example question : cell surface receptors

Question 1

Question

Answer 1

You have discovered a new cell surface receptor. You have evidence that it may have the following properties:
* it appears to have an extra-cellular domain
* it may be a type 1 single pass transmembrane protein
* it is synthesised and appears at the cell surface after a particular stimulus
* it disappears from the cell surface after binding its ligand and is degraded in a lysosome
* it is a glycoprotein
* Discuss the mechanisms that the cell would use to achieve these properties.

Discuss how you would confirm whether the protein has the above properties

Question 2

Analysis of the question

Answer 2

5 parts, For each part there are two questions:
1. What mechanisms does the cell use to produce that property
2. What practical/experimental steps would you take to prove each property
You could break it down into each part and treat them all separately.
Or you could write a more integrated essay because the points are mechanistically related

Question 3

Overview

Answer 3

• Cell surface receptors are transmembrane proteins
• They tend to have a single transmembrane domain
• This information is given in the question but should be restated
• Transmembrane proteins are synthesised in the ER
• This maybe after activation of its gene by, for instance, a growth factor
• Glycosylation starts in the ER
• Cell surface proteins pass through the Golgi Apparatus where they are further modified, by glycosylation
• Then packaged into vesicles for transport to the plasma membrane
• Fusion of the vesicle with the plasma membrane results in its presentation at the cell surface
• Removal of such receptors is usually as a result of clathrin mediated endocytosis (usually triggered by a signal that response to the receptor is no longer needed)
• This results in endocytic vesicles containing the protein being formed
• Then transported to the endosome, which develops into a lysosome
• Once in the lysosome the protein may be degraded by proteolytic enzymes

Question 4

Mechanistic details of synthesis in ER

Answer 4

Synthesis in the ER starts with arrival of a mRNA in the cytoplasm (although you could discuss events prior to this)
* Ribosomal subunits assemble onto the mRNA
* For extra marks you could discuss how the ribosome works
* For a type I membrane protein, the nascent polypeptide will start with a “signal sequence”
* This binds to signal recognition particle (SRP)
* Which binds to the SRP receptor (an ER resident transmembrane protein)
* This docks the ribosome onto a translocon
* And feeds the nascent polypeptide through the translocon channel
* The signal sequence (which resembles a transmembrane domain) remains in the translocon as the rest of the protein is synthesis into the channel and passes into the ER lumen
* Signal sequence is cleaved off by signal peptidase and remains in the membrane
* Transfer continues until a hydrophobic “stop transfer sequence” is synthesized
* This causes the translocon to disassemble leaving the protein in the ER membrane
* As the N-terminus of the protein is synthesised first, this leaves the N-terminus within the ER lumen and the C-terminus in the cytosol
* Therefore when the protein reaches the plasma membrane, the C-terminus will still be in the cytosol and the N-terminus will be in the extra-cellular space
* For extra marks you could discuss the mechanism of signalling that stimulated the expression of the protein (if you have time/words)

Question 5

Is it a transmembrane protein?

Answer 5

• use the gene sequence provided to deduce the proteins primary sequence
• predict whether a stretch of amino acids is likely to be a transmembrane domain – is there a stretch of hydrophobic amino acids of the correct length
• If you have one predicted transmembrane domain, plus an N-terminus hydrophobic domain, this would suggest a Type I membrane protein.
• so predict that N-terminal domain is a signal sequence and therefore would not be present in the mature protein (as it would be cleaved)
• If the gene codes for an N-terminal hydrophobic domain, but you find it is missing from the mature protein, this would be evidence for a cleaved signal sequence
• To prove this: purify the protein and use mass spectrometry sequencing

Question 6

Does it have an extra-cellular domain?

Answer 6

• ways to prove this: raise an antibody against the protein
• do a Western blot of a cell culture, should give you a single band of a specific molecular weight
• You could then treat the intact cells with a protease enzyme such as trypsin
• This would cleave off the extracellular domain of the protein (the rest of the protein would be protected by the membrane)
• The protein would now appear smaller on a Western blot (by the size of the extra cellular domain)
• This would only work if the part of the protein recognised by the antibody was not the extracellular domain
• you could clone the gene into an expression vector that tags it with green fluorescent protein (GFP)
• Express the protein-GFP in a cell and use fluorescence microscopy to see if it is transported to the plasma membrane* You could use FRAP (left) to show that it is dynamic
• If you placed the GFP at one end, you could use this to determine the “topology” of the protein (i.e. which side of the membrane each end of the protein is)
• You could do this using immuno-electron microscopy with an anti-GFP antibody
• Or you could use immuno-fluorescence – an anti-GFP antibody (labelled with a red fluorophore) would be applied to intact cells. As antibodies cannot cross membranes, it would only label the plasma membrane if the GFP was on the outside of the cell

Question 7

How does it get to the plasma membrane? How is it glycosylated?

Answer 7

• To get to the plasma membrane from the ER, it would be trafficked through the secretory system
• In the ER the first steps of N-glycosylation could occur
• then packaged into COPII coated vesicles and transported to the cis Golgi
• next steps of glycosylation occur by progressing through each stack of the Golgi
• facilitated by COPI dependent Cisternal Progression. Explain Cisternal Progression model
• exocytosis from the trans-Golgi network
• could mention transport of vesicles along the cytoskeleton and could mention SNAREs

Question 8

Transfer to cell surface membrane and glycosylation

Answer 8

• not covered this year – speculate, justify and bring in external knowledge.
• There are drugs that inhibit COPI or COPII dependent transport which would prevent specific steps of the secretory pathway
• There are inhibitors of glycosylation and there are probes which bind to specific types glycosylated proteins (such as lectins) which can be tagged with fluorescent or other markers and use show that a protein is glycosylated. Or you could use mass spectrometry
• There are enzymes that cleave off the polysaccharide – this would result in a reduction in the molecular weight of the protein on a SDS PAGE gel

Question 9

removal from the cell surface: endocytosis

Answer 9

• The question states that the protein disappears from the cell surface
• It also states that it is degraded in a lysosome
• This means that it is taken into the cell by clathrin mediated endocytosis
• explain the mechanism of clathrin mediated endocytosis: recruitment of adaptors, recruitment of clathrin triskelion which form a cage that bends the membrane into spherical vesicle, which is pinched off by dynamin. Clathrin is then removed. Actin may help this process. Vesicles may be transported on the microtubules to the endosome, where they fuse with the assistance of SNAREs (which pull membranes together, excluding water and allowing fusion)
• Again, you could follow this process by imaging the location of the GFP tag after addition of the ligand.