STRUCTURE AND PROPERTIES OF BIOMOLECULES - A. E. Zemlyakov - 2017

04. RECEPTORS AND LECTINS

Receptors

❖ Receptor (from Lat. receptio - perception) is a conformationally flexible biopolymer located On the surface or inside a Cell, capable of specifically binding a certain chemical compound (Ligand) and triggering a biological response.

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In biological systems, receptors interact with a wide range of low-molecular-weight BIOREGULATORS, such as Hormones, Neurotransmitters, Prostaglandins, Leukotrienes, and NUCLEOTIDES. These endogenous ligands possess diverse chemical structures. Specifically, they include Amino Acids, Peptides, Proteins, Steroids, Lipids, Alkaloids, and biogenic amines.

Most receptors are Glycoproteins or Lipoproteins. Ligand-receptor interactions are typically non-covalent in nature. Hydrogen Bonds, ionic, dipole-dipole, and hydrophobic interactions play a crucial role in ligand binding. Structure/103.html">Van der Waals forces also make a certain contribution to The formation of the ligand-receptor complex. The binding process induces conformational changes that are subsequently translated into a biological response.

Many ligands bind to a receptor not via a single site on their structure, but through multiple sites, meaning that multicenter interactions take place. In other words, individual Components of the ligand molecule, such as functional groups or hydrophobic regions, are recognized by distinct segments of the receptor.

In A number of cases, a receptor can interact with multiple ligands. Such interactions are classified into several types:

a) non-competitive - the binding of different ligands occurs at different sites of the receptor structure;

b) competitive - different ligands bind to the same site on the receptor;

c) uncompetitive - the second ligand is able to bind only after the Formation of the receptor-first ligand complex.

From the perspective of eliciting a biological response, ligands are subdivided into agonists - substances that, upon interaction with a receptor, induce a biological response (d), and antagonists - chemical compounds that interact with the receptor but do not trigger a biological response (e).

Antagonists can bind either to the agonist recognition site in a different manner that does not lead to the target response, or interact with another part of the receptor structure, shifting it into an inactive state (allosteric inhibitor) (f).

The ability of a ligand to bind to a receptor is referred to as affinity. The affinity of an agonist is assessed using the ED₅₀ value - The amount of the compound that produces a 50% effect or an effect in 50% of experimental animals. For antagonists, the IC₅₀ characteristic is used - the concentration that provides a 50% inhibitory effect.

❖ Classification of receptors. A distinction is made between Membrane Receptors (integrated into the phospholipid cell membrane) and intracellular receptors, including those located on the inner surface of The Cell membrane, in the Cytoplasm, and on the membranes of cell Organelles, such as nuclear membranes. The latter include, in particular, receptors for steroid and THYROID HORMONES.

Table 4. Selected types and subtypes of receptors

Endogenous ligands typically target not a single receptor, but a group of receptors with similar Specificity, which often mediate different biological responses. Various types and subtypes of receptors are localized on Cells of different Tissues. Some of these receptor types and subtypes are presented in the table. Consequently, the action of a single ligand leads to diverse biological effects in different Organs. While by the early 1990s several dozen receptor types had been discovered, decoding The Human Genome has revealed the existence of several hundred receptors, though ligands for most of them have not yet been identified.

There are 4 main groups of receptors:

■ ligand-gated ion channel receptors (ionotropic receptors), for example, nicotinic acetylcholine receptors or GABA_A receptors;

■ metabotropic receptors - coupled to G proteins, which in turn can regulate The activity of either Enzymes or Ion Channels, for example, angiotensin receptors or adrenoceptors;

■ receptors that directly control Tyrosine kinase activity, such as Insulin and Growth Hormone receptors;

■ intracellular receptors that regulate Introduction/24.html">DNA METABOLISM/31.html">Transcription, such as receptors for steroid and thyroid hormones. Let us examine specific Examples from various receptor groups.

Ion Channel Receptors (Ionotropic Receptors)

❖ GABA_A receptor. Receptors of this type consist of five subunits. Several types of subunits have been identified, designated as α, β, γ, δ, ε, π, θ, and ρ. Some subunits form a series of isoforms: α₁-α₆, β₁-β₄, γ₁-γ₄, and ρ₁-ρ₃. The most prevalent are the α₁β₂γ₂ (60%) and α₂β₃γ₂ (up to 20%) receptor types. The molecular mass of these receptors is 220-270 kDa, and their diameter reaches 8 nm.

Each subunit contains over 200 amino acid residues, which form 4 transmembrane α-helical segments (TM1-TM4), each approximately 20 amino acids long.

The hydrophilic N- and C-terminal domains are located on the outer surface of the membrane. Within the membrane, There is a large hydrophilic domain between the TM-3 and TM-4 fragments that contains phosphorylation sites. The TM-2 domain faces the interior of the channel and contains amino acids responsible for ion recognition. Conformational Changes in this domain govern the opening and closing of the ion channel.

When γ-aminobutyric acid (GABA) binds to the recognition sites located at the interfaces of the α- and β-subunits, a conformational change in the protein subunits occurs, forming an ion channel. Chloride ions flow through this channel into the cell, triggering neuronal inhibition.

The receptor features binding sites for benzodiazepines (BZs), barbiturates, steroids, and ethanol, which act as allosteric agonists.

Metabotropic Receptors

Receptors of this type interact with intramembrane G proteins, which in turn can activate or inhibit a variety of enzymes or ion channels.

Structurally, metabotropic receptors are transmembrane proteins or glycoproteins featuring seven α-helical intramembrane domains. The amino acid chain typically contains 450-550 amino acid residues. The N-terminus of the protein is located extracellularly and generally forms the ligand-binding sites. The C-terminal region is situated intracellularly and is capable of interacting with G proteins.

Biological signal Transduction via metabotropic receptors is mediated by specialized signaling molecules known as intracellular mediators or messengers. The Mechanisms of action for such intracellular mediators as cyclic adenosine monophosphate (cAMP), cyclic guanosine monophosphate (cGMP), Inositol trisphosphate, diacylglycerol, nitric oxide(II), and Ca²⁺ ions have been the most thoroughly studied.

G proteins are complexes composed of three subunits: α, β, and γ. At least 20 α subunits, 4 β subunits, and 7 γ subunits have been identified.

Several groups of G proteins are distinguished:

■ G_s proteins contain the α_s subunit and activate the enzyme adenylate cyclase, thereby inducing The production of the intracellular messenger cAMP;

■ G_i proteins include the α_i subunit, which inhibits adenylate cyclase and activates the hydrolytic enzyme cAMP phosphodiesterase, ultimately leading to a decrease in cAMP concentration;

■ G_t proteins of rhodopsin receptors contain the α_t subunit (Transducin), which activates cGMP phosphodiesterase, leading to a reduction in the concentration of the intracellular messenger cGMP;

■ G_o proteins inhibit Ca²⁺ channels and activate K⁺ channels;

■ G_k proteins activate K⁺ channels and inhibit adenylate cyclase;

■ G_q proteins contain the α_q subunit, which promotes the activation of phospholipase C and, consequently, the Generation of the intracellular messengers inositol trisphosphate and diacylglycerol.

For elucidating the MECHANISM OF ACTION of metabotropic receptors, American scientists M. Rodbell and A. Gilman were awarded the Nobel Prize in Physiology or Medicine in 1994.

❖ β-Adrenergic receptors. The interaction of β-adrenergic receptors with norepinephrine and epinephrine leads to the activation of the intracellular enzyme adenylate cyclase and a corresponding increase in the concentration of the intracellular messenger cAMP, which in turn stimulates a series of enzymes.

Let us examine the MAIN STAGES OF this process in more detail.

The Interaction of a ligand with its receptor leads to the formation of a ligand-receptor complex and a conformational change in the receptor. These conformational changes are transmitted to the G_S protein. In the αs subunit, bound guanosine diphosphate (GDP) is converted into guanosine triphosphate (GTP).

The α subunit, activated in this way, dissociates from the βγ subunit complex and targets the enzyme adenylate cyclase. The enzyme is activated and begins to catalyze the synthesis of cAMP from adenosine triphosphate (ATP). An increase in cAMP concentration leads to the Activation of a number of specific intracellular enzymes, such as protein Kinases, which trigger further biochemical transformations.

Signal termination is achieved by The breakdown of the α-subunit–GTP complex, as the protein subunit possesses enzymatic activity and is capable of hydrolyzing GTP to GDP. As a result, the conformation of the subunit changes. The complex is deactivated, and the system returns to its inactive state.

Subsequently, cAMP is degraded by the action of phosphodiesterases and ceases to activate proteins. The hormone dissociates from the receptor and undergoes biodegradation.

The adenylate cyclase system is characterized by signal Amplification at each step. Following interaction with its ligand, a single receptor molecule is capable of activating between 10 and 100 G proteins and, consequently, the same number of adenylate cyclase molecules. Accordingly, each molecule of the activated enzyme synthesizes up to 100 cAMP molecules. Thus, The stimulation of a single receptor can lead to the activation of 10²–10⁴ specific enzymes.

Interaction with a wide range of hormone receptors occurs in a similar manner, for example, dopamine D₁ and D₂ receptors, histamine H₂ receptors, prostaglandin EP₁ and EP₂ receptors, as well as thyrotropin and somatostatin receptors.

✵ The GTP/GDP switch. One of the most important MOLECULAR MECHANISMS OF the process discussed above is The regulation of molecular Conformations through the mutual conversion of GDP ⇄ GTP, the so-called "GTP/GDP switch." The elongation of the molecule by a single phosphate residue leads to the formation of hydrogen bonds with amino acid residues and, consequently, A change in its Spatial Structure. Conversely, the Hydrolysis of the phosphoester bond returns the molecule to its initial state. Another example of the "GTP/GDP switch" mechanism in action will be examined during the Translation process.

External agents, such as diphtheria or cholera toxins, can block this regulatory mechanism. Toxins are defined as poisons of biological origin.

✵ Cholera toxin is produced by the bacterium Vibrio cholerae and consists of two blocks. A block of 5 B subunits, each with a molecular mass of 12 kDa, ensures attachment to intestinal cells.

The A₁ subunit, which penetrates into the cell, binds to αs subunits, blocking their further transformations and thereby increasing the cAMP concentration. It is responsible for the toxic effect by increasing

the concentration of the intracellular mediator cAMP, which in turn leads to the excessive opening of chloride channels and, consequently, to diarrhea.

❖ GABA_B receptor. Receptors of this type are also found in Central Nervous system cells. They consist of two homologous subunits. The receptor itself has a structure analogous to the one discussed above. The transmission of the biological signal involves not only the α₁ subunits, which inhibit adenylate cyclase, but also the βγ subunit complex, which is capable of activating K⁺ channels and inhibiting Ca²⁺ channels.

The α₁ subunits are capable not only of inhibiting adenylate cyclase but also of activating the enzyme phosphodiesterase (PDE), which catalyzes the breakdown of cAMP into AMP. The activation of PDE occurs, as in the case of rhodopsin discussed below, through the binding of the α₁ subunit into a complex with the inhibitory γ subunit of the enzyme.

As a result of the interaction of ligands with such receptors, the concentration of cAMP within the cell decreases.

Receptors that operate in a similar manner include, in particular, the M₂ and M₄ acetylcholine receptors, the α_2A, α_2B, and α_2C noradrenaline receptors, the D₂, D₃, and D₄ dopamine receptors, the H₃ and H₄ histamine receptors, and the 5-HT₁ and 5-HT₅ serotonin receptors.

❖ Rhodopsin is a light-sensitive protein (a chromoprotein) found in the rod photoreceptor Cells of the retina. Similar proteins are also present in halophilic Bacteria.

The receptor protein is composed of 348 amino acid residues. At the N-terminal region, carbohydrate chains are attached to the Asn-2 and Asn-15 residues. The rhodopsin molecule belongs to the class of membrane-integrated receptors. Seven α-helical segments of the peptide chain are embedded in the phospholipid layer of the membrane. The amino group in the side chain of Lys-296 is condensed with the aldehyde 11-cis-retinal.

When a light quantum hits the membrane, a series of transformations is triggered within the polyene chain of the retinal residue, culminating in its isomerization to all-trans-retinal.

This Schiff base is less stable than the initial one, leading to its Cleavage into the protein moiety (opsin) and free aldehyde. The dissociation of retinal alters the conformation of the receptor's protein chain.

These conformational changes are transmitted to the receptor-coupled G_t protein (transducin). The α_t subunit of transducin interacts with the inhibitory γ subunit of phosphodiesterase, thereby activating the enzyme and initiating the hydrolysis of the intracellular messenger cGMP into GMP.

As with the adenylate cyclase system, receptor interaction triggers a signal amplification cascade. An activated rhodopsin molecule is estimated to activate approximately 500 transducin molecules, which in turn stimulate 500 phosphodiesterase molecules, which consequently hydrolyze 10⁵ cGMP molecules. As a result, 250 Na⁺ channels close, preventing the efflux of 10⁶–10⁷ Na⁺ ions per second, ultimately leading to a membrane hyperpolarization of 1 mV, which is then transmitted to the cell's nerve terminal.

The rhodopsin regeneration process is quite complex, involving the reduction of all-trans-retinal to retinol (vitamin A), its Esterification, isomerization accompanied by ester hydrolysis, oxidation of the alcohol to 11-cis-retinal, and the subsequent attachment of the latter to the protein. Most of these chemical transformations take place in the retinal pigment epithelium.

The retinal required by the Organism is also synthesized through The oxidation of vitamin A (retinol).

❖ α₁-Adrenergic receptor. Interaction with noradrenaline receptors (α₁-adrenergic receptors), as well as a number of Peptide Hormones (e.g., thyroliberin, oxytocin, or angiotensin II), biogenic amines (muscarinic acetylcholine receptors M₁, M₃, M₅, histamine H₁ receptor), mononucleotides (purinergic P_2X, P_2Y receptors for ATP), and growth factors (Cytokinins), proceeds via the activation of G_q proteins.

Unlike the adenylate cyclase system, the α_q subunit activates the membrane-bound enzyme phospholipase C (PL-C), which catalyzes the cleavage of phosphatidylinositol 4,5-bisphosphate (PIP₂) into inositol trisphosphate (IP₃) and diacylglycerol (DAG), both acting as intracellular messengers.

In the presence of Ca²⁺ ions, diacylglycerol complexed with phosphatidylserine (PS) activates another membrane enzyme, protein kinase C, which catalyzes the phosphorylation and consequent stimulation of a number of regulatory proteins.

Inositol trisphosphate supplies the Ca²⁺ ions necessary for the transition of protein kinase from its inactive to its active form. It binds to specific receptors on the Ca²⁺ channels of calciosomes—intracellular organelles where Calcium Ions are concentrated—thereby increasing the concentration of these ions within the cell.

Intracellular Ca²⁺ ions are typically transported by a specialized protein called calmodulin. This globular protein contains a high number of glutamic and aspartic acid residues that form 4 calcium-binding sites. Each such domain consists of a loop combined with two mutually perpendicular α-helices. Such domain motifs are referred to in English-language literature as "EF-hands" and have been identified in over 100 calcium-binding proteins.

Tyrosine kinase-linked receptors.

This type includes the IR_A and IR_B receptors for insulin protein molecules, as well as the IGF1R and IGF2R receptors for Insulin-like Growth Factors. The enzyme tyrosine kinase participates in the transmission of the biological signal.

The insulin receptor is a tetramer consisting of two α-subunits with a mass of 135 kDa each and two β-subunits with a mass of 95 kDa, linked by Disulfide Bonds. The extracellular side of the membrane features a dimer of α-subunits, each containing two Cysteine-rich hormone-binding regions.

The β-subunit comprises three domains: extracellular, transmembrane, and intracellular, the latter including the tyrosine kinase enzyme domain. Upon ligand (insulin) binding, this enzyme is activated via autophosphorylation driven by ATP, after which the tyrosine kinase acquires The ability to phosphorylate intracellular proteins.

Initially, tyrosine kinase activates the intracellular signal transducer IRS-1 (insulin receptor substrate) by phosphorylating its tyrosine residues. The tyrosine-phosphate groups of IRS-1 act as a ligand for the Grb-2 protein, which, through another receptor domain, is able to bind to a Proline-rich fragment of the Sos protein.

The resulting Grb-2+Sos complex triggers The conversion of GDP —> GTP in the Ras G-protein, which in turn activates the Raf-1 protein kinase. This enzyme subsequently phosphorylates Serine residues in the mitogen-activated intracellular kinase (MEK), enabling it to catalyze the incorporation of phosphate groups into the tyrosine and Threonine residues of yet another enzyme: the extracellular signal-regulated kinase (ERK).

The activated ERK kinase translocates into the Cell Nucleus and phosphorylates the Elk-1 transcription factor, enabling it to drive the transcription of more than 100 insulin-regulated genes. Ultimately, this leads to the Synthesis of specific proteins within the cell.

Intracellular Receptors

To date, over 50 intracellular receptors with similar mechanisms of action have been identified. The ligands for these receptors include steroids (sex and corticosteroid hormones), thyroid hormones, retinoic acid (an oxidation product of retinol), vitamin D₃, and Other Compounds.

The binding of these compounds to the receptor induces DNA transcription and ultimately results in the synthesis of specific proteins.

❖ Steroid hormone receptors. Let us examine The structure of a steroid hormone receptor as an example. Such receptors are widely distributed, with a single cell containing anywhere from 1,000 to 10,000 steroid receptors.

The receptor structure comprises four distinct domains. The N-terminal region of the chain is responsible for transcription activation. This is followed by a domain containing zinc ions coordinated by cysteine residues, which mediate binding to the DNA molecule (the so-called "zinc fingers").

An adjacent domain performs an inhibitory function, preventing the receptor from interacting with DNA. Closer to the C-terminus lies the domain containing the ligand-binding sites.

Steroid Hormones are typically transported as complexes with carrier proteins. Upon approaching the cell membrane, the steroid leaves its "protein depot" and crosses The Lipid Bilayer of the cell.

Allosteric binding of the steroid hormone alters the conformation of the receptor, causing the inhibitory domain to release its hold and no longer block interaction with the nucleic acid. The DNA-binding domain shifts into an active conformation, coordinating the receptor with the nucleic acid, while the activation domain initiates the transcription process.

❖ Guanylyl cyclase. Enzymes responsible for synthesizing cGMP from GTP exist in both membrane-bound and soluble intracellular forms. The latter can be regarded as an intracellular receptor. Notably, this enzyme belongs to the Chromoproteins and contains a heme group as its prosthetic group.

The molecule that activates guanylyl cyclase is NO. Nitric oxide regulates a range of physiological processes, including Blood vessel relaxation (vasorelaxation), inhibition of platelet aggregation, immune responses, and nervous system function.

The primary pathways for the generation of endogenous nitric oxide involve the biochemical oxidation of Arginine or the reduction of nitrate ester drugs such as nitroglycerin or isosorbide dinitrate.

A significant portion of the aforementioned BIOLOGICAL EFFECTS OF NO is linked to an increase in intracellular cGMP concentration resulting from the stimulation of guanylyl cyclase activity. The induced production of cGMP triggers the phosphorylation of specific serine- and threonine-containing enzymes, thereby propagating the biological signal.

The concentration of cGMP is regulated by cGMP phosphodiesterases, which hydrolyze the cyclic phosphate to GMP. The application of inhibitors targeting these enzymes leads to elevated cGMP levels and, consequently, blood vessel dilation. Viagra is a well-known example of a medication that exerts its effects through this inhibitory mechanism.

Lectins

The specific recognition of various carbohydrate structures in nature is carried out by specialized proteins known as lectins. Such carbohydrate-protein binding occurs in PLANT AND ANIMAL cells, bacteria, and during virus-cell interactions. Lectins can serve functions related to intercellular recognition, adhesion, and the identification of signaling molecules.

For example, the clearance of a number of glycosylated peptide hormones (luteinizing hormone, thyrotropin) is carried out by Liver cells. In this process, hormone uptake occurs through the binding of hepatocyte lectins to the terminal disaccharide fragment GalNAc4-SO₃H (βI —> 4) GlcNAc.

Table 5. Specificity of a number of lectins

Sialic (neuraminic) acid, located at the non-reducing ends of Oligosaccharides, plays an important signaling function in glycoproteins. It "protects" such newly synthesized molecules from degradation in the liver.

Over time, under the action of sialidases (neuraminidases), sialic acid is removed, and the remaining carbohydrate fragments of glycoproteins bind to lectins.

Influenza virus hemagglutinin acts as a lectin, binding to the NeuАс (α2 —> 6) Gаl (β1 —> 4) Glс oligosaccharide fragments on The surface of animal cells, which subsequently facilitates viral entry into the cell. Currently, a number of anti-influenza drugs that inhibit hemagglutinins are under development.

For the bacterium E. coli, lectins located on the outer membrane that are capable of binding to mannose residues act as an "anchor." The binding of these lectins to epithelial mucins ensures the attachment of bacteria to the intestinal surface.

Selectins form a distinct group of Plasma Membrane lectins involved in intercellular recognition and Cell Adhesion processes. Specifically, they facilitate the migration of immune cells through capillaries to tissues affected by infection or inflammation.