细胞技术

G protein-coupled receptors (GPCRs) and receptor tyrosine kinases (RTKs) are transmembrane receptors that initiate intracellular signaling cascades in response to a diverse array of ligands. Recent studies have shown that signal transduction initiated by GPCRs and RTKs is not org ...

The process of signal transduction is dependent on specific protein-protein interactions. In many cases, these interactions are mediated by modular protein domains that confer specific binding activity to the proteins in which they are found. Rapid progress has been made in the biochem ...

Lipid rafts and caveolae are cholesterol- and sphingolipid-rich microdomains of the plasma membrane that concentrate components of certain signal transduction pathways. Interest in and exploration of these microdomains has grown in recent years, especially after the discove ...

The bioluminescence resonance energy transfer (BRET) methodology allows for the study of protein-protein interactions as well as conformational changes within proteins or molecular complexes. BRET is a highly versatile technique that can be applied to in vitro studies using puri ...

Transmembrane-signaling events are mediated and regulated by protein-protein interactions. The yeast two-hybrid screen has proven to be an effective approach for studying interaction between signaling molecules, such as ras and raf. This approach can be used to identify new binding ...

The PDZ domain is a protein-protein interaction module that interacts with a C-terminal short peptide motif in its binding partners. A variety of methods have been used to study PDZ domain interactions. This chapter details the two methods most commonly used in the analysis of PDZ interactions: ...

Microinjection is an excellent technique for studying the acute responses of cells to proteins and can be used to investigate the effects of mutations in proteins on their activity. It has been used widely to study the responses to Rho family guanosine triphosphatases and is particularly use ...

Some membrane proteins must be clustered at target sites to efficiently perform their functions. PDZ domain-containing scaffold proteins bind to the tails of target membrane proteins and promote their localization and clustering on the cell surface. This chapter describes the expe ...

The recognition that Rho guanosine triphosphatases (GTPases) (Rho, Rac, and Cdc42) play important regulatory roles in many areas of cell biology has made the ability to measure their activity in cells an important biological tool. Because Rho GTPases become activated by conversion from g ...

Phospholipase D (PLD) enzymes are present in all animal and plant species and have been linked to many critical cellular processes, including proliferation, differentiation, motility, and secretion. The functional significance of PLD derives from its generation of phosphatidic a ...

Phospholipase D (PLD) hydrolyzes phosphatidylcholine to produce the membraneassociated second messenger, phosphatidic acid (PA) and choline. Two phospholipase D enzymes—PLD1 and PLD2—have been identified, although their regulatory mechanisms are yet to be fully underst ...

Microarrays are made by immobilizing to a solid support thousands of DNA probes that detect soluble complementary target sequences using the hybridization pairing rules of nucleic acids. Receptor triggering induces a cascade of signaling events that often involves the modulation ...

Introducing genes into cells by retroviral transduction has greatly increased the abil-ity to study signal transduction pathways in primary cells. Retroviral transduction has proven to be an efficient method to express genes of interest in cells that are difficult to manipulate using ...

Proteomics is the study of the set of proteins, or proteome, expressed by a cell under specific conditions. Proteomics methodology consists of protein extraction, protein separation, and protein identification. Currently, two-dimensional gel electrophoresis (2DE) and matrix- ...

Stromal-derived factor (SDF)-1, an α-chemokine that binds to G protein-coupled seven transmembrane-spanning receptor, CXCR4, plays an important and unique role in regulating the trafficking of normal hematopoietic stem/progenitor cells and their homing/ retention in bone mar ...

CXCR4, the receptor for stromal cell-derived factor (SDF)-1, was expressed in Saccharomyces cerevisiae coupled to the pheromone response pathway via a chimeric Gα subunit. Engagement of CXCR4 by SDF-1 resulted in expression of reporter genes, HIS3 or lacZ under the transcriptional cont ...

The ability of G protein-coupled receptors to transduce signaling typically is induced by the binding of an appropriate ligand (agonist), resulting in a conformational change of the receptor and the subsequent interaction with the G protein heterotrimer. Some mutants of G protein-cou ...

G protein-coupled receptors are involved in the regulation of many aspects of normal physiology and pathology. Recent research has broadened our view of how the cell trans-duces ligand binding to cellular responses. It is becoming clear that phenomena that take place both at the cell surface, ...

Seven transmembrane-spanning receptors, widely referred to as G protein-coupled receptors (GPCRs), mediate a broad spectrum of extracellular signals at the plasma membrane through G proteins, thereby modulating a variety of biological processes. In addition to G proteins, they al ...

Traditionally, lipid rafts have been defined by their insolubility in ice-cold Triton X-100 and low-buoyant density. These low-density membrane microdomains have been referred to as detergent-resistant membranes, Triton-insoluble membranes, and Tritoninsoluble floati ...