Menu

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 ?

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. 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. 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.

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

Return to Menu

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.

  Temperature is important to the overall patching and capping process.  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

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  Return to Menu

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. 

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. 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

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. 

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.

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).

Receptor-mediated uptake of LDL receptors: a model for studies of trafficking and defects.

Cholesterol bound to Low density lipoproteins (LDL) is taken up by cells so that cholesterol can be used in construction of membranes, etc.  In this case the receptor is recycled and the ligand (LDL-cholesterol) is metabolized so the free cholesterol can be released and used by the cell. There are two genetic mutations that cause either no uptake of LDL receptors or uptake and accumulation of cholesterol in late endosomes.  We will look at these diseases to learn more the importance  of Key elements in receptor mediated endocytosis are.

First, this cartoon summarizes the entire process of endocytosis of LDL-cholesterol bound to LDL receptors. (taken from Alberts et al. Molecular Biology of the Cell, Garland Publishing, N.Y. Third edition, 1994 ). To review, after the ligand and receptors are collected in the coated pits, which then form coated vesicles, the clathrin coat is removed and the vesicle fuses with forming endosomes. The early endosome allows recycling of the LDL receptor.  The late endosome/lysosome is the site of accumulation of cholesterol followed by hydrolysis.  Free cholesterol is then available to be used by the cell.   

How do receptor mediated endocytosis and LDL receptors help reduce our serum levels of cholesterol?

The cartoon shows  the LDL receptors  with their ligand (LDL) being collected in a clathrin coated pit. LDL has been called the "bad cholesterol". High serum LDL's go along with high serum cholesterol However, we can reduce serum cholesterol by taking it up into cells that need it (for membranes, steroid hormone production, etc.) This requires a specific LDL receptor and a working receptor mediated endocytosis process. 

Some families have a defect in the Adaptin-2 binding site on the LDL receptor.  Recall that this site helped concentrate the LDL receptor in the coated pit.  In fact, binding to the Adaptor protein actually helped recruit the clathrin to the site.  The defect is shown in the above cartoon from your text. This genetic deficiency  prevents LDL and its receptor from entering the coated pit or from being taken up. The result very high serum levels of cholesterol and all the problems resulting from that. This cartoon was taken from Alberts et al. Molecular Biology of the Cell, Garland Publishing, Third edition, 1994.
Return to Menu

Do coated pits accommodate only one ligand, or can more than one enter at a time?

Supposing a cell is stimulated with two hormones (and it has receptors for both), or a hormone and a growth factor. Do both ligands enter via the same packages, or is there a selection for one particular type of ligand in a coated pit. This is a perfect question for cytochemistry with different types of labels attached to different ligands. For example, one can use colloidal gold, ferritin, peroxidase and detect as many as 2-4 ligands on a given cell. 

This figure shows that multiple ligands can enter the cell in the same coated pit. Furthermore, the vesicles will carry them to the same receptosomes. The photo shows co-detection of ligands as diverse as Epidermal growth factor, vesicular stomatitis virus, or alpha 2 macroglobulin. Labeling molecules (signalling molecules) included gold, peroxidase, ferritin, or the virus itself. This figure was taken from Endocytosis, Edited by Ira Pastan and Mark C. Willingham, Plenum Press, N.Y., 1985
 

Continue studies of Receptor Mediated Endocytosis