Case 2 - BBS2042
Case 2 - Cell-derived Molecules as Signalling Mediators
1. Learning Goals
Types of Signalling Molecules
Peptides
Purines
Pyrimidines
Proteins
Lipids
Gasses
Neurotransmitters: excitatory and inhibitory
Types of Vesicles and Formation
Signalling Molecule Production
Involves proteins and includes simple steps
Secretion of Signalling Molecules
Primarily proteins
Extracellular Vesicle Induced Signalling
Mechanism of signalling in target cells
Gases as Signalling Molecules
Production, secretion, and function in tissues:
Nitric oxide (NO): role in the cardiovascular system
Carbon monoxide (CO)
Hydrogen sulfide (H₂S)
2. What are Signalling Molecules?
Definition: Signalling molecules (ligands) are substances released by cells that bind to specific receptors on target cells to trigger a biological response.
The Chemical Nature determines:
How they are synthesized
How they are released
Whether they can cross membranes
What type of receptor they bind
How fast and long-lasting their effects are
2.1 Peptide and Protein Signalling Molecules
Description:
Peptides: Short chains of amino acids
Proteins: Longer, folded chains of amino acids
Examples:
Peptides: insulin, glucagon, vasopressin, oxytocin
Proteins: growth hormone, cytokines (e.g., IL-2, TNF-α), erythropoietin
Neuropeptides: substance P, endorphins
Transport & Receptors:
Hydrophilic → cannot cross lipid bilayers
Bind cell-surface receptors such as:
GPCRs
Ionotropic receptors
Receptor tyrosine kinases (RTKs)
Cytokine receptors (JAK/STAT)
Signalling Characteristics:
Fast onset, often short-lived
Amplified via second messengers (cAMP, Ca²⁺, IP₃)
Typical Roles:
Endocrine signalling (hormones)
Paracrine/autocrine signalling (growth factors, cytokines)
Synaptic neuromodulation
2.2 Purines and Pyrimidines (Nucleotide-based Signalling)
Definition: These are nucleotides or nucleosides, not nucleic acids.
Main Molecules:
Purines: ATP, ADP, adenosine
Pyrimidines: UTP, UDP
Where They Act:
Especially important in:
Nervous system
Immune system
Vascular signalling
Receptors:
P1 Receptors (Adenosine Receptors)
GPCRs (A₁, A₂A, A₂B, A₃)
Often inhibitory or modulatory
P2 Receptors (Nucleotides)
P2X: ligand-gated ion channels (fast response)
P2Y: GPCRs (slower, modulatory)
Signalling Features:
Very rapid signalling (especially P2X)
Often short range (paracrine)
Quickly degraded by ecto-nucleotidases
Typical Functions:
Neurotransmission
Pain signalling
Inflammation
Platelet aggregation
Vasodilation/constriction
2.3 Lipid-derived Signalling Molecules
Definition: Hydrophobic or amphipathic molecules derived from membrane lipids
Steroids:
Derived from cholesterol
Examples:
Cortisol
Aldosterone
Estrogen, testosterone
Properties:
Cross membranes freely
Bind intracellular (nuclear) receptors
Act as transcription factors leading to slow onset, long-lasting effects
Eicosanoids:
Derived from arachidonic acid
Examples:
Prostaglandins
Thromboxanes
Leukotrienes
Characteristics:
Made on demand (not stored)
Act locally (paracrine/autocrine)
Bind GPCRs
Functions:
Inflammation
Pain
Fever
Smooth muscle tone
Phospholipid Derivatives:
Examples:
DAG (diacylglycerol)
IP₃ (technically water-soluble, but lipid-derived)
PIP₃
Roles:
Serve as second messengers inside cells
Control Ca²⁺ release, PKC activation, Akt signalling
Endocannabinoids:
Examples:
Anandamide
2-AG
Bind CB₁/CB₂ GPCRs
Involved in retrograde signalling in synapses
2.4 Small-molecule Signalling
Examples:
Gases: NO, CO, H₂S
Amino Acids: glutamate, GABA, glycine
Biogenic Amines: dopamine, serotonin, histamine
Key Properties:
Often very fast
Short half-life
Act via ion channels or GPCRs
2.5 Comparison Overview
Classes of Signalling Molecules:
Class
Membrane Crossing
Receptor Type
Speed
Duration
Peptides / proteins
❌
Cell-surface
Fast
Short
Purines / pyrimidines
❌
GPCRs / ion channels
Very fast
Very short
Steroid lipids
✅
Intracellular
Slow
Long
Eicosanoids
❌ (mostly)
GPCRs
Moderate
Short
Gases
✅
Intracellular
Very fast
Very short
3. What Qualifies as a Neurotransmitter?
A molecule is considered a neurotransmitter if it meets all the following criteria:
Is synthesized in the neuron
Is stored in synaptic vesicles (classically; with some exceptions like NO)
Is released upon presynaptic depolarization (Ca²⁺-dependent)
Binds to specific receptors on the postsynaptic cell
Is terminated by reuptake, degradation, or diffusion
Excitatory vs. Inhibitory Neurotransmitters:
Excitatory: Leads to depolarization (EPSP)
Inhibitory: Leads to hyperpolarization or shunting (IPSP) which increases threshold for excitatory neurotransmitters
3.1 Neurotransmitter Examples
Glutamate:
Function: Excitatory
Accounts for approximately 80% of synaptic transmission in CNS
Essential for learning, memory, and plasticity
Receptor Type: Ionotropic receptor (fast response)
GABA:
Function: Inhibitory (in the brain)
Synthesized from glutamate via glutamate decarboxylase (GAD)
Receptor Types: GABA A, GABA B, GABA C receptors
Glycine:
Function: Inhibitory (in the spinal cord and brainstem)
Major inhibitory transmitter in spinal motor circuits
Mechanism: Ligand-gated Cl⁻ channels; blocked by strychnine
4. What are Vesicles?
Definition: Vesicles are small, membrane-bound compartments that transport cargo (proteins, lipids, neurotransmitters) between cellular compartments or to/from the plasma membrane.
Their identity is defined by:
Coat proteins (how they form)
Cargo they carry
Direction of transport
4.1 COPII Vesicles (ER → Golgi)
Function: Transport newly synthesized proteins from the rough ER to the Golgi.
Formation Steps:
Sar1 Activation:
Sar1 (a small GTPase) binds GTP
Inserts into ER membrane and initiates curvature
Inner Coat Assembly:
Sar1 recruits Sec23/Sec24
Sec24 binds cargo or cargo receptors
Outer Coat Assembly:
Sec13/Sec31 form a cage around the vesicle
Budding and Scission:
The vesicle buds off from ER
Uncoating:
GTP hydrolysis → coat disassembles
Key Idea: COPII moves proteins forward in the secretory pathway.
4.2 COPI Vesicles (Golgi → ER, intra-Golgi)
Function: Retrograde transport; returns ER-resident proteins and redistributes Golgi enzymes.
Formation Steps:
ARF1 Activation:
ARF1-GTP inserts into Golgi membrane
Coatomer Recruitment:
Multi-subunit COPI coat binds ARF1
Cargo Selection:
Recognizes retrieval signals (e.g., KDEL)
Budding and Release:
Vesicle forms and detaches
Uncoating:
ARF1 hydrolyzes GTP → coat falls off
Key Idea: COPI is mainly a recycling vesicle system.
4.3 Secretory Vesicles
Description: These are not coat-defined in the same way as COP or clathrin vesicles.
Constitutive Secretory Vesicles:
Function: Continuous delivery of:
Membrane proteins
Lipids
Extracellular matrix (ECM) components
Characteristics:
Default pathway
No storage
Vesicles fuse with membrane immediately
Regulated Secretory Vesicles:
Function: Store signalling molecules (e.g., hormones, neuropeptides)
Characteristics:
Dense-core vesicles
Cargo often processed inside vesicle
Require Ca²⁺ signal for fusion
4.4 Multivesicular Bodies (MVBs)
Definition: Endosomes containing intraluminal vesicles (ILVs) that sit at a decision point:
Fates:
Degradation
Secretion
MVB Formation Steps
Early endosome formation
From endocytosis
Inward budding
Endosomal membrane buds into the lumen forming ILVs
Requires ESCRT complexes
Cargo sorting
Ubiquitinated proteins preferentially included
Removes receptors from plasma membrane
4.5 ESCRT-Dependent Pathway
Definition: The Endosomal Sorting Complex Required for Transport (ESCRT) is the primary machinery used.
Process:
ESCRT complexes (0, I, II, III) sort cargo, deform the membrane, and abscise (cut) the vesicles.
4.6 Exosomes (Extracellular)
Definition: Small extracellular vesicles (30–150 nm) released when MVBs fuse with the plasma membrane.
Formation Steps:
Endocytosis: Inward budding of membranes → forms early endosome
ILVs Formation: Are formed inside multivesicular bodies (MVB)
MVB Fusion: MVB fuses with plasma membrane
Release: ILVs released as exosomes into the extracellular space
Exosome Cargo:
Proteins
Lipids
mRNA
miRNA
Signalling molecules
Functions:
Intercellular communication
Immune modulation
Development
Cancer signalling
Biomarkers (liquid biopsies)
4.7 Microvesicles (Ectosomes, Extracellular)
Definition: Larger extracellular vesicles (100–1000 nm) formed by direct outward budding of the plasma membrane into the extracellular space.
Process:
Involves blebbing (membrane that draws together) and fission (pinching off the membrane), done by the cytoskeleton
Key Features:
No MVB involvement
Actin-dependent
Different composition from exosomes
Uses regulatory hormones and neurotransmitters
5. Production and Secretion of Signalling Molecules
5.1 Gene Transcription (Nucleus)
Process:
The gene encoding the signalling protein is transcribed into mRNA.
mRNA is processed (capping, splicing, poly-A tail).
mRNA exits the nucleus via nuclear pores.
Vesicles involved: None
5.2 Translation and Targeting to the Rough ER
Process:
Translation begins on a cytosolic ribosome.
An N-terminal signal peptide emerges, and the signal recognition particle (SRP) binds the signal peptide.
Ribosome is targeted to the rough ER and translation continues with the protein entering the ER lumen.
Product: Preproprotein
Vesicles involved: None
5.3 Protein Folding and Early Processing in the ER
Process:
Signal peptide is cleaved → proprotein
Protein folds into its correct conformation.
Disulfide bonds form (if required).
ER quality control removes misfolded proteins.
Vesicles involved: None (still within ER lumen)
5.4 Export from ER via COPII Vesicles
Process:
Properly folded proproteins are selected for transport.
COPII vesicles bud from the ER membrane.
Step-by-step:
ER quality control
Only properly folded proteins can exit the ER; misfolded proteins are retained and degraded (ERAD).
Export signals on cargo
Many secreted proteins contain ER export motifs:
Di-acidic (DXE)
Di-hydrophobic
Cargo receptors
Soluble proteins cannot directly bind coat proteins.
They bind cargo receptors (transmembrane proteins) whose cytosolic tail binds Sec24 (COPII).
COPII coat recruitment
Sar1-GTP initiates curvature; Sec23/24 capture cargo; Sec13/31 complete the vesicle.
Key Idea: COPII vesicles selectively package proteins that are folded, tagged, and receptor-bound.
Function: ER → Golgi transport
Vesicle involved: COPII
5.5 Transport and Processing in the Golgi Apparatus
Process:
COPII vesicles fuse with the cis-Golgi.
Protein moves through the:
cis-Golgi
medial-Golgi
trans-Golgi
Processing in Golgi:
Glycosylation
Sulfation
Proteolytic trimming
Sorting signals added
Vesicles involved:
COPI vesicles recycle Golgi enzymes backward
COPI vesicles return escaped ER proteins
Vesicle involved: COPI (indirectly, recycling)
5.6 Sorting at the Trans-Golgi Network (TGN)
Description: The TGN is the major sorting hub where the protein is sorted into one of two pathways:
Constitutive Secretion (Default pathway for most cells)
Process:
Protein is packaged into constitutive secretory vesicles which bud from the TGN and move directly to the plasma membrane, fusing immediately.
Release: Continuous.
Examples: ECM proteins, growth factors
Vesicle involved: Constitutive secretory vesicle
Regulated Secretion (Used by endocrine and neuronal cells)
Immature Secretory Granules:
Protein is packaged into regulated secretory vesicles, often still in inactive pro-form which bud from the TGN.
Step-by-step:
Sorting Signals in Cargo:
Specific peptide motifs promote entry into regulated vesicles (aggregation-prone domains, pH–dependent interactions).
Cargo Aggregation:
In the TGN (low pH, high Ca²⁺), prohormones aggregate.
Sorting Receptors:
Some proteins bind TGN sorting receptors, clustering cargo into dense-core granules.
Proteolytic Processing:
Proproteins often enter vesicles inactive; proteases generate the active signalling molecule.
Result:
Formation of immature secretory granules, highly enriched in specific cargo.
Lysosomal Sorting:
Proteins destined for lysosomes carry mannose-6-phosphate (M6P) which binds cargo in TGN and is packaged into clathrin-coated vesicles.
This is not used for signalling molecule secretion, but it's an important distinction.
5.7 Vesicle Maturation
Inside regulated secretory vesicles:
Proproteins are cleaved into active forms
Cargo becomes concentrated
Vesicles become dense-core granules
Vesicle involved: Regulated secretory vesicle
5.8 Storage
Description: Mature vesicles are stored near the plasma membrane with release waiting for a stimulus.
5.9 Stimulus-dependent Exocytosis
Process:
External signal triggers Ca²⁺ influx which activates SNARE proteins to mediate membrane fusion.
The vesicle fuses with the plasma membrane and the signalling molecule is released extracellularly.
Vesicle Targeting and Docking
Rab GTPases:
Each vesicle carries specific Rab proteins which interact with:
Tethering complexes on target membrane to ensure vesicles dock at the correct membrane.
Tethering:
Long tethering proteins capture vesicles and bring them close to the plasma membrane.
SNARE-mediated Membrane Fusion
SNARE Proteins:
v-SNAREs (on vesicle)
t-SNAREs (on target membrane)
Step-by-step Fusion:
SNARE Pairing:
v-SNARE and t-SNAREs form a complex that pulls membranes together.
Membrane Destabilization:
Lipid bilayers distort and a fusion pore begins to form.
Fusion Pore Opening:
Vesicle lumen connects to extracellular space.
Regulation of Exocytosis
Constitutive Exocytosis:
SNAREs alone are sufficient, with fusion occurring as soon as the vesicle arrives.
Regulated Exocytosis (key for signalling molecules):
Priming:
Vesicles are docked but restrained with a partially assembled SNARE complex.
Triggering Signal:
Ca²⁺ influx (via voltage-gated channels).
Calcium Sensor:
Synaptotagmin binds Ca²⁺, releasing the fusion clamp.
Rapid Fusion:
Full SNARE zippering and cargo release occurs in milliseconds–seconds.
After fusion:
Vesicle membrane becomes part of the plasma membrane.
SNAREs are recycled.
Membrane excess is removed by endocytosis.
5.10 Post-secretion Fate
Outcomes for the signalling protein:
Binds its receptor
Degraded extracellularly
Or is internalized via clathrin-mediated endocytosis
6. How Extracellular Vesicles Induce Signalling in Target Cells
6.1 Release of Extracellular Vesicles from Donor Cell
Exosomes:
Form inside multivesicular bodies (MVBs) and released when MVBs fuse with the plasma membrane.
Microvesicles (Ectosomes):
Bud directly outward from the plasma membrane.
EV content includes:
Membrane proteins (ligands, receptors)
Lipids
Cytosolic proteins
mRNA, miRNA, other ncRNAs
6.2 EV Transport through the Extracellular Environment
EVs can diffuse locally (paracrine) or travel via blood, lymph, interstitial fluid.
Protection Advantage:
The lipid bilayer protects cargo from:
Proteases
RNases
Harsh extracellular conditions
6.3 Target Cell Recognition and Binding
EVs do not bind randomly; targeting is selective.
Mechanisms of Specificity:
Surface Ligand–Receptor Interactions:
EV membrane proteins bind receptors on target cells such as:
Integrins
Tetraspanins (CD9, CD63, CD81)
Growth factor ligands
Lipid-Based Recognition:
EVs enriched in phosphatidylserine and sphingolipids are recognised by lipid-binding receptors on target cells
Extracellular Matrix Trapping:
EVs can bind ECM components, creating local signalling niches
6.4 Modes of EV-Induced Signalling
Core Concept: EVs can signal in multiple fundamentally different ways.
Surface Signalling (no uptake required):
Receptor activation at the plasma membrane occurs when EV membrane proteins act as ligands, binding to receptors on the target cell and triggering classical signalling cascades.
Examples:
Growth factor signalling
Immune checkpoint signalling
Notch-like juxtacrine signalling (EV-bound ligands)
EVs function as mobile signalling platforms rather than delivery vehicles.
Endocytosis followed by Signalling from Endosomes:
EVs are taken up by clathrin-mediated endocytosis, caveolin-mediated endocytosis, macropinocytosis, or phagocytosis (immune cells).
Endosomal Signalling:
EV membrane proteins remain intact within early or late endosomes, where receptors or ligands signal from endosomal membranes.
Importance: Endosomal signalling is prolonged, spatially regulated, and often differs from surface signalling.
Cargo Release into the Cytosol:
EV membrane fuses with endosomal membrane or becomes leaky, allowing EV contents to enter the cytosol.
Functional Cargo Action:
RNA-Mediated Signalling:
miRNAs repress target mRNAs, altering gene expression and causing long-term phenotypic changes.
Protein Delivery:
EVs may deliver enzymes, transcription factors, and signalling intermediates, constituting horizontal transfer of functional molecules.
Lysosomal Processing followed by Signalling:
EVs delivered to lysosomes can have their cargo degraded, with breakdown products (e.g., lipids, peptides) potentially activating intracellular sensors or modulating metabolism/inflammation.
6.5 Downstream Cellular Responses
Depending on the signalling mode, EVs can induce:
Changes in gene expression
Altered proliferation
Differentiation
Migration
Immune activation or suppression
Metabolic reprogramming
Time Scale:
Surface signalling → fast (minutes)
Endosomal signalling → intermediate
RNA/protein cargo → slow (hours–days)
6.6 Signal Termination
Mechanism:
EV–receptor complexes are internalised, cargo is degraded, miRNAs diluted over time, and EV membranes recycled.
7. General Principles of Gaseous Signalling Molecules
Characteristics of Gaseous Signalling Molecules:
Are small, diffusible gases
Are not stored in vesicles
Synthesized on demand
Diffuse freely across membranes
Act locally (paracrine / autocrine)
Have very short half-lives
Significance of Diffusion:
Freely diffusing gases do not require secretion machinery or classical membrane receptors.
7.1 General Signalling Logic (All Gasotransmitters)
Step-by-step:
Stimulus activates a synthesizing enzyme
Gas is produced in the cytosol
Gas diffuses out of the producing cell
Gas diffuses into neighbouring cells
Gas binds intracellular targets
Gas is rapidly inactivated or degraded
7.2 Nitric Oxide (NO)
Production:
Enzyme: Nitric oxide synthase (NOS)
Isoforms:
nNOS: found in neurons; Ca²⁺-dependent
eNOS: found in endothelium; Ca²⁺-dependent
iNOS: found in immune cells; Ca²⁺-independent
Reaction:
L-arginine + O₂ + NADPH → NO + L-citrullineCofactors: BH₄, FAD, FMN
Release / Secretion:
NO is not secreted but immediately diffuses across membranes acting within ~100–200 µm
Target and Signalling Mechanism:
Primary target: soluble guanylyl cyclase (sGC)
NO binds heme group on sGC
sGC converts GTP to cGMP
cGMP activates:
PKG → intracellular Ca²⁺ decrease → smooth muscle relaxation
Ion channels
Phosphodiesterases
Major Tissue Functions:
Vascular System:
Acetylcholine activates calcium-calmodulin binding, producing NO leading to vasodilation.
Nervous System:
Acts as a retrograde neurotransmitter modulating synaptic plasticity (Long-Term Potentiation, LTP).
Immune System:
iNOS produces large amounts of NO which is cytotoxic to pathogens.
Termination:
Rapid oxidation to nitrate/nitrite, reaction with hemoglobin, or formation of free radicals may occur.
7.3 Carbon Monoxide (CO)
Production:
Enzyme: Heme oxygenase (HO)
Reaction:
Heme → CO + biliverdin + Fe²⁺Release / Secretion:
CO diffuses freely from the producing cell and is present in lower concentrations than NO.
Target and Signalling Mechanisms:
Targets:
Soluble guanylyl cyclase (less potent than NO)
Ion channels (K⁺, Ca²⁺ channels)
Mitochondrial enzymes
Major Tissue Functions:
Nervous System:
Involved in neuromodulation and circadian rhythm regulation.
Vascular System:
Contributes to vasodilation (weaker than NO).
Cytoprotection:
Exhibits anti-inflammatory and anti-apoptotic properties.
Termination:
CO binds to hemoglobin with high affinity and undergoes oxidation in tissues.
7.4 Hydrogen Sulfide (H₂S)
Production:
Enzymes: CBS (brain), CSE (cardiovascular), 3-MST (mitochondria) using substrates like cysteine and homocysteine.
Release / Secretion:
H₂S diffuses freely across membranes and can also be stored in bound sulfane sulfur pools.
Target and Signalling Mechanisms:
Mechanisms:
Ion Channel Modulation:
Opens K_ATP channels and alters Ca²⁺ channels.
Protein Sulfhydration:
Modifies cysteine residues and alters enzyme activity.
Major Tissue Functions:
Cardiovascular System:
Promotes vasodilation and offers cardioprotection.
Nervous System:
Involved in synaptic modulation and neuroprotection.
Metabolism:
Regulates mitochondrial respiration.
Termination:
Occurs through mitochondrial oxidation and binding to metalloproteins.
7.5 Comparison of NO, CO, and H₂S
Features:
| Feature | NO | CO | H₂S |
|---------------------|-------------|-------------|-------------|
| Enzyme | NOS | HO | CBS / CSE / 3-MST |
| Stored? | ❌ | ❌ | Limited |
| Diffusion | Free | Free | Free |
| Main Target | sGC | sGC, ion channels | Ion channels, proteins |
| Speed | Very fast | Fast | Moderate |
| Half-life | Seconds | Minutes | Minutes |
| Toxic at high levels | Yes | Yes | Yes |