4. Vesicular Transport, Glycosylation, and Cell Signaling Mechanisms

Course Overview and Introduction to Vesicular Transport

Topics Overview

The following topics define the structure of the course and the current focus of the material:

  • Chemical Foundations

  • Protein Structure, Function, and Regulations

  • Gene to Protein

  • Protein Targeting and Sorting

  • Vesicular Transport (Primary focus for current materials)

  • Membrane Structure and Transport

  • ATP Synthase

  • Cell Signaling (Secondary focus for current materials)

  • Cytoskeleton, Molecular Motors, and Microtubules

  • Cell Cycle, Mitosis and Meiosis

  • Stem Cells

  • Cell Death (Apoptosis)

  • Cancer

The Golgi Apparatus and Protein Sorting

Proteins move through the Golgi apparatus through a continuous process of maturation and transport. The path generally flows from the Rough Endoplasmic Reticulum (RER) toward the plasma membrane or lysosomes.

The Maturation and Transport Model

  1. Cisternal Maturation: The Golgi is a dynamic structure where cisternae move and change their biochemical identity. The process follows this sequence: cis-Golgi $\rightarrow$ medial-Golgi $\rightarrow$ trans-Golgi $\rightarrow$ trans-Golgi network (TGN).

  2. ER-to-Golgi Transport: Proteins are synthesized on bound ribosomes through cotranslational transport into or across the ER membrane. Transport vesicles move from the ER to the Golgi.

  3. Vesicle Budding and Fusion: Vesicles bud from the ER and fuse to form the cis-Golgi.

  4. Retrograde Transport:     * Golgi-to-ER: COP I vesicles bring cargo and misfolded proteins back to the ER.     * Intra-Golgi: Retrograde transport occurs from later cisternae (trans) back to earlier cisternae (cis) to recycle Golgi-resident proteins.

  5. Exocytosis Pathways:     * Constitutive Secretion: Continuous release of proteins to the plasma membrane.     * Regulated Secretion: Proteins are stored in secretory vesicles and released only in response to a specific signal.

  6. Endocytosis: Materials from the exterior enter the cell via endocytic vesicles, move to the late endolysosome, and eventually the lysosome.

Mechanisms of Vesicle Coating and Transport

Specific Vesicle Types and Their Components

Vesicles are categorized by their protein coats, which define their destination and function. Small GTPases, specifically ARF, are associated with most of these processes.

Table of Vesicle Types (Table 14-1)

Vesicle Type

Transport Step Mediated

Coat Proteins Associated

Associated GTPase

Clathrin + AP1

trans-Golgi to Endosome

Clathrin and AP1 complexes

ARF

Clathrin + GGA

trans-Golgi to Endosome

Clathrin + GGA

ARF

Clathrin + AP2

Plasma membrane to Endosome

Clathrin + AP2 complexes

ARF

AP3 complexes

Golgi to Lysosome, melanosome, or platelet vesicles

AP3 complexes

ARF

COP I

Retrograde Golgi transport

COP I

ARF

COP II

ER to cis-Golgi

COP II

Sar1

Clathrin-Coated Vesicle Mechanism

The formation and release of clathrin-coated vesicles involve a series of coordinated steps and key molecular players:

Key Players

  • Cargo Receptor: Binds the specific protein to be transported.

  • ARF: A small GTPase involved in recruitment.

  • Adaptor Proteins: Link the cargo receptors to the clathrin coat.

  • Clathrin: Forms the structural outer lattice (coat) of the vesicle.

  • Dynamin: A GTPase that polymerizes around the neck of the budding vesicle.

  • Hsp70 and Auxilin: ATPases involved in the uncoating process after the vesicle has pinched off.

The Pinching-Off Process

Dynamin acts as the molecular "scissor." It polymerizes around the vesicle neck. Upon GTP hydrolysis, a conformational change occurs that provides the mechanical force required to pinch the vesicle off from the donor membrane.

Summary of Targeting and Docking

  • Proteins are targeted cotranslationally to the ER via signal sequences.

  • Specific coat proteins are recruited by small GTPases (e.g., Sar1-GTP).

  • Coat proteins recruit cargo and assembly proteins to pinch the vesicle from the membrane.

  • Coat Shedding: The coat must shed before the vesicle can fuse with the target membrane.

  • Specificity Proteins:     * Rabs: Small GTPases that define the specificity of vesicle docking.     * v-SNARE / t-SNARE: Complementary pairs on the vesicle (v) and target (t) membranes that ensure the correct fusion and deposition of cargo.

Protein Modification: ER vs. Golgi Functions

Processing in the Endoplasmic Reticulum (ER)

  1. Protein Folding: Facilitated by molecular chaperones such as BiP.

  2. Quality Control: The Unfolded Protein Response (UPR) monitors the state of folding.

  3. Disulfide Bond Formation: Catalyzed by the enzyme Protein Disulfide Isomerase (PDI).

  4. Initial Glycosylation: The addition of core sugar groups to the protein.

Processing in the Golgi Apparatus

  1. Sugar Refinement: Modification and further addition of complex sugars.

  2. Protease Processing: Cleavage of pro-proteins into functional forms (e.g., the conversion of pre-insulin to functional insulin).

Biological Implications of Glycosylation

Glycosylation, the addition of sugars to amino acid residues in the ER and Golgi lumen, has profound effects on cellular function and physiology.

Types of Glycosylation

  • O-linked: Sugars attached to the oxygen atom of Serine or Threonine residues.

  • N-linked: Sugars attached to the nitrogen atom of Asparagine residues.

Functional Roles

  • Protein Folding: Sugar structures help the protein achieve its correct conformation; this modulates function.

  • Protein Targeting: A specific example is the addition of Mannose-6-phosphate (Man-6 phosphate) in the Golgi, which targets proteins specifically to the lysosome.

  • Cell Migration: Sugars on cell surfaces act as receptors. For instance, they allow axons to innervate muscle cells during embryonic development.

  • Blood Typing: The addition of different sugars by glycosyltransferases determines ABO blood types and plays a role in immune rejection.

  • Immune Surveillance: Mutations in cancer cells can lead to "aberrant glycosylation." This makes cancer cells appear "foreign" to the immune system, potentially allowing the body to identify and kill them daily.

Case Study: Allergic Reactions and Cellular Communication

The Mechanism of an Allergy

  1. Trigger: Proteases (allergens) enter the body from the outside.

  2. Signaling: Allergens trigger signaling mechanisms involving D-cells and T-cells.

  3. Antibody Production: B-cells produce Immunoglobulin E (IgEIgE).

  4. Cell Activation: IgEIgE binds to receptors on Basophils and Mast cells.

  5. Histamine Release: These cells release histamine, leading to common symptoms like sneezing, itchy eyes, and runny noses.

Principles of Cell Signaling

Definitions and Terminology

  • Ligand: The signaling molecule that binds to a receptor.

  • Receptor: The protein that receives the signal and initiates a response.

  • Downstream Pathway: The cascade of events following receptor activation.

  • Agonist: A molecule that binds to a receptor and triggers the same response as the natural ligand.

  • Antagonist: A molecule that blocks the receptor, inhibiting the response.

Characteristics of Signaling Systems

  • Redundancy: Multiple pathways or molecules perform the same function.

  • Pleiotropy: A single signaling molecule has different functions depending on the target cell (e.g., IL-4 affects B-cells, T-cells, and macrophages differently).

  • Synergy: The combined effect of two signals is greater than the sum of their individual effects (2+2=82 + 2 = 8). Example: IL-4 and IL-13.

  • Additivity: The combined effect is equal to the sum of individual effects (2+2=42 + 2 = 4).

  • Feedback Mechanisms:     * Negative Feedback: The output of a pathway inhibits its own production (e.g., the Hypothalamus-Pituitary-Thyroid axis where thyroid hormones inhibit TRH and TSH).     * Positive Feedback: The output enhances the initial signal (e.g., Oxytocin in pregnancy).

Signal Integration and Cell Fate

Cells integrate multiple inputs to decide on a response. The following outcomes are determined by specific signal combinations:

  1. Survive: Basic maintenance signals.

  2. Grow + Divide: Additional signals (A,B,C,D,EA, B, C, D, E).

  3. Differentiate: Specific developmental signals (A,B,C,F,GA, B, C, F, G).

  4. Die (Apoptosis): In the absence of any survival signaling, the cell undergoes programmed cell death.

Categories of Signaling by Distance and Method

Extracellular Signaling Types

  • Endocrine Signaling: Glands secrete hormones into the blood vessel to reach distant target cells.

  • Paracrine Signaling: Secretory cells release signals to affect adjacent target cells (local action).

  • Autocrine Signaling: A cell releases a signal that binds to receptors on its own surface (self-stimulation).

  • Contact-Dependent (Plasma-membrane-attached): Signaling requires physical attachment between the signaling cell and the target cell receptor (e.g., Notch signaling).

Ligand Properties

  1. Small Hydrophobic Molecules (Intracellular): Can cross the plasma membrane.     * Examples: Steroids (Testosterone, Estrogen), Retinoids, Thyroxine, and gases like Nitric Oxide (NO).

  2. Hydrophilic Molecules (Cell-Surface): Cannot cross the membrane; require cell-surface receptors.     * Examples: Proteins (Insulin, Growth factors), Peptides (TRH, LH, FSH), and Amino acid derivatives (Serotonin from tryptophan, Thyroxine from tyrosine).

Major Classes of Cell-Surface Receptors

1. Ion-Channel-Coupled Receptors

  • Example: Nicotinic Acetylcholine Receptor (nAChR). Binding of acetylcholine allows Na+Na^{+} ions to enter the cell.

  • Example: Glycine Receptor. Allows Cl−Cl^{-} ions to enter.

2. G-Protein-Coupled Receptors (GPCRs)

  • Structure: 7-Transmembrane Domain (7-TMD) proteins.

  • Mechanism: Binding of a signal molecule activates a G-protein (GTPase), which then triggers an effector enzyme to produce second messengers.

  • Examples: Glucagon receptor, Histamine receptors (H1,H2,H3,H4H1, H2, H3, H4).

3. Enzyme-Coupled Receptors

  • Mechanism: Ligands often bind as dimers, causing the receptor subunits to come together and activate their catalytic domains.

  • Example: Receptor Tyrosine Kinases (RTKs) like the Epidermal Growth Factor Receptor (EGFR) and the Insulin Receptor.

Signal Transduction and Amplification

Why is signaling complex?

Complexity allows for two vital functions: Signal Amplification and Coordinated Diverse Responses.

Signal Amplification Example: Epinephrine

  1. Epinephrine binds to a transmembrane receptor at low concentration (10−10 M10^{-10}\,M).

  2. Adenylyl Cyclase is activated.

  3. ATP is converted to cAMP (second messenger), increasing the concentration significantly (10−6 M10^{-6}\,M).

  4. cAMP activates Protein Kinase A (PKA).

  5. Activated enzymes produce thousands of final metabolic products.

Diverse Effectors

A single signal can elicit different responses based on the cell type's effector proteins:

  • Metabolic Enzymes: Alter metabolism.

  • Transcription Regulatory Proteins: Alter gene expression (Slow response).

  • Cytoskeletal Proteins: Alter cell shape or movement (Fast response).

Acetylcholine Diversity

  • Heart muscle: Decreases rate and force of contraction.

  • Salivary gland: Causes secretion.

  • Skeletal muscle: Causes contraction.

Signal Termination and Experimental Study

Stopping the Signal

Signaling cannot be continuous; it must be modulated. Termination occurs via:

  1. Removing the signal ligand.

  2. Inactivating receptors (receptor sequestration or degradation).

  3. Reversing intracellular signaling events (e.g., via phosphatases).

  4. Transcriptional negative feedback (long-term control).

Experimental Method: Western Blotting

To study phosphorylation-mediated signaling, researchers use phospho-specific antibodies.

  • Total Antibody: Measures the total amount of a protein (e.g., Total Stat5).

  • Phospho-specific Antibody: Measures only the activated, phosphorylated form (e.g., Phospho-Stat5).

  • Increasing concentrations of a stimulus (like Epo in units/ml) typically show a corresponding increase in the signal intensity of the phospho-specific band on a Western blot.

Overall Summary

  • Signaling involves ligand binding, receptor activation, and downstream pathways.

  • Multiple steps enable amplification and regulation.

  • One signal produces diverse effects depending on cell type and available effectors.

  • Cells integrate multiple signals to generate coordinated responses.

  • Signaling is terminated by ligand removal, receptor inactivation, and signaling reversal.