Reversible phosphorylation is important for G-protein-coupled receptor (GPCR) signaling, desensitization, and endocytosis, yet the precise location and role of in vivo phosphorylation sites is unknown for most receptors. This chapter describes a powerful analytical method ...
It is now clear that nearly all G-protein-coupled receptors (GPCRs) are phosphorylated and palmitolyated. The process of receptor phosphorylation has been extensively studied because it offers a regulatory mechanism that is both rapid and dynamic. However, it has recently become cle ...
Internalization of G-protein-coupled receptors (GPCRs) occurs in response to agonist activation of the receptors and causes a redistribution of receptors away from the plasma membrane toward endosomes. Internalization of lower-affinity small molecule GPCRs such as muscari ...
Fusion constructs between G-protein-coupled receptors and G-protein α-subunits have been used to examine a variety of aspects of the functioning of these signaling systems. Here we describe some of the various techniques and methods used in detail. The process of fusing the two components, ...
The demonstration that many intracellular signaling processes are mediated by a family of closely related guanine nucleotide binding proteins (G-proteins) has led to the development of specific techniques that can be used to identify which of these polypeptide(s) is involved on recep ...
On activation, G-protein-coupled receptors (GPCRs) exert many of their cellular actions through promotion of guanine nucleotide exchange on the Gα-subunit of heterotrimeric G-proteins to release free Gα-GTP and βγ-subunits. In membrane preparations, GTP can be substituted by 35S ...
The activation of heterotrimeric G-proteins is tightly regulated by the exchange of GTP for GDP in the α-subunit; mostly—but not exclusively—seven-transmembrane receptors function as the guanine nucleotide exchange factors (GEFs). A research goal may be to determine which G-prote ...
Although methods for expressing foreign proteins in clonal cell lines are well established, mature neurons remain a difficult preparation for the introduction of foreign genes. Microinjection is a reliable method for producing robust targeted expression in neurons that has advan ...
This chapter describes the protocol for preparation of recombinant adenoviruses and infection of target cells to express transiently G-protein-coupled receptors or other proteins of interest. Adenoviruses are nonenveloped viruses containing a linear double-stranded DNA ...
The molecular cloning of the cDNA sequences encoding most G-protein-coupled receptors, including those from humans, allows their study in a variety of recombinant systems. In this respect, transfected mammalian cell lines constitute the most frequently used model for investigati ...
The localization and density of G-protein-coupled receptors (GPCRs) on the cell surface is a critical factor for specifying signaling within and between neurons. GPCRs and their associated signaling components are localized to specific neuronal compartments that give rise to a vari ...
G-protein-coupled receptor mRNAs are expressed at low levels and therefore present a challenge for the study of their sites and levels of expression. In situ hybridization (ISH) and Northern blotting are powerful methods for the localization of mRNAs and the study of regulation of mRNA expre ...
Epitope tagging of a receptor involves introducing a defined amino acid sequence, to which an antibody has already been produced, into the primary amino acid sequence of the receptor. The new sequence can be as short as 10–15 amino acids and the method allows the receptor to be monitored without having ...
Immunocytochemistry exploits the incomparable specificity of the antibody-antigen interaction to form the basis of a flexible approach to the study of expression and localization of proteins both in model systems and their physiological context. This chapter details the theory a ...
Antibodies have proved invaluable in the study of G-protein-coupled receptors (GPCRs). The utility of these immunoglobulin probes for investigation of protein structures and functions arises from their selectivity as well as their versatility. Antibodies can be used to analyze GP ...
Methods are presented for identifying and quantifying allosteric interactions of G-protein-coupled receptors with labeled and unlabeled ligands using radioligand-binding assays. The experimental designs and analyses are based on the simplest ternary complex alloster ...
The radioligand-binding assay is a relatively simple but powerful tool for studying G-protein-coupled receptors. There are three basic types of radioligand-binding experiments: (1) saturation experiments from which the affinity of the radioligand for the receptor and the bindi ...
The misexpression and overexpression of proteins by injection of synthetic mRNA into Xenopus oocytes and, more particularly, embryos has provided the experimental basis for a considerable portion of our current knowledge of early vertebrate development (1, and references there ...
Immunohistochemistry is a very powerful technique for determining both the tissue-specific and subcellular location of endogenous and exogenous proteins within an embryo. The technique is relatively simple and when used on its own or in conjunction with other immunological techn ...
Immunohistochemistry is a powerful technique for determining both the presence of and the subcellular location of proteins within tissues. Zebrafish are particularly amenable to this technique and it is possible to localize proteins both in whole embryos and larvae, as well as section ...
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