James S. Davidson

Reversible inhibition of intercellular junctional communication by glycyrrhetinic acid

Intercellular gap-junctional communication was measured using metabolic co-operation in co-cultures of argininosuccinate synthetase-deficient and argininosuccinate lyase-deficient human fibroblasts. 18-alpha-glycyrrhetinic acid (AGA) was found to inhibit communication by more than 95% at concentrations as low as 2 microM. Concentrations up to 100 microM were not cytotoxic over a period of 2 hours. Communication inhibition was of rapid onset and was readily reversible. Communication remained continuously yet reversibly blocked in cells cultured in the presence of AGA for 20 days. The related compounds 18-beta-glycyrrhetinic acid and carbenoxolone also caused communication inhibition. The effect is probably not mediated via mineralocorticoid or glucocorticoid receptors since aldosterone and glucocorticoids had no effect on communication. AGA thus has properties of a useful inhibitor in the study of intercellular junctional communication.

Cloning and characterization of the human GnRH receptor

A cDNA encoding the human GnRH receptor (GnRHR) has been cloned and functionally expressed in both Xenopus oocytes and COS-1 cells. The 2160 bp cDNA encodes a 328 amino acid protein with a predicted amino acid sequence that is 90% identical to that of the mouse GnRHR (Tsutsumi et al. (1992) Mol. Endocrinol. 6, 1163-1169). Injection of synthetic RNA transcript into oocytes led to the development of a depolarizing response to agonists when assayed by voltage-clamp electrophysiology. Consistent with the expression of a mammalian GnRHR, the response was blocked by GnRH antagonists. Following expression of the human GnRHR in COS-1 cells, agonists and an antagonist displaced [125I]GnRH agonist from membrane isolates with nanomolar range dissociation constants similar to those described for displacement from human pituitary membranes. Transfected COS-1 cells manifested a GnRH-stimulated increase in phosphoinositol turnover, with an EC50 of approximately 3 nM, which was inhibited by GnRH antagonists. Northern blot analysis revealed a single band of approximately 4.7 kb expressed in human pituitary which was not detected in testis. The predicted structure of the human GnRHR is similar to that previously reported for the mouse receptor. Although the mammalian GnRHR is a seven transmembrane domain receptor, it differs from other G-protein coupled receptors in several respects, most notably the lack of a cytoplasmic C-terminal domain. The present study demonstrates that the cDNA isolated encodes the human GnRHR and suggests that several unique features conserved among mammalian GnRHRs may be essential for receptor function and/or regulatory control.

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.

Sclerosteosis - an autosomal recessive disorder.

Sclerosteosis is a rare, potentially lethal skeletal disorder in which massive bony overgrowth leads to facial distortion, cranial nerve compression and progressive rise in intra‐cranial pressure. Gigantism and syndactyly of the 2nd and 3rd fingers are associated features. In a nationwide investigation in South Africa, 25 affected individuals in 15 Afrikaner kindreds have been studied. The minimum prevalence of the condition in this community is 1 in 75,000. Analysis of pedigree data confirms that sclerosteosis is an autosomal recessive condition. The gene frequency in the Afrikaner people is estimated at 0.0035, with 10,000 clinically normal heterozygotes in this population. Heterozygote detection may be possible on a basis of recognition of minor changes which are apparent on skull radiographs.

A new mutation, R563Q, of the beta subunit of the epithelial sodium channel associated with low-renin, low-aldosterone hypertension.

Objective To determine the relationship between R563Q, a mutation of the renal epithelial sodium channel, and hypertension. Methods Hypertensive patients with low renin and aldosterone, hypokalemia or resistant hypertension were selected for DNA analysis. Genomic DNA encoding the C-terminal domain of the epithelial sodium channel beta subunit from hypertensives and controls was amplified by polymerase chain reaction and screened for the R563Q mutation by digestion with Sfc1 restriction enzyme, or sequenced. Results A previously undescribed mutation, R563Q, of the beta epithelial sodium channel was found in 10 of 139 black hypertensives, but was not present in any of 103 black normotensives, a significant (P = 0.0058) difference in frequency. The frequency of the mutation in the subgroup of black low-renin, low-aldosterone hypertensives (four of 14) was significantly (P = 0.0001) greater than in normotensives, and was also greater (P = 0.041) than in normal-high renin hypertensives, suggesting that R563Q is an activating mutation of the epithelial sodium channel. R563Q was also found in seven out of 250 mixed ancestry hypertensives, and was significantly (P = 0.017) associated with low-renin, low-aldosterone hypertension in this population group. The mutation was found in one of 100 mixed ancestry normotensives but not in any of 136 white hypertensives. Of the 18 R563Q patients, 11 had severe hypertension, leading to renal failure in two cases, while only two had hypokalaemia. Conclusions R563Q, a new variant of the beta epithelial sodium channel, is associated with low-renin, low-aldosterone hypertension, in South African black and mixed-ancestry patients. Only a minority of individuals with the R563Q allelle fully express the Liddles syndrome phenotype.

[8] Ligand binding and second-messenger assays for cloned Gq/G11-coupled neuropeptide receptors: The GnRH receptor

Publisher Summary This chapter discusses ligand binding and second-messenger assays for cloned Gq/G11-coupled neuropeptide receptors. The identification and characterization of cloned G protein-coupled neuropeptide receptors require methodology for monitoring ligand binding and coupling to intracellular signaling pathways in transfected cells. The availability of both monitoring systems is a prerequisite for mutagenesis studies directed at the molecular dissection of ligand-binding sites, molecular switches mediating agonist activation of the receptor, and domains involved in G protein binding and activation for signal transduction. Neuropeptidergic neurons and their targets in the central and peripheral nervous systems represent a major regulatory component, which is even more prevalent than classical biogenic amine neurons and their targets. Neuropeptides also encompass a far more diverse array of molecular signaling structures, whose importance in the regulation of neural and endocrine pathways is reflected in the increasing quest for neuropeptide analogs by the pharmaceutical industry. The essential components of a neuropeptide receptor assay are a high specific activity and high-affinity radiolabeled ligand, a relatively rich source of receptor (membrane-associated or solubilized), and a method for separating bound and free ligand.

Extracellular adenosine triphosphate activates phospholipase C and mobilizes intracellular calcium in primary cultures of sheep anterior pituitary cells

In primary cultures of sheep anterior pituitary cells extracellular ATP (ED50 0.4–0.8 μM) stimulated efflux of 45Ca2+ from a slow‐turnover intracellular pool. ADP was also effective whereas AMP and adenosine were not. The ATP effect was not due to cell permeabilization as 100 μM ATP did not elicit efflux of 2‐deoxy[3H]glucose metabolites. This 45Ca2+ mobilization may be mediated by inositol trisphosphate, since ATP (ED50 1 μM) stimulated inositol phosphate generation. These results demonstrate P2‐purinoceptors in sheep anterior pituitary cells which are coupled to phospholipase C activation and intracellular Ca2+ mobilization, and raise the possibility of a regulatory role for extracellular ATP in the anterior pituitary.

The initial phase of GnRH-stimulated LH release from pituitary cells is independent of calcium entry through voltage-gated channels

Kinetic studies on gonadotropin‐releasing hormone (GnRH)‐stimulated luteinizing hormone (LH) release were undertaken using rat and chicken pituitary cell cultures. In response to continuous GnRH stimulation, a biphasic pattern of LH release was demonstrated. The two phases showed different susceptibility to the voltage‐gated Ca2+ channel blockers D600 and nifedipine. The first (transient) phase of LH release was unaffected by the Ca2+ channel blockers whereas the second (sustained) phase was inhibited by both drugs. These results indicate that the initial phase of LH release is independent of Ca2+ entry through voltage‐gated Ca2+ channels and may depend on mobilisation of intracellular Ca2+ or entry of extracellular Ca2+ through another mechanism.

Incorporation of an additional glycosylation site enhances expression of functional human gonadotropin-releasing hormone receptor

Mutation ofN-glycosylation sites in the mouse gonadotropin-releasing hormone receptor was previously shown to impair its expression in COS-1 cells. We therefore investigated the effects of adding an extra glycosylation site to the human gonadotropin-releasing hormone receptor, as a means for increasing its expression. Covalent labeling of the mutant receptor expressed in COS-1 cells with a gonadotropin-releasing hormone (GnRH) photoreactive analog demonstrated a shift in apparent molecular weight, indicating that the new site was in fact glycosylated. The receptor with extra glycosylation site displayed normal binding affinities for agonists buserelin and [d-Ala6-Pro9-NHEt]-GnRH, and the antagonist antide, and a slightly increased affinity for GnRH. Receptor number was increased by 1.7-fold in membrane preparations from cells expressing the mutant receptor, compared with wild-type. Photoaffinity labeling of cell-surface receptors in intact cells demonstrated a 1.8-fold increase in binding sites on the cell surface. The GnRH receptor (GnRHR) with extra glycosylation site conferred a markedly enhanced signaling response to agonist. Dose-response curves for GnRH-stimulated inositol phosphate production were left-shifted by an average of 4.4-fold, and maximal inositol phosphate responses were increased by 1.2 fold, in cells transfected with mutant compared with wild-type receptor, indicating that the increase in binding sites represented functional receptors. These results demonstrate that addition of an extra glycosylation site enhances expression of the human GnRHR, a strategy that may be applicable to other cell-surface receptors.

Inhibition of pituitary hormone exocytosis by a synthetic peptide related to the rab effector domain

GTP‐binding proteins of the rab family are believed to function at several steps in intracellular vesicular transport. We examined the effects of a rab‐related peptide in permeabilized pituitary cells, in which exocytosis can be triggered by distinct Ca2+‐dependent or Ca2+‐independent pathways. We report that a synthetic peptide of 18 amino acids related to the rab effector domain, rab3AL(30–47) inhibited luteinizing hormone (LH) and growth hormone (GH) exocytosis triggered by either pathway. Ca2+‐stimulated LH and GH release were inhibited by more than 80% and 50%, respectively, by 100 μM peptide. The peptide (100 μM) also inhibited LH and GH exocytosis stimulated by phorbol myristate acetate plus cAMP by more than 45% and 80%, respectively. The effect was sequence‐specific since a second peptide, lacking the first 3 amino acids but otherwise identical failed to inhibit exocytosis. These results suggest that a protein of the rab family is involved in regulated pituitary hormone exocytosis, and they identify 3 amino acids of the putative rab effector domain which may be functionally important in exocytosis.