Fu-Zon Chung

Transcriptional downregulation of stromelysin by tetracycline.

We investigated the role of tetracycline in the transcriptional regulation of matrix metalloproteinases. Using interleukin‐1β (IL‐1) induced stromelysin as a model system, we describe the repression of the endogenous stromelysin RNA accumulation, as well as the transcriptional inhibition of various stromelysin promoter/chloramphenicol‐acetyltransferase constructs in transient transfection assays. The inhibition occurred in a dose‐dependent fashion, with an IC50 of about 1 μM. Our results suggest that the transcriptional inhibition by tetracycline is not due to a block of activity of the activating protein complex 1 (AP‐1) but is mediated by sequences upstream of the AP‐1 binding site.

Characterisation of [125I][MePhe7]neurokinin B binding to tachykinin NK3 receptors : evidence for interspecies variance

Human tachykinin NK3 receptors expressed in Chinese hamster ovary (CHO-K1) cells were characterised using the novel radioligand [125I]iodohistidyl,[MePhe7]neurokinin B ([125I][MePhe7]neurokinin B). [125I][MePhe7]neurokinin B was shown to label human NK3 binding sites with high affinity in a saturable and reversible manner. The rank order of affinity of a range of tachykinin ligands confirmed that the tachykinin receptor expressed was the NK3 receptor type. An interspecies comparison of NK3 binding sites revealed pharmacological differences between human, guinea pig and rat tachykinin NK3 receptors. The NK2 selective antagonist SR 48968, inhibited binding of [125I][MePhe7]neurokinin B to NK3 binding sites with Ki values of 287 nM and 205 nM in human and guinea pig respectively, but was > 30-fold less active in the rat.

The Unpredicted High Affinities of a Large Number of Naturally Occurring Tachykinins for Chimeric NK1/NK3 Receptors Suggest a Role for an Inhibitory Domain in Determining Receptor Specificity

Three chimeric receptors were constructed by exchanging exon sequences between human NK1 and NK3 receptor genes. The resulting chimeric receptors not only retained high affinities for their natural ligands substance P and neurokinin B but also exhibited surprisingly high affinities for other naturally occurring tachykinins including neurokinin A, neuropeptide K, neuropeptide γ, eledoisin, kassinin, physalaemin, and phyllomedusin. In contrast, these chimeric receptors displayed a wide range of variability in their affinities for non-naturally occurring ligands including selective agonists and antagonists of NK1, NK2, and NK3 receptors. Since the only common feature among these naturally occurring neurokinin peptides is the conserved C-terminal sequences, our data suggest that these conserved sequences must play the major role in conferring high affinity binding to the chimeric receptors. To explain the apparently “improved” affinities of these naturally occurring ligands for the chimeric receptors as compared with their affinities for the parent NK1 and NK3 receptors, we are proposing that certain inhibitory domains that are present in the NK1 and/or NK3 receptors are compromised in these chimeric receptors. Upon disruption of these inhibitory domains during the formation of chimeras, the naturally occurring ligands can interact more favorably with chimeric receptors through their conserved C-terminal sequences. Based on this hypothesis, the binding affinities of natural tachykinin ligands may be largely determined by their conserved C-terminal sequences, whereas receptor selectivities of these ligands are influenced more by the presence or absence of inhibitory domains rather than specific binding domains on their target receptors.

Characterization of tachykinin mediated increases in [Ca2+]i in Chinese hamster ovary cells expressing human tachykinin NK3 receptors

The nature of the senktide response of the human NK3 receptor expressed in Chinese hamster ovary cells was characterised using the Ca2+ sensitive dye Fura-2 and imaging methods. Application of the NK3 receptor agonist senktide caused an increase in [Ca2+]i in the cells. The profile for NK3 receptor agonists was that senktide was more potent than [beta-Ala8]neurokinin A-(4-10) which was more potent than [Sar9,Met(O2)11]substance P. SR 48968 was a poor antagonist of the senktide response in intact cells confirming the weak affinity of this agent for the NK3 receptor (IC50 of approximately 1 microM) shown in binding assays. The NK3 receptor mediated increase in intracellular Ca2+ was independent of [Ca2+]o, blocked by the microsomal Ca2+ ATPase inhibitor thapsigargin and the phospholipase C inhibitor U73122 but not by ryanodine. Thus the source of the Ca2+ was probably a ryanodine insensitive, inositol triphosphate sensitive intracellular store.

Structural Motifs Encoded by Individual Exons of the Human Neurokinin-1 Receptor Gene Interact Differentially with Selective Agonists and Antagonists

Abstract: Three chimeric receptors were constructed by exchanging exons between human neurokinin NK1 and NK3 receptor genes. The N‐terminal sequences of these chimeric receptors are encoded by exon 1, exon 1–2, or exon 1–3 of the NK1 receptor gene, whereas the remaining C‐terminal sequences of these chimeric receptors are encoded by corresponding exons of the human NK3 receptor gene. Substance P bound with high affinities to all three chimeric receptors, suggesting that in addition to the common structures composed of conserved amino acid residues among neurokinin receptors, structural elements encoded by the first exon of the human NK1 receptor gene may also play an important role for substance P binding. On the contrary, potent NK1 antagonists L703,606 and SR140,333 did not show any detectable binding to these chimeric receptors. In accordance, sequences encoded by exon 4, and possibly exon 5, are likely to contain important structural motifs that may directly or indirectly influence the binding of these antagonists. Further comparison of the binding affinities of highly selective NK1 agonists, [Sar9,Met(O2)11]substance P, substance P methyl ester, and septide, revealed that each agonist may interact differently with the human NK1 receptor. These results show that the exon‐exchanging technique can be a useful tool for studying structure‐function relationships of receptors in which exon‐intron junctions are fully conserved among receptor subtypes.

Cloning, expression and characterization of a human dopamine D402 receptor (CHO K1 cells) and various D4.2/D2L chimeras (COS-7 cells)

Abstract 1. 1. Using the gene splicing technique a synthetic human dopamine (DA) D 4.2 gene was constructed and subsequently stably expressed in CHO K1 cells. 2. 2. Binding of [ 3 H]spiperone to membranes prepared from human DA D 4.2 CHO Kl cells was saturable with a K d of 93 ± 0.51 pM and a B max of 768 ± 22 fmol per mg protein. 3. 3. Clozapine, apomorphine, and S(+)-NPA were more selective for D 4.2 than for D 2L receptors, with c ratios of 5.7, 7.1, and 19.6, respectively. 4. 4. Functional studies indicated that DA D 4.2 receptors expressed in CHO K1 cells inhibited forskolin stimulated cAMP levels showing coupling to G-proteins. 5. 5. Two reciprocal human D 2L and D 4.2 chimeric receptors (D 2L /D 4.2 and D 4.2 /D 2L ) were constructed by exchanging the amino-terminal end to the third transmembrane (TM) of one receptor with the counter part of the other receptor and expressing them transiently into COS-7 cells. The chimeric D 2L /D 4.2 receptor displayed non-detectable specific binding of [ 3 H] spiperone and other ligands. The chimeric D 4.2 /D 2L receptor binding affinities of DA agonists were more affected than that of antagonists, suggesting that binding affinities of agonists are more sensitive to changes in receptor conformation than that of antagonists. 6. 6. This study characterized the pharmacology of a novel synthesized DA D 4.2 receptor that provides a useful model for screening of potential D 4.2 receptor agonist and antagonist .

High Throughput 96-Well Plate Assay for Receptor-Mediated Phosphatidylinositol Turnover

The G-protein coupled receptor family represents a large number of neurotransmitter receptors. Among the diverse signal transduction pathways mediated via G-proteins, phospholipase C mediated phosphatidylinositol hydrolysis represents one of the best characterized signal transduction mechanisms. Accordingly, the measurement of agonist-induced phosphatidylinositol turnover has been used as a convenient functional assay for receptor activation. Assays currently used for this purpose, however, are not suitable for high throughput screening. In this article, an improved technique using 96-well microtiter plate format for measuring phosphatidylinositol turnover is introduced. Anion exchange columns were prepared on fiber glass 96-well multiscreen filter plate. Separation and detection of released inositol phosphates were conducted in a 96-well format. Cells expressing certain neurotransmitter receptors were challenged with agonists and the receptor-mediated PI turnover was measured by the new technique and the results obtained were compared to that obtained from traditional assays. The results indicate that the 96-well assay is 10 to 20 times more efficient than the traditional method and is, furthermore, suitable for high throughput drug screening. Our data also indicate that this method is particularly useful for characterizing multiple antagonists by Schild analysis.

Gene mapping of mouse IL-4 receptor : the loci of interleukin 4 (IL-4) receptor gene and lymphocyte function associated antigen 1 (LFA-1) gene are closely linked on chromosome 7

1 Department of Medicine, School of Medicine, University of Colorado Health Sciences Center, Denver, CO 80262, USA 2 Denver Veterans Administration Medical Center, Denver, CO 80220, USA 3 Department of Biotechnology, Parke-Davis Pharmaceutical Research Division, Warner Lambert Company, Ann Arbor, MI 48105, USA 4 National Jewish Center for Immunology and Respiratory Diseases, Denver, CO 80206, USA 5 The Jackson Laboratory, Bar Harbor, ME 04609, USA

A Screening Strategy Based on Differential Binding of Ligand to Receptor and to Binding Proteins: Screening for Compounds Interacting with Corticotrophin-Releasing Factor-Binding Protein

The ligand-receptor interaction has been commonly used in development of high throughput screening assays for new drugs. In some cases, an endogenous ligand interacts not only with membrane receptors but also with soluble binding proteins. Corticotrophin-releasing factor (CRF) is an important stress neurotransmitter/hormone involved in both the central and peripheral nervous systems. CRF exerts its function by interacting with CRFR1 and CRFR2 receptors. In addition, CRF-binding protein (CRF-BP) binds CRF with high affinity. Accordingly, CRF-BP has been suggested to play an important role in modulating CRF function. Based on the potential involvement of CRF-BP in many neurological disorders, it is desirable to develop a screening assay to look for drugs that either mimic or interfere with CRF binding to CRF-BP. An assay was developed to monitor the interactions of radiolabeled CRF with human/rat CRF-BP and the mouse CRFR1 (mCRFR1) receptor. By carefully examining the binding characteristics of radiolabeled CRF to mCRFR1, the assay was able to identify compounds that bind to CRF-BP with high affinity and have little or no affinity for mCRFR1 receptors. Based on a mathematical model, we have verified the screening system with several well-characterized CRF ligands that all have different affinities for CRF receptors and CRF-BP.