University of Texas Medical Branch

Cell Biology Graduate Program

Read pages 618-633 and p 640 in your text: Alberts et al, Molecular Biology of the Cell, Garland publishing, N.Y., Third Edition, 1994.

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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 ?
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?
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 ?
How does receptor mediated endocytosis reduce our serum levels of cholesterol?
Do coated pits accommodate only one ligand, or can more then one enter in the same pit?
How are proteins destined for secretion sorted and packaged?
What part of the Golgi complex produces secretory vesicles?
Describe the process of Regulated exocytosis?
What happens to the secretory vesicle membranes after exocytosis? Does the cell simply get larger and larger because of the new membranes?
In an electron micrograph, how would you distinguish profiles showing exocytosis and endocytosis? Could both events be occurring at the same time?

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 extend processes and bring cells or soluble material into the cell in a vacuole. In the presentation on lysosomes , we learned that the vacuole formed in the cell by phagocytosis or pinocytosis often became a lysosome after hydrolases were brought to it and the pH was adjusted. The vacuoles formed are called phagosomes or macropinosomes. This cartoon was taken from Endocytosis, Edited by Ira Pastan and Mark C. Willingham, Plenum Press, N.Y., 1985

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. The process depends first on specific binding to the receptor, which is a subject worthy of a lecture in itself. This figure shows this process as "coated pit endocytosis". The coated pit is a specialized region of the membrane that is coated with clathrin (for stability, to aid the transport process). The coated pit forms a coated vesicle and then loses its clathrin coat. It then joins with other coated pits to form a receptosome.

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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

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

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How does the process work?  An overview:

Receptors are brought to the plasma membrane by vesicles from the trans region of the Golgi complex . Review the definition of transmembrane proteins. Where and how are these receptor proteins inserted into the membrane? How does the Golgi complex maintain the fluidity of the plasma membrane , the receptors can move laterally in the membrane and collect in the specialized regions called clathrin coated pits.

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. Cytochemically, this appears as patches of label on the cell surface (patching) Eventually, the patches coalesce to form a cap at one pole of the cell (capping). Not all cells form caps, but most do form patches. Why would this process be an advantage for the cells? Imagine the large amounts of extracellular fluid that would be taken up if the cells endocytosed the ligand receptor complex all over its surface. Thus, the pre-concentration process minimizes the amount of fluid that is taken up in the vesicle.

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Recall the studies of membranes and Membrane fluidity . Receptors are moving in the plane of the membrane as long as the temperature is 37 C. In the presentation on Membrane fluidity, we talked about photobleaching with a laser beam. This allows you to study the lateral diffusion coefficients of the ligand-receptor complexes. Fluorescent molecules signal the site of the complexes and they are bleached after the laser exposure. Then, the photobleaching system measures the speed of the recovery in the bleached area (return of the fluorescence) as the label returns by lateral diffusion. This table was taken from Endocytosis, Edited by Ira Pastan and Mark C. Willingham, Plenum Press, N.Y., 1985 An example of some measurements for different receptors is found in this table. The objective of the lateral movement is to collect the ligand-receptor complex in the clathrin coated pits. So, some receptors appear to be moving faster than others. One might speculate that this may be related to size of the receptor or the ligand. 

Temperature may affect the binding of the ligand (rate) as well as the lateral mobility of the ligand-receptor complex. Some ligands will not bind well at low temperatures. However, others will bind, but not be taken in. This photograph shows the peroxidase (HRP) detection of a ligand that is distributed on the membrane at 4 C. Note the right hand control panel that shows absence of label in the presence of competing unlabeled ligand. Note the presence of the coated pit, even in the control. So, the formation of these is not temperature dependent. However, after warming for a few minutes, the formation of vesicles and endosomes is evident. It is important to note that Receptor mediated endocytosis is much faster than phagocytosis or pinocytosis. If one were to simply have a non-binding ligand present, it might take hours for the ligand to enter via pinocytosis. Thus, this rapid uptake coupled with the absence of label in the presence of competing ligand is a sign that this is receptor mediated. This figure was taken from Endocytosis, Edited by Ira Pastan and Mark C. Willingham, Plenum Press, N.Y., 1985

Some coated vesicles may be configured with a deep invagination, called a "neck". These were discovered by Willingham and Pastan and can be seen in serial sections as a thin region connection between the outside of the membrane and the vesicle. The vesicle contains labeled ligand attached to receptor. Formation of these necks is definitely temperature dependent as can be seen in the following table.

This figure and Table were taken from Endocytosis, Edited by Ira Pastan and Mark C. Willingham, Plenum Press, N.Y., 1985

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Finally, temperature is important to the overall patching and capping process discussed during the lecture on membranes. There we showed that, after they are bound, Membrane Receptors move laterally in the plane and groups of receptor-ligand complexes may actually coalesce in a patch and eventually in a cap. Antibodies are good examples of receptors that react this way. This figure shows what happens if the temperature is 4 C. There is a diffuse labeling. Warming the cells immediately produces patching. This figure was taken from Endocytosis, Edited by Ira Pastan and Mark C. Willingham, Plenum Press, N.Y., 1985

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Clathrin coats surround the pit as diagrammed in the above cartoon. The assembly of the clathrin molecules on the pit appears to drive the pit to invaginate. This cage-like molecule may help stabilize the vesicle as it buds from the membrane. 

Clathrin coated pits may move in the plane of the membrane, however recent studies show that there is an "organized movement" as if the pits are tethered to cytoskeletal elements. The following paper studies coated pits in living cells that were transfected with a plasmid carrying a cDNA for green fluorescent protein (GFP) attached to the light chain of clathrin.  Gaidarov I, Santini, F, Warren, RA and Keen, JH Spatial control of coated-pit dynamics in living cells. Nature, Cell Biology 1: 1-7. 1999. The cells made GFP-clathrin and were able to insert the protein into coated pits.  This was tested via antibodies to coated pit proteins as well as studies of the endocytosis of transferrin. When time lapse photography was used to learn if the coated pits moved, they found that the pits appeared and disappeared at intervals.  Studies of regional spacing showed that appearance of new pits was often close to sites of old pits, suggesting regional organization. Superimposed images showed a linear pattern as if the pits were organized. 

The studies showed that the coated pits were resistent to detergents (Triton-X).   And, they were able to show that the retraction of cellular processes that followed triton-X treatment produced linear movement of each coated pit in the plasma membrane as if it was organized by or on cytoskeletal elements.  See the paper by this group (above citation) for the photographs and movies of these findings.

Formation of Endocytic vesicles.

Once the vesicle has formed, the clathrin coat is lost (perhaps via a chaperone protein of the heat shock protein 70 family). The loss of the coat is an energy requiring process. After the coat is lost, the vesicles join with other vesicles to form endosomes or receptosomes. The following electron micrograph shows clathrin coated pits forming a vesicle. It is taking up lipoprotein particles. Note how thick and well defined the clathrin coat is. This cartoon was taken from Alberts et al, Molecular Biology of the Cell, Garland Publishing, N.Y. 1994, Third Edition

This micrograph was taken from Endocytosis, Edited by Ira Pastan and Mark C. Willingham, Plenum Press, N.Y., 1985 Return to Menu

Role of adaptin in the transport of the receptor-ligand complex

How do receptors know how to get to the coated pits? Specific coat proteins, which are really a multisubunit complex, called adaptins, trap specific receptors that are destined for the clathrin coated pits. One end binds to a signal sequence in the receptor molecule. The adaptins that work at the cell surface are called Adaptor Protein-2. Consult Figure 13-53 in your text (Alberts et al, Molecular Biology of the Cell, Garland Publishing, N.Y. Third edition, 1994), The signal sequence on the receptor molecule has a tyrosine-X-Arginine-Phenylalanine linkage near the Carboxy terminus. This signal sequence binds to the top of adaptin while the other pole of adaptin binds to clathrin. So, we have a very clever concentrating device that insures that the receptors stay in the coated pit.

Adaptin-receptor binding stimulates the attachment of clathrin.  This assembly then quickly drives the formation of the vesicle. The vesicle then buds from the plasma membrane and forms an endocytic vesicle which fuses with other endocytic vesicles to form the early endosome (Rab5-GTP is instrumental in this homotypic fusion of the endocytic vesicles to form endosomes).

The fusion events also use tethering and docking proteins called EEA-1 and Rabaptin-5 which help align the vesicles and bring them close enough together to effect the fusion. A pair of SNARE (v-snares and t-snares) also function in this process. For more information, see the recent mini-review by:  Pfeffer, SR  Transport vesicle targeting: Tethers before Snares: Nature Cell Biology 1; E17-E22, 1999.

Note: Adaptins can be found in other parts of the cell. For example, review the process of sorting of lysosomal enzymes. This also involves a signal sequence on the enzyme that binds to a mannose 6 phosphate receptor. It also is shunted by a clathrin coated pit that buds from the trans Golgi complex. The above figure shows this diagrammatically. The important thing to know about adaptins is that not all are alike. The adaptins that bind the cytoplasmic tail of plasma membrane receptors are different from those that bind the mannose 6 phosphate receptors found in lysosomes. The latter group recognize a different set of signals unique to those receptors.

Adaptins capture the receptors as they move laterally through the membrane. This sequesters them in the coated pits. Receptors that do not have the "right" tail region (signal), or other types of molecules are allowed to pass through. Therefore, in disease states, where the receptors cannot enter coated pits, one might speculate that there may be a defect in the tail region (at least).