BIOCHEMISTRY: A TEXTBOOK FOR UNIVERSITIES - E. S. Severin - 2004

SECTION 5. BIOLOGICAL MEMBRANES

V. Transmembrane Signal Transduction

An essential property of membranes is their ability to perceive and transmit signals from the external environment into The Cell. The "recognition" of signaling molecules is carried out by receptor Proteins embedded in The cell membrane of target Cells or located within the cell. A target cell is defined by its ability to selectively bind a given signaling molecule via a receptor.

If a signal is perceived by Membrane Receptors, the information transfer pathway can be represented as follows:

✵ interaction of the receptor with the signaling molecule (primary messenger);

✵ activation of the membrane enzyme responsible for the generation of The secondary messenger;

✵ Formation of the secondary messenger cAMP, cGMP, IP₃, DAG, or Ca²⁺;

✵ activation of specific proteins (primarily protein Kinases) by messengers, which in turn phosphorylate Enzymes and thereby influence The activity of intracellular processes.

Despite the immense diversity of signaling molecules, receptors, and the processes they regulate, there are only a few mechanisms of transmembrane information transfer: utilizing the adenylate cyclase system, the Inositol phosphate system, catalytic receptors, cytoplasmic receptors, or nuclear receptors.

A. Signaling molecules —Hormones, Neurotransmitters, Eicosanoids, growth factors, nitric oxide (NO)

Signaling molecules can be either nonpolar or polar substances. Nonpolar substances, such as Steroid Hormones, enter the cell by passing directly through The Lipid Bilayer. Polar signaling molecules do not enter the cell, but instead bind to specific receptors on the cell membrane. This interaction triggers a cascade of sequential events within the membrane itself and inside the cell. Polar signaling molecules include protein hormones (e.g., Glucagon, Insulin, parathyroid hormone), neurotransmitters (e.g., acetylcholine, Glycine, γ-aminobutyric acid), growth factors, cytokines, and eicosanoids.

B. Receptors

Based on their localization, receptors are classified as membrane, cytoplasmic, or nuclear. According to another Classification, all receptors can be divided into fast-responding (within milliseconds) and slow-responding (within minutes or even hours, which is characteristic of hormones that transmit signals to intracellular receptors). Receptors of the first type are integral Oligomeric Proteins containing a subunit with a binding site for the signaling molecule and a central ion channel (Fig. 5-29).

Class="center">Fig. 5-29. Participation of receptors in Transmembrane Signal Transduction. Receptors: 1 — ion channel-linked, e.g., the GABA receptor; 2 — with catalytic activity (insulin receptor); 3 — coupled to phospholipase C, e.g., the α₁-adrenergic receptor; 4 — with catalytic activity (guanylate cyclase, ANP receptor); 5 — coupled to adenylate cyclase, e.g., β-adrenergic receptors; 6 — binding hormones in the Cytosol or Nucleus, e.g., the cortisol receptor.

Receptors of the second type, localized in membranes and not associated with channels, are subdivided into two major groups: catalytic receptors, possessing intrinsic Tyrosine kinase or guanylate cyclase activity, and receptors that interact via a G protein with a membrane enzyme. The binding of a Ligand (e.g., a hormone) to a receptor on the outer surface of the cell membrane alters the activity of a cytoplasmic enzyme, which in turn initiates a cellular response. In other words, information—rather than electrical charges or dissolved molecules—is transferred across the membrane.

In the case of cytoplasmic receptors, the hormone itself crosses the membrane, and information regarding the presence of the hormone in the cell is transmitted to The Nucleus via the receptor.

Depending on their specific Functions, various cells in the Organism possess a defined set of receptors. The membrane of a single cell may contain over a dozen Different types of receptors. By interacting with receptors, extracellular chemical messengers influence the METABOLISM and functional state (proliferation, secretion, etc.) of target cells.

1. Adrenaline receptors — adrenergic receptors

Adrenergic receptors are classified according to their distribution in the organism into central and peripheral. Central adrenergic receptors, localized in various Regions of the Brain, participate in The regulation of CNS functions, whereas peripheral receptors control the activity of Internal Organs.

All adrenergic receptors are classified into two types — α and β — but each type has several subtypes, the most common being α₁-, α₂-, β₁-, and β₂-receptors. Depending on their anatomical Location, Cells of the same type, such as vascular smooth Muscle cells or adipocytes, may contain different types of receptors.

Despite significant similarities between α- and β-receptors and their subtypes, they are encoded by different genes. Adrenergic receptors belong to a family of proteins characterized by seven transmembrane α-helices (conventionally referred to as domains). The lengths of the N- and C-termini, as well as the lengths of domains 1 through 4, vary among different receptor types and subtypes (Fig. 5-30).

Fig. 5-30. Membrane Organization OF THE β₂-adrenergic receptor. 1 — receptor fragment involved in G_s protein binding; 2, 3 — sites of potential phosphorylation by protein kinase A (2) and β-adrenergic receptor kinase (3); 4 — glycosylation site; 5 — adrenaline-binding site.

Adrenoceptors are Glycoproteins that contain various carbohydrate moieties. Aspartate residues located in the N-terminal region undergo glycosylation.

β-Adrenoceptors are found in virtually all Tissues of the body. The number of β-adrenoceptors per cell ranges from 300 to 4,000.

The adrenaline-binding site is formed by amino acid residues of the third, fifth, and sixth domains. Another functionally important region is the interaction site with G proteins, which are involved in generating the cellular response. Serine and Threonine residues in the third intracellular domain and the C-terminus of the adrenoceptor can be phosphorylated by protein kinase A or a specific β-adrenoceptor kinase. Phosphorylation leads to a conformational change in the receptor and a decrease in affinity for the G protein, or it prevents G protein binding.

α-Adrenoceptors are distinguished by their localization (e.g., hepatocytes have α₁ receptors, while adipocytes have α₂ adrenoceptors) and The Mechanism of biological signal transduction. Effector systems associated with α₁ and α₂ adrenoceptors involve different types of G proteins — G_plc proteins (stimulatory G proteins) and G_i proteins (inhibitory G proteins) — and, accordingly, the enzymes phospholipase C or adenylate cyclase.

2. Receptors with Tyrosine Kinase Activity

Tyrosine protein kinases are enzymes that phosphorylate specific proteins at tyrosine residues and are divided into two types: membrane (receptor) and cytoplasmic. Intracellular tyrosine protein kinases participate in signal transduction pathways to the nucleus. Receptor tyrosine protein kinases are involved in transmembrane signal transmission.

An example of a receptor tyrosine protein kinase is the insulin receptor (Fig. 5-31). The insulin receptor is a tyrosine protein kinase that phosphorylates proteins at tyrosine OH groups.

Fig. 5-31. Activation of the insulin receptor, a tyrosine protein kinase.

The receptor consists of two α and two β subunits linked by Disulfide Bonds and non-covalent interactions. Both α and β subunits are glycoproteins with carbohydrate moieties on the outer surface of the membrane. The α subunits are located extracellularly on the membrane surface. The insulin-binding site is formed by the N-terminal domains of the α subunits. The β subunits span the membrane bilayer and do not participate in insulin binding.

The catalytic center of the tyrosine protein kinase is located on the intracellular domains of the β subunits. In the absence of the hormone, insulin receptors exhibit no tyrosine kinase activity. The binding of insulin to the site on the α subunits activates the enzyme, with the tyrosine protein kinase itself (the β subunits) acting as the substrate; that is, the β subunit is phosphorylated at multiple tyrosine residues. Phosphorylation of the β subunits occurs via an intermolecular transphosphorylation mechanism, meaning one β chain phosphorylates another β chain of the same receptor molecule. This, in turn, alters the substrate Specificity of the tyrosine protein kinase, enabling it to phosphorylate other intracellular proteins. Activation and changes in specificity are driven by Conformational Changes in the insulin receptor following hormone binding and autophosphorylation.

The key protein phosphorylated by tyrosine protein kinase is insulin receptor substrate-1 (IRS-1). Phosphorylated IRS-1 activates enzymes, such as tyrosine phosphoprotein phosphatase, and proteins involved in the Regulation of cellular processes.

Dephosphorylation of the receptor by tyrosine phosphoprotein phosphatase returns it to an inactive state. The affinity of the receptor for insulin decreases when it is phosphorylated by protein kinase A at serine and threonine amino acid residues.

3. Receptors with Guanylate Cyclase Activity

Guanylate cyclase catalyzes The formation of cGMP from GTP, serving as one of the key messengers in intracellular signal transduction (Figs. 5-32, 5-33). Guanylate cyclase exists in both membrane-bound and cytosolic forms within the cell.

Fig. 5-32. Formation of 3',5'-cyclic GMP (cGMP).

Fig. 5-33. Regulation of the activity of membrane-bound (1) and cytosolic (2) guanylate cyclase.

The ratio of these two enzyme forms varies across different tissues. For example, in Small Intestine cells, 90% of guanylate cyclase is membrane-bound, whereas in the Lungs and Liver, it is only 20%. Cytosolic and membrane-bound guanylate cyclases differ not only in their cellular localization but also in molecular weight, activity, and regulatory mechanisms.

The cytosolic form of guanylate cyclase consists of two subunits (α and β) and contains a heme prosthetic group. The activator of this form of guanylate cyclase, nitric oxide (NO)—which is synthesized from Arginine by the enzyme nitric oxide synthase (see Section 9)—binds at the heme site.

Membrane-bound guanylate cyclase is a transmembrane glycoprotein. The intracellular domain of guanylate cyclase exhibits catalytic activity, while the extracellular domain acts as a receptor. The binding of an activator to the receptor induces a conformational change in both the membrane and cytosolic domains, resulting in the activation of guanylate cyclase. Human tissues contain three Types of Membrane-bound guanylate cyclases, which are activated by specific regulators: atrial natriuretic factor (ANF), brain natriuretic peptide, and the intestinal peptide guanylin.

Three MAIN TYPES OF intracellular receptor proteins that interact with cGMP have been identified in tissue cells: cGMP-dependent protein kinase (protein kinase G), cGMP-regulated Ion Channels, and a cGMP-regulated phosphodiesterase specific for cAMP (which catalyzes The conversion of cAMP to AMP).

cGMP plays a crucial role in regulating Ca²⁺ Homeostasis across various cell types. An increase in cGMP concentration leads to a decrease in intracellular Ca²⁺ levels, both by activating Ca²⁺-ATPases and by inhibiting the receptor-mediated influx of this ion into the Cytoplasm. These effects are mediated by the Action of Protein kinase G on Membrane Proteins involved in Ca²⁺ turnover.

C. Structural and functional Organization of G Proteins

G proteins (GTP-binding proteins) are universal mediators in signal transduction from receptors to cell membrane enzymes, which catalyze the Formation of secondary messengers of hormonal signals. G proteins are oligomers consisting of α, β, and γ subunits. The composition of βγ dimers varies slightly across different tissues, but within a single cell, all G proteins typically possess an identical set of βγ subunits. Therefore, G proteins are generally classified according to their α subunits. Sixteen genes encoding various G protein α subunits have been identified, some of which encode more than one protein due to alternative RNA splicing.

Each α subunit within a G protein contains specific sites for:

✵ binding GTP or GDP;

✵ interacting with the receptor;

✵ binding to βγ subunits;

✵ phosphorylation by protein kinase C;

✵ interacting with the enzyme adenylate cyclase or phospholipase C.

G protein structures lack α-helical membrane-spanning domains, classifying them as membrane-anchored proteins (Fig. 5-34).

Fig. 5-34. Membrane localization of G proteins. For G protein association, the acylation of α-protomers with aliphatic residues of Fatty acids, such as myristic acid (M) or isoprene, is essential. The γ subunit of the G protein possesses a geranylgeranyl group (G) linked via a thioester bond to the C-terminal Cysteine residue.

Regulation of G Protein Activity

The inactive form of a G protein is the αβγ-GDP complex, while the activated form is αβγ-GTP. G protein activation occurs upon interaction with the activator-receptor complex; conformational Changes in the G protein decrease the affinity of the α subunit for GDP while increasing it for GTP. The replacement of GDP with GTP in the active center of the G protein disrupts the complementarity between α-GTP and the βγ subunits. A receptor bound to a signaling molecule can activate A large number of G protein molecules, thereby amplifying the extracellular signal at this stage (Fig. 5-35).

Fig. 5-35. G protein functional cycle. — receptor; Г — hormone; АЦ — adenylate cyclase.

The activated α subunit of the βγ protein (α-GTP) interacts with a specific cell membrane protein and alters its activity. Such proteins may include adenylate cyclase, phospholipase C, cGMP phosphodiesterase, Na⁺ channels, and K⁺ channels.

The next stage in the G protein functional cycle is the dephosphorylation of GTP bound to the α subunit, with The enzyme catalyzing this reaction being the α subunit itself.

Dephosphorylation leads to the formation of the α-GDP complex, which is not complementary to the specific membrane protein (e.g., adenylate cyclase), but exhibits a high affinity for βγ protomers. The G protein returns to its inactive form, αβγ-GDP. Upon subsequent receptor activation and the exchange of GDP for GTP, the cycle repeats. Thus, G protein α subunits undergo a shuttling motion, carrying stimulating or inhibiting signals from the receptor—activated by a primary messenger (such as a hormone)—to the enzyme that catalyzes the formation of the secondary messenger.

Certain forms of protein kinases can phosphorylate G protein α subunits. A phosphorylated α subunit is not complementary to specific membrane proteins, such as adenylate cyclase or phospholipase C, and therefore cannot participate in signal transduction.

A. Adenylate Cyclase

The enzyme adenylate cyclase, which catalyzes the conversion of ATP to cAMP (Fig. 5-36), is a key enzyme in the adenylate cyclase signaling pathway. Adenylate cyclase has been detected in all cell types.

Fig. 5-36. Formation of cyclic adenosine monophosphate (cAMP).

The enzyme belongs to the group of integral cell membrane proteins and features 12 transmembrane domains. The extracellular fragments of adenylate cyclase are glycosylated. The cytoplasmic domains of adenylate cyclase contain two catalytic centers responsible for The production of cAMP, a secondary messenger involved in regulating the activity of protein kinase A.

Adenylate cyclase activity is influenced by both extracellular and intracellular regulators. Extracellular regulators (hormones, eicosanoids, biogenic amines) exert their effects through specific receptors that transmit signals to adenylate cyclase via G protein α subunits. The α_s subunit (stimulatory) activates the enzyme upon interaction, whereas the inhibitory α subunit inhibits it. In turn, adenylate cyclase stimulates the GTP phosphatase activity of the α subunits. As a result of GTP dephosphorylation, α-GDP and related inactive subunits are formed that lack complementarity to adenylate cyclase.

Out of 8 studied isoforms of adenylate cyclase, 4 are Ca²⁺-dependent (activated by Ca²⁺). The regulation of adenylate cyclase by intracellular calcium enables the cell to integrate the activities of the two primary secondary messengers, cAMP and Ca²⁺.

D. Phospholipases

Phospholipases are Enzymes of the hydrolase class that catalyze the Catabolism of Glycerophospholipids. They are divided into secretory phospholipases, which are part of pancreatic juice, and cellular phospholipases. Cellular phospholipases A₁, A₂, D, and C differ in their specificity for the cleaved group. All phospholipases are calcium-dependent enzymes (Fig. 5-37).

Fig. 5-37. Action of phospholipases.

Phospholipase C — an enzyme that hydrolyzes the phosphodiester bond in glycerophospholipids. In human cells, 10 isoforms of phospholipase C have been identified, differing in molecular weight, localization, regulation mechanism, and substrate specificity. The Structure of all phospholipase C isoforms lacks hydrophobic domains that could mediate their interaction with the membrane. However, certain forms of phospholipase C are anchored to the membrane via a hydrophobic anchor—a myristic acid acyl residue—or through interaction with the bilayer surface. The catalytic activity of all phospholipase C isoforms depends on Calcium Ions.

Most phospholipases C are specific for phosphatidylinositols and practically do not hydrolyze Other types of Phospholipids. The active enzyme can hydrolyze up to 50% of the total phosphatidylinositols in the cell membrane. The Hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP₂) yields diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP₃), which serve as secondary messengers in transmembrane signal transduction via the inositol phosphate pathway.

E. Protein Kinases

All polar signaling molecules acting on target cells through membrane receptors exert their biological function by phosphorylating specific Proteins and Enzymes that regulate cellular metabolism. Phosphorylation alters (either increases or decreases) their activity. Protein kinases catalyze the phosphorylation of proteins along serine, threonine, and tyrosine amino acid residues. Protein kinases can function as a subunit of a membrane receptor, such as the tyrosine protein kinase of the insulin receptor, whose activity is regulated by the hormone. Another group consists of protein kinases regulated by secondary messengers of the hormonal signal (cAMP, cGMP, Ca²⁺, DAG), such as protein kinase A, protein kinase C, protein kinase G, calmodulin-dependent protein kinases, etc.

1. Protein Kinases A

Protein kinases A (cAMP-stimulated) are involved in the adenylate cyclase signaling pathway. Protein kinase A consists of four subunits, R₂C₂, comprising two Regulatory Subunits (R₂) and two catalytic subunits (C₂) (see Fig. 5-41). The R₂C₂ complex lacks enzymatic activity.

The R₂C₂ complex attaches to the membrane through various mechanisms. Some forms of protein kinase A are anchored via the aliphatic myristic acid residue of their catalytic subunits. In many tissues, protein kinase A associates with anchored AKAP proteins (cAMP-dependent protein kinase anchoring proteins). AKAPs possess a binding site for the regulatory subunits of protein kinase A. Via AKAP proteins, protein kinase A binds to the membrane in regions containing enzymes that catalyze cAMP formation (adenylate cyclase) or its hydrolysis (phosphodiesterase), as well as proteins whose activity the enzyme regulates, such as voltage-gated Ca²⁺ channels.

The regulatory subunits of protein kinase A feature specific cAMP-binding sites. The binding of cAMP to the regulatory subunits induces a conformational change and decreases their affinity for the catalytic subunits C, leading to dissociation According to the scheme:

cAMP₄ + R2C2 —> cAMP₄ + C + C

The C subunits represent the active form of protein kinase A, which catalyzes the phosphorylation of proteins at serine and threonine residues. The catalytic subunits C of different protein kinase A types are not identical; they differ primarily in their substrate protein specificity.

2. Protein Kinases C

Protein kinases C are involved in the inositol phosphate signaling pathway. The enzyme consists of two functionally distinct domains: regulatory and catalytic. The regulatory domain contains two structures (zinc fingers) formed by cysteine-rich peptide chain fragments binding two zinc ions (see Section 1). These zinc fingers participate in diacylglycerol binding. Another segment of the regulatory domain exhibits a high affinity for Ca²⁺. An increase in cytosolic calcium concentration enhances the affinity of protein kinase C for membrane phosphatidylserine. The translocation of protein kinase C to the membrane enables the enzyme to bind DAG, which further increases the affinity of protein kinase C for calcium ions (Fig. 5-38). The most common isoforms of protein kinase C are activated by Ca²⁺, diacylglycerol, and phosphatidylserine.

Fig. 5-38. Regulation of Protein kinase C (PKC) activity. PS — phosphatidylserine; DAG — diacylglycerol.

The catalytic domain contains a binding site for ATP and the substrate protein. The active form of protein kinase C phosphorylates proteins at serine and threonine residues. A drop in cellular calcium ion concentration disrupts the binding of protein kinase C to phosphatidylserine and diacylglycerol, causing the enzyme to transition into an inactive form and detach from the membrane.

3. Protein Kinases G

Unlike protein kinase A, protein kinase G is not ubiquitous; it is found in the lungs, Cerebellum, smooth muscle, and platelets. Protein kinase G isoforms may be membrane-bound or cytosolic. Soluble protein kinase G consists of two identical subunits, each possessing two cGMP-binding sites. The binding of cGMP to these regulatory sites induces conformational changes in the subunits and enhances the catalytic activity of the enzyme (Fig. 5-39). Similar to protein kinases A and C, protein kinase G is specific for certain protein substrates, which it phosphorylates at serine and threonine residues.

Fig. 5-39. Regulation of protein kinase G (PKG) activity.

F. Phosphodiesterases

Phosphodiesterases are enzymes that catalyze the conversion of cAMP (Fig. 5-40) or cGMP into inactive AMP or GMP metabolites. By lowering the concentrations of secondary messengers, phosphodiesterases terminate the cascade of reactions triggered by the receptor activator.

Fig. 5-40. Conversion of cAMP to AMP.

Phosphodiesterases exist in tissue cells in two forms: soluble and membrane-bound. Membrane-bound enzyme forms account for 5–40% depending on the tissue. The same tissue may contain different forms of phosphodiesterase that differ in substrate affinity, molecular weight, electrical charge, regulatory properties, and intracellular localization.

Cyclic nucleotide phosphodiesterases do not exhibit absolute specificity; therefore, as a rule, a single enzyme form is capable of hydrolyzing both cAMP and cGMP. However, the rates of hydrolysis of these two NUCLEOTIDES by the same phosphodiesterase can vary significantly. This depends on whether the cell contains a more cAMP-specific or cGMP-specific phosphodiesterase, the intracellular ratio of cAMP to cGMP concentrations, and the action of phosphodiesterase regulators.

Most tissues contain phosphodiesterase-1, which is more specific for cAMP and is activated by Ca²⁺, the 4 Ca²⁺-calmodulin complex, and cGMP.

3. The Adenylyl Cyclase System

The effects of hundreds of structurally diverse signaling molecules—such as hormones, neurotransmitters, and eicosanoids—are mediated through the adenylyl cyclase system.

The transmembrane signaling system functions through the coordinated action of several proteins: R_s, the receptor for the signaling molecule that activates adenylyl cyclase, and R_i, the receptor for the signaling molecule that inhibits adenylyl cyclase; G_s (stimulatory) and G_i (inhibitory) G proteins; and the enzymes adenylyl cyclase (AC) and protein kinase A (PKA) (Fig. 5-41).

Fig. 5-41. The adenylyl cyclase system.

Sequence of events leading to adenylyl cyclase activation:

✵ binding of an adenylyl cyclase system activator, such as a hormone (H), to its receptor (R_s) induces a conformational change in the receptor and increases its affinity for the G_s protein, resulting in the formation of the [H][R][G-GDP] complex;

✵ association of [H][R] with G-GDP decreases the affinity of the G_s protein α subunit for GDP while increasing its affinity for GTP, leading to the exchange of GDP for GTP;

✵ this triggers the dissociation of the complex; the released α subunit bound to a GTP molecule exhibits high affinity for adenylyl cyclase:

[H][R][G-GTP] —> [H][R] + α-GTP + βγ;

✵ interaction of the α subunit with adenylyl cyclase alters the conformation of the enzyme and activates it, thereby increasing The rate of cAMP synthesis from ATP;

✵ conformational changes in the [α-GTP][AC] complex stimulate an increase in the GTPase activity of the α subunit. GTP dephosphorylation proceeds, and one of the reaction products—inorganic phosphate (P_i)—dissociates from the α subunit, leaving the [α-GDP] complex intact; the rate of this hydrolysis determines the signal duration;

✵ formation of a GDP molecule within the active center of the α subunit decreases its affinity for adenylyl cyclase while increasing its affinity for the βγ subunits, thereby returning the G_s protein to its inactive form;

✵ if the receptor remains bound to an activator, such as a hormone, the protein functional cycle repeats.

Activation of Protein Kinase A (PKA)

✵ cAMP molecules can reversibly bind to the regulatory subunits of PKA.

✵ Binding of cAMP to the regulatory subunits (R) causes the dissociation of the C₂R₂ complex into a cAMP₄R₂ complex and free catalytic subunits (C + C).

✵ Active protein kinase A phosphorylates specific target proteins at serine and threonine residues, altering the conformation and activity of the phosphorylated proteins, which in turn leads to changes in the rate and direction of regulated cellular processes.

✵ Intracellular cAMP concentration is subject to regulation and depends on the balance between the activities of Adenylyl Cyclase and phosphodiesterase.

AKAPs (A-kinase anchor proteins) play a major role in regulating the Intracellular Signaling system. These anchored proteins participate in the assembly of multienzyme complexes that include not only protein kinase A, but also phosphodiesterase and phosphoprotein phosphatase.

Cascade mechanism of signal Amplification and suppression. The transmission of a signal from a membrane receptor through a G protein to the enzyme adenylate cyclase serves as a classic example of a signal amplification cascade. A single receptor-activating molecule can "turn on" several G proteins, each of which subsequently activates multiple mol-

ecules of adenylate cyclase, resulting in the production of thousands of cAMP molecules. At this stage, the signal is amplified by a factor of 10² to 10³. The generated cAMP then "turns on" another enzyme, protein kinase A, amplifying the signal by an additional 1000-fold. Subsequent phosphorylation of target enzymes by protein kinase A further enhances the signal, yielding a total amplification factor of 10⁶ to 10⁷. Thus, through the mechanism of cascade amplification, a single regulatory molecule is capable of altering the activity of millions of other molecules.

However, for every transmembrane signaling system, the cell possesses a counter-regulatory mechanism designed to suppress the signal. Each step in the enzymatic cascade is tightly controlled by specific dampening mechanisms. For instance, prolonged hormone exposure leads to the desensitization of membrane receptors, causing them either to be inactivated or internalized into the cell via endocytosis along with the hormone. Receptor desensitization consequently reduces the activation level of the adenylate cyclase system. If intracellular cAMP concentrations remain elevated for an extended period (resulting in sustained protein kinase A activity), calcium channels may undergo phosphorylation, leading to a rise in intracellular Ca²⁺ levels. Calcium, in turn, activates a Ca²⁺-dependent phosphodiesterase, which catalyzes the conversion of cAMP to AMP. The resulting inactivation of protein kinase A (R₂C₂) decreases the rate of specific enzyme phosphorylation. Finally, the "switch-off" process is completed by phosphoprotein phosphatase, which dephosphorylates the Phosphoproteins.

Effects of Bacterial toxins on adenylate cyclase activity (ADP-ribosylation of G proteins)

To investigate the function of G proteins within the adenylate cyclase system, researchers have utilized exogenous bacterial poisons, specifically cholera and pertussis toxins. Under experimental conditions, these toxins elevate adenylate cyclase activity in virtually all types of somatic cells. For example, cholera toxin can stimulate the secretion of THYROID HORMONES by thyroid follicular cells, steroid hormones by adrenal cortex cells, and promote lipolysis in adipocytes. The diverse cellular responses to cholera toxin are fundamentally driven by elevated intracellular cAMP levels.

Cholera toxin is an oligomeric protein. One of its subunits functions as the enzyme ADP-ribosyltransferase; upon penetrating the cell, it catalyzes the attachment of ADP-ribose to the α-subunit of the [α_s-GTP][AC] complex (the adenylate cyclase activation step).

NAD⁺ + [α_s-GTP][AC] —> [ADP-ribosyl-α_s-GTP][AC] + nicotinamide + H⁺.

ADP-ribosylation inhibits the intrinsic GTPase activity of the α-subunit, preventing GTP dephosphorylating. Consequently, the functional cycle of the G_s protein arrests at the stage of activating adenylate cyclase—the enzyme responsible for generating cAMP from ATP. The adenylate cyclase enzyme thus maintains heightened activity over an extended duration.

Upon entering the cell, the active subunit of pertussis toxin catalyzes the ADP-ribosylation of the α_i-subunit of the activated G_i protein (α_iβγ-GTP).

NAD⁺ + [α_iβγ-GTP] —> [ADP-ribosyl-α_iβγ-GTP] + nicotinamide + H⁺.

The modified α-subunit retains a high affinity for the βγ-subunits, meaning the G_i protein loses its ability to dissociate into α-GTP and βγ subunits. As a result, the inhibitory signal (α-GTP) fails to reach adenylate cyclase, leaving the enzyme open exclusively to activation upon binding with α_s-GTP. The action of pertussis toxin on tissue cells invariably leads to elevated cAMP levels.

The Clinical symptoms of cholera and pertussis develop as a direct consequence of the toxins produced by their respective causative microorganisms.

II. Inositol Phosphate System

The functioning of the inositol phosphate transmembrane signaling system (Fig. 5-42) involves: R (receptor), phospholipase C, G_plc (the protein that activates phospholipase C), along with various membrane and cytosolic proteins and enzymes.

Fig. 5-42. The inositol phosphate system.

Sequence of events leading to the activation of phospholipase C:

✵ binding of a signaling molecule (such as a hormone) to the receptor (R) induces a conformational change and increases its affinity for the G_plc protein.

✵ formation of the [H][R][G_plc-GDP] complex leads to a reduced affinity of the G_plc protein's α-protomer for GDP and an increased affinity for GTP, resulting in the replacement of GDP with GTP.

✵ this process triggers the dissociation of the complex; the released α-subunit, bound to a GTP molecule, acquires a high affinity for phospholipase C.

✵ α-GTP interacts with and activates phospholipase C. Activated phospholipase C catalyzes the hydrolysis of the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP₂).

✵ this hydrolysis yields the hydrophilic molecule inositol 1,4,5-trisphosphate (IP₃), which is released into the cytosol. The second reaction product, diacylglycerol (DAG), remains anchored in the membrane and assists in activating the enzyme protein kinase C (PKC).

✵ inositol 1,4,5-trisphosphate (IP₃) binds to specific recognition sites on The Endoplasmic reticulum (ER) Ca²⁺ channel, triggering a conformational protein change that opens the channel and allows Ca²⁺ to flood into the cytosol. In the absence of cytosolic IP₃, the channel remains closed.

Activation of Protein Kinase C

✵ An elevation in cytosolic Ca²⁺ concentration accelerates the interaction of calcium ions with the inactive cytosolic enzyme protein kinase C (PKC) and the protein calmodulin, thereby branching the signal initially received by the cell receptor.

✵ The binding of protein kinase C to calcium ions enables the enzyme to engage in calcium-mediated interactions with molecules of acidic membrane phospholipids, specifically phosphatidylserine (PS). Diacylglycerol, by binding to specific sites within protein kinase C, further enhances its affinity for calcium ions.

✵ An enzymatic complex is formed on the inner leaflet of the membrane — [PKC] [Ca²⁺] [DAG] [PS] — representing active protein kinase C, which phosphorylates specific enzymes at serine and threonine residues.

The Role of Calmodulin in Inositol Phosphate Signal Transduction

Cells of many tissues contain the protein calmodulin, which functions as an intracellular receptor for Ca²⁺ and possesses 4 binding sites for Ca²⁺. The [calmodulin] - [4 Ca²⁺] complex lacks enzymatic activity, but its interaction with various proteins and enzymes leads to their activation.

System Self-Regulation

Like most transmembrane signaling systems, the inositol phosphate pathway incorporates not only a signal amplification mechanism but also a mechanism for signal attenuation. Inositol 1,4,5-trisphosphate (IP₃) and diacylglycerol (DAG) present in the cytosol can be converted back into phosphatidylinositol 4,5-bisphosphate (PIP₂) within the membrane through a series of reactions. The enzymes catalyzing the regeneration of this phospholipid are activated via phosphorylation by protein kinase C.

The intracellular concentration of Ca²⁺ decreases to basal levels through the action of Ca²⁺-ATPases located in The Plasma Membrane and endoplasmic reticulum, as well as Na⁺/Ca²⁺ and H⁺/Ca²⁺ translocases (active antiporters) in the plasma and mitochondrial membranes.

The functioning of Ca²⁺ translocases and Ca²⁺-ATPases can be activated by:

✵ the [calmodulin] [4 Ca²⁺] complex;

✵ protein kinase A (via phosphorylation);

✵ protein kinase C (via phosphorylation).

A decrease in the concentrations of Ca²⁺ in the cell and diacylglycerol in the membrane triggers a conformational change in protein kinase C, reducing its affinity for phosphatidylserine and causing the enzyme to dissociate into the cytosol in its inactive form.

Enzymes and proteins phosphorylated by protein kinase C are converted back to their dephosphorylated form through the action of phosphoprotein phosphatase.

K. Signal Transduction via Intracellular Receptors

Signal transmission by lipid-soluble steroid hormones and thyroxine requires these hormones to cross the plasma membrane of target cells (Fig. 5-43).

Fig. 5-43. Signal transduction via intracellular receptors.

Hormone Receptors may be localized in the cytosol or the nucleus. Cytosolic receptors are associated with chaperone proteins (often a complex of several chaperones). Both nuclear and cytosolic receptors for steroid and thyroid hormones contain a DNA-binding domain characterized by two zinc finger motifs.

Sequence of events leading to Transcription activation:

✵ the hormone diffuses across the lipid bilayer of the cell membrane.

✵ interaction of the hormone with the receptor (R) induces a conformational change in the receptor, decreasing its affinity for chaperone proteins, which dissociate from the hormone-receptor complex.

✵ the hormone-receptor complex translocates into the nucleus and interacts with regulatory nucleotide sequences in the DNA, known as enhancers or silencers.

✵ the accessibility of the promoter to RNA polymerase increases (upon interaction with an enhancer) or decreases (upon interaction with a silencer).

✵ the transcription rate of structural genes increases or decreases accordingly.

✵ the rate of Translation increases or decreases.

✵ the amounts of proteins capable of influencing cell metabolism and functional state are altered.

The effects of hormones that transmit signals via intracellular receptors cannot be observed immediately, as template processes (transcription and translation) require hours to take place.

L. Signaling Specificity

Given the vast number of signaling molecules, corresponding receptors, transmembrane signal transduction systems, and second messengers, it remains a puzzle to researchers how protein kinases select the appropriate metabolic pathway enzyme for phosphorylation. To explain this phenomenon, researchers have proposed the "targeting hypothesis". According to this hypothesis, the Specificity of protein kinases and phosphoprotein Phosphatases is achieved through the formation of membrane compartments containing not only the protein kinases and phosphoprotein phosphatases themselves, but also specific substrate proteins. The presence of a myristic or palmitic acid residue in the structure of substrate proteins is a prerequisite for their "anchoring" within the corresponding membrane compartment.

However, in most cases, the activation of any given metabolic process is controlled not by a single intracellular signaling system, but by several, making the crosstalk among these systems an important factor in the cellular response.