cAMP stimulates protein kinase

Active protein kinase

FIGURE 1-37 Activation of protein kinase A. Binding of hormone (H) to the membrane-bound receptor on the outside of the cell results in a signal transduction using the G protein to the inside of the cell so as to catalyze the enzymatic conversion of ATP into cAMP (second messenger). The cAMP then diffuses to the soluble tetrameric inactive protein kinase A moiety. The tetramer dissociates to generate two active catalytic (C) subunits. These kinase subunits then affect the phosphorylation of other proteins involved in the generation of a biological response related to the initiating hormone. The triangles bound to the R-R dimer represent molecules of cAMP.

a switch to sequentially turn on and turn off the signal transduction actions of a. This represents the essence of the signal transduction process. Thus, the alternate binding of GTP induced by the hormone occupancy of its receptor, followed by the movement of a within the membrane bilayer to AC, followed by a slow appearance of GTPase activity resulting in the conversion of a-GTP (active) —» a-GDP (inactive) describes the molecular details of the transduction process.

It has been possible to biochemically characterize Gs and G, through the use of cholera toxin, pertussis toxin, and the reagent NaF; the effects of the consequences of their presence on adenylate cyclase activity are summarized in Table 1-8. Cholera toxin transfers an ADP-ribosyl moiety from NAD+ to the a-subunit of Gs, which has the consequence of interrupting the Gs cycling and GTP hydrolysis so that GTP permanently activates Gs, resulting in the permanent activation of AC and an accumulation of cAMP. In contrast, pertu-sussis toxin transfers an ADP-ribosyl moiety to the a-subunit of G;; this does not interrupt the hydrolysis of GTP (see Figure 1-34), but does reduce the affinity of Gi for GTP, which effectively blocks the ability of Gj to become activated such that the Grprotein cycling is interrupted, thus blocking the action of hormones that couple to Gi. When NaF is added to preparations of Gs or Gj, activation of both proteins ensues. Finally, the diterpene, forskolin, has been found to have great utility in studying the properties of adenylate cyclase. Forskolin apparently does not interact directly with either Gs or Gj, but binds directly to the catalytic subunit of AC, thus generating permanent activation.

2. cAMP Protein Kinase A (PKA)

Figure 1-36 presents a model describing the sequence of events that ensues after a hormone occupies its cognate membrane-bound receptor, leading to the activation (or inhibition) of adenylate cyclase. Step 1 defines the resting state where the transmembrane receptor is unoccupied and the G-protein trimeric oligomer is not in physical contact with either the receptor or the adenylate cyclase. Also note that the a-subunit has a bound GDP molecule. As a consequence of hormone occupancy of the receptor's ligand-binding domain (step 2), a conformational change ensues, the end result of which is the probable physical interaction of the a-, f3-, and y-subunits with the unoccupied receptor. As a consequence, the affinity of the a-subunit for GDP is decreased while its affinity for GTP is increased (step 3). Then, in accordance with the model of Figure 1-36, the a-subunit with bound GTP moves within the domain of the membrane lipid bilayer (step 4) to interact physically with the membrane-bound adenyl-

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