[Analysis of GTP-binding protein function with a photoaffinity GTP analog].
The mechanism whereby hormones or neurotransmitters activate G proteins and their intracellular effectors can be studied in reconstituted systems using purified components. However, the regulation of receptor-G protein signaling appears to be substantially more complex in the cell and several additional components participate in this event. To study the relationship among G proteins receptors, and effectors in complex systems, such as membranes of permeable cells, it is necessary to employ methods that selectively allow the examination of G protein activation. One such method is photoaffinity labeling using a hydrolysis-resistant, photoaffinity GTP analog, P3 (4-azidoanilido)-P1-guanosine 5'-triphosphate (AAGTP). Here we describe the synthesis and purification of [32P]AAGTP as well as a procedure suitable studying the G protein function in membrane preparations. Photoaffinity labeling in rat cerebral cortex membranes showed that at least four G proteins (GsH, GsL, Gi, and Go) were labeled by [32P]AAGTP. [32P]AAGTP labeling on Gs and Gi was altered in concert with the activation states of those G proteins. An agonist-specific increase in [32P]AAGTP labeling of the G protein alpha-subunit in a membrane preparation has also been demonstrated. Thus, the photoaffinity labeling method with [32P]AAGTP makes it possible to investigate the behavior of individual G proteins in complex systems such as membrane preparations.
Hatta
S
S
Department of Pharmacology, School of Medicine, Sapporo Medical University, Japan.
Ohshika
H
H
D000345:Affinity Labels; D000818:Animals; D001345:Autoradiography; D001386:Azides; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D019204:GTP-Binding Proteins / Q000032:analysis* / Q000502:physiology; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives*
D000345:Affinity Labels; D001386:Azides; C031907:GTP gamma-4-azidoanilide; D006160:Guanosine Triphosphate; D019204:GTP-Binding Proteins