Analysis of Signal Transduction: Formation of cAMP, cGMP, and Ins(1,4,5)P3 In Vivo and In Vitro

This chapter describes biochemical assays with which to analyze signal transduction from surface cAMP receptors via G proteins to the effector enzymes adenylyl cyclase, guanylyl cyclase, and phospholipase C. The cAMP-mediated formation of the second messengers cAMP, cGMP, and Ins(1,4,5)P₃ in cells will provide information on the entire pathway; here, details on isotope-dilution assays, through which the concentrations of these second messengers can be determined, are offered. Subsequently, several assays that analyze specific parts of the sensory transduction pathway are described. The assays include equilibrium and nonequilibrium binding to surface cAMP receptors on cells and on isolated membranes. The interaction between the cAMP receptor and G proteins can be measured as GTPγS-mediated inhibition of cAMP-binding to the receptor, or as cAMP-mediated stimulation of GTPase activity or GTPγS-binding to G proteins. Finally, assays for the in vitro G protein-mediated activation of the effector enzymes adenylyl cyclase, guanylyl cyclase, and phospholipase C are described.