Biomedical subjects

W R Moyle

Publications and source records attributed to W R Moyle.

At least 19 recordsLinked to original sources

PubMed2001

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.

Alanine↗

PubMed2001

Threading of a glycosylated protein loop through a protein hole: implications for combination of human chorionic gonadotropin subunits.

Chorionic gonadotropin (hCG) is a heterodimeric placental glycoprotein hormone essential for human reproduction. Twenty hCG beta-subunit residues, termed the seatbelt, are wrapped around alpha-subunit loop 2 (alpha 2) and their positions "latched" by a disulfide formed by cysteines at the end of the seatbelt (Cys 110) and in the beta-subunit core (Cys 26). This unique arrangement explains the stability of the heterodimer but raises questions as to how the two subunits combine. The seatbelt is latched in the free beta-subunit. If the seatbelt remained latched during the process of subunit combination, formation of the heterodimer would require alpha 2 and its attached oligosaccharide to be threaded through a small beta-subunit hole. The subunits are known to combine during oxidizing conditions in vitro, and studies described here tested the idea that this requires transient disruption of the latch disulfide, possibly as a consequence of the thioredoxin activity reported in hCG. We observed that alkylating agents did not modify either cysteine in the latch disulfide (Cys 26 or Cys 110) during heterodimer formation in several oxidizing conditions and had minimal influence on these cysteines during combination in the presence of mild reductants (1--3 mM beta-mercaptoethanol). Reducing agents appeared to accelerate subunit combination by disrupting a disulfide (Cys 93--Cys 100) that forms a loop within the seatbelt, thereby increasing the size of the beta-subunit hole. We propose a mechanism for hCG assembly in vitro that depends on movements of alpha 2 and the seatbelt and suggest that the process of glycoprotein hormone subunit combination may be useful for studying the movements of loops during protein folding.

Alkylating Agents↗

PubMed2001

Partial restoration of lutropin activity by an intersubunit disulfide bond: implications for structure/function studies.

Gonadal function is controlled by lutropins and follitropins, heterodimeric cystine knot proteins that have nearly identical alpha-subunits. These heterodimeric proteins are stabilized by a portion of the hormone-specific beta-subunit termed the "seatbelt" that is wrapped around alpha-subunit loop 2 (alpha 2). Here we show that replacing human chorionic gonadotropin (hCG) alpha 2 residue Lys51 with cysteine or alanine nearly abolished its lutropin activity, an observation that implies that alpha Lys51 has a key role in hormone activity. The activity of the heterodimer containing alpha K51C, but not that containing alpha K51A, was increased substantially when beta-subunit seatbelt residue beta Asp99 was converted to cysteine. As had been reported by others, heterodimers containing alpha K51C and beta D99C were crosslinked by a disulfide. The finding that an intersubunit disulfide restored some of the activity lost by replacing alpha Lys51 suggests that this residue is not crucial for receptor binding or signaling and also that hCG and related hormones may be particularly sensitive to mutations that alter interactions between their subunits. We propose the unique structures of hCG and related family members may permit some subunit movement in the heterodimer, making it difficult to deduce key residues involved in receptor contacts simply by correlating the activities of hormone analogs with their amino acid sequences.

Animals↗

PubMed2000

Bifunctional hCG analogs adopt different conformations in LH and FSH receptor complexes.

Human reproduction requires specific interactions between follitropin (hFSH) and its receptor (FSHR) and between lutropin (hLH) or choriogonadotropin (hCG) and the lutropin receptor (LHR). Substitution of hFSH residues between hCG beta-subunit cysteines 11-12 creates a bifunctional analog that binds both receptors. To understand the basis of this observation, we used antibody probes to compare the conformations of bifunctional analogs before and after they were complexed with each receptor. Introduction of hFSH residues between cysteines 11-12 changed a distant conformation-sensitive region created by the juxtaposition of the subunit aminotermini. This region, found not to contact either receptor, was altered further when bifunctional ligands bound FSHR. All other surfaces, detected in LHR complexes, were also recognized in FSHR complexes, an indication that bifunctional ligands bind both receptors in similar orientations. These observations suggest that unlike hCG or hFSH, bifunctional gonadotropins can acquire "lutropin" and "follitropin" conformations, a phenomenon accentuated by receptor contacts.

Amino Acid Sequence↗

PubMed2000

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.

Antibodies, Monoclonal↗

PubMed1999

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.

Amino Acid Sequence↗

PubMed1999

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.

Amino Acid Sequence↗

PubMed1999

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.

Adrenal Glands↗

PubMed1998

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.

Amino Acid Sequence↗

PubMed1998

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.

Amino Acid Sequence↗

PubMed1998

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.

Amino Acid Sequence↗

PubMed1997

Influence of subunit interactions on lutropin specificity. Implications for studies of glycoprotein hormone function.

Bovine lutropin (bLH) and human chorionic gonadotropin (hCG) are heterodimeric glycoprotein hormones required for reproduction. Both bind rat LH receptors (rLHRs), but hCG binds human LH receptors (hLHRs) 1000-10,000 fold better than bLH. We tested the premise that this difference in affinity could be used to identify lutropin receptor contacts. Heterodimers containing hCG/bLH alpha- or beta-subunit chimeras that bound hLHR like hCG (or bLH) were expected to have hCG (or bLH) residues at the receptor contact sites. Analogs containing one subunit derived from hCG bound hLHR much more like hCG than bLH, indicating that each bLH subunit contains all the residues sufficient for high affinity hLHR binding. Indeed, the presence of bovine alpha-subunit residues increased the activities of some hCG analogs. The low hLHR activity of bLH was due primarily to an interaction between its alpha-subunit and beta-subunit residue Leu95. Leu95 does not appear to contact the hLHR since it did not influence the hLHR activity of heterodimers containing human alpha-subunit. These observations show that interactions within and between the subunits can significantly influence the activities of lutropins, thereby confounding efforts to identify ligand residues that contact these receptors.

Amino Acid Sequence↗

PubMed1997

Chimeric proteins can exceed the sum of their parts: implications for evolution and protein design.

Chimeric analogs derived from pairs of homologous proteins routinely exhibit activities found in one or both parents. We describe chimeras of two glycoprotein hormones, human chorionic gonadotropin (hCG) and human follitropin (hFSH), that exhibit activity unique to a third family member, human thyrotropin (hTSH). The results show that biological activity can be separated from hormone-specific amino acid residues. This is consistent with a model for the evolution of homologous ligand-receptor pairs involving gene duplication and the creation of inhibitory determinants that restrict binding. Disruption of these determinants can unmask activities characteristic of other members of a protein family. Combining portions of two ligands to create analogs with properties of a third family member can facilitate identifying key determinants of protein-protein interaction and may be a useful strategy for creating novel therapeutics. In the case of the glycoprotein hormones, this showed that two different hormone regions (i.e., the seat-belt and the intersubunit groove) appear to limit inappropriate contacts with receptors for other members of this family. These observations also have important caveats for chimera-based protein design because an unexpected gain of function may limit the therapeutic usefulness of some chimeras.

Amino Acid Sequence↗

PubMed1996

hCG beta residues 94-96 alter LH activity without appearing to make key receptor contacts.

The ability of human chorionic gonadotropin (hCG) to distinguish lutropin (LHR) and follitropin (FSHR) receptors is controlled principally by beta-subunit residues 94-117. To learn how residues 94-96 (Arg-Arg-Ser) influence LHR binding, we studied the effects of replacing them on the LH and FSH activities of a bifunctional hCG analog in which residues 101-109 were derived from FSH. Analogs containing 1-3 arginines and no aspartates at residues 94-96 bound LHR with 25-400% the potency of hCG. When residues 94-96 were neutral or contained 1-3 aspartates, LHR binding was reduced 6-100 fold but remained at least ten-fold greater than the negative control analog containing residues 94-117 derived from FSH. Residues 94-96 had little influence on FSHR binding. These observations support a model [Moyle et al. (1995) J. Biol. Chem. 270:20,020] in which residues 94-96 influence LHR binding specificity primarily through an effect on hormone conformation rather than by direct participation in essential high affinity receptor contacts.

Amino Acid Sequence↗

PubMed1995

The groove between the alpha- and beta-subunits of hormones with lutropin (LH) activity appears to contact the LH receptor, and its conformation is changed during hormone binding.

Gonadotropins are heterodimeric glycoprotein hormones that control vertebrate fertility through their actions on gonadal lutropin (luteinizing hormone, LH) and follitropin (follicle-stimulating hormone, FSH) receptors. The beta-subunits of these hormones control receptor binding specificity; however, the region of the beta-subunit that contacts the receptor has not been identified. By a process of elimination we show this contact to be the portions of beta-subunit loops one and three found in a hormone groove created by the juxtaposition of the alpha- and beta-subunits. Most other regions of the beta-subunit can be recognized by antibodies that bind to human chorionic hormone (hCG)-receptor complexes or replaced without disrupting hormone function. Using a series of bovine LH/hCG and human FSH/hCG beta-subunit chimeras we identified key hCG beta-subunit residues in the epitopes of two antibodies that bind to hCG-receptor complexes. These epitopes include the surfaces of beta-subunit loops one and three near residue 74 on the outside of the hormone groove and parts of the C-terminal end of the "seat belt" that holds the two subunits together. The antibody that recognized residue 74 bound to receptor complexes containing most mammalian lutropins better than to the free hormones, an indication that the outside surface of the beta-subunit groove is altered during hormone binding. This region of the beta-subunit is furthest from the alpha-subunit and is recognized equally well in the free beta-subunit and in the heterodimer. Thus, the receptor associated increase in antibody binding appears due to an interaction of this portion of the beta-subunit with the receptor and not to an effect of the receptor on the relative positions of the alpha- and beta-subunits. Unlike most previous studies designed to identify portions of the beta-subunit likely to contact the LH receptor, this indirect approach provides data that are more easily interpreted because it does not rely on the use of mutations that disrupt hormone function. The approach described here should be valuable for studying the receptor interactions of other complex ligands.

Amino Acid Sequence↗

PubMed1995

Model of human chorionic gonadotropin and lutropin receptor interaction that explains signal transduction of the glycoprotein hormones.

The goal of these studies was to devise a model that explains how human chorionic gonadotropin (hCG) interacts with lutropin (LH) receptors to elicit a hormone signal. Here we show that alpha-subunit residues near the N terminus, the exposed surface of the cysteine knot, and portions of the first and third loops most distant from the beta-subunit interface were recognized by antibodies that bound to hCG-receptor complexes. These observations were combined with similar data obtained for the beta-subunit (Cosowsky, L., Rao, S.N.V., Macdonald, G.J., Papkoff, H., Campbell, R.K., and Moyle, W.R. (1995) J. Biol. Chem. 270, 20011-20019), information on residues of hCG that can be changed without disrupting hormone function, the crystal structure of deglycosylated hCG, and the crystal structure of a leucine-repeat protein to devise a model of hCG-receptor interaction. This model suggest that the extracellular domain of the LH receptor is "U-" or "J"-shaped and makes several contacts with the transmembrane domain. High affinity hormone binding results from interactions between residues in the curved portion of the extracellular domain of the receptor and the groove in the hormone formed by the apposition of the second alpha-subunit loop and the first and third beta-subunit loops. Most of the remainder of the hormone is found in the large space between the arms of the extracellular domain and makes few, if any, additional specific contacts with the receptor needed for high affinity binding. Signal transduction is caused by steric or other influences of the hormone on the distance between the arms of the extracellular domain, an effect augmented by the oligosaccharides. Because the extracellular domain is coupled at multiple sites to the transmembrane domain, the change in conformation of the extracellular domain is relayed to the transmembrane domain and subsequently to the cytoplasmic surface of the plasma membrane. While the model does not require the hormone to contact the transmembrane domain to initiate signal transduction, small portions of both subunits may be near the transmembrane domain and assist in initiating the hormonal signal. This is the first model that is consistent with all known information on the activity of the gonadotropins including the amounts of the hormone that are exposed in the hormone-receptor complex, the apparent lack of specific contacts between much of the hormone and the receptor, and the roles of the oligosaccharides in signal transduction.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗

PubMed1995

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.

Amino Acid Sequence↗

PubMed1994

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.

Amino Acid Sequence↗