Gonadotropin-releasing hormone binding sites in human breast carcinoma.

Gonadotropin-releasing hormone analogs can cause regression of hormone-dependent breast carcinomas. These effects are thought to be mediated through the inhibition of gonadotropic and steroid hormones. These analogs may also act directly on the tumor because they are effective in treating breast cancer in some postmenopausal women. The presence of specific binding sites for gonadotropin-releasing hormone was demonstrated in human breast carcinomas by means of a novel approach of ligand immunoblotting. The results indicate a possible mechanism by which the peptide has direct effects on this tissue. These binding proteins were not detectable in non-neoplastic breast tissue.

Phosphatidylinositol 4-kinase: gene structure and requirement for yeast cell viability.

Phosphatidylinositol (PtdIns) 4-kinase catalyzes the first step in the biosynthesis of PtdIns-4,5-bisphosphate (PtdIns[4,5]P2). Hydrolysis of PtdIns[4,5]P2 in response to extracellular stimuli is thought to initiate intracellular signaling cascades that modulate cell proliferation and differentiation. The PIK1 gene encoding a PtdIns 4-kinase from the yeast Saccharomyces cerevisiae was isolated by polymerase chain reaction (PCR) with oligonucleotides based on the sequence of peptides derived from the purified enzyme. The sequence of the PIK1 gene product bears similarities to that of PtdIns 3-kinases from mammals (p110) and yeast (Vps34p). Expression of PIK1 from a multicopy plasmid elevated PtdIns 4-kinase activity and enhanced the response to mating pheromone. A pik1 null mutant was inviable, indicating that PtdIns4P and presumably PtdIns[4,5]P2 are indispensable phospholipids.

Genetics of ncHH: from a peculiar inheritance of a novel GNRHR mutation to a comprehensive review of the literature.

BACKGROUND: Normosmic congenital hypogonadotropic hypogonadism (ncHH) is caused by the deficient production, secretion, or action of gonadotropin-releasing hormone (GnRH). Its typical clinical manifestation is delayed puberty and azoospermia. Homozygous and compound heterozygous mutations in the GNRHR gene (4q13.2) are the most frequent genetic causes of ncHH. OBJECTIVES: (i) Characterization at the molecular level (genetic origin and functional effect) of a unique homozygous mutation (p.Gly99Glu) in a ncHH man; (ii) to provide a comprehensive catalog of GNRHR mutations with genotype-phenotype correlation and comparison of in vitro studies vs. in silico prediction tools. MATERIAL AND METHODS: A ncHH man and his parents, in whom we performed the following: (i) Sanger sequencing, qPCR of the GNRHR gene; (ii) chromosome 4 SNP array; and (iii) competition binding assay and inositol phosphate signaling assay. PubMed and Human Genome Mutation Database (HGMD) search for GNRHR mutations. Bioinformatic analysis of 55 reported variants. RESULTS: qPCR showed two GNRHR copies in the index case. SNP array revealed the inheritance of two homologous chromosomes 4 from the mother (maternal heterodisomy; hUPD) with two loss of heterozygosity regions, one of them containing the mutated gene (maternal isodisomy; iUPD). Functional studies for the p.Gly99Glu mutation demonstrated a right-shifted GnRH-stimulated signaling response. Bioinformatic tools show that commonly used in silico tools are poor predictors of the function of ncHH-associated GNRHR variants. DISCUSSION: Functional analysis of the p.Gly99Glu mutation is consistent with severely decreased GnRH binding affinity (a severe partial loss-of-function mutation). Complete LOF variants are associated with severe and severe/moderate phenotype, whereas partial LOF variants show wide range of clinical manifestations. CONCLUSION: This is the first ncHH patient carrying a novel causative missense mutation of GNRHR with proven 'severe pLOF' due to maternal hUPD/iUPD of chromosome 4. Our literature review shows that functional studies remain essential both for diagnostic and potential therapeutic purposes.

Insertional mutagenesis of the arginine cage domain of the gonadotropin-releasing hormone receptor.

The pattern of side-chain conservation at the cytoplasmic side of the third transmembrane domain of rhodopsin family G protein-coupled receptors, Asp/Glu-Arg-Tyr/X-X-X-Ile/Val, defines a structural "arginine cage" domain. Previous computational and mutagenesis studies of the GnRH receptor indicated an important contribution of local interactions to the function of this domain. We have investigated the functional importance of the intrahelical position and orientation of the arginine cage using insertional mutagenesis. Introduction of a single Ala proximal to the conserved Asp-Arg of this domain caused loss of detectable ligand binding. Inserting a second Ala, however, restored high-affinity agonist binding. Further insertion of three or four Ala residues at this site generated receptors that bound agonist with an affinity 3- to 10-fold higher than that of the wild-type receptor. Loss of detectable coupling to inositol phosphate turnover in all these mutant receptors confirms that the structure required in this region for efficient signaling is highly constrained. In contrast, the recovery of agonist binding with the progressive insertion of two to four Ala residues indicates that specific orientations of this segment can stabilize high-affinity receptor conformations that are uncoupled from signal transduction.

Cloning and functional expression of a mouse gonadotropin-releasing hormone receptor.

GnRH plays a pivotal role in the reproductive system, and GnRH analogs have wide therapeutic applications ranging from the treatment of prostatic cancer to infertility. Determination of the predicted structure of the GnRH receptor (GnRHR) would illuminate the mechanisms of receptor activation and regulation and allow directed design of improved GnRH analogs. We report the cloning of a cDNA representing the mouse GnRHR and confirm its identity using Xenopus oocyte expression. Injection of sense RNA transcript leads to the expression of a functional, high affinity GnRHR. Expression of the GnRHR using gonadotrope cell line RNA, however, is blocked by an antisense oligonucleotide. In situ hybridization in the rat anterior pituitary reveals a characteristic GnRHR distribution. The nucleotide sequence encodes a 327-amino acid protein which has the seven putative transmembrane domains characteristic of G protein-coupled receptors, but which lacks a typical intracellular C-terminus. The unusual structure and novel potential regulatory domain of the GnRHR may explain unique aspects of its signal transduction and regulation.

A high affinity gonadotropin-releasing hormone (GnRH) tracer, radioiodinated at position 6, facilitates analysis of mutant GnRH receptors.

Cloning of GnRH receptors from several animal species has made it possible to investigate receptor function using site-directed mutagenesis. However, many mutant GnRH receptors exhibit decreased ligand binding, which makes analysis of their ligand binding characteristics technically difficult. To increase the affinity of binding to the GnRH receptor, a novel tracer ligand, 125I-[His5,D-Tyr6]GnRH, was designed and synthesized to allow radioiodination at position 6 rather than the usual position 5. In competition binding assays, total binding of 125I-[His5,D-Tyr6]GnRH was higher than binding of a conventional tracer ligand, 125I-[D-Ala6,N-MeLeu7,Pro9NHEt]GnRH. The bindable fractions and specific activities of both peptides were similar, and the receptor binding affinities of the unlabeled peptides were indistinguishable. However, comparison of the radiolabeled peptides in saturation binding assays showed that the affinity of the peptide, 125I-[His5,D-Tyr6]GnRH, (Kd, 0.19 nM), was approximately 2-fold higher than that of the conventional tracer. The increased binding of 125I-[His5,D-Tyr6] GnRH has allowed the development of a sensitive GnRH receptor binding assay for analysis of mutant GnRH receptors that exhibit decreased ligand binding.

Gonadotropin-releasing hormone (GnRH)-binding sites in human breast cancer cell lines and inhibitory effects of GnRH antagonists.

GnRH-binding sites have previously been described in human breast tumors, and a GnRH agonist has been shown to inhibit growth of the MCF-7 human breast cancer cell line. We have investigated the presence of GnRH-binding sites in ZR-75-1, MDA-MB-231, Sk Br 3, MDA-MB-157, and MCF-7 human breast cancer cell lines and the effect of GnRH analogs on the incorporation of [3H]thymidine and 14C-labeled amino acids into DNA and protein. Specific GnRH-binding sites were present in membrane preparations of all five human breast carcinoma cell lines. Studies in three cell lines indicated low affinity (Kd, 1.6-3.0 X 10(-6) M) GnRH binding similar to that reported in human placenta and corpus luteum. In contrast, human pituitary GnRH receptors were of high affinity (Kd, 4.8 X 10(-9) M). Breast carcinoma cell GnRH-binding sites also differed from the pituitary receptor in their inability to discriminate between GnRH and superactive analogs. Binding of a [125I]GnRH analog to ZR-75-1 breast cancer cells and pituitary membranes was affected similarly by various cations. GnRH antagonists rapidly inhibited [3H]thymidine incorporation into DNA (within 3 hr), and this effect was reversible. GnRH antagonists also inhibited cell growth, but only after 6 days. GnRH agonists did not alter either thymidine incorporation or growth. The present observations of low affinity GnRH-binding sites in breast cancer cell lines and inhibitory effects of GnRH antagonists point to the possibility of an autocrine regulatory role of GnRH-like peptides in mammary cells.

Agonist activity of mammalian gonadotropin-releasing antagonists in chicken gonadotropes reflects marked differences in vertebrate gonadotropin-releasing receptors.

The pharmacology of mammalian and avian gonadotropin-releasing (GnRH) receptors differs for agonist analogues. We have therefore compared the activities of mammalian-based GnRH antagonists in sheep and chicken gonadotropes to further elucidate the different structural requirements of the receptors. The antagonist activities of ten GnRH analogues were compared in cultured sheep and chicken pituitary cells by determining the dose required to cause a 50% inhibition of luteinizing hormone secretion (IC50) induced by GnRH at its half-maximal concentration (EC50). Nine analogues showed high antagonist activity in the sheep bioassay. Analogue IC50s varied between half and twice ((1.22-6.06) x 10(-10) M) the GnRH EC50 (3 x 10(-10) M). One of these peptides exhibited partial agonist activity. In contrast, eight of the analogues showed low antagonist activity in chicken pituitary cells, with IC50s varying from 46 to 1490 times ((1.4-44.7) x 10(-7) M) the GnRH EC50 (3 x 10(-9) M) and had a different order of potencies compared with that in the sheep. Furthermore, two analogues did not display antagonist activity at all in the chicken bioassay, but acted as pure agonists, stimulating LH secretion. These findings demonstrate marked differences in pharmacology between the avian and mammalian pituitary GnRH receptors and emphasize that GnRH antagonists, selected for their efficacy in mammals, cannot necessarily be used for physiological studies in non-mammalian vertebrates. The distinctly different pharmacology of the receptors and structural requirements of analogues for agonist/antagonist activity establish a basis for identifying receptor features involved in ligand-induced signal propagation using chimaeras of cloned sheep and chicken receptors.

Identification of N-glycosylation sites in the gonadotropin-releasing hormone receptor: role in receptor expression but not ligand binding.

The asparagine residues of the three N-glycosylation consensus sequences in the mouse gonadotropin-releasing hormone receptor were mutated to determine which residues were glycosylated and the function of glycosylation. Photoaffinity labelled Gln4 and Gln18 receptor mutants exhibited lower apparent molecular weight on SDS polyacrylamide gel electrophoresis, while the Gln102 receptor showed wildtype mobility. This indicates that the receptor is glycosylated at Asn4 and Asn18 but not at Asn102. Binding affinities of all the mutant receptors were normal, indicating that carbohydrate moieties are not involved in ligand binding interactions. However, expression of the Gln4 and Gln18 receptors were substantially decreased, indicating a role for glycosylation in receptor expression or stability. All the glycosylation site mutants were capable of normal signal transduction, as indicated by their ability to stimulate inositol phosphate production.

A new photoreactive antagonist cross-links to the N-terminal domain of the gonadotropin-releasing hormone receptor.

A new photoreactive gonadotropin-releasing hormone (GnRH) antagonist [Ac-(4-azidobenzoyl)-D-Lys1, D-4-Cl-Phe2, D-Trp3, D-Arg6, D-Ala10]GnRH (PAnt-1) was synthesized and shown to bind covalently to mouse and human GnRH receptors after ultraviolet irradiation. PAnt-1 exhibited high binding affinity (Ki = 3.1 +/- 0.8 nM), and high crosslinking efficiency as shown by loss of 78% of binding sites following crosslinking at saturating concentration. Crosslinking resulted in irreversible receptor blockade as shown by inhibition of GnRH-stimulated inositol phosphate production. PAnt-1 has a photoreactive group at residue 1 of the peptide, a region believed to be critical in determining antagonist versus agonist properties of GnRH analogues. The attachment site of PAnt- to the receptor was localized between residues 11 and 19 of the extracellular N-terminal domain of the receptor by peptide mapping studies using natural sequence differences between human, mouse and sheep GnRH receptors, as well as a panel of GnRH receptor constructs with a series of engineered protease cleavage sites. A disulphide bridge between Cys14 and Cys200 was cleaved during crosslinking, suggesting that Cys14 is the crosslinked residue. These results suggest that peptide GnRH antagonists bind to the receptor with the N-terminal end of the peptide positioned in a site comprising the constrained regions of the N-terminal domain and second extracellular loop in the vicinity of the Cys14-Cys200 disulphide bridge.

A single amino acid substitution in transmembrane helix VI results in overexpression of the human GnRH receptor.

OBJECTIVE: Construction of constitutively active mutants of the GnRH receptor, a member of the G-protein coupled receptor superfamily, would facilitate investigation of the mechanism of receptor activation. DESIGN: Point mutations were introduced in the human GnRH receptor in positions corresponding to those which caused constitutive activity in other G-protein coupled receptors. The effects of these mutations on ligand binding, receptor intracellular signaling and receptor expression were determined. METHODS: Wild type and mutated receptor cDNAs were expressed in COS-1 cells. Basal and agonist-stimulated inositol phosphate production and ligand binding were determined. In addition, receptor mRNA levels, cell surface receptor stability and rate of internalization were measured. RESULTS AND CONCLUSIONS: Although none of the mutant receptors exhibited constitutive activity, mutation of Phe-2 72 in transmembrane helix VI to Leu increased cell surface receptor numbers, with unchanged affinities for radiolabeled agonist, superagonist and antagonist peptides compared with wild type receptor. The cell surface receptor stability and rate of internalization were similar for wild type and F272L GnRH receptors. Thus a single amino acid mutation in transmembrane helix VI causes an increase in cell surface receptor numbers, which appears to result from an increased rate of receptor protein translation, processing or insertion into membranes.

Adolescents' explanations for political issues: concordance with their views of self and society.

The relationships between adolescents' explanations for unemployment, poverty, and homelessness and their beliefs about opportunity, reports of family values, and personal aspirations were tested for 434 teenagers (mean age = 16 years 4 months). Explanations were coded for references to individual causes, societal causes, or both. Higher maternal education and average household income in the adolescent's school district were positively related to the likelihood of attributing all three problems to societal causes. When explaining unemployment, older adolescents noted both causes, and boys mentioned individual factors whereas girls mentioned societal factors. After adjustment for background factors, those endorsing individual causes were more likely to believe that all Americans enjoyed equal opportunity and that government support encouraged dependency, and they were more committed to materialist goals. In contrast, youth endorsing societal or situational causes had more altruistic life goals and reported that compassion was emphasized in their families.

Change in family work status: effects on parent-adolescent decision making.

The interactions of parental work status, family integration, and sex of child on parent-adolescent decision making were examined in a 4-wave study of 504 adolescents and their mothers. 3 work status groups were compared. Deprived families reported a layoff or demotion at Time 1 and no recovery by Time 4. Recovery families reported similar work status losses at Time 1 and reemployment by Time 4. Nondeprived families reported stable employment at both times. Adolescents in deprived households, especially boys, reported the highest conflict with parents. Adolescents in recovery families reported high conflict when parents were unemployed, but levels declined when parents were reemployed. According to mothers, daughters in deprived households enjoyed the highest level of autonomy of any adolescent group.

Spatially and temporally regulated expression of the MADS-box gene AGL2 in wild-type and mutant arabidopsis flowers.

AGL2 is one of several Arabidopsis floral MADS-box genes that were isolated based on sequence similarity to the homeotic gene AGAMOUS. To investigate its possible role in flower development, we have characterized in detail the expression pattern of AGL2 in both wild-type and mutant flowers using RNA in situ hybridization. We find that AGL2 is floral-specific; it is not expressed in the inflorescence meristem. Within the floral meristem, AGL2 is first expressed very early in development, after the floral meristem has emerged from the inflorescence meristem but before any of the organ primordia emerge. The AGL2 transcript is very abundant and uniform throughout the floral meristem and in the primordia of all four floral organs: sepals, petals, stamens and carpels. Thus, AGL2 represents a new class of MADS-box genes which is expressed in all four whorls of the flower. The AGL2 transcript remains abundant in each organ during morphological differentiation, but diminishes as each organ undergoes the final maturation phase of development. AGL2 expression is high in developing ovules and, after fertilization, in developing embryos and seed coats, abating as seeds mature. In the floral organ identity mutants ag-1, ap3-3 and ap2-2, the AGL2 expression pattern is organ- and stage-dependent. These results indicate that AGL2 may play a fundamental role in the development of all floral organs, and of seeds and embryos, and that AGL2 ultimately depends upon the organ identity genes for proper expression.

Purification and characterization of a soluble phosphatidylinositol 4-kinase from the yeast Saccharomyces cerevisiae.

A phosphatidylinositol (PI) 4-kinase was purified 25,000-fold from the cytosolic fraction of extracts from the yeast Saccharomyces cerevisiae. The purification consisted of an ammonium sulfate fractionation followed by chromatography on sulfonated-agarose (S-Sepharose), phosphocellulose, threonine-agarose, and quaternary amino (Mono Q), and sulfonated (Mono S) beads. Major contaminants in the purification, Hsc82 and Hsp82 (yeast homologs of the mammalian heat shock protein Hsp90), were eliminated by using a combination of molecular genetics (to construct a null mutation in HSC82), altered growth conditions (to minimize expression from the inducible HSP82 gene), and high ionic strength fractionation conditions (to remove the residual Hsp82). The purified enzyme had an apparent subunit molecular weight of 125,000, much larger than any other well characterized PI-4-kinase reported previously. Like mammalian PI-4-kinases, the yeast enzyme specifically phosphorylated PI on position 4 of the inositol ring and was stimulated by Triton X-100. However, activity was not inhibited by adenosine, a potent inhibitor of certain (type II) mammalian PI-4-kinases. The enzyme displayed typical Michaelis-Menten kinetics with apparent Km values of 100 microM for ATP and 50 microM for PI. To date, this yeast enzyme is the first soluble PI-4-kinase purified from any source.

Changes in parents' work status and adolescents' adjustment at school.

The effects of change in parental work status on early adolescents' school adjustment before and after the transition to junior high school were examined in a 2-year longitudinal study. Data were gathered from 883 adolescents, their mothers, and teachers. Based on patterns of change or stability in parental work status during the 2 years of the study, 4 groups were compared: deprived, declining, recovery, and stable families. With parents' education controlled, teachers said that adolescents in deprived and declining families were less competent than their peers in stable or recovery families. In addition, adolescents whose parents experienced a decline in work status were the most disruptive in junior high school. While most students had difficulty adjusting to junior high school, the transition was particularly difficult for those students whose parents were simultaneously dealing with changes in work status.

Specific expression of the AGL1 MADS-box gene suggests regulatory functions in Arabidopsis gynoecium and ovule development.

Several members of the MADS-box gene family have been shown to be important regulators of flower development, controlling such well-studied early events as the formation of the floral meristem and the specification of floral organ identity. Other floral-specific MADS-box genes, of as yet unknown function, have been isolated by homology and are proposed to be part of a regulatory hierarchy controlling flower development. Some of these genes might regulate later aspects of flower development, such as development of individual floral organs, which is less well studied at the molecular level. This paper presents a detailed analysis of the expression pattern of one such gene from Arabidopsis, AGL1, using RNA in situ hybridization. It is found that AGL1 is specifically expressed in particular regions of the gynoecium and ovule, only during and after floral development stage 7. AGL1 expression at the tip of the growing carpel primordia, along the margins of the ovary valves in developing and mature gynoecia and in specific regions of developing and mature ovules provides important insights into the possible roles of AGL1. It is proposed that AGL1 may have regulatory functions in the structural definition and/or function of the valve margins, in axis maintenance during ovule development, in nutritional supply to the growing ovule and embryo sac, and in pollen tube guidance. In the floral homeotic mutants ag-1, ap3-3 and ap2-2, AGL1 mRNA is expressed in an organ-dependent manner, suggesting that AGL1 is a carpel-specific gene and as such ultimately depends upon the carpel identity gene AG for proper gene expression.

Advances in understanding gonadotrophin-releasing hormone receptor structure and ligand interactions.

Gonadotrophin-releasing hormone (GnRH) is the central regulator of the reproductive system and its analogues are used widely in the treatment of diverse diseases. The GnRH receptor is a member of the large family of G-protein-coupled receptors (GPCRs) which have seven transmembrane domains. Knowledge of these receptors has assisted the development of molecular models of the GnRH receptor that allow prediction of its three-dimensional configuration and the way GnRH binds and activates its receptor. Comparison with other GPCRs led to the discovery that Lys121, in the third transmembrane domain, has a role in agonist binding. The history of GnRH structure-activity studies has allowed the identification of an acidic residue in the third extracellular loop of the receptor that is required for binding of mammalian GnRH, while synthetic GnRH analogues have showed that Asn102, in the second extracellular loop, may interact with the carboxy-terminus of GnRH. These residues can now be incorporated into the receptor models that are being used to design orally active non-peptide GnRH analogues for contraception and treatment of a variety of reproductive disorders.

Chimeric analogues of vertebrate gonadotropin-releasing hormones comprising substitutions of the variant amino acids in positions 5, 7, and 8. Characterization of requirements for receptor binding and gonadotropin release in mammalian and avian pituitary gonadotropes.

Variant forms of mammalian gonadotropin-releasing hormone (GnRH) (pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly.NH2) are present in chicken ([Gln8] GnRH and [His5, Trp7, Tyr8]GnRH), salmon ([Trp7, Leu8]GnRH), and lamprey ([Tyr3, Leu5, Glu6, Trp7, Lys8] GnRH). To delineate the functional importance of the variant amino acids in positions 5, 7, and 8, the natural peptides and chimeric analogues were tested for gonadotropin-releasing activity and receptor-binding activity in rat, sheep, and chicken pituitaries. The results demonstrate that (i) the mammalian receptor has a high fidelity for Arg8 while the chicken receptor is less discriminatory and accepts basic or neutral amino acids in this position. Arg8 may contribute to conformational stabilization, and conformational constraint with D-Trp6 restored activity to analogues lacking Arg8 in the mammalian systems. D-Trp6 incorporation did not generally enhance activity in the chicken pituitary. (ii) His5 accompanying Arg8 in analogues markedly diminished activity in the chicken while gonadotropin-releasing activity was retained in the sheep pituitary. Receptor-binding activity was increased in the sheep indicating an uncoupling of receptor occupancy and activation. (iii) Substitution in position 7 is tolerated by the mammalian and chicken receptor. With Trp7-substituted analogues receptor-binding activity was relatively lower than gonadotropin-releasing activity in the sheep pituitary, suggesting an enhanced receptor activation by these analogues or the existence of different GnRH receptors.