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) G_s and G₁ , and Ca²⁺ -calmodulin (1 ). Binding of a hormone to a stimulatory receptor induces coupling of the guanosine diphosphate (GDP) liganded heterotrimeric G_s protein to the receptor. Under these conditions, guanosine triphosphate (GTP) replaces GDP on the G_s protein, which then dissociates into the G_s α-GTP subunit and the βγ complex (2 ). The G_s 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 G_i class of proteins, except that it is unclear exactly how inhibition of the adenylyl cyclase enzyme occurs. Evidence exists that G_l α-GTP directly inhibits adenylyl cyclase, whereas other studies implicate a role for βγ complexes liberated from G_l in combining with and functionally inactivating free G_s α-GTP (1 , 3 ). Both mechanisms probably operate under different conditions.