibit binding of radiolabelled hCG to the human LHR than to the rat LHR, a phenomenon that proved useful for identifying regions of the extracellular domain that contact lutropins. Previous studies using truncated receptors and lutropin/follitropin receptor chimaeras localized most, if not all, high-affinity ligand contacts to the N-terminal three-fifths of the rat LHR extracellular domain. We report here that 10-fold more bLH was needed to inhibit binding of labelled hCG to rat/human LHR chimaeras containing the N-terminal three-fifths of the human LHR extracellular domain than to the rat LHR. Unexpectedly, 100-fold more bLH was required to inhibit binding of labelled hCG to chimaeras containing the C-terminal one-fifth of the human LHR extracellular domain than to the rat LHR. The ability of the C-terminal portion of the human LHR extracellular domain to inhibit bLH binding suggests this region of the receptor also contacts the ligand even though it is not needed for ligand binding. The extracellular domains of all the glycoprotein hormone receptors are thought to be horseshoe-shaped, a consequence of their leucine-rich repeat motifs. Portions of the ligand that become located within the cavity created by the concave surface of the horseshoe would have the opportunity to contact residues in the C-terminal portion of the extracellular domain. Changes to the ligand or receptor that influence this interaction would be expected to alter binding and confound efforts to identify residues in key ligand-receptor contacts.

Functional homodimeric glycoprotein hormones: implications for hormone action and evolution.

BACKGROUND: Human chorionic gonadotropin (hCG), lutropin, follitropin, and thyrotropin act as alpha beta heterodimers to control reproduction and thyroid function. The alpha and beta subunits of these proteins are divided into three loops (alpha 1,alpha 2,alpha 3; beta 1,beta 2,beta 3) by cysteine knots and the heterodimer is stabilized by 20 beta-subunit residues wrapped around alpha 2 like a seatbelt. Understanding how these hormones interact with their receptors, a matter of considerable dispute, would facilitate design of pro- and anti-fertility agents. RESULTS: By swapping alpha 2 for beta 2 and vice versa and, in some cases, adding an amino-terminal coiled-coil dimerization domain, we prepared homodimeric analogs that have the conformation found in each 'half' of hCG. Homodimers containing loops beta 1,alpha 2,beta 3 and none, part, or all of the seatbelt stimulated signal transduction to the same extent as hCG, albeit with lower potency. Those containing alpha 1,beta 2,alpha 3 were inactive. CONCLUSIONS: The activities of homodimers containing the beta 1,alpha 2,beta 3 groove exceed those of other minimized analogs more than 100-1000-fold, suggesting this portion of the hormone forms the major receptor contact. The discovery that glycoprotein hormone heterodimers can be converted to functional homodimers supports the proposal that this protein family evolved from an active homodimeric ancestor by gene duplication and acquisition of mutations to loop 2 that prevent homodimerization. This approach to protein minimization should be applicable to other proteins composed of architecturally related subunits, including those that might have arisen by gene duplication.

Epitope-specific focusing of the immune response to a minimized human chorionic gonadotropin analog.

Minimized proteins have long been used to elicit an immune response to particular regions of a protein antigen. Most efforts to derive minimized proteins have employed synthetic peptide fragments. This approach works well for linear epitopes but poorly for conformational epitopes. Here we describe a homodimeric human chorionic gonadotropin (hCG) analog that retains the conformation of related parts of hCG and elicits high affinity specific antibodies. This novel immunogen displays the tertiary structure of selected loops of the protein but lacks structures that could elicit potentially undesirable antibodies.

The lutropin beta-subunit N-terminus facilitates subunit combination by offsetting the inhibitory effects of residues needed for LH activity.

Human chorionic gonadotropin (hCG) contains a beta-subunit N-terminal amino acid extension that contacts the alpha-subunit and is needed for efficient alpha and hCG beta-subunit combination. Here we report that an hCG beta-subunit analog, lacking residues 2-8, combined with the alpha-subunit more efficiently when positively charged residues between beta-subunit cysteines 10 and 11 were replaced with negatively charged residues found in the corresponding portion of follitropin. Residues 2-8 had no influence on binding of hCG to lutropin receptors. Positive charges between cysteines 10 and 11 are essential for high affinity binding of lutropins to their receptors. Therefore, the N-terminal extension found in all lutropin beta-subunits appears to have evolved to offset the inhibition of subunit combination by beta-subunit residues that are essential for lutropin activity. This beta-subunit extension is not found in follitropins or thyrotropins, hormones that have negatively charged residues between cysteines 10 and 11.

Co-evolution of ligand-receptor pairs.

Specific receptors for lutropin (luteinizing hormone; LH) and follitropin (follicle-stimulating hormone; FSH) mediate the actions of human chorionic gonadotropin (hCG) and FSH5 on the gonads. Here we report that short independent sequences of the beta-subunit enable hCG to distinguish between the receptors for FSH and LH. Residues between the 11th and 12th cysteines restrict FSH receptor binding; residues between the 10th and 11th cysteines and, to a much lesser extent, residues carboxy-terminal to the 12th cysteine also affect LH receptor binding. CF101-109, an hCG analogue containing hFSH beta residues between the 11th and 12th cysteines, had high affinity for both LH and FSH receptors. Modifications to CF101-109 that reduce binding to either LH or FSH receptors yield gonadotropin analogues having differing ratios of LH:FSH activity. Ligand-binding specificity of the LH receptor is determined by residues encoded by parts of exons 2-4 and 7-9 which prevent hFSH binding but have little effect on hCG binding. FSH receptor specificity is controlled primarily by residues encoded by exons 5 and 6 that prevent hCG binding but have little effect on hFSH binding. These determinants can be interchanged to create receptor analogues that bind hCG and hFSH. Our observations support a model in which distinct negative determinants restrict ligand-receptor interaction. This explains coevolution of binding specificity in families of homologous ligands and their receptors. Natural or designed manipulation of these determinants leads to the 'evolution' of new, specific protein-protein interactions.

Leutropin/beta-adrenergic receptor chimeras bind choriogonadotropin and adrenergic ligands but are not expressed at the cell surface.

In some G-protein-coupled receptors (e.g. beta-adrenergic receptor (beta 2 AR)), the ligand-binding pocket is contained within the hydrophobic transmembrane domain. In others (e.g. luteinizing hormone receptor (LHR)), the relative roles of the extracellular N-terminal domain and the transmembrane region in hormone binding are unknown. To study the roles of these domains, we prepared vectors encoding the rat LHR N-terminal domain alone (L- -), the LHR N-terminal domain fused to the transmembrane and C-terminal domains of the vesicular stomatitis virus-G protein (LVV), the LHR N-terminal domain fused to the transmembrane and C-terminal domains of the hamster beta 2 AR (LAA), and the beta 2 AR N-terminal domain fused to the transmembrane and C-terminal domains of the rat LHR (ALL). Membrane preparations obtained from COS-7 cells expressing the beta 2 AR or LAA bound the beta-adrenergic antagonist 125I-cyanopindolol with equal affinity, confirming the observation that the beta 2 AR transmembrane domain forms the hormone-binding site. Membranes from COS-7 cells transfected with LHR bound 125I-human choriomic gonadotropin (hCG). However, membranes from LAA-, L(- -)-, and LVV-transfected cells had low capacity to bind 125I-hCG unless they were solubilized with Triton X-100. The affinity of the detergent-solubilized receptors for 125I-hCG was similar to that of the LHR. We were unable to detect binding of 125I-hCG to ALL in the presence or absence of detergent. These observations suggest that, whereas the transmembrane region of the beta 2 AR is sufficient to bind adrenergic ligands, the N-terminal region of the LHR is required for binding of hCG. Although the N terminus of the LHR is sufficient to bind hCG, both the N terminus and the transmembrane domains of the LHR are required for receptor expression on the cell surface.

Cloning of rat lutropin (LH) receptor analogs lacking the soybean lectin domain.

cDNAs coding for rat ovarian luteinizing hormone receptor analogs lacking three of the leucine repeats were detected in a library which had been prepared from rat luteal tissue undergoing human chorionic gonadotropin-induced luteinization. These leucine repeats correspond to amino acids 206-267 and contain the portion of the receptor that is homologous to the soybean lectin. The cDNA library also contained a receptor analog lacking amino acids 321-700 which code for the transmembrane domain. S-1 mapping suggests that this latter form constitutes approximately half of all receptor-related mRNA.