Master’s-Level Cell Biology & Advanced Molecular Biology Notes

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1. Definition

Clathrin-mediated trafficking is a highly regulated vesicular transport system in which clathrin coats assemble on cytoplasmic membrane surfaces to help generate and sort transport vesicles.

Clathrin-mediated pathways are especially important in:

• Endocytosis from the plasma membrane
• Transport from the trans-Golgi network
• Endosomal sorting
• Receptor recycling
• Lysosomal delivery
• Regulation of cell-surface proteins

Important: Clathrin is a coat protein, not the entire trafficking machinery. Cargo selection, membrane curvature, vesicle scission, uncoating, targeting, docking and fusion require many additional proteins.

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2. Basic Concept

             CARGO
               ↓
        Cargo receptor
               ↓
          Adaptor protein
               ↓
        Clathrin assembly
               ↓
       Membrane curvature
               ↓
       Budding vesicle
               ↓
            Scission
               ↓
           Uncoating
               ↓
        Target compartment

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3. Major Clathrin-Mediated Pathways

Clathrin participates in several trafficking routes:

                    CLATHRIN
                       │
          ┌────────────┼────────────┐
          ↓            ↓            ↓
     Plasma          TGN          Endosome
     membrane         │             │
          ↓            ↓             ↓
     Endocytosis   Lysosomal      Sorting/
                    pathway       recycling

Major examples

1. Plasma membrane → early endosome
2. Trans-Golgi network → endosome
3. Endosome → selected recycling/degradative pathways

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4. Clathrin-Mediated Endocytosis

The best-studied pathway is clathrin-mediated endocytosis (CME).

It allows cells to selectively internalize:

• LDL
• Transferrin
• Activated receptors
• Nutrient receptors
• Membrane proteins
• Signaling molecules

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5. Overall Mechanism

Plasma membrane
       ↓
Cargo recognition
       ↓
Adaptor recruitment
       ↓
Clathrin recruitment
       ↓
Coat assembly
       ↓
Membrane bending
       ↓
Clathrin-coated pit
       ↓
Neck formation
       ↓
Dynamin-dependent scission
       ↓
Clathrin uncoating
       ↓
Early endosome

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6. Step 1 — Cargo Recognition

The process begins when specific cargo molecules interact with membrane receptors.

For example:

Extracellular LDL
       ↓
LDL receptor
       ↓
Cytoplasmic receptor tail
       ↓
Adaptor protein
       ↓
Clathrin

The cytoplasmic tails of many receptors contain sorting motifs recognized by adaptor proteins.

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7. Cargo-Sorting Signals

Cargo receptors contain short cytoplasmic sequence motifs.

Important motifs include:

• YXXΦ motifs
• D/EXXXLL motifs

where Φ represents a bulky hydrophobic amino acid.

These motifs can be recognized by adaptor complexes.

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8. Adaptor Proteins

Adaptor proteins connect cargo receptors to clathrin.

At the plasma membrane, an important adaptor complex is:

AP2

Simplified organization:

Extracellular
     │
   Cargo
     │
 Receptor
     │
 Cytoplasmic tail
     │
     ↓
    AP2
     │
     ↓
  Clathrin
     │
 Cytoplasm

Therefore:

Adaptors provide a molecular bridge between cargo and the clathrin coat.

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9. AP2

AP2 = adaptor protein complex 2

It is a major adaptor complex involved in clathrin-mediated endocytosis at the plasma membrane.

AP2 helps:

• Recognize cargo
• Interact with phosphoinositides
• Recruit clathrin
• Organize the developing coated pit

A key membrane lipid involved is:

PI(4,5)P₂

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10. Role of PI(4,5)P₂

PI(4,5)P₂ is an important plasma-membrane phosphoinositide.

It helps recruit and organize proteins involved in endocytosis.

Conceptually:

Plasma membrane
      │
 PI(4,5)P₂
      ↓
   AP2 recruitment
      ↓
Cargo concentration
      ↓
Clathrin assembly

Thus, membrane lipids act as molecular landmarks for trafficking machinery.

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11. Clathrin Structure

The fundamental structural unit of clathrin is the:

triskelion

A clathrin triskelion contains:

• 3 heavy chains
• 3 light chains

             Heavy chain
                  \
                   \
        Heavy ─────●───── Heavy
                   /
              Clathrin
              triskelion

Many triskelia assemble into a lattice.

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12. Clathrin Coat

Clathrin triskelia form a curved polygonal lattice.

This promotes membrane curvature.

Flat membrane
──────────────────────
       ↓
     Curvature
        ╲
         ╲____
        /    \
       /      \

The coat therefore contributes to formation of a budding vesicle.

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13. Clathrin Does Not Directly Determine Everything

A common misconception is:

“Clathrin makes the vesicle.”

More accurately:

Clathrin contributes to coat formation and membrane curvature, while other proteins determine cargo selection, membrane remodeling, scission, targeting and fusion.

Important components include:

• Adaptors
• Accessory proteins
• Actin
• Dynamin
• Rab proteins
• SNARE proteins

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14. Clathrin-Coated Pit

A typical sequence is:

Early pit
   ↓
Growing pit
   ↓
Deep invagination
   ↓
Narrow neck
   ↓
Scission

The membrane becomes progressively curved until a nearly spherical vesicle is formed.

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15. Dynamin and Vesicle Scission

Dynamin is a large GTPase involved in scission of many clathrin-coated vesicles.

It assembles around the neck of the budding vesicle.

       Plasma membrane
───────────────┐
               │
             ╱─┴─╲
            │Dynamin│
             ╲─┬─╱
               │
               ↓
        Vesicle released

GTP hydrolysis contributes to the remodeling/constriction process leading to scission.

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16. Role of Actin

Actin is especially important when substantial membrane deformation or mechanical force is required.

Actin can contribute to:

• Membrane invagination
• Pit maturation
• Vesicle movement
• Force generation

This is particularly important in cells with high membrane tension or specialized membrane architecture.

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17. Vesicle Uncoating

Immediately after scission, the clathrin coat must be removed.

This process involves:

• Hsc70
• Auxilin
• Associated co-chaperone machinery

Clathrin-coated vesicle
          ↓
       Uncoating
          ↓
Uncoated transport vesicle
          ↓
       Endosome

Uncoating allows the vesicle to interact with subsequent trafficking machinery.

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18. Hsc70

Hsc70 is a molecular chaperone involved in clathrin uncoating.

Its activity helps disassemble the clathrin lattice so that clathrin can be recycled for another round of vesicle formation.

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19. Early Endosome

After uncoating, the vesicle is delivered toward the early endosome.

The early endosome acts as a major sorting station.

Clathrin-coated vesicle
          ↓
       Uncoating
          ↓
    Early endosome
          │
     ┌────┼────┐
     ↓    ↓    ↓
 Recycle Signal Degrade

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20. Endosomal Sorting

Internalized cargo may have several possible fates.

Recycling

Return to the plasma membrane.

Degradation

Transport toward lysosomes.

Signaling

Continue or modify receptor signaling from endosomal compartments.

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21. Clathrin and Receptor Recycling

Example:

Transferrin receptor

Plasma membrane
       ↓
Clathrin-mediated endocytosis
       ↓
Early endosome
       ↓
Cargo/receptor sorting
       ↓
Recycling endosome
       ↓
Plasma membrane

This allows the receptor to be reused.

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22. Clathrin and Receptor Downregulation

Some receptors are internalized and subsequently degraded.

Activated receptor
       ↓
Ubiquitination
       ↓
Endocytosis
       ↓
Early endosome
       ↓
Late endosome
       ↓
Lysosome
       ↓
Degradation

This reduces the number of receptors at the cell surface.

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23. Clathrin and Lysosomal Delivery

Clathrin also participates in trafficking from the trans-Golgi network (TGN) toward endosomal/lysosomal compartments.

A simplified pathway:

Golgi
  ↓
Trans-Golgi network
  ↓
Clathrin-coated vesicle
  ↓
Endosome
  ↓
Lysosome

This pathway is important for delivery of lysosomal enzymes.

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24. Mannose-6-Phosphate Pathway

Many lysosomal hydrolases receive a:

mannose-6-phosphate (M6P)

sorting signal.

The pathway can be simplified as:

Lysosomal enzyme
       ↓
Golgi processing
       ↓
M6P recognition
       ↓
M6P receptor
       ↓
Clathrin-coated vesicle
       ↓
Endosome
       ↓
Lysosome

The M6P receptor subsequently participates in recycling.

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25. Clathrin at the Trans-Golgi Network

At the TGN, clathrin works with different adaptor proteins than those used at the plasma membrane.

Important adaptor complexes include:

• AP1
• GGA proteins

These recognize specific cargo and promote sorting into transport vesicles.

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26. AP1

AP1 = adaptor protein complex 1

It is important in trafficking involving:

• Trans-Golgi network
• Endosomes

Conceptually:

TGN
 │
Cargo
 │
AP1
 │
Clathrin
 ↓
Transport vesicle
 ↓
Endosome

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27. GGA Proteins

GGA = Golgi-localized, γ-ear-containing, ARF-binding proteins

GGAs are important clathrin-associated sorting proteins at the TGN.

They participate in the transport of selected cargo, particularly lysosomal enzymes.

They interact with:

• ARF GTPases
• Cargo sorting signals
• Clathrin

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28. AP2 vs AP1

Feature	AP2	AP1
Major location	Plasma membrane	TGN/endosomes
Main role	Endocytosis	Intracellular sorting
Clathrin association	Yes	Yes
Cargo selection	Yes	Yes
Important lipid context	PI(4,5)P₂	TGN phosphoinositide environment

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29. ARF GTPases

ARF proteins are small GTPases that regulate membrane trafficking.

At the Golgi/TGN they help recruit:

• Adaptors
• Coat proteins
• Other trafficking machinery

Conceptually:

ARF-GTP
   ↓
Adaptor recruitment
   ↓
Clathrin recruitment
   ↓
Cargo sorting
   ↓
Vesicle formation

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30. Rab GTPases vs ARF GTPases

Both are small GTPases but have different major roles.

Feature	Rab	ARF
Major role	Vesicle identity/targeting	Coat/adaptor recruitment and membrane trafficking
Important process	Docking/fusion	Budding/coat recruitment
Molecular state	GDP/GTP switch	GDP/GTP switch

There is substantial functional integration between these systems.

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31. Clathrin Trafficking Is a Multi-Step Process

A useful master’s-level framework is:

1. Cargo recognition
        ↓
2. Adaptor recruitment
        ↓
3. Clathrin assembly
        ↓
4. Membrane curvature
        ↓
5. Pit maturation
        ↓
6. Dynamin-mediated scission
        ↓
7. Uncoating
        ↓
8. Vesicle targeting
        ↓
9. Tethering
        ↓
10. Docking
        ↓
11. SNARE-mediated fusion
        ↓
12. Cargo sorting

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32. Coat Assembly Is Highly Regulated

Clathrin coat formation depends on:

• Cargo availability
• Adaptor proteins
• Phosphoinositides
• Accessory proteins
• Membrane curvature
• Cytoskeletal forces

Thus, cells can regulate when and where clathrin-coated vesicles form.

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33. Accessory Proteins

Many accessory proteins regulate clathrin-coated pit formation.

Examples include:

• AP180
• Epsin
• CALM
• Intersectin
• Endophilin
• Amphiphysin

Their functions include:

• Membrane curvature
• Coat organization
• Recruitment of dynamin
• Actin regulation
• Pit maturation

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34. Epsin

Epsin is an accessory protein involved in clathrin-mediated endocytosis.

It contains domains that interact with:

• PI(4,5)P₂
• Ubiquitinated cargo
• Clathrin
• Other endocytic proteins

Its membrane-binding properties can contribute to membrane curvature.

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35. BAR-Domain Proteins

BAR-domain proteins are important membrane-remodeling proteins.

They can sense or generate membrane curvature.

Flat membrane
     ↓
BAR protein recruitment
     ↓
Curvature
     ↓
Deep membrane invagination

They help coordinate membrane shape with the endocytic machinery.

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36. Cargo-Specific Trafficking

Different cargoes use different receptors and adaptors.

Therefore:

Different cargo
       ↓
Different receptor
       ↓
Different sorting signal
       ↓
Adaptor recognition
       ↓
Specific trafficking route

This allows the cell to maintain precise control over membrane composition.

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37. Clathrin-Mediated Trafficking and Cell Signaling

Many signaling receptors are internalized through clathrin-dependent pathways.

Example:

Growth factor
     ↓
Receptor activation
     ↓
Clathrin-mediated internalization
     ↓
Endosome
     ↓
Signal continues/modifies
     ↓
Recycling or degradation

Thus, clathrin trafficking can regulate both:

signal intensity and signal duration.

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38. GPCR Internalization

Many GPCRs undergo ligand-induced internalization.

A simplified pathway:

Ligand
 ↓
GPCR activation
 ↓
Receptor phosphorylation
 ↓
β-arrestin recruitment
 ↓
AP2 recruitment
 ↓
Clathrin-coated pit
 ↓
Endocytosis
 ↓
Endosome

The receptor may then be recycled or degraded.

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39. β-Arrestin

β-arrestin has multiple functions.

It can:

• Reduce GPCR coupling to G proteins
• Link receptors to clathrin/AP2 machinery
• Promote receptor internalization
• Participate in signaling from endosomes

Thus, receptor trafficking and signaling are closely interconnected.

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40. Clathrin and Membrane Homeostasis

Clathrin-mediated endocytosis removes selected components from the plasma membrane.

Exocytosis adds membrane components.

              Plasma membrane
                    │
          ┌─────────┴─────────┐
          ↓                   ↑
     Clathrin CME          Exocytosis
          ↓                   ↑
      Endosomes           Secretory
                            vesicles

The balance contributes to maintenance of:

• Surface area
• Lipid composition
• Receptor density
• Membrane protein distribution

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41. Clathrin-Mediated Trafficking and Disease

Defects in endocytic trafficking can affect:

• Neurotransmission
• Cholesterol metabolism
• Receptor signaling
• Immune function
• Lysosomal function

Examples of biologically important pathways include:

Familial hypercholesterolemia

Defects in LDL receptor function impair LDL uptake.

Neurodegenerative disease

Defective endosomal trafficking can contribute to abnormal neuronal homeostasis.

Cancer

Altered receptor trafficking can influence growth-factor signaling.

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42. Clathrin and Familial Hypercholesterolemia

Normal pathway:

LDL
 ↓
LDL receptor
 ↓
Clathrin-mediated endocytosis
 ↓
Endosome
 ↓
LDL processing

If LDL receptor function or trafficking is defective:

↓ LDL uptake
       ↓
↑ circulating LDL
       ↓
Atherosclerotic risk

This illustrates the physiological importance of receptor-mediated trafficking.

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43. Clathrin-Coated Vesicle: Complete Molecular Model

                 EXTRACELLULAR
                      │
                    Cargo
                      │
                  Receptor
                      │
──────────────────────┼──────────────────
                Plasma membrane
                      │
                   AP2
                      │
               Accessory proteins
                      │
                  Clathrin
                 /    │    \
                /     │     \
             Curved clathrin coat
                    ↓
              Coated pit
                    ↓
               Deep pit
                    ↓
              Dynamin ring
                    ↓
                 Scission
                    ↓
           Clathrin-coated vesicle
                    ↓
                Uncoating
                    ↓
              Early endosome

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44. Advanced Concept: Coat and Targeting Are Different Problems

An important conceptual distinction:

Coat machinery

Determines:

How and where the vesicle buds.

Targeting machinery

Determines:

Where the vesicle goes.

Fusion machinery

Determines:

Which membrane it fuses with.

Coat
 ↓
Budding
 ↓
Rab/tethering
 ↓
Target recognition
 ↓
SNAREs
 ↓
Fusion

This division of labor is fundamental to intracellular trafficking.

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45. Advanced Concept: Clathrin Is Recycled

Clathrin is not consumed during each trafficking event.

After uncoating:

Clathrin-coated vesicle
        ↓
     Uncoating
        ↓
Free clathrin
        ↓
Cytoplasmic pool
        ↓
New coated vesicle

This makes clathrin a reusable component of the trafficking machinery.

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46. Advanced Concept: Endocytosis Is Coupled to the Cytoskeleton

Clathrin-mediated trafficking is coordinated with:

• Actin polymerization
• Microtubule transport
• Motor proteins

This allows cells to connect:

membrane remodeling → vesicle formation → intracellular transport

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47. Integrated Pathway

                 PLASMA MEMBRANE
                        │
                 Cargo receptor
                        ↓
                      AP2
                        ↓
                   Clathrin
                        ↓
                Coated pit
                        ↓
                     Dynamin
                        ↓
                 Vesicle scission
                        ↓
                    Uncoating
                        ↓
                  Early endosome
                        │
             ┌──────────┼──────────┐
             ↓          ↓          ↓
         Recycling   Signaling  Degradation
             │          │          │
             ↓          ↓          ↓
       Plasma membrane Endosome  Lysosome

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48. High-Yield Table

Component	Main function
Clathrin	Coat formation and membrane curvature
AP2	Plasma-membrane cargo/adaptor complex
AP1	TGN/endosomal sorting
GGA	TGN cargo sorting
PI(4,5)P₂	Plasma-membrane recruitment platform
Dynamin	Vesicle scission
Hsc70	Clathrin uncoating
Auxilin	Helps recruit/activate Hsc70 during uncoating
Rab proteins	Vesicle identity and targeting
ARF proteins	Coat/adaptor recruitment at Golgi/TGN
SNAREs	Membrane fusion
Epsin	Cargo/coat interaction and membrane curvature
Actin	Mechanical support/remodeling

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49. Important Differences

Clathrin vs Dynamin

Clathrin → coat formation

Dynamin → scission

Clathrin vs SNARE

Clathrin → vesicle budding

SNARE → membrane fusion

Rab vs Clathrin

Rab → trafficking/targeting

Clathrin → vesicle formation

AP2 vs Clathrin

AP2 → cargo selection/adaptation

Clathrin → coat assembly

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50. Examination Short Note

Clathrin-Mediated Trafficking

Clathrin-mediated trafficking is a regulated vesicular transport pathway in which clathrin coats assemble on cytoplasmic membrane surfaces to facilitate cargo sorting and vesicle formation. At the plasma membrane, cargo receptors interact with adaptor proteins such as AP2, which also interact with phosphoinositides such as PI(4,5)P₂. Clathrin triskelia then assemble into a lattice, promoting membrane curvature and formation of a clathrin-coated pit.

As the pit matures, accessory proteins and the actin cytoskeleton contribute to membrane remodeling. Dynamin, a GTPase, participates in scission of the budding vesicle. Following scission, the clathrin coat is removed by an uncoating machinery involving Hsc70 and auxilin. The uncoated vesicle subsequently enters the endosomal trafficking system.

Clathrin also participates in transport from the trans-Golgi network to endosomes, where adaptor proteins such as AP1 and GGA proteins help select cargo. Clathrin-mediated trafficking therefore contributes to receptor internalization, nutrient uptake, lysosomal enzyme delivery, receptor recycling, signaling regulation and plasma-membrane homeostasis.

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51. Viva Questions

Q1. What is clathrin?
A major coat protein involved in formation of transport vesicles.

Q2. What is a clathrin triskelion?
A structural unit containing three clathrin heavy chains and three light chains.

Q3. What is the role of AP2?
Cargo selection and recruitment of clathrin at the plasma membrane.

Q4. What is the role of PI(4,5)P₂?
It provides an important membrane-binding platform for endocytic proteins.

Q5. What is the role of dynamin?
GTP-dependent membrane scission.

Q6. What happens after vesicle scission?
The clathrin coat is removed and the vesicle proceeds toward its target compartment.

Q7. Which proteins are important in uncoating?
Hsc70 and auxilin-associated machinery.

Q8. What is the role of Rab proteins?
They regulate vesicle identity, targeting, docking and trafficking.

Q9. What is the role of SNARE proteins?
They mediate membrane fusion.

Q10. Name two clathrin-associated adaptor systems at the TGN.
AP1 and GGA proteins.

Q11. What is the major sorting station following plasma-membrane endocytosis?
The early endosome.

Q12. How does clathrin trafficking regulate receptor signaling?
By controlling receptor internalization, recycling, endosomal signaling and degradation.

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52. One-Minute Revision

                 CLATHRIN TRAFFICKING
                         │
                    Cargo selection
                         ↓
                    Adaptor/AP2
                         ↓
                      Clathrin
                         ↓
                  Coated pit
                         ↓
                 Membrane curvature
                         ↓
                      Dynamin
                         ↓
                     Scission
                         ↓
                  Uncoating
                         ↓
                   Early endosome
                         │
              ┌──────────┼──────────┐
              ↓          ↓          ↓
          Recycling   Signaling  Degradation
              ↓          ↓          ↓
           Surface    Endosome   Lysosome

Core concept

Clathrin-mediated trafficking is a coordinated process of cargo selection, coat assembly, membrane remodeling, vesicle scission, uncoating, targeting and membrane fusion. Clathrin generates the transport carrier, whereas adaptors select cargo, dynamin assists scission, Rab proteins regulate targeting, and SNAREs mediate fusion.