Gene expression systems in the development of high-throughput screens.

Recent advances in the development of combinatorial automated chemical synthesis, robotic sample handling, and data collection and analysis have significantly increased the number of compounds available for screening against potential therapeutic targets. The implementation of highly sensitive in vitro biochemical and cell-based high-throughput screening assays is essential to facilitate the rapid identification of selective and potent lead molecules from compound libraries. The ability to easily produce functional proteins in sufficient quantities for in vitro biochemical assays and to devise useful cell-based systems is dependent on the successful application of a variety of gene expression systems.

Functional expression of olfactory-adrenergic receptor chimeras and intracellular retention of heterologously expressed olfactory receptors.

Replacing the G-protein-coupling domains of the beta 2-adrenergic receptor with homologous domains of putative olfactory receptors produced chimeric receptors which were able to stimulate pigment dispersion in Xenopus melanophores, a G-protein-mediated pathway. A multiple replacement chimera containing the second, third and C-terminal cytoplasmic domains of receptor OR5 elevated cyclic adenosine 3':5'-monophosphate (cAMP) and suppressed production of inositol phosphates. Co-expression of G alpha olf did not alter the strength of response of this chimera. A novel rat olfactory receptor cDNA (U131) was isolated and sequenced. Expression of U131 and OR5 constructs containing an N-terminal epitope-tag or C-terminal fusion to green fluorescent protein occurred in an intracellular network but not in the plasma membrane of heterologous cells. Similarly treated beta 2-adrenergic receptors were functional and were observed in the plasma membrane and the intracellular network. These results demonstrate that the putative cytoplasmic domains of olfactory receptors are capable of functional interaction with heterologous G-proteins of the G alpha s subtype. Instead, the absence of these receptors from the plasma membrane of heterologous cells appears to explain our inability to determine if odorants can activate the olfactory receptor clones. We hypothesize that the olfactory receptors have requirements for maturation and targeting to the plasma membrane that are different from most other G-protein-coupled receptors.

Use of a cell-based, lawn format assay to rapidly screen a 442,368 bead-based peptide library.

A cell-based, lawn format assay utilizing an in situ photocleavage method has been developed that allows the rapid examination of large bead-based compound libraries as discrete molecules. The format uses frog melanophore cells in a contiguous, adherent, confluent layer in small petri dishes covered with a 0.5-1-mm layer of agarose containing 130 micron diameter TentaGel beads at a density of 2-20 beads/mm2. Employing this technique a 9-mer, 442,368-member peptide library (designed around the 13 amino acid alpha-MSH peptide sequence) made up of 12 separate pools of 36,864 peptides/pool was assayed. Initially, a fraction (approximately 10%) of each pool was scanned (approximately 3700 beads from each pool) in 60-mm petri dishes to identify the most active pools. Upon direct photocleavage of the beads with UV light (365 nm), each petri dish was photographed over a 60-min period with a CCD camera to record changes in light intensity as an index of melanosome dispersion. Active beads were those that were surrounded by a localized decrease in light transmittance indicating melanosome dispersed cells. Upon examination with a dissecting microscope, single beads centrally located to a circular array of dispersed cells were identified and removed from the agarose and sequenced by Edman degradation to determine the peptide sequence. Re-synthesized peptides were re-examined against alpha-MSH receptor to confirm and quantify the activity. Several 9-mer peptides were identified with potencies similar to the natural 13-mer peptide. This method allows for the rapid screening of large bead-based photo-cleavable peptide libraries with the advantage that each compound is screened as a discrete molecule in a well-less format.

Studies on zinc- and cadmium-bound proteins in bovine kidneys by biochemical and neutron activation techniques.

Bovine kidneys were found to contain about 78 ppm Zn and 0.78 ppm Cd. Approximately 45% of Zn and 60% of Cd were present in the cytosol fraction. More than 95% of these two metals were bound to macromolecules. Both Zn- and Cd-protein complexes were observed to be stable between pH 7 and 10.5. Separation and characterization of these proteins were carried out using several chromatographic and electrophoretic techniques in conjunction with instrumental neutron activation analysis (INAA). The results showed the presence of at least four Zn-binding proteins with mol wt greater than 300,000, 260,000, 89,000, and 27,000 and at least three Cd-binding proteins of mol wt greater than 300,000, 32,000, and 13,000.

The G-protein-coupled receptor 40 family (GPR40-GPR43) and its role in nutrient sensing.

Recent deorphanization efforts have paired the G-protein-coupled receptors GPR40, GPR41 and GPR43 with fatty acids as endogenous ligands. While carboxylic acids have been historically known to serve as fuel sources and biomarkers of disease, these studies demonstrate that fatty acids can act as signalling molecules at the cell-surface level. This receptor subfamily shares approx. 30% identity among members, with some limited cross-over between ligand activities. Generalized expression patterns within the pancreatic beta-cell, adipose depots and the gastrointestinal tract infer involvement in energy source recognition, absorption, storage and/or metabolism. GPR40, activated by medium and long-chain fatty acids, has been shown to potentiate insulin secretion at the beta-cell, and has been hypothesized to participate in the detrimental effects of chronic fatty acid exposure on beta-cell function. GPR41 and GPR43 have been reported to stimulate leptin release and adipogenesis respectively via activation by short-chain fatty acids. These common themes implicate GPR40, GPR41 and GPR43 in playing significant roles in metabolic diseases, such as diabetes, obesity and the metabolic syndrome.

Cloning and characterization of an endothelin-3 specific receptor (ETC receptor) from Xenopus laevis dermal melanophores.

We report here the presence of a receptor specific for endothelin-3 (termed ETc receptor or ETcR) on Xenopus laevis dermal melanophores. Activation of ETcR causes the dispersion of the pigment granules within the melanophores. The EC50 for ET-3 to induce the pigment dispersion is 24 +/- 7 nM, compared to greater than 10 microM for both ET-1 and -2. This effect desensitizes in a manner that is dependent on both time and the concentration of ET-3 used to stimulate the cells. A cDNA encoding for ETcR was isolated by a polymerase chain reaction-mediated DNA amplification strategy using degenerate oligonucleotides prepared based on conserved regions of other known G-protein-coupled receptor sequences and by the subsequent screening of a frog melanophore cDNA library. The cloned cDNA consists of 2,240 nucleotides, with an open reading frame coding for 444 amino acids containing an initial 20-amino acid signal sequence. The predicted mature peptide consists of 424 amino acids with a heptahelical structure common to the G-protein-coupled receptor surperfamily. Its deduced amino acid sequence is 47 and 52% identical to ETA and ETB receptors, respectively, while ETA and ETB are 48% identical to each other. Expression of cDNA in HeLa cells, which do not contain endothelin receptors, enables the cells to specifically bind [125I]ET-3. Competition binding experiments performed on HeLa cells transiently expressing pETc show that the apparent Ki values for ET-3 and ET-1 to displace [125I]ET-3 are 45.5 +/- 16 and 114 +/- 22 nM, respectively.

Combinatorial diffusion assay used to identify topically active melanocyte-stimulating hormone receptor antagonists.

alpha-Melanocyte-stimulating hormone (alpha-MSH) is implicated in pigmentation, central nervous system and immune system functions, growth, mitogenesis, and melanoma. Evaluation of these roles has been hindered by the lack of alpha-MSH antagonists. A combinatorial chemistry-based diffusion assay is used to find random tripeptides that antagonize normal frog and human melanoma MSH receptors and to identify pharmacological groups responsible for receptor interaction. The alpha-MSH antagonist D-Trp-Arg-Leu-NH2 is used to demonstrate directly the contribution of MSH to normal skin tone in frogs following injection or topical application.

Functional characterization of a receptor for vasoactive-intestinal-peptide-related peptides in cultured dermal melanophores from Xenopus laevis.

A receptor for vasoactive-intestinal-peptide (VIP)-related peptides was functionally characterized in a cell line derived from Xenopus melanophores using a recently described microtiter-plate-based bioassay. Activation of the melanophore VIP receptor by VIP or the peptides pituitary-adenylate-cyclase-activating polypeptide (PACAP 38), PACAP 27, and helodermin stimulated intracellular 3'-5' cyclic adenosine monophosphate (cAMP) accumulation and pigment dispersion in the cells. Helodermin, with an EC50 (concentration of peptide inducing half-maximal melanosome dispersion) of 46.5 pM, was the most potent activator of pigment dispersion, followed by PACAP 38 > VIP > PACAP 27. A similar order of potencies was observed for the peptides to induce cAMP accumulation. The responses to VIP agonists were selectively inhibited by the VIP antagonists PACAP-(6-27) and (N-Ac-Tyr(1)-D-Phe2)-growth-hormone-releasing factor[GRF](1-29)-NH2. Taken together, the results suggest that the melanophores express a VIP receptor that shares certain characteristics of, but also differs significantly from, other previously identified VIP receptors.

Pigment dispersion in frog melanophores can be induced by a phorbol ester or stimulation of a recombinant receptor that activates phospholipase C.

Pigment dispersion in frog melanophores is classically mediated by receptors that activate protein kinase A via an elevation of intracellular cyclic AMP. Here, 12-O-tetradecanoylphorbol-13-acetate (TPA), an activator of protein kinase C, is found to induce pigment dispersion. To demonstrate that an increase in cAMP is not required for the melanosome movement, a murine bombesin receptor was expressed in the melanophores. When these cells were treated with bombesin, they accumulated intracellular inositol phosphates but not cAMP and dispersed their pigment. Four agonists, one partial agonist, and two antagonists for the bombesin receptor were compared for their ability to induce or block bombesin-induced pigment dispersion. In all cases, the degree of pigment dispersion followed simple equilibrium reactions. The resulting dose-response curves allowed for the determination of the effective concentration for half-maximal pigment dispersion (EC50) or half-maximal inhibition of bombesin-stimulated pigment dispersion (IC50) for the peptides. As the pigment dispersion assay can rapidly evaluate chemicals for their effects on receptors that activate phospholipase C via a functional assay, it has potential utility for investigations of ligand-receptor interactions and for massive drug screening.

Discovery and structure-function analysis of alpha-melanocyte-stimulating hormone antagonists.

Structure-function relationships of alpha-melanocyte-stimulating hormone (alpha-MSH, alpha-melanotropin) were investigated and novel alpha-MSH receptor antagonists were identified. Based on the alpha-MSH-[5-13] peptide sequence, a multi-use peptide library consisting of 31,360 structurally different candidates was generated, and approximately 40% of the peptides were individually screened for their ability to block receptor function. This led to the identification of antagonists with a range of potencies and revealed structural requirements necessary for receptor inactivation. The most potent antagonist Met-Pro-D-Phe-Arg-D-Trp-Phe-Lys-Pro-Val-NH2 has an IC50 value of 11 +/- 7 nM. Analysis revealed that D-Trp5 and Phe6 were crucial to its antagonistic properties which could be potentiated by D-Phe3. This study demonstrates that residues in positions 5-6, 7-9, and 10 of the alpha-MSH sequence constitute crucial determinants for potent antagonist activity.

Creation and functional screening of a multi-use peptide library.

Studies of functional interactions between transmembrane proteins such as G-protein-coupled receptors and ligands would benefit from the ability to utilize synthetic molecule libraries. This is realized here by the construction and application of a multi-use combinatorial peptide library (MUPL). Peptides are liberated from their supports in a dry state so that the problem of signal interference due to mixing of peptide molecules, particularly agonists and antagonists, is avoided. In addition, the peptides are released from their supports in a controlled manner so that fractions are available for multiple independent tests, thus eliminating the need for iterative library analysis and resynthesis. The MUPL concept was validated with a functional screen which detects agonists to G-protein-coupled receptors and led to the discovery of new ligands. It is expected that combining MUPLs with functional assays will enhance both basic scientific research and the rates of drug discovery and development.

A new tool for investigating G protein-coupled receptors.

Vertebrate olfactory receptors are members of the seven-transmembrane-domain G protein-coupled receptor family. They utilize intracellular signal transduction pathways which are activated by stimulation of odorant receptors and use the second messengers cAMP and/or inositol 1,4,5-trisphosphate and diacylglycerol. Studies of how odorants bind to and activate the receptors can be considered part of the more general problem of how chemicals interact with G protein-coupled receptors. This review describes the development of a new technique for assessing functional interactions between chemicals and these receptors in only minutes. Predicted uses of the system include structure-function analyses of both G protein-coupled receptors and their ligands, studies of receptor coupling to G proteins and cloning of cDNAs for these receptors.

Functional expression of recombinant G-protein-coupled receptors monitored by video imaging of pigment movement in melanophores.

Xenopus laevis melanophores are capable of functionally expressing recombinant receptors which couple via G-proteins to adenylate cyclase or phospholipase C (PLC). Receptor-mediated stimulation of either of these enzymes causes dispersion of melanosomes while receptor stimulation leading to inhibition of adenylate cyclase induces their aggregation. Translocation of melanosomes within thousands of individual pigment cells was simultaneously tracked by capturing gray scale video images before and after receptor activation. Digital subtraction of poststimulation from prestimulation images was performed on a microcomputer, generating bitplane images containing pixels with nonzero gray scale values wherever melanosome movement had occurred. Movement in both centripetal and centrifugal directions was detectable. The assay was tested using four receptors: a human beta 2-adrenergic receptor which stimulates adenylate cyclase, murine substance P and bombesin receptors which stimulate PLC, and a human D2 dopamine receptor which inhibits adenylate cyclase. Based on melanosome translocation following application of ligands, expression of functional receptors could be consistently detected in melanophores which received only single copies of a plasmid encoding any of the four receptors. By imaging fields containing up to 11,000 melanophores, the presence of a plasmid coding for a receptor could be detected when its frequency was one per 10,000 plasmids transfected. Combining receptor-mediated pigment translocation in melanophores with the rapid data-handling ability of video technology should provide a bioassay useful for investigating the function of G-protein-coupled receptors and for screening cDNA libraries for clones encoding new receptors.

Use of constitutive G protein-coupled receptor activity for drug discovery.

This article describes the behavior of transiently transfected human receptors into melanophores and the potential use of constitutive receptor activity to screen for new drug entities. Specifically, transient transfection of melanophores with different concentrations of receptor cDNA presumably leads to increased levels of receptor expression. This leads to an increased response to agonists (both maxima and potency) and, in some cases, an agonist-independent constitutive receptor activity. Transfections with increasing concentrations of the G(s) protein-coupled human calcitonin receptor type 2 (hCTR2) cDNA produced sufficient levels of constitutively activated receptor to cause elevated basal cellular responses. This was observed as a decrease in the transmittance of light through melanophores (consistent with G(s) protein activation) and increased response to human calcitonin. The receptor-mediated nature of this response was confirmed by its reversal with the hCTR2 peptide inverse agonist AC512. A collection of ligands for hCTR2 either increased or decreased constitutive hCTR2 activity, suggesting that the constitutive system was a sensitive discriminator of positive and negative ligand efficacy. Similar results were obtained with G(i)-protein-coupled receptors. Transient transfection of NPY1, NPY2, NPY4, CXCR4, and CCR5 cDNA produced increased light transmittance through melanophores (consistent with G(i)-protein activation). NPY1 cDNA produced little constitutive response on transfection, whereas maximal levels of constitutive activity ranging from 30 to 45% were observed for the other G(i)-protein-coupled receptors. Responses to agonists for these receptors increased (both maxima and potency) with increasing cDNA transfection. The receptor/G(i)-protein nature of both the constitutive and agonist-mediated responses was confirmed by elimination with pertussis toxin pretreatment. These data are discussed in terms of the theoretical aspects of constitutive receptor activity and the applicability of this approach for the general screening of G protein-coupled orphan receptors.