生物化学技术

One of the many physiological functions of cyclic guanosine 3′,5′ monophosphate (cGMP) signalling is the regulation of a specific mode of axonal branching. The bifurcation of axons from dorsal root ganglion (DRG) neurons at the dorsal root entry zone of the embryonic spinal cord is triggered by a c ...

Obesity has reached pandemic dimensions with more than half a billion adults affected worldwide. Detailed knowledge of adipose biology is required for the development of urgently needed novel therapies directed against obesity. Two types of adipose tissue can be distinguished in hum ...

cGMP-dependent protein kinase, also known as protein kinase G (PKG), is activated independently of cGMP by a novel thiol-reactive mechanism involving the formation of an intermolecular disulfide. This oxidative modification within PKG is generally not detected by conventional We ...

Cyclic nucleotides (cAMP and cGMP) as second messengers regulate a wide variety of biological processes such as cellular growth, secretary signaling, and neuroplasticity. These processes can be regulated by increasing the synthesis of cyclic nucleotides (cyclases), by regulati ...

Radioimmunoassay is an established method to determine the amount of a specific substance in a given cell or tissue sample. Commercially available RIA or Elisa are very cost intensive. Here, we describe the generation of radioactive cGMP tracer and the quantification of cGMP. Although work ...

In recent years a variety of fluorescent probes for measurement of cGMP signals have been developed (Nikolaev et al., Nat. Methods 3:23–25, 2006; Honda et al., Proc Natl Acad Sci USA 98:2437–42, 2001; Nausch et al., Proc Natl Acad Sci USA 105:365–70, 2008). The probes are comprised of known cGMP binding sites—e. ...

Real-time and noninvasive imaging of intracellular second messengers in mammalian cells, while �preserving their in vivo phenotype, requires biosensors of exquisite constitution. Here we provide the methodology for utilizing the single wavelength cGMP-biosensor δ-FlincG ...

The cGMP-dependent protein kinases (cGK), which belong to the family of serine/threonine kinases, exhibit their diverse functions in cells through interaction with a variety of substrate proteins. Several substrates were identified and the interactions studied using different ...

F�rster resonance energy transfer (FRET)-based biosensors are powerful tools for real-time monitoring of signaling events in intact cells using fluorescence microscopy. Here, we describe a highly sensitive method which allows FRET-based measurements of the second messenger c ...

Cyclic guanosine 3′–5′-monophosphate (cGMP) is an important signaling molecule in physiology, pathophysiology, and pharmacological therapy. It has been proposed that the functional outcome of an increase of cGMP in a given cell largely depends on the existence of global versus local cG ...

G protein coupled receptors (GPCRs) are highly flexible and dynamic proteins, which are able to interact with diverse ligands, effectors, and regulatory proteins. Site-directed mutagenesis (SDM) is a powerful tool for providing insight into how these proteins actually work, both in its o ...

The addition of one or more epitope tags to G-protein-coupled receptors (GPCRs) has facilitated a wide variety of studies on their structure and function. Epitope-tagging is achieved using relatively straightforward molecular techniques but requires careful consideration ab ...

The development of a cell-based system that allows the integration of a gene of interest (GOI), such as a G protein-coupled receptor (GPCR), into a specific site on the genome, has made the generation of mammalian cell lines able to express such proteins easy and efficient. Flp-In™ stable cell lines are is ...

This chapter describes the protocol for the preparation of recombinant adenoviruses and infection of target cells to transiently express G protein-coupled receptors (GPCRs) or other proteins of interest. Adenoviruses are non-enveloped viruses containing a linear double-st ...

Protein networks and their dynamic regulation play a fundamental role in biological systems. Seven transmembrane-spanning G protein-coupled receptors (GPCRs) constitute the largest family of membrane receptors controlling the flow of information from the extracellular e ...

G protein-coupled receptors (GPCRs) play a key role in the regulation of physiological functions. Deregulation of their activities often results in pathological disorders and therefore these receptors constitute major targets for drug development. The emergence of new concepts ...

Bioluminescence resonance energy transfer (BRET) has become an extremely valuable technology for the real-time study of protein–protein interactions in live cells. This technique is highly amenable to the monitoring of G protein-coupled receptor (GPCR)–protein interactio ...

Numerous recent studies have suggested that the predicted cytosolic domains of G protein-coupled receptors represent a surface for association with proteins that may serve multiple roles in receptor localization, turnover, and signaling beyond the well-characterized inter ...

Heterotrimeric G proteins are composed of α, β, and γ subunits. G proteins can be activated by a large number of cell-surface hepathelical receptors and can transduce signals from these receptors to various intracellular signaling molecules. When G protein-coupled receptors are bound by t ...

The ability to visualize the subcellular localization of proteins by labeling them with fluorescent proteins is a powerful tool in cell biology. In the G protein-coupled receptor signaling field, this technique has been utilized to examine the various aspects of receptor behavior, incl ...