Lan-Hsin Wu

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.

New reporter cell lines to study macrophage-tropic HIV envelope protein-mediated cell-cell fusion.

The infection of human cells by HIV-1 virus can be mimicked by a fusion process between cells expressing the HIV envelope protein (Env) and cells expressing both human CD4 (huCD4) and appropriate human chemokine receptors. In this study, a macrophage-tropic (M-tropic) HIV cell-cell fusion assay was established that utilized huCD4, human CCR5 (huCCR5), and HIV ADAgpl60 as fusion components and a Gal4/VP16-activated luciferase as a reporter system. By combining CHO cells expressing huCD4 and huCCR5 with CHO cells expressing HIV ADAgpl60, a 300-fold increase in luciferase activity could be elicited relative to control. No luciferase activity was detected when HXB2gpl60 (T-tropic) was used instead of ADAgpl60 (M-tropic) as the fusion partner in the assay. Addition of anti-huCD4 (RPA-T4) or anti-huCCR5 (2D7) monoclonal antibodies in the assay significantly inhibited the fusion event; in contrast, an anti-CXCR4 (12G5) monoclonal antibody had little effect, indicating that the fusion assay was huCD4 and huCCR5 dependent. The cell-cell fusion occurred in a time-dependent manner; the maximum luciferase activity was detected about 8 hr after mixing the cells. The fusion events could also be monitored by another reporter system in which Gal4/VP16 activated green fluorescent protein (GFP) was used as the reporter instead of luciferase. In combination with fluorescence microscopy, the GFP reporter system allowed visualization of the fusion events in real time. Compared with previously described HIV fusion models, this system has several advantages, including simplicity, sensitivity, and the ability to allow continuous monitoring of the HIV cell-cell fusion event. Finally, this cell-cell fusion system is easily adapted to study other HIV fusion events.

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.

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.

Substitution of lysine-181 to aspartic acid in the third transmembrane region of the endothelin (ET) type B receptor selectively reduces its high-affinity binding with ET-3 peptide

Summary: In the G protein‐coupled receptor family, a highly conserved aspartic acid located within the third transmembrane domain has been shown to be involved in ligand binding. Within the endothelin (ET) peptide receptor family, this aspartic acid has been replaced by a lysine. To assess the importance of this residue in ET binding, the lysine (position 181) of rat ET type B receptor was replaced by an aspartic acid. The effects on ligand binding and phosphoinositide turnover of both the wildtype and K181D mutant receptors were examined using transient receptor expression in COS‐7 cells. Using [125I]ET‐1 as the radioactive peptide ligand in displacement binding studies, the wild‐type receptor displayed a typical non‐isopeptide‐selective binding profile with similar IC50 values (0.2‐0.6 nM) for all three ET peptides (ET‐1, ET‐2, and ET‐3). The mutant receptor showed an increase in IC50 values for ET‐1 (5 nM), ET‐2 (27 nM), and ET‐3 (127 nM).The K181D mutant receptor still elicited full inositol phosphate (IP) accumulation responses in the presence of saturating concentrations of ETs (10 nM of ET‐1, 100 nM of ET‐2, or 1 μM of ET‐3). indicating that the mutation did not affect G protein coupling.

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.