Chapter 3: Recognition and Response - Comprehensive Study Notes

Fundamental Principles of Receptor-Ligand Interactions

• Nature of Binding Interaction:

  • Receptor-ligand binding occurs exclusively via multiple noncovalent bonds.

  • Types of noncovalent interactions include:

    • Hydrogen bonds.

    • Ionic bonds.

    • Van der Waals interactions.

    • Hydrophobic interactions.

  • While individual noncovalent bonds are weak, their cumulative effect delivers a strong binding affinity.

  • Least Likely Bond: Covalent bonds are the least likely interaction to be observed between receptors and ligands because they are permanent and difficult to break, which would interfere with the reversible nature of immune signaling.

• Quantitative Measure of Binding Strength ($K_d$):

  • The dissociation constant ($K_d$) is the primary measure used to determine the strength of ligand binding.

  • Lower $K_d$ values indicate higher affinity, as less ligand is required to saturate the receptors.

  • Aggregation and clustering of receptors after ligand binding can enhance the overall binding $K_d$.

• Affinity vs. Avidity:

  • Affinity: Refers to the strength of an individual, single bond between a receptor and its ligand.

  • Avidity: Refers to the combined, overall strength of binding resulting from multiple interactions between multivalent receptors and ligands.

  • Multivalency: Receptor-ligand interactions may be multivalent (having multiple binding sites), which significantly increases the avidity of the interaction.

  • An interaction can possess a weak affinity but still exhibit high overall avidity due to multiple binding points.

Molecular Consequences of Ligand Binding

• Primary Changes in Receptors:

  • Binding induces molecular changes in the receptor, including:

    • Conformational changes (structural shifts).

    • Dimerization or clustering (multimerization).

    • Altered location within the cell membrane (e.g., movement into lipid rafts).

    • Covalent modifications.

• Induced Intracellular Cascades:

  • Alterations in receptor structure or location trigger intracellular events, such as:

    • Activation of specific enzymes.

    • Changes in the intracellular location of various molecules.

• Receptor Variability through Combination:

  • Combinatorial Protein Chain Use: Multiple different protein chains can contribute to a single ligand-binding site. Different combinations of these chains recognize different ligands, effectively increasing receptor variability.

• Cell-Cell Interactions and Signal Transduction:

  • Extended contact between cells facilitates signal transduction and the exchange of cytokine signals.

  • Immune cells rely on binding affinity to maintain contact over long periods.

  • Extended binding may lead to cytoskeletal reorganization within the cell.

  • Scientific Example: Pulecio et al. (Journal of Experimental Medicine, 2010) demonstrated "Cdc42-mediated MTOC polarization in dendritic cells" to control the targeted delivery of cytokines at the immune synapse, such as the polarized secretion of IL-12.

B-Cell Receptor (BCR) and Antibody Structure

• General Features:

  • Immune receptors bear immunoglobulin (Ig) domains.

  • The BCR contains an antibody of a defined specificity.

  • Antibodies are quaternary proteins consisting of two identical heavy (H) chains and two identical light (L) chains.

• Antibody Domains and Functions:

  • Variable Regions ($V_H$ and $V_L$): Antigen specificity is determined by the interaction between the variable regions of the light and heavy chains.

  • Constant Regions ($C_H$ and $C_L$): Antibody effector activity (e.g., phagocytosis, complement fixation) is a function of the constant regions of the heavy chain.

  • Specific Sub-regions:

    • Complementarity-Determining Regions (CDRs): There are three hypervariable regions (CDR1, CDR2, and CDR3) in both the $V_H$ and $V_L$ domains. These regions come together to form the specific antibody combining site.

    • Framework Regions: Invariant amino acids interspersed near each CDR. They are responsible for the proper folding of the CDRs into the combining site.

• Secreted vs. Membrane-Bound BCR:

  • The major difference between membrane-bound and secreted forms of the BCR is the carboxyl (C) terminus of the heavy chain.

  • Membrane-bound BCR: Includes a hydrophobic segment at the C-terminus for membrane anchoring.

  • Secreted antibody: Lacks this hydrophobic carboxyl terminus transmembrane segment.

• Isotypes and Subclasses:

  • Antibody isotypes are defined by the sequence of the constant region of the heavy chain.

  • Heavy Chain Isotypes:

    • Alpha (̑) : IgA.

    • Delta (̔) : IgD.

    • Epsilon (̕) : IgE.

    • Gamma (̓) : IgG.

    • Mu ($μ$) : IgM.

  • Light Chain Isotypes: Kappa ($κ$) and Lambda ($λ$).

  • Subclasses: Further diversity exists, specifically within IgG through the use of subclasses.

• BCR Coreceptors:

  • The BCR complex includes molecules required for signal transduction beyond the antibody itself.

  • Ig̑ and Ig̒: Transduce signals via Immunoreceptor Tyrosine Activation Motifs (ITAMs).

  • Additional Coreceptors: CD19, CD81, and CD21 transmit and relay signals to the cell interior.

T-Cell Receptor (TCR) Structure and Coreceptors

• Antigen Recognition:

  • TCRs bind to peptides derived from antigens that have been degraded and presented by Antigen-Presenting Cells (APCs) within Major Histocompatibility Complex (MHC) molecules.

  • The TCR recognizes both the antigen peptide and the MHC molecule as a complex.

  • Peptides can be sourced from endogenously or exogenously processed antigens.

• TCR Structure:

  • Composed of two subunits, usually an alpha (̑) chain and a beta (̒) chain.

  • Alternative TCR type uses gamma (̓) and delta (̔) chains.

  • Each chain has a constant region (containing the transmembrane domain) and a variable region.

  • Variable regions contain three CDRs forming the peptide-specific binding site.

• Table: T-Cell Accessory Molecules and Coreceptors:

  • CD4: Binds to Class II MHC; functions in adhesion and signal transduction; member of Ig superfamily.

  • CD8: Binds to Class I MHC; functions in adhesion and signal transduction; member of Ig superfamily.

  • CD2 (LFA-2): Binds to CD58 (LFA-3); functions in adhesion and signal transduction; member of Ig superfamily.

  • CD28: Binds to CD80 or CD86; functions in signal transduction to fully activate naive T cells.

  • CTLA-4: Binds to CD80 or CD86; functions in inhibitory signal transduction.

  • CD45R: Binds to CD22; functions in adhesion and signal transduction.

  • CD5: Binds to CD72; functions in signal transduction.

• TCR Signaling Components:

  • CD3: Contains ITAMs that transmit the signal from the TCR to the cell.

  • CD4 and CD8 Functions: Increase the avidity of peptide binding by the TCR.

Pattern-Recognition Receptor (PRR) Families

• General Concepts:

  • Pathogen-Associated Molecular Patterns (PAMPs): Recurring motifs on bacteria, yeast, and parasites.

  • Pattern-Recognition Receptors (PRRs): Receptors for PAMPs. Unlike BCRs/TCRs, they are not clonally distributed; they are expressed equally on all cells of the same type.

  • Locations: PRRs can be integral membrane proteins or localized intracellularly.

• Table: PRR Families and Descriptions:

  • Toll-like receptor (TLR): Located in the plasma membrane, endosomes, and lysosomes. Ligands include microbial carbohydrates, lipoproteins, fungal mannans, flagellin, and viral RNA. Functions in producing antimicrobials and cytokines; inflammation.

  • C-type lectin receptor (CLR): Located in the plasma membrane. Ligands include carbohydrate components of fungi, mycobacteria, viruses, and parasites. Functions in phagocytosis and inflammation.

  • RIG-I-like receptor (RLR): Located in the cytosol. Recognizes viral RNA. Functions in interferon and cytokine production.

  • Nucleotide oligomerization domain (NOD)-like receptor (NLR): Located in the cytosol. Recognizes fragments of intracellular or extracellular bacterial cell wall peptidoglycans. Functions in inflammation and antimicrobial production.

  • Absent-in-melanoma (AIM)-like receptor (ALR): Located in the cytosol and nucleus. Recognizes viral and bacterial DNA. Functions in interferon and cytokine production.

  • cGAS/STING: DNA sensor in the cytosol. Recognizes viral and bacterial DNA. Functions in interferon production.

Cytokines and Chemokines

• Core Concepts:

  • Cytokine Signal: An event instructing a cell to change its metabolic or proliferative state.

  • Binding: Usually noncovalent and high affinity; often induces changes in the target cell's transcriptional program.

  • Chemokines: A subset of cytokines specifically designed to attract cells (chemoattractants).

• Modes of Action:

  • Endocrine: Released into the bloodstream to effect distant cells.

  • Paracrine: Effect nearby cells.

  • Autocrine: Bind to receptors on the cell that produced them.

• Biological Characteristics of Cytokines:

  • Pleiotropy: One cytokine induces different biological effects depending on the target cell.

  • Redundancy: Two or more cytokines mediate similar functions (e.g., IL-2, IL-4, and IL-5 all causing B-cell proliferation).

  • Synergy: Combined activity of two cytokines is greater than the sum of their individual effects.

  • Antagonism: One cytokine inhibits the action of another (e.g., IFN-̓ blocking the IL-4 induced class switch to IgE).

  • Cascade Induction: A cytokine action on one target cell causes that cell to produce additional cytokines.

• Major Cytokine Families:

  • Interleukin-1 (IL-1) Family: Includes IL-1̑, IL-1̒, IL-18, and IL-33. Important inflammatory mediators; act locally on capillary permeability and systemically on the liver to produce acute phase proteins.

  • Class 1 (Hematopoietin) family: Diverse members (IL-2, IL-4, IL-6, IL-12, G-CSF, etc.). receptors share common subunits:

    • ̓_c subunit: Recognized by IL-2, IL-4, IL-7, IL-9, IL-15, IL-12.

    • ̒_c subunit: Recognized by IL-3, IL-5, GM-CSF.

    • $gp130$ subunit: Recognized by IL-6, IL-11, LIF, OSM, CNTF, IL-27.

  • Class 2 (Interferon) family:

    • Type I: IFN-̑ and IFN-̒ ($18$ to $20\,kDa$ dimers). Antiviral effects; inhibit protein synthesis.

    • Type II: IFN-̓. Produced by T and NK cells; modulates adaptive immunity.

    • Type III: IFN-$λ$. Secreted by plasmacytoid dendritic cells; regulates viral replication.

  • Tumor Necrosis Factor (TNF) family: Regulate development and homeostasis. Includes soluble (TNF-̑, TNF-̒) and membrane-bound members (Lymphotoxin-̒, BAFF, APRIL, CD40L, FasL/CD95).

  • Interleukin-17 family: Proinflammatory molecules; monomers range from $17.3$ to $22.8\,kDa$. Typically exist as homodimers. IL-17A induces neutrophil recruitment.

  • Chemokines: Small ($7.5$ to $12.5\,kDa$) proteins with conserved disulfide bonds. Use G-protein–coupled receptors (seven-pass transmembrane) to direct leukocyte migration.

Intracellular Signaling Pathways

• Early Events in Signaling:

  • Antigen-Mediated Receptor Clustering: Initiates signaling; clustered receptors localize in lipid rafts.

  • Tyrosine Phosphorylation: An early step where Src-family kinases phosphorylate ITAMs on CD3 (T cells) or Ig̑/̒ (B cells).

  • Adapter Proteins: Help gather members of signaling pathways into a complex.

• Phospholipase C (PLC) Pathway:

  • Signals induce breakdown of phosphatidyl bisphosphate ($PIP_2$) by PLC̓.

  • This generates Inositol trisphosphate ($IP_3$), causing an increase in cytoplasmic calcium ($Ca^{2+}$).

  • $Ca^{2+}$ binds to Calmodulin (CaM), changing its conformation and altering activity.

• The Ras/MAP Kinase Cascade:

  • Ras: A G-protein activated when GTP is exchanged for GDP.

  • GEFs (Guanine-nucleotide exchange factors): Activate Ras by inducing the exchange.

  • GAPs (GTPase activating proteins): Inhibit Ras by stimulating GTP breakdown.

  • Nuclear Events: Phosphorylated ERK (pERK) activates Elk-1, leading to Fos protein production. pFos binds with pJun to form the transcription factor AP-1. AP-1 facilitates transcription of the IL-2 gene.

• Nuclear Factor Kappa B ($NF-κ B$) Pathway:

  • $NF-κ B$ is held inactive in the cytoplasm by $Iκ B$.

  • Protein Kinase C (PKC) activation leads to the phosphorylation and ubiquitination of $Iκ B$.

  • The destruction of $Iκ B$ allows $NF-κ B$ to translocate to the nucleus to enhance transcription.

• JAK/STAT Pathway:

  • Associated with Class 1 and 2 cytokine receptors.

  • Cytokine binding leads to receptor apposition and activation of Janus kinases (JAKs).

  • JAKs phosphorylate the receptor and then phosphorylation of Signal Transducers and Activators of Transcription (STATs).

  • Phosphorylated STATs dimerize and translocate to the nucleus for gene activation.

Questions & Discussion

• Q: Which would be the LEAST likely bond or interaction observed when receptors interact with their ligands?

  • A: Covalent bonds.

• Q: The major difference between the membrane bound and the secreted form of the BCR is the…?

  • A: Hydrophobic segment of the heavy chain.

• Q: Match each heavy chain to the appropriate isotype.

  • A: ̑: IgA; μ: IgM; ̓: IgG; ̔: IgD; ̕: IgE.

• Q: Match each PRR to the correct description.

  • A: TLR: Plasma membrane/endosomes, lipoproteins/carbs; CLR: Plasma membrane, mycobacteria/parasites; RLR: Cytosol, viral RNA; NLR: Cytosol, bacterial cell wall fragments; ALR: Cytosol/nucleus, viral/bacterial DNA.

• Q: IL-2, IL-4, and IL-5 are produced by T cells and cause B cell proliferation. They are considered to be…?

  • A: Redundant.