Studies on Receptor-mediated Activation of Adenylyl Cyclases

Field: Biochemistry, Genetics and Molecular Biology (Life Sciences)

Abstract Beef renal medullary neurohypophyseal hormone (NHH)-sensitive adenylyl cyclase was found to exist in several discrete states of activity depending on the previous history of the membranes, on the time of incubation, and on the presence of GTP and NHH. Thus, the time course of cyclic adenosine 3':5'-monophosphate (cAMP) accumulation of membranes incubated at about 0.08 mm ATP exhibits a burst phase that lasts between 2 and 4 min, after which time the reaction proceeds linearly. Both the initial transient state and the final stable state of activity are stimulated by NHH (arginine-vasopressin and oxytocin). The burst in the progress curve is not observed in the presence of 10-5 m GTP or high concentrations of ATP (above 1.0 mm). In the absence of hormone, the GTP-induced state is less active than the initial transient state and more active than the stable state. In the presence of hormone, the GTP-induced state is less active than either one of the states observed in the absence of GTP. Both NHH and GTP are active when added after the stable state of activity has been formed. Removal of adenylyl cyclase assay reagents after the NHH-stimulated stable state of activity has formed, followed by washing with NHH-free medium, results in formation of a basal state of activity that resembles the stable state of activity obtained in the absence of hormone; it is stimulated by NHH and affected by GTP, and does not exhibit a burst in the time course of cAMP accumulation. Removal of adenylyl cyclase assay reagents after the NHH-stimulated GTP state has formed, followed by washing with NHH-free and GTP-free medium results in formation of a basal state of activity that resembles the original one: it exhibits a burst in its progress curve, is stimulated by NHH, and is affected by GTP. It is concluded that hormonal stimulation of the NHH-sensitive adenylyl cyclase system in beef renal medullary membranes is reversible, that GTP action is reversible and that the stable state of activity requires the presence of GTP to revert to a state whose existence is transient upon incubation under adenylyl cyclase assay conditions. These findings are discussed in the light of Frieden's histeretic concept (Frieden, C. (1970) J. Biol. Chem. 247, 5788–5899). Stimulation of adenylyl cyclase activity by hormone is significantly lowered by GTP. This inhibitory effect is seen over a wide range of magnesium concentrations (0.5 to 20 mm) and is half-maximal at about 2 x 10-7 m GTP. Adenylyl cyclase activities exhibit pH optima that are higher in the presence than in the absence of GTP. 5'-Guanylyl-imidophosphate, a GTP analogue, mimics the actions of GTP in that it stimulates basal activity and leads to the formation of a state of activity of the enzyme that is much less stimulated by NHH. This suggests that the observed actions of GTP are probably of an allosteric nature and unrelated to its phosphorylating capacity. Stimulation of adenylyl cyclase activity determined at 1 to 10 µm ATP is low (2.5- to 3.5-fold). Stimulation by NHH can be enhanced either by increasing the concentration of ATP or by addition of adenosine or AMP. Addition of adenosine at 0.08 mm ATP does not further increase the response of the system to NHH. It is concluded that ATP can interact with this adenylyl cyclase system at three sites: (a) catalytic site, serving as substrate; (b) regulatory site I, enhancing the response of the system to NHH (an effect mimicked by adenosine and AMP); and (c) regulatory site II, mimicking the action of GTP in inducing the formation of a stable state that is less susceptible to stimulation by occupied NHH receptor.