Biological Chemistry - Berezov T. T., Korovkin B. F. 1998

Hormones
Molecular mechanisms of hormonal signal transduction
Adenylate cyclase messenger system

The adenylate cyclase signaling pathway is the most extensively studied mechanism of hormonal signal Transduction. It involves at least five well-characterized Proteins: 1) the hormone receptor; 2) the enzyme adenylate cyclase, which is responsible for synthesizing cyclic AMP (cAMP); 3) a G protein that couples the receptor to adenylate cyclase; 4) a cAMP-dependent protein kinase that catalyzes the phosphorylation of intracellular Enzymes or target proteins, thereby altering their activity; and 5) phosphodiesterase, which degrades cAMP and thus terminates (shuts off) the signal (Fig. 8.5).

Pure preparations of alpha- and beta-adrenergic receptors have been isolated from the Plasma Membranes of Liver, Muscle, and adipose tissue Cells. Hormone binding to the beta-adrenergic receptor has been shown to induce structural Changes in the receptor's intracellular domain, which in turn enables the receptor to interact with the second protein of the signaling pathway—the GTP-binding protein.

The GTP-binding protein, or G protein, is a mixture of two protein types: the stimulatory G protein, G_s, and the inhibitory G protein, G_i, both with a Molecular Weight of 80,000–90,000. Each of these consists of three distinct subunits (alpha, beta, and gamma), making them heterotrimers. The beta subunits of G_s and G_i have been shown to be identical (molecular weight 35,000). Meanwhile, the alpha subunits—which are products of different genes (molecular weights 45,000 and 41,000)—are responsible for the stimulatory and inhibitory activities of the G protein, respectively. The hormone-receptor complex not only confers upon the G protein The ability to readily exchange bound endogenous GDP for GTP, but also shifts the G_s protein into an active state. In the presence of Mg²⁺ ions, the active G protein dissociates into beta and gamma subunits and the alpha subunit-G_s complex in its GTP-bound form. This active complex then migrates to the adenylate cyclase molecule and activates it. Subsequently, the complex undergoes self-inactivation driven by the energy of GTP Hydrolysis and the reassociation of the beta and gamma subunits to regenerate the initial GDP-bound form of G_s.

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Fig. 8.5. The adenylate cyclase pathway of hormonal signal transduction.

Rec – receptor; G – G protein; AC – adenylate cyclase.

Adenylate cyclase is an integral Cell/33.html">Plasma Membrane protein whose Active Site is oriented toward the Cytoplasm and catalyzes the synthesis of cAMP from ATP:

The catalytic component of adenylate cyclase, isolated from various animal Tissues, consists of a single polypeptide chain with a molecular weight of 120,000–150,000. In the absence of G proteins, it is practically inactive and contains two SH groups: one is involved in coupling with the G_i protein, while the other is essential for catalytic activity. The enzyme molecule possesses several allosteric sites through which its activity is regulated by low-molecular-weight compounds, including Mg²⁺, Mn²⁺, and Ca²⁺ ions, as well as adenosine and forskolin. Under the action of phosphodiesterase, cAMP is hydrolyzed to yield inactive 5'-AMP.

Protein kinase is the intracellular enzyme through which cAMP exerts its effects. Protein kinase can exist in two forms. In the absence of cAMP, protein kinase occurs as a tetrameric complex composed of two catalytic (C₂) and two regulatory (R₂) subunits with molecular weights of 49,000 and 38,000, respectively; in this form, the enzyme is inactive. In the presence of cAMP, the protein kinase complex reversibly dissociates into one R₂ subunit and two free catalytic C subunits. The latter exhibit enzymatic activity, catalyzing the phosphorylation of Proteins and Enzymes and thereby modulating cellular activity.

Fig. 8.6. Covalent Regulation of Glycogen phosphorylase.

It should be noted that a large class of cAMP-dependent protein Kinases*, designated as protein kinases A, has been discovered in cells; they catalyze The transfer of a phosphate group to the OH groups of Serine and Threonine (so-called serine-threonine kinases). Another class of protein kinases, notably the one activated by the Insulin Receptor (see above), acts exclusively on the OH group of Tyrosine. However, in all cases, The addition of a highly charged and bulky phosphate group not only induces Conformational Changes in the phosphorylated proteins but also alters their activity or kinetic properties.

The activity of many enzymes is regulated by cAMP-dependent phosphorylation; accordingly, most protein-Peptide Hormones stimulate this process. However, A number of hormones exert an inhibitory effect on adenylate cyclase, thereby reducing cAMP levels and protein phosphorylation. Specifically, the hormone Somatostatin binds to its specific receptor—an inhibitory G protein ($G_i$, which is a structural homolog of the $G_s$ protein mentioned earlier)—inhibiting adenylate cyclase and cAMP synthesis, thus producing an effect directly opposite to that of adrenaline and Glucagon. In several Organs, Prostaglandins (particularly $ ext{PGE}_1$) also exert an inhibitory effect on adenylate cyclase, although in the same organ (depending on The Cell type) this very same $ ext{PGE}_1$ can activate cAMP synthesis.

The Mechanism of activation and Regulation of Muscle Glycogen phosphorylase, which stimulates glycogen breakdown, has been studied in greater detail. There are two forms: the catalytically active phosphorylase a and the inactive phosphorylase b. Both phosphorylase forms are composed of two identical subunits (with a molecular weight of 94,500), in each of which a serine residue at position 14 undergoes phosphorylation-dephosphorylation, leading to activation and inactivation, respectively (Fig. 8.6).

Under the action of phosphorylase b kinase, whose activity is regulated by cAMP-dependent protein kinase, both subunits of the inactive phosphorylase b molecule undergo covalent phosphorylation and are converted into active phosphorylase a. Subsequent dephosphorylation of the latter by a specific phosphorylase a phosphatase leads to Enzyme inactivation and restoration of the initial state.

Three types of regulation of glycogen phosphorylase have been discovered in Muscle tissue. The first type is covalent regulation, which is based on hormone-dependent phosphorylation-dephosphorylation of the phosphorylase subunits (see Fig. 8.6).

* For the Discovery of the class of protein kinases and Phosphatases, E. Krebs and E. Fischer were awarded the Nobel Prize in 1992.

Fig. 8.7. Allosteric Regulation of glycogen phosphorylase.

The second type is allosteric regulation. It is based on adenylylation-deadenylylation reactions of glycogen phosphorylase b subunits (activation and inactivation, respectively). The direction of these reactions is determined by the concentration ratio of AMP and ATP, which bind not to the active site, but to the allosteric site of each subunit (Fig. 8.7).

In contracting muscle, the accumulation of AMP resulting from ATP consumption leads to the adenylylation and activation of phosphorylase b. Conversely, at rest, high concentrations of ATP displace AMP, resulting in allosteric inhibition of this enzyme via deadenylylation.

cAMP and protein kinase play a central role in the Hormonal Regulation of Glycogen Synthesis and degradation in the liver (Fig. 8.8). For details on the Chemical transformations of glycogen, see Chapter 10*.

The third type is calcium regulation, which is based on the allosteric activation of phosphorylase b kinase by $ ext{Ca}^{2+}$ ions. The concentration of these ions increases During Muscle contraction, thereby promoting The formation of active phosphorylase a.