Analysis of function of receptor-G-protein and receptor-RGS fusion proteins.
Fusion constructs between G-protein-coupled receptors and G-protein alpha-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 receptor and the G-protein alpha-subunit, result in a construct that acts as an agonist stimulated GTPase enzyme whose expression level can be accurately determined by radioligand binding. The effects of different ligands, mutations, and other signaling system components can thus be analyzed by a series of relatively simple assays. Recently we have begun to supplement this approach with the use of fusions between G-protein-coupled receptors and regulator of G-protein signaling proteins to examine the function of this important group of signaling molecules.
Ward
Richard J
RJ
Molecular Pharmacology Group, University of Glasgow, Scotland, United Kingdom.
Milligan
Graeme
G
D000215:Acylation; D002462:Cell Membrane / Q000378:metabolism; D020555:GTP-Binding Protein Regulators / Q000235:genetics / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008024:Ligands; D011233:Precipitin Tests / Q000379:methods; D043562:Receptors, G-Protein-Coupled / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D013462:Sulfur Radioisotopes / Q000378:metabolism; D014162:Transfection / Q000379:methods
D020555:GTP-Binding Protein Regulators; D008024:Ligands; D043562:Receptors, G-Protein-Coupled; D011993:Recombinant Fusion Proteins; D013462:Sulfur Radioisotopes; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D019204:GTP-Binding Proteins