Vasopressin-Sensitive Adenylate Cyclase: Subunit Interactions Assessed by Target Analysis and Computer Modelling

Title: Vasopressin-Sensitive Adenylate Cyclase: Subunit Interactions Assessed by Target Analysis and Computer Modelling
Authors: Verkman, Alan S.; Ausiello, M.D., Dennis A.; Skorecki, Karl L.
Publisher: Kidney International Supplement
Date Published: December 01, 1987
Reference Number: 215

This translation by the NDI Foundation is to assist the lay reader. To provide a clear, accessible interpretation of the original article, we eliminated or simplified some technical detail and complicated scientific language. We concentrated our translation on those aspects of the article dealing directly with NDI. The NDI Foundation thanks the researchers for their work toward understanding and more effectively treating this disorder.
© Copyright NDI Foundation 2007 (JC)

Adenylate cyclase (AdC) is a complex enzyme system that catalyzes the formation of cyclic adenosine monophosphate (cAMP). But for AdC to be able to do this, it must first be activated by a hormone. AdC exists in different cells and can be activated by a few different hormones. One of the cells AdC is located in is the principal cell of the kidney collecting duct, and there it is activated by the antidiuretic hormone, vasopressin (VP). The molecular sequence is thought to be as follows: VP binds with one of its receptors, the vasopressin-2 receptor (V2R). The V2R is coupled to a stimulatory guanine nucleotide regulatory protein (Gs). The Gs protein activates AdC, which in turn activates cyclic adenosine phosphate (cAMP).

The Gs protein consists of three subunits: an alpha unit, a beta unit and a gamma unit. Researchers have questioned how all these subunits are structured in the cell membrane both before and after exposure to the stimulating hormone (e.g. VP). What is the sequence of subunit interactions involved in the activation of AdC?

There are two opposite mechanisms which can be involved in the enzyme activation: associative or dissociative. In associative mechanisms the activating hormone induces the coming together (association) of the subunits of the G protein to form an active enzyme. In dissociative mechanisms the activating hormone induces some of the subunits to separate (dissociate) from other subunits in order to form an active enzyme.

Much of the research on enzyme systems involve dissolving the cells in which they exist into a liquid. This method has advantages, but it can disrupt the subunit assembly which the researchers want to study. Recently, researchers have taken advantage of a research methodology called target analysis by radiation inactivation. Radiation inactivation allows researchers to study enzyme activation systems in whole cell culture, i.e., it allows the cells to stay intact and operate much as they would inside the body.

According to target theory, the biological activity of the molecule being studied decreases as a result of being "hit" by a dose of radiation. There is a mathematical relation between the size of the radiation dose and the degree of biological activity which helps researchers pinpoint the molecular size of the molecule being studied. Using target analysis, computer modeling on the adenylate cyclase system and combining this with known biochemical characteristics of each of the subunits involved in the AdC system, Ausiello, et al., were able to present a comprehensive model for the stimulation and inhibition of AdC.

They found AdC was stimulated by a dissociative activation mechanism. Before stimulation by VP, the alpha, beta and gamma units are coupled together to form a single unit, the Gs protein. Guanosine diphosphate (GDP) is bound to the nucleotide binding site on the alpha unit. When VP binds with V2R, guanosine triphosphate (GTP) replaces GDP on the alpha unit. Then the Gs protein dissociates into the alpha unit and the beta/gamma unit. The alpha unit then is able to connect with and activate the AdC system. When GTP undergoes a chemical process which converts it back to GDP, the alpha unit deactivates and then returns to and recombines with the beta/gamma unit to form the complete Gs protein.

That is how AdC is stimulated. How is it inhibited?

Just as AdC can be stimulated by a Gs; it can be inhibited by an inhibitory G protein (Gi). The Gi is also comprised of an alpha unit, a beta unit and a gamma unit. But the alpha unit has a different effect, namely it inhibits AdC when it connects with it. Whereas VP binds with the Gs-coupled V2R to stimulate AdC, alpha-2-adrenergic agents bind with Gi-coupled alpha-2-adrenergic receptors, and certain opiates bind with their Gi-coupled receptors to inhibit AdC.

It was thought that Gi can inhibit AdC by another means, namely that when the alpha unit of the Gi dissociates from the beta/gamma unit, the beta/gamma unit is free to couple to the stimulatory alpha unit so it is not free to connect with AdC.

Ausiello, et al., studied the effect of Gi on AdC after seeing that adding GTP to intact membranes stimulated AdC, but inhibited the maximal VP-stimulated activity. When VP was in low concentrations, GTP enhanced AdC activity. When VP was in higher concentrations, GTP inhibited AdC activity. They found Gi was the effector of this inhibitory response, but direct interaction of the receptor with Gi was not required. However, a direct inhibitory interaction of the inhibitory alpha with AdC was required.

The authors offer their model as a framework from which to probe the molecular mechanisms involved in enzyme activation.