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Biomedical subjects

W R Moyle

Publications and source records attributed to W R Moyle.

At least 37 records · Page 2Linked to original sources
PubMed1992

Assembly and expression of a synthetic gene encoding the bovine glycoprotein hormone alpha-subunit.

The glycoprotein hormones are a family of alpha beta heterodimeric proteins which are responsible for gonadal and thyroid function. In previous studies we employed chimeric glycoprotein hormone beta-subunits to identify amino acid residues critical for binding to receptors and antibodies. To facilitate similar studies of the alpha-subunit of these hormones, we assembled a 406 bp synthetic gene which encodes the human alpha-subunit leader sequence and the secreted portion of the bovine alpha-subunit. It contains unique restriction sites that can be used for cassette mutagenesis or for making human/bovine alpha-subunit chimeras. The gene was assembled from eight long oligodeoxynucleotides in a single ligation and its structure verified by DNA sequencing. Co-transfection of COS-7 cells with the synthetic gene and the cDNA for human chorionic gonadotropin (hCG) beta-subunit resulted in the secretion of a functional alpha beta heterodimer which bound to luteinizing hormone receptors. The protein was recognized by several monoclonal antibodies including B109, an antibody to a conformational epitope which binds hCG but not the free bovine alpha-, human alpha-, or hCG beta-subunits. This suggests that the binding site for B109 may be formed by residues located primarily within the hCG beta-subunit and that formation of this epitope requires a change in conformation of the beta-subunit when it combines with the alpha-subunit.

Amino Acid Sequence↗
PubMed1991

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.

Amino Acid Sequence↗
PubMed1991

Conversion of human choriogonadotropin into a follitropin by protein engineering.

Human reproduction is dependent upon the actions of follicle-stimulating hormone (hFSH), luteinizing hormone (hLH), and chorionic gonadotropin (hCG). While the alpha subunits of these heterodimeric proteins can be interchanged without effect on receptor-binding specificity, their beta subunits differ and direct hormone binding to either LH/CG or FSH receptors. Previous studies employing chemical modifications of the hormones, monoclonal antibodies, or synthetic peptides have implicated hCG beta-subunit residues between Cys-38 and Cys-57 and corresponding regions of hLH beta and hFSH beta in receptor recognition and activation. Since the beta subunits of hCG or hLH and hFSH exhibit very little sequence similarity in this region, we postulated that these residues might contribute to hormone specificity. To test this hypothesis we constructed chimeric hCG/hFSH beta subunits, coexpressed them with the human alpha subunit, and examined their ability to interact with LH and FSH receptors and hormone-specific monoclonal antibodies. Surprisingly, substitution of hFSH beta residues 33-52 for hCG beta residues 39-58 had no effect on receptor binding or stimulation. However, substitution of hFSH beta residues 88-108 in place of the carboxyl terminus of hCG beta (residues 94-145) resulted in a hormone analog identical to hFSH in its ability to bind and stimulate FSH receptors. The altered binding specificity displayed by this analog is not attributable solely to the replacement of hCG beta residues 108-145 or substitution of residues in the "determinant loop" located between hCG beta residues 93 and 100.

Amino Acid Sequence↗
PubMed1990

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.

Amino Acid Sequence↗
PubMed1990

Localization of residues that confer antibody binding specificity using human chorionic gonadotropin/luteinizing hormone beta subunit chimeras and mutants.

The glycoprotein hormones are a family of conserved heterodimeric proteins which share a common alpha subunit but differ in their hormone-specific beta subunits. We used chimeras of human chorionic gonadotropin (hCG) and luteinizing hormone (hLH) beta subunits to identify residues which enable monoclonal antibodies (mAb) to distinguish the two hormones. The LH beta-CG beta chimeras appeared to fold similar to hCG beta, since they combined with hCG alpha and, depending on their sequences, were recognized by hCG-selective mAbs. Amino acid residues Arg8-Arg10,Gly47-Ala51, and Gln89-Leu92 form a major epitope region and appear to be adjacent to each other on the surface of hCG beta. Gly47-Ala51 and Gln89-Leu92 are recognized by dimer-specific mAbs while Arg8-Arg10 is recognized by mAbs which have highest affinity for the free beta subunit. These observations suggest that the conformation of this region of the beta subunit changes when the alpha and beta subunits combine. Residues which are C-terminal of Asp112 form a second epitope domain. mAbs to the third domain distinguish hCG beta and hLH beta by the presence of Asn77 in hCG beta and can be detected after hCG binds to receptors. These findings were used to develop a model of hCG beta which predicts the locations of these residues and their positions relative to the alpha subunit and receptor interfaces.

Amino Acid Sequence↗
PubMed1988

Bioimmunoassay (BIA): a sandwich immunoassay scheme employing monoclonal antibodies and hormone receptors to quantify analytes.

When some antigens bind to receptors, a portion of the antigen remains exposed and can be recognized by labeled monoclonal antibodies. By measuring the amount of antibody bound to the antigen-receptor complex, one can quantify the amount of antigen that is present. Since this assay procedure depends on simultaneous receptor recognition of a biologically active site and antibody recognition of a distal epitope on the analyte, we call it a bioimmunoassay. Bioimmunoassays have many of the advantages of radioligand receptor assays (RRA) used to quantify biological activity and, depending on the choice of antibodies employed, may be more specific than RRA. In addition, since they are sandwich assays, they are usually more sensitive than RRA. Bioimmunoassays can be performed in several different modes and in the case described here we used a radiolabeled antibody to detect hormone-receptor complexes. Hence we term this example a bio-immunoradiometric assay or BIO-IRMA. We illustrate the properties of various assay procedures using a monoclonal antibody to the beta subunit of hCG which recognizes an epitope common to all other mammalian LH/hCG-like gonadotropins and which is capable of detecting 10 pg of hCG standard. In principle, this assay can be applied to any material capable of binding to a receptor, enzyme, etc. which can also be recognized by an antibody. Since it is a sandwich type of assay, it is subject to the same advantages and limitations of other sandwich assays except that it can be used to discriminate some biologically active and inactive analytes. Monoclonal antibodies which are prepared from spleen cells of animals immunized with antigen-receptor complexes and selected for their ability to bind antigen-receptor complexes should prove most useful for bioimmunoassay procedures.

Animals↗
PubMed1987

Detection of conformational changes in human chorionic gonadotropin upon binding to rat gonadal receptors.

After binding to rat testicular or ovarian luteinizing hormone (LH) receptors, human chorionic gonadotropin (hCG) and mammalian LH can be detected with monoclonal antibodies directed against a conserved epitope on the beta subunit of the hormones. Two such anti-hCG/anti-LH monoclonal antibodies, known as B105 and B110, compete with one another for binding to this epitope region on free and receptor-bound hormone. By comparing the affinities of B105 and B110 for these two forms of hCG, we have detected apparent changes in the structure of the hormone which develop subsequent to receptor binding. Whereas the affinity of B105 for receptor-bound hCG is approximately 10-fold lower than that for free hCG, the affinity of B110 for receptor-bound hCG is nearly 20-fold greater than that for free hCG. Both B105.hCG and B110.hCG complexes bind to the receptor; however, they have approximately 25 and 50% lower affinity than hCG. Thus, although B110 binds better to the form of hCG which is bound to receptors, binding of B110 to hCG does not appear to induce a conformational change in the hormone which facilitates hormone-receptor binding. Consequently, both B105 and B110 partially inhibit binding of hCG to its receptors. Fab fragments of B105 and B110 are as effective as intact B105 and B110 in inhibiting the binding of labeled B105 and B110 to hCG-receptor complexes, suggesting that circular complexes which might be formed by the interaction of divalent antibody, two molecules of hCG, and two membrane-bound receptors or one divalent receptor are not contributing to the affinity of the antibodies for receptor-bound hCG. Alternatively, formation of circular complexes can explain an increase in apparent affinity of B105 for ovine or bovine LH-receptor complexes. Data obtained with B105 suggest either that the structure of the epitope is altered following binding or that a portion of the epitope is partially obscured when hCG binds to the receptor. In contrast, the data obtained using B110 are not explained by models in which steric factors reduce the affinity of the antibody for the hormone-receptor complex. Therefore, as a minimal explanation for these observations, we postulate that the conformation of the B105/B110 epitope region is altered following binding of the hormone to receptors. The nature of the conformational change and its relationship to LH/hCG action is unknown.

Animals↗
PubMed1987

Rat cholesterol side-chain cleavage enzyme (P-450scc): use of a cDNA probe to study the hormonal regulation of P-450scc mRNA levels in ovarian granulosa cells.

A rat ovarian cDNA library was constructed and screened by differential colony hybridization to detect cDNA clones specific for mRNA induced by follicle-stimulating hormone (FSH). The cDNA clone which demonstrated the greatest degree of induction contained a 766-bp insert which was characterized and sequenced. We conclude that this cDNA is specific for the rat gene coding for cholesterol side-chain cleavage enzyme (P-450scc) by virtue of nucleotide sequence homology to the bovine and human P-450scc cDNA sequences. Southern blotting of rat genomic DNA suggests the presence of a single P-450scc gene. Northern blot analysis indicates that P-450scc mRNA is present in steroidogenic tissues (ovary, adrenal, testis), but not in brain, kidney, liver, lung, or heart. The rat P-450scc mRNA is induced by FSH or pregnant mare's serum gonadotropin in ovaries of estrogen-treated immature rats in vivo. In cultured granulosa cells, estradiol treatment alone did not increase P-450scc mRNA levels, but in combination with FSH or 8-Br-cAMP resulted in three- to four-fold increase in this mRNA.

Amino Acid Sequence↗
PubMed1987

Nonreceptor binding of human chorionic gonadotropin (hCG): detection of hCG or a related molecule bound to endometrial tissue during pregnancy using labeled monoclonal antibodies that bind to exposed epitopes on the hormone.

When hCG adsorbs to surfaces, including membranes from tissues that lack specific hCG receptors, it adsorbs with a particular orientation. Some sites on the alpha- and beta-subunits project away from the surface and can be detected with radiolabeled monoclonal antibodies. Other epitopes, which are located on a region on the hormone that presumably contacts the surface, lose their ability to bind antibody. Using antibodies specific for epitopes on hCG which remain exposed and can be detected when the hormone is adsorbed to rat brain homogenates, we found hCG or closely related substances bound to progestational decidual tissues. Immunologically reactive material adsorbed to the decidual tissue increased and decreased in parallel with the serum levels of hCG throughout pregnancy. Binding of labeled monoclonal antibody to substances similar or identical to hCG in other tissues, including placenta and fetal lung, but not red cells, also was identified. Unlike material adsorbed to decidual tissues, receptor-bound hCG was not recognized by any of our alpha-subunit-specific antibodies. This finding suggests either that the adsorbed hormone has a different orientation than receptor-bound hormone or that the adsorbed hormone has dissociated into subunits. These studies represent the first detection of nonreceptor binding of hCG or related molecules to tissues lacking receptors or presumed not to synthesize the hormone. The role of nonreceptor-bound hCG, if any, is unknown. Other than its effects on stimulation of luteal steroidogenesis during early pregnancy, the role of hCG during most of pregnancy has not been determined. Conceivably, the nonreceptor binding we identified is related to a role for hCG in pregnancy that is not associated with an action on the ovarian LH receptor.

Adsorption↗
PubMed1985

Use of the average antibody-antigen bond concept and probability theory to simplify modeling of linear and circular antibody-antigen complex formation.

We illustrate use of a simple approach to describe the equilibrium interactions of mixtures of monoclonal antibodies and antigens. This procedure is based on elementary concepts in probability theory and is readily suited to describing interactions of antibodies and antigens which form circular as well as linear complexes. The method is also suited to describing the inhibitory effects of antibodies which compete for overlapping epitopes and an example is provided to show how the procedure can be used to describe the interactions of antibodies which inhibit circular complex formation. We also outline simple strategies for preparing computer programs to simulate binding of antigens to defined antibody mixtures. The methods described should facilitate design of immunoassay procedures based on the use of defined mixtures of monoclonal antibodies.

Antibodies, Monoclonal↗
PubMed1985

Potential of a quantal response as a mechanism for oscillatory behavior: implications for our concepts of hormonal control mechanisms.

This article describes the potential of a quantal (i.e., all-or-none) response as a model for understanding the interactions between endocrine, paracrine and autocrine hormones. We review the general features of continuous and discontinuous (i.e., oscillating) quantal models including the role of a threshold. In addition, we also describe a few of the many different biochemical mechanisms which may give rise to quantal behavior. One of the more attractive schemes involves the coordinate regulation of opposing biochemical pathways resulting from phosphorylation of hormone receptors and/or rate-limiting enzymes. At least one hormone receptor (i.e., that for insulin) and many rate-limiting enzymes which control the flow of metabolites through a variety of metabolic pathways can be phosphorylated at multiple sites by one or more protein kinases. Phosphorylation may enhance or inhibit the activities of these proteins depending on which sites are modified. Furthermore, since phosphorylation of some sites on a protein may enhance the ability of phosphoprotein phosphatases to dephosphorylate other sites responsible for biological activity of the protein, phosphorylation also has the potential to produce a discontinuous quantal response. Quantal response mechanisms may alter our notions of endocrine regulation. When a quantal response mechanism is applied to a simple negative feedback model similar to that which was originally postulated to explain the interactions between gonadotropin and steroid hormonal levels, the model can account for the oscillations in hormone levels even when the input is constant. Conversely, when a graded mechanism is applied to the same negative feedback model, the model will almost certainly result in constant hormone levels. Further, the model illustrates that small changes in rate constants and thresholds of response, amplification of hormonal signals, and degradation of intermediate regulators can produce large shifts in the output of the system. These may account for the variability in hormonal levels observed in some endocrine systems. Finally, the high sensitivity of the quantal response mechanism accounts for the data which suggest that gonadotropins may play permissive rather than causal roles in regulation of gonadal function. Since increasing evidence suggests that all cells of a given type may not be equal in terms of hormonal responsiveness, measurements of response in single cells over short time periods will be needed before the role of a quantal response can be determined and endocrine regulation will be fully understood.

Animals↗
PubMed1985

Use of antisera to follicle-stimulating hormone (FSH) to detect non-FSH factors in human serum which modulate rat granulosa cell steroidogenesis.

We measured the ability of serum from women to stimulate steroidogenesis in cultured granulosa cells. Serum promoted estradiol and progesterone synthesis in proportion to its FHS content measured by RIA [i.e. serum from postmenopausal women (PM) greater than serum from the midcycle at the time of the gonadotropin surge (MC) greater than serum from the first day of the menstrual cycle (D1) greater than serum from a hypophysectomized woman (AP)]. The FSH activity of these sera was reduced but not eliminated when we included excess antisera to ovine or human FSH in the culture medium (i.e. PM greater than MC greater than D1 greater than AP). These antisera completely neutralized the actions of ovine FSH, human FSH, and menopausal gonadotropin (Pergonal) added to serum. In contrast to the stimulation seen with 5% or lower concentrations of serum in the culture medium, we observed that 10-20% serum inhibited FSH-induced androgen aromatization and progesterone accumulation. The degree of stimulation or inhibition of steroidogenesis depended on the number of granulosa cells added to each culture. High initial cell concentrations inhibited the ability of the cells to respond to either serum or PMSG. In addition to factors which stimulate or inhibit FSH-induced steroidogenesis, human serum contains factors distinct from FSH which cause the cells to flatten and adhere more tightly to the culture dishes. Although progesterone synthesis was increased in cells which had flattened on the surface of the culture dishes, this phenomenon was not a prerequisite for serum-induced steroidogenesis. We conclude that serum contains factors immunologically distinct from FSH, possibly of pituitary origin, which induce granulosa cell steroidogenesis. In addition, serum contains inhibitory substances which block hormone-induced steroidogenesis and which tend to obscure the stimulatory effects of FSH. Detection of both factors depends in part on the number of granulosa cells used to innoculate the cell cultures.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1984

Specificity considerations in cooperative immunoassays.

Mixtures of some monoclonal antibodies form circular antibody-antigen complexes, which facilitates their ability to bind antigen. This effect forms the basis of a potentially very sensitive assay procedure, the cooperative immunoassay (CIA). Unlike other immunoassays, in which binding can be characterized by a simple "binding constant," the binding of antigen by two antibodies in a CIA depends on several binding parameters, including the affinity of each antibody for antigen as well as the tendency of the reactants to form a circular complex. The ability of the CIA to distinguish between two similar molecules depends on the relative affinity of the antibodies for each antigen and on the ability of the antigens to participate in forming a circular complex. To study the binding of antibody mixtures to cross-reacting antigens, we devised a mathematical model to account for all possible antibody-antigen complexes, including those composed of circular complexes; however, we limited this model to the case in which one antibody was adsorbed to a solid phase. We illustrate here both theoretically and experimentally that a mixture of two antibodies in a CIA may have increased or decreased specificity, if circular complexes containing one or two molecules of cross-reacting antigen are formed. We discuss simple practical considerations that can help optimize specificity and sensitivity of solid-phase assays involving two monoclonal antibodies.

Antibodies, Monoclonal↗
PubMed1983

Cooperative immunoassays: ultrasensitive assays with mixed monoclonal antibodies.

Mixtures of certain monoclonal antibodies appear to bind human chorionic gonadotropin in a "cooperative" fashion because they form circular complexes with the hormone. Experiments illustrate how this property might be exploited to develop very sensitive immunoassays for human chorionic gonadotropin or any other antigen. Since the assays are not based on competitive inhibition between radiolabeled and unlabeled antigen, they are much more sensitive than a traditional radioimmunoassay in which either one of the same antibodies is used alone.

Antibodies, Monoclonal↗
PubMed1983

Quantitative explanation for increased affinity shown by mixtures of monoclonal antibodies: importance of a circular complex.

Mixtures of some but not all monoclonal antibodies which bind to separate epitopes on human chorionic gonadotropin (hCG) show an increased affinity for the hormone. To find an explanation for the increase in affinity, we developed a mathematical model which predicts the quantities of intermediates formed when pairs of IgG1 mouse monoclonal antibodies having affinities of approximately 10(8) M-1 for hCG are mixed with the hormone. At low antibody concentrations (i.e. less than 1 nM or 0.15 micrograms/ml) analysis of possible antibody-hormone combinations, including linear and circular chains composed of less than 12 molecules of antibody and 12 molecules of hCG, suggests the increase in affinity is due to formation of a circular complex containing two molecules of antibody and two of hCG. Further, the model predicts that the circular complex will be the major species formed at antibody-antigen equivalence. This prediction is supported by experimental observations on the molecular weight of a new complex formed in the presence of hCG and the mixture of the monoclonal antibodies. In addition, based on experimental values of binding constants for individual antibodies to hCG, the model correctly quantifies the loss in complex observed in the presence of excess hCG antigen. At high antibody concentrations (i.e. greater than 10 nM or 1.5 micrograms/ml) the formation of linear chains of antibody hCG pairs becomes appreciable and contributes to the increase in apparent affinity of the mixture for hCG. These results suggest that the observed affinity of complex mixtures of antibody for antigens containing multiple epitopes calculated from Scatchard plots may not be related to the affinity or avidity of any of the antibody species for a given epitope.

Antibodies, Monoclonal↗
PubMed1983

A circular antibody-antigen complex is responsible for increased affinity shown by mixtures of monoclonal antibodies to human chorionic gonadotropin.

Mixtures of some pairs of monoclonal antibodies that have separate epitopes on the beta-subunit of hCG have increased affinity for the hormone relative to that of either antibody alone. A mathematical model developed to explain the phenomenon predicted that a circular tetrameric complex composed of each antibody and two molecules of hCG was responsible for the effect. This structure has now been identified experimentally by the following criteria: 1) the m.w. of the complex observed by electrophoresis (370,000 g/mol) and gel filtration (440,000 g/mol) was in agreement with the m.w. expected for a tetramer composed of two molecules of antibody and two molecules of hCG (i.e., 376,000 g/mol); 2) the ratio of individual antibodies to hCG measured with the use of 131I and 125I-labeled antibodies and/or hCG was 1:1:2; and 3) the complex failed to adhere to affinity columns containing either antibodies or hCG covalently coupled to Sepharose. These columns adsorbed B101, B102, hCG, and mixtures of B101 plus hCG or B102 plus hCG. The observations made with the affinity resins are compatible with a circular model for antigen-antibody complex in which the epitopes of the antigen and the binding site of the antibodies were mutually and completely obscured. Although not studied in detail, a similar complex was formed when the beta-subunit of hCG was substituted for the intact hormone. In addition, a mixture of antibodies that bound to the alpha- and beta-subunits of hCG (i.e., A102 and B102) and that had a higher affinity for the hormone than either antibody also gave rise to a similar species that could be detected after electrophoresis. A pair of antibodies that bind to separate epitopes on the beta-subunit (i.e., B101 and B103) and do not show enhanced affinity for hCG failed to form a stable complex that could be identified as a separate species after electrophoresis. Thus, the studies reported here confirm earlier theoretical predictions linking the increase in affinity observed on mixing monoclonal antibodies to the formation of a circular complex.

Antibodies, Monoclonal↗