Study guide
Receptor Mediated Endocytosis
Cell Biology 2003
Gwen Childs, Ph.D.
Objectives
At the end of this unit, the student should be able to:
1.
Compare and contrast major types of internalization
events.
2.
Categorize the variety of ligands that enter by
receptor mediated endocytosis.
3.
Describe necessary conditions and each step in the
process of receptor mediated endocytosis.
a.
Describe the mechanisms
behind the operation of the clathrin coated pit.
b.
Distinguish the functions and environment of the
early endosome from those of the late endosome
c.
Describe the routes of trafficking from the Golgi
complex to the late endosomes
d.
Demonstrate how a late endosome converts to a lysosome.
4.
Identify each domain in the receptor mediated
endocytosis pathway by its characteristic cytochemical or pH markers.
Competencies
Given a clinical scenario and
the appropriate cytochemical markers (such as pH or other identifiers), the
student should be able to deduce which domain is involved in an interrupted
receptor-mediated endocytosis event.
A Guide to studying this unit
Read pp 492-500. In particular, study the clinical scenario
presented on page 494.
B. Lecture outline
I. How do cells internalize molecules
and other cells? Compare and contrast major types of internalization events.
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In this unit, we will learn about a third set of structures: the endosomes Endosomes are formed by receptor-mediated endocytosis.
In this case, cells bring in proteins and other types of ligands attached to
the plasma membrane via receptors |
This figure diagrams the
major internalization events. In the two views on
the right, receptors are not needed for internalization. During phagocytosis,
cells may simply internalize particles or cells, like bacteria (cell eating).
In the second, called pinocytosis, cells internalize soluble material
(cell drinking). In both types of internalization, the cells bring particles,
cells or soluble material into the cell in a vacuole. When we study lysosomes,
we learn that the vacuole formed in the cell by phagocytosis or pinocytosis
often became a lysosome after hydrolases are brought to it and the pH was
adjusted. The vacuoles formed are called phagosomes or macropinosomes.
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II.
Categorize the variety of ligands that enter by receptor mediated endocytosis
Hormones and Growth Factors: Insulin, Epidermal Growth Factor, Growth Hormone,
Thyroid stimulating hormone, Nerve Growth Factor, Calcitonin,Glucagon,
Prolactin, Luteinizing Hormone, Thyroid hormone, Platelet Derived Growth Factor
, Interferon, Catecholamines.
Toxins and lectins: Diptheria Toxin,
Pseudomonas toxin, Cholera toxin, Ricin, Concanavalin A.
Viruses: Rous sarcoma virus, Semliki forest virus, Vesicular
stomatitis virus, Adenovirus
Serum transport proteins and antibodies: Transferrin, Low
density lipoprotein, Transcobalamin, Yolk proteins, IgE, Polymeric IgA,
Maternal IgG, IgG, via Fc receptors
III. How does the process work? Describe each step in the process of receptor mediated endocytosis.
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A. Step 1. Ligand binding Receptors are brought to
the plasma membrane by vesicles from the trans region of the Golgi complex .
Receptors are transmembrane proteins. Review the definition of transmembrane
proteins. Where and how are these receptor
proteins inserted into the membrane? The receptors can move laterally in the
membrane and collect in the specialized regions called clathrin coated pits.
This is seen as Patching and capping. B. Step 2. Patching and capping When the ligand binds to
its specific receptor, the ligand-receptor complex accumulates in the
coated pits. In many cells, these pits and complexes begin to concentrate in
one area of a cell |
C.
Process can be used to bring in multiple ligands
D. Effect of temperature on the process:
Warming promotes patching, capping, and endocytosis. May not be needed for binding, however.
IV.
Describe the mechanisms behind the
selectivity of the clathrin coated pit
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Recycled clathrin adds to membrane at the
site of the pit. Invaginates into the
cytoplasm (if temperature is 37 C. ). Receptors are collected in pits. Pits
pinch to form vesicles. Vesicles then
lose clathrin coat. Receptors are transmembrane proteins that may span the membrane. They have a signal sequence at the end of
their cytoplasmic domain (carboxy terminus): Tyrosine-X-
Arginine-Phenylalanine. Signal sequence binds to adaptin molecules in the
clathrin coat. (Adaptor protein AP-2) . This stops and concentrates the
receptor. It stays inside the pit.
Signal sequence even stimulates more clathrin to accumulate. Why is this important? Sequestration
concentrates ligand to conserve intake of fluid. Also, serves as a gateway to entry. There is a genetic defect in LDL receptors which prevents them from binding to Adaptin-2 . Thus, they do not enter clathrin coated pits and cannot be brought into the cell via receptor mediated endocytosis. The result is
high serum cholesterol, because LDL provides a critical mechanism for
reducing cholesterol levels and getting it into the cells.
Hypercholesterolemia. There is a familial form that comes from a mutation in the LDL receptor. It binds cholesterol, but never lets it enter the cells. In our lecture, Mr. Murphy and 2/4 of his children had this disease. Also, father and 2/5 siblings had it. What kind of inheritance? Will cause heart attacks (early) and atherosclerosis.
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V,
Distinguish the functions and environment of the early endosome from those of
the late endosome
A. How are early endosomes formed?
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To understand what
happens to cholesterol, need to know about trafficking through the endosomes. Vesicles lose
clathrin coat and then fuse to form early endosome. (pH 5.9-6.0). In order to fuse, they carry a rab5 sorting
signal linked to guanosine diphosphate (GDP). Also have “v-snares and t-snares” Review this from your sorting and trafficking lectures.
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B.
Early endosome functions and characteristics. |
Early endosomes
can release some receptors from their ligand. Low pH of 6 allows
release. Receptors are then recycled
back to the plasma membrane (pH >6). Then, early endosome may become a
late endosome. Thus, markers for early endosome compartment include:
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Apply this to LDL
receptors.
Cartoon below shows that LDL receptors are an example of the class that are
recycled. The LDL is released in the
early endosome to allow return of the receptor and its re-use
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Early endosome transition to late
endosome Cholesterol is released and eventually transported out of the late endosome to be used in membranes, steroid hormones, etc.
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C.
Late endosomes; Describe the routes of trafficking from the Golgi complex to
the late endosomes.
Late endosomes are formed as the pH continues
to drop to 5-6.0. Clathrin-coated
vesicles from the Trans Golgi Network carry digestive enzymes to the late
endosome and fuse with these structures, releasing their contents. The late
endosome thus becomes a degradative body and also acquires the marker for
mannose 6 phosphate receptor "MPR+.
It changes its rab surface marker to rab7-GDP, probably to accommodate
the new targeting vesicles with which it will fuse. This means that the late
endosome can be identified by the presence of the rab7 GDP
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To
summarize, markers for late endosomes include:
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Late endosomes include multivesicular bodies and contain whorls or vesicles of membranes inside. They also contain an unusual lipid which has become another marker for this stage. The lipid is called lysobisphosphatidic acid (LBPA) which has a larger head group than tail. Its structure enables it to be inserted into highly curved membranes, like the membrane whorls. It is believed that this allows retention and binding of specific molecules in the whorls by lipid-protein; lipid-lipid interactions. One type of molecule is cholesterol and it is believed that this is an important site for cholesterol accumulation. Late endosomes function to degrade many proteins and lipids. They also are responsible for returning the MPR receptors back to the Trans Golgi network. They recycle these by budding off membranes that carry back the receptors and target the Trans Golgi membranes for fusion. After fusion, the MRP receptors are available to capture and sort new degradative enzymes for future trafficking to the late endosomes. |
D.
Demonstrate how a late endosome converts to a lysosome
Finally, late endosomes may not be
able to digest all the material that needs to be destroyed. Therefore, the next
step is the fusion of late endosomes and lysosomes creating a hybrid
organelle. The pH continues to drop.
Residual heavily glycosylated lysosomal associated membrane proteins
(LAMPs) may thus be transmitted to lysosomes. LAMPs then become a marker
for a late endosome or a lysosome. Since lysosomes do not have MPR receptors
(they have all been sent to the Golgi), one could distinguish the lysosome and
the late endosome on the basis of labeling for MPR. Thus, fusion between
the late endosome and lysosomes begins after the MPR have been sent back to the
Trans Golgi. The steps involved in
forming late endosomes and lysosomes are drawn in the above cartoon. Note that
lysosomes continue to communicate with late endosomes and may deliver important
material back to this group of organelles. Lysosomes are considered the
end product of endocytosis. Thus, lysosomes do not communicate
directly with the Trans Golgi (and hence the plasma membrane). However,
they could communicate with upstream structures by way of the retrograde
transport to the late endosome.
To summarize, markers for lysosomes include:
- LAMP+,
- acid hydrolases,
- MPR negative,
- NPC1 (in normal cells) (Neiman
Pick Complex protein).
E. Importance of late endosome-lysosome compartments:
They
regulate trafficking of critical nutrients. They also regulate cellular stores
of different molecules, by enzymatically degrading them. If there is a failure
in the trafficking or degradation, the lysosome will build up the product and
eventually this will damage the cells. There may be rather wide-spread effects
throughout the body.
V.
Three examples of lysosomal storage diseases: Nieman Pick A, B, C
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Type C Niemann-Pick
usually affects children of school age, but the disease may strike at any time
from early infancy to adulthood. Always fatal. Some of the symptoms may
include: Jaundice at (or shortly after) birth;
An enlarged spleen and/or liver. Difficulty with upward and downward eye
movements (Vertical Supranuclear Gaze
Palsy). Slurred, irregular speech ("dysarthria") Learning
difficulties and progressive intellectual decline ("dementia");
Sudden loss of muscle tone which may lead to falls ("cataplexy")
VI. What have we learned about receptor
mediated recognition events?
Receptor must have
recognition sites for ligand. Binding may activate second messengers.
Receptor must also have recognition site for clathrin coated pit (for
adaptin). Clinical
significance—hypercholesterolinemia.
Endocytic vesicles must have specific rab5-GTP’s to fuse and form early
endosome.
Early endosome will recycle some receptors
Late endosome must have rab7 GDP recognition sites for fusion and communication
with the Trans Golgi network.; Site
for release of cholesterol—Clinically significant for Nieman Pick C
Site for recycling of Mannose 6 phosphate receptors.
Lysosomes are the final stop in the endocytic pathway.
Site for degradation of membranes, proteins, and lipids. Clinically significant for Neiman Pick A
and B
Test
yourself!! How much do you know about receptor mediated endocytosis?
List
the major types of internalization events?
What
types
of ligands enter by receptor mediated endocytosis?
Describe
each step in the process
?
Do
coated pits accommodate
only one ligand, or can more then one enter in the same pit?
What
is the advantage
of the patching and capping process to the cell?
What
is the role of the clathrin
around the coated pit?
What
guides the ligand and receptor into the coated
pit? What would happen if one mutated the signal sequence on the receptor
molecule?
How
does receptor mediated endocytosis reduce
our serum levels of cholesterol?
What
is the effect of temperature
on the process?
How do early
endosomes form? What is the Early Endocytic recycling pathway and how
is it used?
What
happens to the receptor
in the endosome?
How do late
endosomes form?
How
do late endosomes
become lysosomes ?
Sample question.
Ms. Jones brought her
daughter to your ER because of persistent cough and fever. She was diagnosed with pneumonia and her
spleen was enlarged. An analysis of her
bone marrow cells showed abnormal large, foamy macrophages which were
distinguished by LAMP +, mannose 6 phosphate negative (MPR-) bodies, which
contained many membrane whorls. A defective enzyme involved in break down of
lipids was the diagnosis. This defect
was the result of a problem in the following domain:
1) Early endosome
2) Clathrin coated pits
3) Lysosomes
4) Trans Golgi complex
5) Rab5 GDP vesicles
Answer: 3
You suspect that your cardiac
patient, Mr. Murphy, has hypercholesterolemia. You are waiting for blood tests from family members. In the meantime, your lab can take his blood
cells and test trafficking of the LDL receptors. You use dual immunolabeling for LDL receptors and rab5 GDP
sorting signal vesicles (pH 6.0) and find labeling for both constituents, but
no contiguous labeling. In other words,
LDL receptors are not in rab5 GDP vesicles.
This confirms your hypothesis because.
1)
LDL must enter by
receptors stored and recycled in rab7 GDP coated vesicles
2)
LDL receptors are
defective and therefore not sorted to the plasma membrane
3)
Trafficking of all
vesicles containing mannose-6 phosphate receptors from the Golgi complex is
blocked
4)
There is a defect in
adaptin carried by rab5 GDP vesicles.
5)
Before they can get to rab5 GDP
vesicles, LDL receptors must enter clathrin-coated pits.
Answer 5