Carson R. Loomis

Cloning and expression of multiple protein kinase C cDNAs

Three different protein kinase C related cDNA clones were isolated from a rat brain cDNA library and designated PKC-I, PKC-II, and PKC-III. These each encode very similar, but distinct, polypeptides that contain a region homologous with other protein kinases. COS cells transfected with either PKC-I or PKC-II specifically bind at least 5-fold more 3H-PDBu (phorbol ester) than control cells. An increase in Ca2+, phosphatidylserine, and diacylglycerol/phorbol-ester-dependent protein kinase activity is also observed in COS cells transfected with either PKC-I or PKC-II. The physiological implications of the discovery of three protein-kinase-C-related cDNAs are discussed.

Quantitative measurement of sn-1,2-diacylglycerols.

Publisher Summary The hypothesis that sn-l, 2-diacylglycerols function as the intracellular second messengers of growth factors, hormones, and neurotransmitters has gained wide acceptance. This method has been used successfully to measure basal and stimulated diacylglycerol levels in a variety of tissues and cells, including human platelets, rat hepatocytes, sis- and ras-transformed Normal Rat Kidney (NRK) cells, a human leukemia cell line (HL60), and human neutrophils. sn-l, 2-Dioleoylglycerol is prepared from sn-l,2-dioleoyl-glycerol-3-phosphocholine (Avanti) by phospholipase digestion, purified by extraction in ether, and quantitated by ester analysis using cholesteryl acetate as a standard. Bovine heart cardiolipin in chloroform is from Avanti and should be stored under N 2 after opening. This chapter discusses the preparation of membranes containing diacylglycerol kinase. With the recent explosion of interest in diacylglycerols as the in vitro activator of the Ca 2+ -activated phospholipid dependent protein kinase (protein kinase C) and the implication that diacylglycerols function as intracellular second messengers, this assay method provides a useful tool to explore the role of diacylglycerols as the second messengers of neurotransmitters, hormones, and growth factors in a number of biological settings.

Advancing Biological Understanding and Therapeutics Discovery with Small-Molecule Probes

Small-molecule probes can illuminate biological processes and aid in the assessment of emerging therapeutic targets by perturbing biological systems in a manner distinct from other experimental approaches. Despite the tremendous promise of chemical tools for investigating biology and disease, small-molecule probes were unavailable for most targets and pathways as recently as a decade ago. In 2005, the NIH launched the decade-long Molecular Libraries Program with the intent of innovating in and broadening access to small-molecule science. This Perspective describes how novel small-molecule probes identified through the program are enabling the exploration of biological pathways and therapeutic hypotheses not otherwise testable. These experiences illustrate how small-molecule probes can help bridge the chasm between biological research and the development of medicines but also highlight the need to innovate the science of therapeutic discovery.

The ligand-independent translocation assay: an enabling technology for screening orphan G protein-coupled receptors by arrestin recruitment.

Finding natural and/or synthetic ligands that activate orphan G protein-coupled receptors (oGPCRs) is a major focus in current drug discovery efforts. Transfluor is a cell-based GPCR screening platform that utilizes an arrestin-green fluorescent protein conjugate (arrestin-GFP) to detect ligand interactions with GPCRs. The assay is ideally suited for oGPCRs because binding of arrestin-GFP to activated receptors is independent of the interacting G protein. Before embarking on a high-throughput screen, it is important to know that the target oGPCR can actually bind arrestin-GFP. This information was thought to be inaccessible, however, as arrestin-GFP recruitment is an agonist-driven process. This chapter describes an assay that enables GPCRs to be validated in Transfluor in the absence of ligand. This assay, termed the ligand-independent translocation (LITe) assay, utilizes a modified G protein-coupled receptor kinase to bypass the requirement of ligand for initiating arrestin-GFP translocation. Using the LITe assay, one can determine if an oGPCR binds arrestin-GFP and if the response is quantifiable by high-content screening instruments. In addition, the assay expedites the development and identification of oGPCR stable cell lines with the best Transfluor properties. In this way, the assay provides criteria for selecting the best oGPCRs to move forward for a Transfluor screening campaign. Moreover, the assay can be used for quality control purposes during the orphan receptor screen itself by providing positive translocation responses for calculation of Z prime values. In summary, the LITe assay is a powerful new technology that enables a faster and more reliable path forward in the deorphanization of GPCRs with Transfluor.

High‐Content Screening of Known G Protein‐Coupled Receptors by Arrestin Translocation

G protein-coupled receptors (GPCRs) have proven to be one of the most successful target classes for drug discovery. Accordingly, many assays are available to screen GPCRs, including radioactive-binding assays, second messenger signaling assays, and downstream reporter assays. One of the more novel approaches is the Transfluor technology, a cell-based assay that uses a detectable tag on a cytosolic protein, called arrestin, that is involved in the desensitization or inactivation of GPCRs. Monitoring the translocation of GFP-tagged arrestin from the cytosol to activated GPCRs at the plasma membrane measures the pharmacological effect of test compounds that bind the receptor target. Moreover, the Transfluor assay provides further, high-content information on the test compound itself and its effects on cell processes due to the fluorescent imaging of whole cells used in this screen. Screening known GPCRs with Transfluor against large compound libraries is best accomplished in cell lines stably expressing an optimum level of the target receptor. This chapter describes how to generate a clonal cell line stably expressing the known GPCR with suitable Transfluor properties. It then describes the steps involved in performing a Transfluor screen and discusses high content data resulting from the screen.

Regulation of Protein Kinase C by Lipid Cofactors

Hormone-stimulated biological responses in a number of tissues and cell types are associated with turnover of inositol phospholipids. This phenomenon was first observed over 30 years ago by Hokin and Hokin (1953) and, since then, has been demonstrated in a wide variety of systems (reviewed by Michell, 1975; Berridge, 1984; Hirasawa and Nishizuka, 1985; Hokin, 1985). The significance of phosphatidylinositol turnover remained a mystery until two relatively recent discoveries helped clarify the role of inositol phosphatides in transmembrane signaling.

Automation and validation of the Transflour technology: a universal screening assay for G protein-coupled receptors

G protein-coupled receptors (GPCRs) are historically the richest targets for drug discovery, accounting for nearly 60 percent of prescription drugs. The ligands and functions of only 200 out of possibly 1000 GPCRs are known. Screening methods that directly and accurately measure GPCR activation and inhibition are required to identify ligands for orphan receptors and cultivate superior drugs for known GPCRs. Norak Biosciences utilizes the redistribution of a fluorescently-labeled protein, arrestin, as a novel screen for monitoring GPCR activation. In contrast to the present methods of analyzing GPCR function, the power of the Transfluor technology is in its simplicity, large signal to noise ratio, and applicability to all GPCRs. Here, we demonstrate that the Transfluor technology can be automated and quantitated on high throughput image analysis systems. Cells transfected with an arrestin-green fluorescent protein conjugate and the neurokinin-1 GPCR were seeded on 96-well plates. Activation of the NK-1 receptor with Substance P induced translocation of arrestin-GFP from the cytosol to the receptor. Image quantitation of the arrestin-GFP translocation was used to generate dose dependent curves. These results reveal that the Transfluor technology combined with an image analysis system forms a universal platform capable of measuring ligand-receptor interactions for all GPCRs.

Protein Kinase C Regulation by Diacylglycerols: Structure-Function Relationships and Mechanism

A mixed micellar assay for protein kinase C was developed to investigate the specificity and stoichiometry of activation by phospholipids, diacylglycerols, and diacylglycerol analogues. Triton X-100 micelles containing 8 mol% phosphatidylserine (PS) and 2.5 mol% sn-1,2-dioleoylglycerol (diC18:1) activated rat brain protein kinase C in the presence of Ca2+ to the same degree as sonicated PS/diC18:1. Protein kinase C activity was almost totally dependent on diC18:1 in the mixed micellar assay. At 8 mol% PS, diC18:1 stimulated maximally at 1 mol%. At 2.5 mol% diC18:1, PS did not activate until 3 mol% and then did so cooperatively with maximal stimulation occurring at 6–8 mol%. Molecular sieve chromatography demonstrated that monomeric protein kinase C interacts with Triton X-100 micelles in a PS and Ca2+ dependent manner. Interpretations follow: 1) a single molecule of diC18:1 activates monomeric protein kinase C; 2) a phospholipid bilayer is not required; 3) four or more molecules of PS are required. In addition, several diacylglycerol analogues were synthesized to determine the exact structural features required for activation. The data suggest that both carbonyls of the oxygen esters and the 3-hydroxyl are required. A model of protein kinase C activation by PS and diacylglycerol was formulated.