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F Libert

Publications and source records attributed to F Libert.

At least 19 recordsLinked to original sources
PubMed1992

Cloning and functional characterization of a human A1 adenosine receptor.

A human brain hippocampus cDNA library was screened by hybridization with a dog A1 adenosine receptor cDNA probe. Sequencing of the resulting clones identified a 978 residue open reading frame encoding a 326 amino acid polypeptide showing 95.7% similarity with the dog A1 adenosine receptor. Individual clones of stably transfected CHO cells expressing the human A1 receptor were obtained and tested for their response to the A1 agonist CPA [N6-cyclopentyladenosine] in the presence of forskolin. One clone was further characterized with respect to membrane binding of various adenosine agonists and antagonists. The rank order of affinities observed was typical of an A1 adenosine receptor. A Kd value of 2.28 nM was determined using [3H]DPCPX [dipropylcyclopentyl-xanthine], an A1 selective antagonist.

Adenosine↗
PubMed1992

Expression of members of the putative olfactory receptor gene family in mammalian germ cells.

A series of genomic and complementary DNA clones encoding new putative members of G protein-coupled receptors were isolated using homology cloning and low-stringency polymerase chain reaction. Among the unidentified receptors ('orphan receptors'), a human genomic clone (HGMP07) was characterized by the presence of its transcripts in the testis and by its belonging to a large subfamily of genes sharing extensive sequence similarities. Sequence comparison demonstrated that this gene subfamily is the human counterpart of the putative rat olfactory receptors cloned recently. Another 48 members of the family were cloned. Northern blotting further demonstrated the presence of olfactory receptor transcripts in germ cells. Our finding suggests that a common receptor gene family encodes olfactory receptors and sperm cell receptors that could be involved in chemotaxis during fertilization.

Amino Acid Sequence↗
PubMed1991

The orphan receptor cDNA RDC4 encodes a 5-HT1D serotonin receptor.

The cDNA of RDC4, a putative receptor of the G protein-coupled receptor family, has been cloned by PCR methodology. The primary structure of this receptor showed homology with the serotonin 5-HT1A receptor. In this work, RDC4 mRNA has been injected in Y1 adrenal cells and Xenopus oocytes and RDC4 cDNA has been transfected transiently in cos-7 cells. In all these systems serotonin elicited a rise in cyclic AMP levels. Binding studies on membranes of the transfected cos-7 cells using [3H]-LSD showed a pattern of drug affinities consistent with the known properties of a 5-HT1D receptor. RDC4 therefore codes for a 5-HT1D receptor which in the studied systems is positively coupled to adenylate cyclase.

8-Hydroxy-2-(di-n-propylamino)tetralin↗
PubMed1991

Distribution of adenosine A2 receptor mRNA in the human brain.

The distribution of the messenger RNA coding for the recently cloned adenosine A2 receptor was studied in the human brain using in situ hybridization histochemistry. A2 receptor mRNA is exclusively detected in the medium-sized neurons of the caudate, putamen and accumbens nuclei but not elsewhere in the brain. This highly selective distribution of adenosine A2 receptor mRNA in human dorsal and ventral striatum, similar to that of adenosine A2 binding sites reported in rodents, suggests a major role in the basal ganglia physiology.

Adult↗
PubMed1991

The orphan receptor cDNA RDC7 encodes an A1 adenosine receptor.

The extensive amino acid sequence conservation among G protein-coupled receptors has been exploited to clone new members of this large family by homology screening or by PCR. Out of four such receptor cDNAs we cloned recently, RDC7 corresponds to a relatively abundant transcript in the brain cortex, the thyroid follicular cell and the testis. We have now identified RDC7 as an A1 adenosine receptor. The A1 agonist CPA [N6-cyclopentyladenosine] decreased by 80% cAMP accumulation in forskolin-stimulated CHO cells stably transfected with RDC7. Specific binding of another A1 adenosine agonist, [3H]CHA [N6-cyclohexyladenosine], was demonstrated on membranes from Cos cells transfected with a pSVL construct harbouring the RDC7 cDNA insert. The binding characteristics were similar to those of the natural brain A1 receptor. The recombinant and the natural receptors behaved also in the same way in displacement experiments involving a series of A1 adenosine agonists. The binding characteristics of RDC7 were compared to those of RDC8, another orphan receptor recently identified as an A2 adenosine receptor. The two molecular species RDC7 and RDC8 correspond clearly to the A1 and A2 receptor entities defined hitherto on a purely pharmacological basis.

Adenosine↗
PubMed1991

Chromosomal mapping of A1 and A2 adenosine receptors, VIP receptor, and a new subtype of serotonin receptor.

cDNA clones encoding four new receptors of the G-protein-coupled receptor family were obtained by selective amplification and cloning from thyroid cDNA and termed RDC1, RDC4, RDC7, and RDC8. RDC7 and RDC8 have recently been identified as A1 and A2 adenosine receptors, respectively. These cDNAs were utilized for chromosomal in situ hybridization to establish the genomic location of the corresponding genes in man. The results indicate that human RDC1, RDC4, RDC7, and RDC8 are in regions 2q37, 1p34.3-1p36.3, 22q11.2-22q13.1, and 11q11-11q13, respectively.

Chromosome Banding↗
PubMed1991

Current developments in G-protein-coupled receptors.

The rate at which receptors have been cloned has recently increased dramatically--existing families have been extended and new families created. The rapid cloning by homology of 'orphan receptors' has also stimulated the development of a new reverse pharmacology.

Animals↗
PubMed1991

Thyroperoxidase, but not the thyrotropin receptor, contains sequential epitopes recognized by autoantibodies in recombinant peptides expressed in the pUEX vector.

The sequential epitopes on the human thyroperoxidase (TPO) recognized by antibodies in the sera of patients with autoimmune thyroid disease were investigated using a recombinant DNA technique. Previous studies led to the isolation of two overlapping cDNA clones that encode polypeptides of TPO (85 residues, C2; 100 residues, C21) recognized by sera from several patients with autoimmune disease that contained antimicrosomal autoantibodies. In this report the vector pUEX1 was used to clone and express small random fragments of TPO cDNA in Escherichia coli as a beta-galactosidase fusion protein. Colonies were screened with a serum from a patient with Hashimoto's thyroiditis, and immunoreactive peptides were identified by sequencing the corresponding DNA inserts. Two linear epitopes of human TPO (amino acids 590-622 and 710-722) were recognized by the autoantibodies. This confirmed our previous results and provide a more precise localization of the antigenic determinants involved. The same approach has been applied in an attempt to identify the binding site(s) for autoantibodies on the human TSH receptor. In contrast to the data obtained with TPO, sera from patients with blocking (from idiopathic myxoedema) or stimulating (from Graves' disease) activity did not recognize the linear TSH receptor peptide fragments generated in our libraries.

Amino Acid Sequence↗
PubMed1990

RDC8 codes for an adenosine A2 receptor with physiological constitutive activity.

The cDNA of an unidentified recently cloned G protein-coupled receptor, RDC8, has been expressed in Y1 adrenal cells, in dog thyrocytes in primary culture and in Xenopus oocytes. In all these systems this resulted in the activation of adenylyl cyclase and of the cyclic AMP cascade in the absence of any added external signal. However, this physiologically constitutive activator was inhibited by adenosine deaminase and by inhibitors of the adenosine A2 receptor. Cos 7 cells transfected with RDC8 cDNA constructs acquired binding characteristics of an adenosine A2 receptor. Moreover, RDC8 mRNA and adenosine A2 receptors display a very similar distribution in the brain. RDC8 therefore codes for an A2 adenosine receptor. Whether the physiologically constitutive activation of this receptor is entirely explained by endogeneously produced adenosine is as yet unknown.

Adenosine↗
PubMed1990

Use of the recombinant human thyrotropin receptor (TSH-R) expressed in mammalian cell lines to assay TSH-R autoantibodies.

We report on two assays for autoantibodies to the TSH-R which have been developed using materials from mammalian cells transfected with the cDNA for the human TSH-R. In the first, a particulate fraction has been prepared from COS cells, transiently expressing the human TSH-R and used in a radioreceptor assay in conjunction with bovine 125I-TSH. Immunoglobulins (IgGs) from patients with Graves' disease (n = 11) and idiopathic myxoedema (n = 2) have been used as competitors of 125I-TSH binding to the COS TSH-R membranes and the results have been compared with those obtained with a commercially available kit for measuring TSH-R autoantibodies, which uses solubilised porcine TSH-R. Both assays showed similar performance, being particularly sensitive to antibodies from patients with idiopathic myxoedema. In the second assay system we have used a CHO cloned cell line (JP26) stably transfected with the human TSH-R. A selection of IgG preparations from patients with Graves' disease and of six normal controls was used to test the ability of this cell line to detect thyroid stimulating immunoglobulins (TSAb) by increasing its cAMP production. The assay was performed under two conditions: in standard (isotonic) medium or in hypotonic medium. Freshly thawed human thyrocytes incubated in hypotonic medium served as a reference method. Only five patients scored positive when tested in the JP26 cell line under isotonic conditions. When the assay was performed in a hypotonic medium, a significant positive correlation was observed between the results given by JP26 cells and human thyrocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Stable expression of the human TSH receptor in CHO cells and characterization of differentially expressing clones.

The human thyrotropin receptor cDNA was transfected in CHO cells and individual clones were isolated. They were tested for their response to thyrotropin, forskolin and antibodies from a patient with high levels of thyroid stimulating antibodies. Several clones were characterized extensively with respect to membrane binding of labeled thyrotropin, cAMP accumulation in response to thyrotropin and kinetics of cAMP production. Data for three representative clones are presented. Receptor number as assessed by membrane binding of labeled thyrotropin, and cAMP production, measured in a thyrotropin response bioassay, are correlated. The Kd value for the human thyrotropin receptor expressed in CHO was estimated to be 50 pM.

Animals↗
PubMed1990

A cloned G protein-coupled protein with a distribution restricted to striatal medium-sized neurons. Possible relationship with D1 dopamine receptor.

RDC8, a recently cloned new putative member of the G protein-coupled receptor family, is exclusively present in the medium-sized neurons of the dorsal and ventral striatum in the rat and dog brains. The existence of a striatum-restricted putative G protein-coupled receptor is of major importance for the understanding of basal ganglia physiology and degenerative diseases pathogeny such as Huntington's and Parkinson's disease. This striatal restricted localization mimics the major striatal dopamine D1 receptor localization. RDC7, another putative G protein-coupled receptor presenting a close sequence similarity with RDC8, is mainly distributed in the cerebral cortex, the amygdala, the hippocampus and the claustrum. This localization is also compatible with that expected from a subtype of dopamine D1 receptors.

Animals↗