Cross Talk Between Stimulatory and Inhibitory Guanosine 5'-Triphosphate Binding Proteins: Role in Activation and Desensitization of the Adenylate Cyclase Response to Vasopressin

Title: Cross Talk Between Stimulatory and Inhibitory Guanosine 5'-Triphosphate Binding Proteins: Role in Activation and Desensitization of the Adenylate Cyclase Response to Vasopressin
Authors: Ausiello, M.D., Dennis A.; Verkman, Alan S.; Skorecki, Karl L.
Publisher: Biochemistry
Date Published: January 27, 1987
Reference Number: 216
The inhibitory GTP-binding protein (Gi) is known to mediate the effects of a number of hormones that act through specific receptors to inhibit adenylate cyclase. In this study we examined the mechanism whereby Gi modulates the response of adenylate cyclase to a stimulatory hormone and its role in desensitization. In membranes prepared from the cultured renal epithelial cell line LLCPK1, adenylate cyclase activity was stimulated 16-fold by 1-2 microM lysine vasopressin. Addition of GTP (1-100 microM) resulted in stimulation of basal activity but inhibition of hormone-stimulated activity (approximately 40% inhibition at 100 microM GTP). This contrasts with the usual effect of GTP to support or augment activation by stimulatory receptors. The inhibitory effect was abolished by pertussis toxin, which had little effect on basal activity in the absence or presence of added GTP or on vasopressin-stimulated activity in the absence of added GTP. GTP-mediated inhibition was vasopressin concentration dependent. At concentrations of vasopressin below the K1/2 for enzyme activation (approximately 0.6 nM), GTP was stimulatory, and at higher concentrations, GTP was inhibitory. The inhibitory effect of GTP was also observed for a V2-receptor agonist and was not abolished by a V1-receptor antagonist, indicating that a distinct V1 receptor did not mediate inhibition of adenylate cyclase. Using the known subunit structure of adenylate cyclase, we developed the minimal mechanism that would incorporate a modulatory role for Gi in determining net activation of adenylate cyclase by a stimulatory hormone. The predicted enzyme activities for basal and maximal hormone stimulation in the presence and absence of GTP were generated, and model parameters were chosen to match the experimental observations.(ABSTRACT TRUNCATED AT 250 WORDS)

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)

In certain cells such as the principal cells of the kidney collecting duct, a complex enzyme system called adenylate cyclase (AdC) is stimulated by a molecular response to the antidiuretic hormone, vasopressin (VP). A model developed by Verkman, et al., explains the VP stimulated activation of AdC: VP arrives at the basolateral membrane of the collecting duct cells. There it binds with its receptor, the vasopressin-2 receptor (V2R). The V2R is coupled to a stimulatory G protein (Gs). Gs is made up of three subunits, an alpha stimulatory unit (alpha s), a beta unit and a gamma unit. The alpha s has a nucleotide binding site to which guanosine diphosphate (GDP) is bound.

When VP binds with V2R, the GDP is converted to guanosine triphosphate (GTP). This allows the Gs to separate into the alpha s unit and a beta/gamma unit. When alpha s separates from the beta/gamma unit, it connects with AdC, thereby activating it. Then the GTP bound to the alpha s unit is converted back to GDP and the alpha s unit disconnects from AdC and rejoins the beta/gamma unit to form the whole Gs.

Verkman, et al., refined and expanded their model of VP-activated AdC to account for the various ways AdC activation can be inhibited. Just as there are stimulatory G proteins (Gs), there are inhibitory G proteins (Gi). They too are comprised of three subunits: an inhibitory alpha unit (alpha i), a beta unit and a gamma unit. Gi's are coupled to receptors which accept hormones which inhibit AdC (such as alpha-2-adrenergic agents).

Upon the binding of the AdC inhibiting hormone with its receptor, the GDP bound to the alpha i unit converts to GTP and the Gi separates into the alpha i unit and the beta/gamma unit. The alpha i unit connects with AdC, inhibiting its action. Then GTP converts back to GDP and the alpha i unit disconnects from AdC and rejoins the beta/gamma unit to form the whole Gi.

But there is another proposal stating that Gi could inhibit AdC in another way as well. In this scenario, when alpha i separates from the beta/gamma unit, the beta/gamma unit joins with the separated alpha s unit, preventing it from connecting with AdC. In short, Gi was thought to be able to tone down the response of AdC to VP by having its beta/gamma unit occupy the alpha s unit of the Gs. This meant that Gi could down-regulate AdC activation without having to directly interact with the vasopressin-2 receptor.

Verkman, et al., explored this possibility in two ways:

  1. by using their computer model of VP-induced AdC activation to predict the outcome of various manipulations of the variables involved in the activation and inhibition process; and
  2. by using a special laboratory cell culture in their laboratory experiments that kept the AdC system intact, functioning as it would in a living body. They were able to compare their computer model predictions with the observed results of their experiments.

When they added VP to their cell culture, they observed the expected result: AdC activity increased 16-fold. When the authors added low concentrations (1-100uM) of GTP to the culture, they observed opposing effects: in the absence of VP, GTP increased AdC activity, but when these same concentrations were added to the culture when it was infused with VP, the GTP inhibited VP's ability to fully activate AdC. GTP could stimulate AdC, but it inhibited VP from stimulating AdC as fully as it ordinarily could have. This was puzzling because ordinarily GTP augments hormone/receptor stimulation of AdC.

Pertussis toxin (PT) interrupts the VP-initiated molecular sequence from activating AdC by uncoupling the V2R from the G protein. The authors pretreated their cell culture with PT. They found the added GTP could no longer inhibit VP-induced AdC activity. This indicated that GTP's inhibitory effect was dependent on VP being present in the cell. In low concentrations of VP, GTP acted to stimulate AdC; in high concentrations of VP, it inhibited AdC. The fact that PT could reverse the inhibition indicated that GTP needed functional Gi's to carry out its inhibiting effect. This indication was strengthened after the author's experiments eliminated the possibility that the vasopressin-1 might have mediated GTP's inhibitory effects.

These results led the authors to refine their ATP activation model, allowing it to incorporate Gi with Gs in the overall AdC activation mechanism. Verkman et al., tested their model by predicting the outcomes of several interactions within the VP-induced AdC activation system and comparing them to observed effects of experiments designed to test the same interactions in the laboratory cell culture. Specifically, they examined:

  1. whether Gi had to directly interact with the vasopressin-2 receptor for the inhibition of AdC to occur; and
  2. whether the action of the beta/gamma unit from Gi binding with alpha i was sufficient to account for GTP-mediated inhibition of AdC activity.

Their model indicated that Gi did not directly have to interact with the vasopressin-2 receptor in order for AdC activity to be lowered. What it did need was to have its alpha i subunit interact directly with AdC itself. The model indicated that the Gi and Gs engaged in a type of cross talk resulting in the alpha i, in the presence of added GTP, to be able to interact with AdC more frequently than alpha s. This conclusion was strengthened when their model predicted that the beta/gamma unit from the Gi did not have the ability to occupy enough alpha s units in order to inhibit AdC active.

The authors' experimental observations confirmed their model's predictions. Thus, their model provides a mechanism to explain how Gi can inhibit AdC activity without directly interacting with the vasopressin-2 receptor.