Measurement of Adenylyl Cyclase Activity in Cell Membranes

In mammals, adenylyl cyclase is a family of membrane-bound enzymes that catalyze the conversion of adenosine triphosphate (ATP) to adenosine 3′:5′-cyclic monophosphate (cAMP). cAMP is an ubiquitous intracellular signaling molecule that modifies cell function by activating cAMP-dependent protein kinase A (PKA). The adenylyl cyclase enzyme is itself regulated by various proteins, including the G-proteins (guanine nucleotide binding regulatory proteins) Gs and G1 , and Ca2+ -calmodulin (1 ). Binding of a hormone to a stimulatory receptor induces coupling of the guanosine diphosphate (GDP) liganded heterotrimeric Gs protein to the receptor. Under these conditions, guanosine triphosphate (GTP) replaces GDP on the Gs protein, which then dissociates into the Gs α-GTP subunit and the βγ complex (2 ). The Gs α-GTP subunit then interacts with and activates the adenylyl cyclase enzyme. Activation of this enzyme ceases when the GTP is hydrolyzed back to GDP by GTPase activity intrinsic to the α-subunit of G-proteins. Binding of a hormone to an inhibitory receptor leads to a similar train of events involving the Gi class of proteins, except that it is unclear exactly how inhibition of the adenylyl cyclase enzyme occurs. Evidence exists that Gl α-GTP directly inhibits adenylyl cyclase, whereas other studies implicate a role for βγ complexes liberated from Gl in combining with and functionally inactivating free Gs α-GTP (1 , 3 ). Both mechanisms probably operate under different conditions.