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cell lecture 11
Chemical Signalling
Key Points:
Inter-cellular communication:
This means communication between different cells.How do cells communicate?
They release chemicals like hormones or other molecules.
These chemicals can either act on nearby cells (local) or far away cells (distant).
Sometimes, cells can show signals on their surface so that nearby cells can detect them.
How does the signal work?
Ligand binding:
A ligand (the signal molecule, like a hormone) binds to a receptor (a special protein) on the surface of a target cell.Signal transduction:
After the ligand binds, the receptor sends the message inside the cell. Sometimes this happens through second messengers.Cellular responses:
The cell reacts to the message. It could change how it behaves, for example, by moving, dividing, or making new proteins.Gene expression:
Sometimes, the signal even changes which genes are turned on or off in the nucleus.
Short and Long Range Signals
Different types of signalling, based on how far the signal has to travel:
Endocrine signalling (Long Distance)
Hormones are released into the bloodstream.
They travel a long distance to reach target cells.
Example: Insulin, adrenaline.
Paracrine signalling (Short Distance)
The signal is released locally and affects nearby cells.
It diffuses through tissue fluid.
Example: Immune system signals.
Juxtacrine signalling (Direct Contact)
No chemical travels far.
The signal is sent by direct contact between two cells.
Example: Touching membranes of two adjacent cells.
Autocrine signalling (Self-signalling)
The cell releases a signal that binds to its own receptors.
It’s like the cell is talking to itself.
Example: Negative feedback loops, like when a cell tells itself to stop producing something.
Receptor-Ligand Interactions (How the signal starts)
Receptor-ligand interactions are specific.
Think of it like a lock and key.
The ligand (the signal molecule) binds to a specific receptor on the surface of a target cell.
This is similar to how an enzyme binds a substrate.
What happens after binding?
When the ligand binds to the receptor, the receptor changes shape (conformational change).
This change helps send the message inside the cell.
Signal transduction cascade:
After the receptor is activated, it starts a chain reaction (cascade) inside the cell.
This often involves second messengers (small molecules that carry the signal deeper inside the cell).
End result:
The cell reacts by either changing its behavior (cellular response) or changing its gene activity (gene expression).
Key Properties of Receptor-Ligand Interactions
This slide explains 3 important characteristics of how ligands and receptors interact:
Saturability
Each cell has a limited number of receptors.
Once all receptors are occupied by ligands, adding more ligand won’t increase the effect (maximum response is reached).
Specificity
The ligand binds only to a specific receptor.
This ensures the correct message is sent to the right cell.
Reversibility
The ligand can unbind from the receptor.
This makes sure the signal is temporary and can stop when needed.
Common Themes in Signal Transduction
This slide talks about common things you’ll find in most cell signalling pathways:
Receptor types (types of "antenna" on cells):
Ion channels: Receptors that allow ions to pass in or out.
G-protein coupled receptors: Very common, involved in senses (like smell, vision).
Enzyme-linked receptors: Receptors that activate enzymes inside the cell.
Cytosolic receptors: Inside the cell, usually bind ligands that can cross the membrane (like steroid hormones).
Signaling cascades and Second messengers:
These help carry the signal inside the cell:
cAMP: A common messenger.
IP3 & DAG: Lipid-based messengers.
Ions: Like calcium (Ca²⁺) that trigger responses.
Gases & free radicals: Sometimes used as messengers.
Signal integration (how signals are controlled and connected):
Scaffolding: Organizing proteins to make signalling efficient.
Cross talk: Different signalling pathways can interact.
Cross activation/inhibition: One pathway can activate or block another.
Feedback inhibition: The signal can stop itself once enough response is made.
Main Types of Receptors
This slide shows the 4 main types of receptors in cells:
Ion Channel Receptors
When a ligand binds, the channel opens or closes.
Allows ions (like Ca²⁺) to move in or out of the cell.
The movement of ions causes a response inside the cell.
G-protein Coupled Receptors (GPCRs)
Ligand binds to the receptor → activates a G-protein.
G-protein activates enzymes → enzymes create second messengers (cAMP, IP₃).
These second messengers trigger a response.
Kinase-linked Receptors
Ligand binding activates an enzyme or a kinase (a protein that adds phosphate groups).
This leads to a cascade activating other proteins, causing a response.
Hormone (Cytosolic) Receptors
Ligands (like steroid hormones) enter the cell and bind to receptors inside the cytosol.
This complex enters the nucleus and changes gene expression.
Second Messengers
Second messengers are small molecules inside the cell that carry the signal forward after the receptor is activated.
Examples:
cAMP (cyclic AMP)
DAG & IP₃ (lipid-derived messengers)
Ca²⁺ ions (calcium ions)
NO (Nitric Oxide gas)
Why are they important? They amplify the signal and make sure the message is passed efficiently inside the cell.
Signal Transduction Cascades
Signal transduction cascade = a chain reaction inside the cell. Think of it like a domino effect:
One signal starts.
It activates many proteins, one after the other.
Leads to a large, amplified cellular response.
These cascades:
Can be complex (many steps).
Involve many different proteins.
Allow for regulation (can turn the signal on or off).
Allow amplification (one signal → many responses).
Allow crosstalk (signals can interact).
Complexity of Signal Cascades
This slide shows three ways signalling can happen:
One receptor activates multiple pathways
→ One signal can cause different responses.Different receptors activate the same pathway
→ Multiple signals can lead to the same result.Different receptors activate different pathways that influence each other
→ Crosstalk: One pathway can affect another.
Why is this important?
Because the cell can regulate, integrate, and fine-tune its responses depending on many signals.
Amplification of Signal Cascades
This slide shows an example of signal amplification:
It starts with 1 ligand binding to a receptor.
This activates a G-protein.
That activates an enzyme (adenylyl cyclase).
The enzyme makes many molecules of cAMP.
Each cAMP activates many kinases.
Kinases activate even more proteins.
End result:
One signal molecule → millions of final response molecules.
This is how a small signal outside the cell can cause a big change inside the cell.
Calcium Signalling (Regulation and Transport)
Calcium inside cells is tightly controlled because even small changes in calcium levels can trigger big changes in cell behavior.
Calcium is actively transported out of the cytosol (fluid inside the cell) to keep its concentration low.
How is it removed?
Ca²⁺-ATPase (P-type ATPase):
Uses energy from ATP to move Ca²⁺ across membranes (like the plasma membrane, or ER/SR membrane).
Na⁺/Ca²⁺ exchangers:
These use the movement of sodium (Na⁺) into the cell to help push calcium out.
Storage:
Ca²⁺ is stored in places like the endoplasmic reticulum (ER), sarcoplasmic reticulum (SR), or mitochondria, keeping cytosolic levels very low.
Ca²⁺ Release in Signal Transduction
When a signal is received (e.g., from a hormone or neurotransmitter), Ca²⁺ is released from storage areas to act as a messenger.
Where does Ca²⁺ get released from?
From the ER/SR or through plasma membrane channels.
Key types of calcium channels:
Voltage-gated & ligand-gated Ca²⁺ channels (on the plasma membrane)
IP₃ receptors (in the ER):
These are ligand-gated and open when IP₃ binds, releasing Ca²⁺.
Ryanodine receptors (in SR):
Sensitive to Ca²⁺ itself and help in muscle contraction.
Overall idea:
The release of Ca²⁺ allows it to act as a signal inside the cell, which can then trigger things like muscle contraction, secretion, gene expression, etc.
Manipulating and Measuring Ca²⁺
Scientists use special tools to study calcium inside cells.
How is Ca²⁺ measured?
Ca²⁺ indicators:
These are fluorescent molecules that light up when Ca²⁺ is present.
Many are based on green fluorescent proteins (GFP) from jellyfish.
The image shows Ca²⁺ signaling in plants using one of these indicators.
How is Ca²⁺ manipulated?
Ca²⁺ ionophores:
These are chemicals that allow Ca²⁺ to pass through membranes, artificially raising calcium levels to study what happens.
What Are GPCRs?
GPCRs (G-protein coupled receptors) are proteins that sit in the plasma membrane of a cell.
They have 7 transmembrane helices (they snake through the membrane 7 times).
They detect signals from outside the cell (like hormones) and pass the message inside using G-proteins.
When a signal binds (called a ligand), the GPCR activates a G-protein inside the cell.
Why Are GPCRs Important?
They’re involved in tons of processes: vision, smell, mood, heart rate, etc.
There are about 900 GPCRs in humans.
They’re the largest family of drug targets — over 50% of drugs work by affecting GPCRs (examples shown in the drug table).
What Are G-Proteins?
G-proteins act as on/off switches.
They’re either:
Inactive when bound to GDP (like a dead battery),
Or active when they switch GDP for GTP (full battery).
Two key regulators:
GEFs (Guanine Exchange Factors) turn them on by swapping GDP for GTP.
GAPs (GTPase Activating Proteins) turn them off by hydrolyzing GTP to GDP.
Types of G-proteins
Heterotrimeric G-proteins have three parts:
Gα (alpha) – binds GTP or GDP, does most of the signaling.
Gβ and Gγ (beta and gamma) – usually stay together as a complex and help signal too.
There are also small monomeric G-proteins, like Arf, but these slides focus on the big heterotrimeric ones.
GPCR + G-protein = GPCR Complex
When the GPCR is not active, it stays near a G-protein with GDP bound (inactive state).
A signal (ligand) binds
Causes conformational change and promotes interaction of Gα with receptor
Receptor has GEF activity, and so Gα exchanges GDP for GTP
Gα-GTP seperates from Gβγ.
Gα-GTP and Gβγ can now both activate other proteins (effectors) in the cell
AN example: adenylyl cyclase, which makes cAMP, a second messenger.
What Is cAMP?
cAMP (cyclic AMP) is made from ATP by adenylyl cyclase.
It’s a second messenger — it spreads the signal inside the cell.
It activates Protein Kinase A (PKA).
PKA phosphorylates proteins to change their activity.
It also adds negative feedback by phosphorylating and inactivating the GPCR.
Immunoprecipitation & Co-IP
This is a lab method to study protein interactions:
Steps:
Make a cell lysate (break open cells).
Add antibodies that bind your protein of interest.
Add secondary antibodies (or protein A) attached to Sepharose beads.
Centrifuge to pull down the protein and anything attached to it.
Co-IP (Co-immunoprecipitation):
Pulls down not just one protein, but also anything interacting with it (like Gα, Gβ, Gγ in a complex).
You can study protein–protein interactions.
What Are Receptor Kinases?
These are special receptors that:
Sit in the cell membrane,
And also act as enzymes (specifically, protein kinases).
When a ligand (like a growth factor) binds to them, they change shape, which activates their internal (cytoplasmic) kinase domain.
This leads to phosphorylation cascades – chains of proteins adding phosphate groups to each other to pass along a signal.
Two key types:
Tyrosine kinases (e.g., EGFR)
Serine/threonine kinases
Growth Factor Signalling & RTKs
Receptor Tyrosine Kinases (RTKs) are activated by growth factors and hormones.
Function: They tell the cell to grow, divide, or differentiate.
Each RTK has:
A ligand-binding domain outside the cell.
A tyrosine kinase domain inside the cell (cytosolic side).
A regulatory tail with tyrosine (Tyr) residues that get phosphorylated.
EGFR Activation
EGFR is a well-known RTK.
When EGF binds, the receptors dimerize (pair up).
This causes autophosphorylation – each receptor phosphorylates the other’s tyrosine tail.
The phosphorylated tails now act like docking stations for other proteins with SH2 domains (a special binding domain that recognizes phosphorylated tyrosines).
EGF Signal Transduction Cascade
One key SH2-domain protein is PLCγ (Phospholipase C gamma):
PLCγ binds to the phosphorylated EGFR tail.
It becomes activated by phosphorylation.
PLCγ cleaves PIP₂ (a lipid) into:
DAG (stays in membrane)
IP₃ (goes to ER and releases Ca²⁺)
This triggers activation of PKC (Protein Kinase C) and Ca²⁺ signaling.
Ras Signalling
Ras is a small G-protein, different from the heterotrimeric ones from GPCRs.
EGFR activates Ras using a protein called GRB2, which:
Has an SH2 domain (binds the phosphorylated EGFR).
Recruits Sos, which acts as a GEF (helps Ras swap GDP for GTP).
Now Ras is active (Ras-GTP), and it can pass on the signal.
Ras Signaling and MAPK Cascade
Activated Ras-GTP starts a MAPK (Mitogen-Activated Protein Kinase) cascade:
Ras → Raf → MEK → MAPK
This ultimately activates transcription factors like Jun and Ets, which turn on genes for growth, survival, and division.
Ras is turned off by GAPs (GTPase Activating Proteins), which help it hydrolyze GTP to GDP.
Serine/Threonine Kinase Receptors
These work similarly to RTKs but phosphorylate serine/threonine residues.
Example: TGF-β receptor family
When TGF-β binds, Type I and Type II receptors come together.
The complex phosphorylates Smad proteins, which then enter the nucleus and affect gene expression.
Plays a big role in cell differentiation (deciding what kind of cell it becomes).
What is Phosphate?
Phosphate (PO₄³⁻) is:
Naturally abundant
Very soluble in water
Chemically versatile, meaning it can form many different types of bonds (especially with oxygen, carbon, or nitrogen).
What is Phosphorylation?
Phosphorylation = the process of adding a phosphate group to a molecule.
In biology, it’s the most common type of post-translational modification — this means it happens after a protein is made, and changes its function.
It usually happens on amino acids that have -OH (hydroxyl) groups:
Serine (S)
Threonine (T)
Tyrosine (Y)
Why Phosphorylation Matters
Adding a phosphate group drastically changes the amino acid:
The phosphorylated version no longer resembles any natural amino acid.
It brings negative charges and new bonding possibilities (like forming hydrogen bonds or ionic bonds).
This changes how the protein folds, functions, or interacts with other molecules.
What Is a Western Blot?
It's a technique used to identify specific proteins in a complex mix.
Here's the basic process:
Proteins are denatured (unfolded) and run on a gel using SDS-PAGE, which separates them by size.
Proteins are transferred from the gel to a membrane (e.g., nitrocellulose or PVDF).
Membrane is probed with antibodies that specifically recognize your protein of interest.
Note on Coomassie Blue:
A non-specific stain used to show all proteins on a gel.
Useful to check:
If protein extraction/loading was even
Purification progress
Overall band patterns
Western Blot Workflow (with Diagram)
Start with your SDS-PAGE gel.
Transfer proteins to a membrane.
Block the membrane to prevent non-specific binding (e.g., with milk or BSA).
Add primary antibody (Ab) — this binds your target protein.
Wash away unbound Ab.
Add secondary antibody — this binds to the primary and is linked to a detection enzyme or fluorophore.
Detect signal (chemiluminescence or fluorescence).
Example Western Blot
Control HeLa cells (Lane 1) vs. EGFR knockout HeLa cells (Lane 2).
Top blot: Uses an antibody for EGFR.
Lane 1 shows a band = EGFR present.
Lane 2 has no band = EGFR is knocked out.
Bottom blot: β-actin loading control (used to show equal protein loading in both lanes).
EGF-Induced Phosphorylation Example
What’s tested? Whether EGFR is phosphorylated (activated) after EGF stimulation.
Lanes:
Lane 1: Untreated cells → no phospho-EGFR signal.
Lane 2: EGF treated cells → strong signal for phosphorylated EGFR (top band).
Lane 3: EGF treated + phosphatase → phosphate is removed → signal disappears.
Lower band shows total EGFR (detected with a different antibody) — used as a control to show EGFR protein is still present in all lanes.
What Are Hormones?
Hormones are secreted signals that act over long distances to coordinate responses between different tissues/organs.
In animals, hormones are made by endocrine glands and released into the bloodstream.
Their effects are usually short-acting (lasting seconds to hours).
Plants also use hormone-like molecules for coordination.
🩺 Example: Epinephrine (a hormone from the adrenal gland):
Targets heart cells → increases heart rate.
Targets liver cells → breaks down glycogen to release glucose.
Types of Hormones
Four main types:
Amino acid-derived:
From amino acids like tyrosine
Examples: epinephrine, norepinephrine
Peptide hormones:
Short chains of amino acids
Examples: vasopressin (ADH), ghrelin
Protein hormones:
Longer peptide chains
Example: insulin
Lipophilic (fat-soluble) hormones:
Examples: testosterone, estrogen, cortisol
Receptors:
Amino acid, peptide, protein hormones → bind surface receptors (e.g., GPCRs, RTKs).
Lipophilic hormones → cross the membrane and bind intracellular receptors.
Adrenergic Hormones
Includes epinephrine and norepinephrine, made by the adrenal glands.
Trigger fight-or-flight response:
Increase heart rate
Mobilize glucose by breaking down glycogen in the liver/muscles
Steroid Hormones
Steroid hormones (like cortisol) are lipophilic, so they:
Diffuse through the plasma membrane without needing a channel.
Bind cytosolic receptors inside the cell.
Once bound:
The hormone–receptor complex enters the nucleus.
Binds specific DNA sequences (like glucocorticoid response elements).
Acts as a transcription factor to turn on gene expression.
What Is Signal Integration?
Cells are constantly receiving multiple signals from different receptors.
These pathways can interact via:
Crosstalk: One pathway affects another.
Cross-activation or inhibition: One pathway can amplify or suppress another.
Feedback: Output from one pathway loops back to regulate itself or others.
Scaffolding (like protein scaffolds or lipid rafts):
Helps organize signaling molecules into efficient, localized platforms.
Ensures that signals are transmitted precisely and efficiently.
Yeast Mating Example (Scaffolding in Action)
In yeast, GPCRs detect mating factors.
The Gβγ subunit recruits a scaffolding protein Ste5, which assembles all components of a MAPK cascade.
This leads to gene expression changes that prepare the cell for mating.
Key concept: Scaffolds like Ste5 organize enzymes spatially → faster and more targeted signaling.
Insulin Signaling Example
Insulin binds to its insulin receptor (IR).
This leads to receptor dimerization and activation.
IRS (insulin receptor substrate) is phosphorylated.
IRS then triggers Glut-4 vesicles to move to the membrane → glucose uptake increases.
Rafts (special membrane domains) organize this system to make it more efficient.
Integration Web
Pathways don’t work in isolation — they’re interconnected.
One receptor can activate many paths.
Multiple receptors can converge on one protein, like cAMP or Ca²⁺.
Crosstalk allows fine-tuned coordination and regulation.
Example: A signal might increase calcium → activate calmodulin → activate kinases → change gene expression.
Analogy: Like piano keys — limited inputs (keys) can make endless outputs (melodies).