Abstract:
:High-content screening (HCS), a combination of fluorescence microscopic imaging and automated image analysis, has become a frequently applied tool to study test compound effects in cellular disease-modeling systems. This chapter describes the measurement of G protein-coupled receptor (GPCR) internalization in the HCS format using a high-throughput, confocal cellular imaging device. GPCRs are the most successful group of therapeutic targets on the pharmaceutical market. Accordingly, the search for compounds that interfere with GPCR function in a specific and selective way is a major focus of the pharmaceutical industry today. This chapter describes methods for the ligand-induced internalization of GPCRs labeled previously with either a fluorophore-conjugated ligand or an antibody directed against an N-terminal tag of the GPCR. Both labeling techniques produce robust assay formats. Complementary to other functional GPCR drug discovery assays, internalization assays enable a pharmacological analysis of test compounds. We conclude that GPCR internalization assays represent a valuable medium/high-throughput screening format to determine the cellular activity of GPCR ligands.
journal_name
Methods Enzymoljournal_title
Methods in enzymologyauthors
Haasen D,Schnapp A,Valler MJ,Heilker Rdoi
10.1016/S0076-6879(06)14008-2subject
Has Abstractpub_date
2006-01-01 00:00:00pages
121-39eissn
0076-6879issn
1557-7988pii
S0076-6879(06)14008-2journal_volume
414pub_type
杂志文章abstract::Guanine nucleotide dissociation inhibitor (GDI) is a central regulator of Rab GTPase family members. GDI recycles Rab proteins from the membrane and sequesters the inactive GDP-bound form of Rab in the cytosol for use in multiple rounds of transport. The balance between the membrane-bound form of Rab and the cytosolic...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(05)03029-6
更新日期:2005-01-01 00:00:00
abstract::Quantitative real-time polymerase chain reaction (qRT-PCR) is a flexible and scalable method for analyzing transcript abundance that can be used at a single gene or high-throughput (>100 genes) level. Information obtained from this technique can be used as an indicator of potential regulation of glycosylation at the t...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(10)79004-2
更新日期:2010-01-01 00:00:00
abstract::Peptides are multidentate chiral ligands capable of coordinating different metal ions. Nowadays, they can be obtained with high yield and purity, thanks to the advances on peptide/protein chemistry as well as in equipment (peptide synthesizers). Based on the identity and length of their amino acid sequences, peptides ...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2016.04.007
更新日期:2016-01-01 00:00:00
abstract::Regulators of G protein signaling (RGS proteins) are a diverse family of proteins that act to negatively regulate signaling by heterotrimeric G proteins; however, recent data have implied additional functions for RGS proteins. Previously, we employed the yeast two-hybrid system and identified the microtubule-destabili...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(04)90004-3
更新日期:2004-01-01 00:00:00
abstract::Protein (poly-)ubiquitination is a posttranslational modification that plays a key role in almost all cellular processes. It involves the installment of either single ubiquitin (Ub) moieties or one of eight different polyUb linkage types, each giving a distinct cellular outcome. Deubiquitinating enzymes (DUBs) reverse...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2018.12.037
更新日期:2019-01-01 00:00:00
abstract::This chapter outlines methods that can be applied to determine the levels of lipids in cells and tissues. In particular, the methods focus upon the extraction and analysis of those lipids critical for monitoring signal transduction pathways. The methods address the analysis of the phosphoinositides, the lipid agonists...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(07)32010-7
更新日期:2007-01-01 00:00:00
abstract::In the least-squares fitting of data, there is a unique answer to the question of how the data should be weighted: inversely as their variance. Any other weighting gives less than optimal efficiency and leads to unreliable estimates of the parameter uncertainties. In calibration, knowledge of the data variance permits...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(08)03810-X
更新日期:2009-01-01 00:00:00
abstract::Significant progress has been made in discovering and cloning a host of proteins, including a range of glycoproteins. The availability of their predicted amino acid sequences provides useful information, including potential N-linked glycosylation sites. However, only a limited number of protein structures have been so...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(06)15007-7
更新日期:2006-01-01 00:00:00
abstract::The unexpected discovery of graphene and especially the follow-up explosion of interest in its properties and applications marked the beginning of a new carbon era. Graphene-based nanostructured materials are highly useful because they show great promise in the field of biotechnology and biomedicine. Owing to their un...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2018.05.013
更新日期:2018-01-01 00:00:00
abstract::The findings in this article illustrate the complexity residing in the regulation of reversible S-glutathionylation of proteins, such as GAPDH. This is clearly reflected in the design of suitable experimental approaches designed to cope with the interaction of several redox-dependent factors. Clear interactions are de...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/s0076-6879(02)48636-3
更新日期:2002-01-01 00:00:00
abstract::This chapter described the preparation and fractionation of libraries of M13 phage displaying proteins as fusions to the major coat protein. High titer (10(13) pfu/ml) phage libraries can readily be generated using a single vector and the level of display surpasses that of gene III fusion phage. Since the synthetic VI...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/s0076-6879(96)67006-2
更新日期:1996-01-01 00:00:00
abstract::Accurate protein structure determination by solution-state NMR is challenging for proteins greater than about 20kDa, for which extensive perdeuteration is generally required, providing experimental data that are incomplete (sparse) and ambiguous. However, the massive increase in evolutionary sequence information coupl...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2018.11.004
更新日期:2019-01-01 00:00:00
abstract::Synthetic hydrogen sulfide (H2S) donors are useful research tools as well as potential therapeutic agents. In this chapter, we report the detailed protocols for the synthesis and evaluation of a series of phosphorodithioate-based H2S donors. Fluorescence assays were used to determine H2S release from the donors in bot...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2014.11.032
更新日期:2015-01-01 00:00:00
abstract::This chapter discusses experimental methods and protocols for the purification and preliminary characterization of DNA polymerases that are specialized for the replicative bypass (translesion DNA synthesis) of base or other types of DNA damage that typically arrest high-fidelity DNA synthesis, with particular emphasis...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(06)08022-0
更新日期:2006-01-01 00:00:00
abstract::In mice with lupus nephritis qualitative changes in anti-DNA antibodies occur, such as IgG switch and increased cationic charge, to render these antibodies pathogenic. Pathogenic anti-DNA idiotypes can be encoded by genes of a normal mouse strain such as SWR, where they remain dormant. When the normal mice are crossed...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/0076-6879(88)62094-5
更新日期:1988-01-01 00:00:00
abstract::Posttranslational modification of circadian clock proteins by phosphorylation is an essential regulatory process in the control of eukaryotic circadian clocks. In the Neurospora circadian clock, the key clock protein FREQUENCY (FRQ) is progressively phosphorylated. The phosphorylation of FRQ is regulated by both kinas...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(05)93017-6
更新日期:2005-01-01 00:00:00
abstract::Protein kinases transfer a phosphoryl group from ATP onto target proteins and play a critical role in signal transduction and other cellular processes. Here, we review the kinase kinetic and chemical mechanisms and their application in understanding kinase structure and function. Aberrant kinase activity has been impl...
journal_title:Methods in enzymology
pub_type: 杂志文章,评审
doi:10.1016/B978-0-12-397918-6.00001-X
更新日期:2014-01-01 00:00:00
abstract::Increasing numbers of compounds, previously classified as antagonists, were shown to inhibit this spontaneous or constitutive receptor activity, instead of leave it unaffected as expected for a formal antagonist. In addition, some other antagonists did not have any effect by themselves, but prevented the inhibition of...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/B978-0-12-381296-4.00003-8
更新日期:2010-01-01 00:00:00
abstract::Omics approaches have become popular in biology as powerful discovery tools, and currently gain in interest for diagnostic applications. Establishing the accurate genome sequence of any organism is easy, but the outcome of its annotation by means of automatic pipelines remains imprecise. Some protein-encoding genes ma...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/bs.mie.2016.09.019
更新日期:2017-01-01 00:00:00
abstract::Since the early days of cell volume regulation research, the role of actin cytoskeleton organization and rearrangement has attracted specific interest. Rapid modifications in actin dynamics and architecture have been described. They were shown to regulate cell volume changes, as well as regulatory volume decrease in a...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(07)28012-7
更新日期:2007-01-01 00:00:00
abstract::Numerous aspects of the immune system operate on the basis of complex regulatory networks that are amenable to mathematical and computational modeling. Several modeling frameworks have recently been applied to simulating the immune system, including systems of ordinary differential equations, delay differential equati...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(09)67004-X
更新日期:2009-01-01 00:00:00
abstract::Glass microfluidic devices have been fabricated to monitor the secretion of glycerol or fatty acids from cultured murine 3T3-L1 adipocytes. In the current studies, adipocytes are perfused in a reversibly sealed cell chamber, and secreted products are analyzed by enzyme assay on either a single- or dual-chip device. Th...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/B978-0-12-800280-3.00011-6
更新日期:2014-01-01 00:00:00
abstract::Glycoproteins are a major class of glycoconjugates displaying a variety of mutual interactions between glycan and protein moieties that ultimately affect molecular organization. Modulation of the pendant glycan structures is important in tuning the functions of glycoproteins. Here we discuss structural aspects and som...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(10)78018-6
更新日期:2010-01-01 00:00:00
abstract::Site-directed mutagenesis is a PCR-based method to mutate specified nucleotides of a sequence within a plasmid vector. This technique allows one to study the relative importance of a particular amino acid for protein structure and function. Typical mutations are designed to disrupt or map protein-protein interactions,...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/B978-0-12-418687-3.00019-7
更新日期:2013-01-01 00:00:00
abstract::Mass spectrometry (MS) has gradually replaced classical methods as a major tool in protein sequencing and characterization. However, the sample preparation repertoire has not changed very much; it has just been adjusted to the needs of the new analytical method. In this chapter frequently used in-solution enzymatic di...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(05)05003-2
更新日期:2005-01-01 00:00:00
abstract::G-protein-coupled receptors (GPCRs) form a superfamily of membrane proteins that play a crucial role in mediating physiological processes as well as pathogenesis of many critical diseases. They are one of the most successful drug targets, accounting for more than 30% of prescription drugs on the market today. Three-di...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(09)05213-6
更新日期:2009-01-01 00:00:00
abstract::Intracellular protein degradation is one of the most precisely regulated processes in living cells. The main component of the degradation machinery is the 20S proteasome present in eukaryotes as well as in prokaryotes. We have developed successful purification protocols for the 20S proteasome in its native state using...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(05)98027-0
更新日期:2005-01-01 00:00:00
abstract::Reviewing the circumstances that have led to the first monoclonal antibody against the disease-associated form of PrP, we consider the availability of PrP knockout mice and recombinant PrP, as well as a reliable conformational screening protocol as being important prerequisites for a successful immunization approach. ...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/s0076-6879(99)09010-2
更新日期:1999-01-01 00:00:00
abstract::Acetyl CoA:arylamine N-acetyltransferase (NAT; E.C. 2.3.1.5) enzymes play a key role in the metabolic activation of aromatic amine and nitroaromatic mutagens to electrophilic reactive intermediates. We have developed a system in which the activation of mutagens by recombinant human NAT2, expressed in Escherichia coli,...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/S0076-6879(05)00011-X
更新日期:2005-01-01 00:00:00
abstract::Glycosylphosphatidylinositol-anchored proteins can be specifically identified by several methods. PI-PLC digestion analyses, the most widely used technique, can be performed more reliably when conducted with purified protein and phase partitioning to exclude steric effects and when combined with alkaline hydrolysis to...
journal_title:Methods in enzymology
pub_type: 杂志文章
doi:10.1016/0076-6879(95)50099-5
更新日期:1995-01-01 00:00:00