Adenylyl cyclase catalytic activity

In addition to the selective responses of adenylyl cyclases to GPy subunits, it is likely that different forms of Gp and Gy subunits influence the various forms of adenylyl cyclase in different ways [19]. There are five known forms of Gp subunits and 11 known forms of Gy subunits (see Ch. 19). Differential expression and regulation of these subunits could provide still additional mechanisms for selectively controlling adenylyl cyclase catalytic activity in specific neuronal cell types. 

This assay is used to measure adenylyl cyclase activity downstream of the receptor, by directly activating lysates with GTPyS - a non-hydrolyzable form of GTP - or MnSO - an ATP chelator that provides a measurement of the intrinsic adenylyl cyclase catalytic activity. MnSO measurements are therefore used as an alternative readout of AGA expression levels. 

Biochemical and molecular cloning studies indicate the existence of nine separate and unique forms of adenylyl cyclase (AC), which comprise a distinct enzyme family, referred to as AC1-AC9 [1, 2]. These members of the adenylyl cyclase superfamily are all membrane-bound. There is also an additional soluble form, sAC, that has catalytic activity similar to the others but is genetically the most divergent member of the family. All the membrane-bound forms of adenylyl cyclase are activated by the stimulatory G protein Gas (see Ch. 19), and all with the exception of AC9 are stimulated by forskolin. The soluble form sAC is not stimulated by either Gas or forskolin but is sensitive to bicarbonate levels. All known forms of... 

First, the binding of one hormone molecule to one receptor catalytically activates several Gs molecules. Next, by activating a molecule of adenylyl cyclase, each active Gsa molecule stimulates the catalytic synthesis of many molecules of cAMP. The second messenger cAMP now activates PKA, each molecule of which catalyzes the phosphorylation of many molecules of the target protein—phosphorylase b kinase in Figure 12-16. This... 

Fig. 5. The open (inactive) and closed (active) conformations of the adenylyl cyclase catalytic domains bound to GocGTPyS. Helices are shown as cylinders and [j strands as ribbons. The C2 domains of the two mAC molecules are superimposed. The open state of mAC, which was crystallized in the absence of an ATP analogue, is colored gray (PDB ID, 1AZS) the closed state, colored blue, was crystallized with ATPaS bound to the AC catalytic site, shown as a stick model (1CJT). Forskolin is present in both complexes. Switch II of Gas is colored red. Note that binding of ATP is accompanied by segmental movement of residues in both domains toward the catalytic site.

To understand how binding of Gscj-GTP promotes adenylyl cyclase activity, scientists will first have to solve the structure of the adenylyl cyclase catalytic domains in their unactivated conformations (i.e., in the absence of bound Gs -GTP). One hypothesis is that binding of the switch II helix to a cleft In one catalytic domain of adenylyl cyclase leads to rotation of the other catalytic domain. This rotation is proposed to lead to a stabilization of the transition state, thereby stimulating catalytic activity. 

Probably all adenylyl cyclases are inhibited competitively by substrate analogs, which bind at the site and to the enzyme configuration with which cation-ATP binds (cf Fig. 4). One of the best competitive inhibitors is (3-L-2, 3 -dideoxy adenosine-5 -triphosphate ( 3-L-2, 3 -dd-5 -ATP Table 4) [4], which allowed the identification of the two metal sites within the catalytic active site (cf Fig. 4) [3]. This ligand has also been labeled with 32P in the (3-phosphate and is a useful ligand for reversible, binding displacement assays of adenylyl cyclases [4]. The two inhibitors, 2, 5 -dd-3 -ATP and 3-L-2, 3 -dd-5 -ATP, are comparably potent... 

There are also many neurotransmitter and hormone receptors that contribute to the fine control of cAMP formation by inhibition of adenylyl cyclase. The action of inhibitory receptors is mediated by several different forms of the Gai family, specifically the Gail, Gai2, Gai3, Gao and Goa subtypes. The Ga subunits of these isoforms can inhibit the catalytic activity of adenylyl cyclase when the enzyme is activated by either Gas or forskolin. The inhibition of catalytic activity does not occur via competition with Gas but appears to occur by an interaction at a symmetric site on the AC molecule. Gai-mediated inhibition of adenylyl cyclase is most dramatic for AC5 and AC6. A few other forms of adenylyl cyclase, most notably AC1, can be inhibited by Gao but this effect is not as potent as the inhibition of AC5 and AC6 by Gai isoforms. The GTPase activity of Gai family members can be accelerated by a large family of RGS proteins (see Chapter 19). 

Regulation of protein kinase A by cAMP takes place by the following mechanism. An increase in cAMP concentration, triggered by activation of adenylyl cyclase, leads to binding of cAMP at specific binding sites on the regulatory subunit. The R subunits dissociate from the tetramer, the catalytic subunits are released from inhibition by the regulatory subunits and can thus phosphorylate substrate proteins. 

Mechanism of action of glucagon. [Note For clarity, G-protein activation of adenylyl cyclase has been omitted.] R = regulatory subunit C = catalytic subunit. 

In general, the MC2 receptor is Gs protein-coupled (Table 1), and two studies in the rabbit pulmonary artery indicate that this is also true for the presynaptic receptor. The evidence is, indirect, however, in that it suggests activation of adenylyl cyclase, the typical transduction step downstream from Gs. In the study by Gothert and Hentrich (1984), the facilitatory effect of ACTH was increased by simultaneous administration of forskolin, an activator of the catalytic subunit of adenylyl cyclase, and AH 21-132, a phosphodiesterase inhibitor. In the study by Costa and Majewski (1988), the facilitatory effect of ACTH was occluded when the vessel was super-fused with a lipid-soluble cAMP analogue. 

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