Welcome to lecture 10. This lecture will be on the T-cell receptor, it's structure, it's genes and it's activation. Now, really before people had a good idea of what the T-cell receptor was, they had some very bad ideas of what it was, which is to say, they had a hard time identifying the T-cell receptor and its genes because they knew something that wasn't true. T-cell receptors do in some ways resemble immunoglobulin receptors and it's easier to study an immunoglobulin receptor because you can get tons and tons of antibodies which are essentially soluble receptors, study them and then go back and look for the membrane-bound versions, but with the T-cell receptor, they're always bound in the membrane, and for that reason, there's only about 10 to the fifth of them in any one cell. Ten to the fifth might sound like a whole lot but compared to an antibody, it isn't. So, with these guys, they were looking for a very small needle in a very large haystack. The other difficulty was that because antibodies recognize the overall tertiary structure of proteins, they thought that that would be true of the T-cell receptor as well, but it's not. A T-cell receptor identifies a short string of peptides and that means that it can recognize a denatured protein and in fact, the protein has to be denatured. Well, scientists didn't notice the T-cell receptors were responding to non native proteins and to their credit, they didn't suppress that information but they didn't know what to do about it. So, one of the first steps that lead to a better understanding of the function and structure of the T-cell receptor was a series of experiments that actually located, identified and studied the genes that code for. This was a series by Hedrick and Davis and that's what is the next part of this clip will be about. Because it's a little bit complicated, I'm going to just introduce it and I want to also point out that this is also a good example of how the separation process works in many cell biology isolation, which is to say the first thing that we're going to do is isolate messages from membrane-bound proteins. So, a T-cell receptor protein is membrane-bound, an MHC II protein is membrane-bound, even things like a CD4 protein are membrane-bound. So, after a while, you might think that every interesting protein we'll ever look at, is going to be stuck in the membrane. But as it turns out, when we first do our separation and separate messages for membrane-bound proteins, we're going to get rid of 97 percent of all the messages. I think that's cool. So, it says that most of the messages that are being produced by a cell at any one time, are messages for things that are not going to be either secreted or windup incorporated into the membrane. Now, the next thing that we do is even cooler. We're going to look at proteins that are found in T cells but not P cells. Both of these cells are adaptive immune cells, they're both lymphoid, they're very similar in some ways but the differences are what the things are that we're really interested in. There will be things on the surface of a T cell that are specific and unique to that cell and one of those better be the T-cell receptor. Then finally, we're going to look at the collection of proteins that are unique to T cells but come from genes that had been rearranged. Remember we don't rearrange MHC I or II, we don't rearrange the genes for CD4, but we do rearrange the genes for T-cell receptors and immunoglobulins. But because we're looking only at proteins found on T cells, we will not be looking at an immunoglobulin proteins and we should be able to identify that receptor. So, it's a little bit of complication, but here we go. Here's a quick reminder of the structure of the B cell receptor, the immunoglobulin receptor and the T-cell receptor. You can also see the coast signaling molecules that are associated with these two peptide receptors and we're going to try to get the genes for these two peptides. Our first step is going to be like burning down the huge haystack that holds this tiny needle of messages that bind to its gene. So, what we're going to do is pick up the messages that are synthesized on the rough endoplasmic reticulum and throw away all of those synthesized on cytoplasmic ribosomes, and this is going to actually eliminate 97 percent of the mRNA. Now, we're going to do this by isolating mRNA from membrane-bound poly salts and that is the RNA for proteins that are put into the membrane or secreted from the cell. So, what kind of messages are we going to pick up? Well, obviously, we're hoping to pick up the receptor message on the T cell and of course we will also in the B cell fraction pick up that receptor. In addition to that, we had messages for the cell adhesion molecules, for the membrane pumps, for the MHC I in the B cell, we also have MHC II, we have hormone and cytokine receptors, we have aquaporins to let water in and out and of course, there's all of those mRNAs for all in the many different signals that that B cell and that T cell secrete. When we get the microsomes from these two different cell fractions, we're going to extract the messages from it. So here we have a whole bunch of messages that are common to both B cells and T cells. Now, the B cells will also have its messages for the MHC II and for its receptors and we're really not interested in that, but what we're really interested in is that is the messages in this fraction that code specifically for the Alpha-beta receptor. Our next step is the equivalent of taking everything left over after we burned down the haystack, that is all the solid things like broken glass and rocks and lead fishing weights and somewhere in there is our needle, these messages. So, the process that we're about to do which would be essentially equivalent to fishing around with a magnet, is this one, we're going to do something called subtraction hybridization, that is we're going to take the isolated T Cell mRNA and we are going to make a copy of it and when we copy it, we're going to make a complementary strand of DNA and we're going to use radioactive precursors. So, when we're done, what we have is cDNA that matches the messages for the T cell RNA and those messages are radioactive so we can identify them. Because what we're going to do is compare this to all those similar rough endoplasmic particulum messages that we isolated from the B cell. So, we're going to put these guys together and when we do that, we going to mix them and the parts of the radioactive DNA that are complimentary to the B cell will be subtracted out, they will bind to the messages from the B cells. We can separate out the double strands of DNA RNA hybrids and we are left with radioactive cDNA from the T cells. We're down to just those messages you find in T cells and only in T cells. Now, we head into the final stage, the equivalent of picking needles off the magnet. Was the message from a rearranged gene or a gene that was not rearranged? Because, if you remember, we had a T-cell receptor that had the Alpha and Beta peptides but it also had a number of other peptides associated with it, that were unique to the T cell. When we take out these messages and look at them, the ones that are not subtracted out, we find that there are 10 messages that are not subtracted out in the process and so we have to look at each one of these messages individually by hybridizing it with the DNA from various sources. If we look at message one and we compare message one to the DNA of liver and B cells, we will see that the resulting hybrid has the same length and that's exactly what we would expect, this is a control really. Because we think we know, that the T-cell receptors shouldn't be arranged in liver cells or B cells and therefore any message for it should essentially separate out in the same place. So, but what we're really interested in now is where this hybrid separates out depending on whether it came from one of six different lineages of T cells, here they are numbered. So, when we hybridized one to the DNA from each one of these six T cell lineages, we find this. Boring. They all come out in the same place and what that tells you is that the gene for this message is the same in the liver, it's the same in the B cell and it's the same in every single clone of the different T cells that we picked out to compare it with. That tells you that number one is not the message you are looking for. We're looking for a message that comes out in different places depending on which T cell and therefore, which form of rearrangement of the gene that this message will then bind to. So, let's try the next one. We're going to try message two. So, here's message two and when we do the same experiments with two, we find out that the liver in the B cell happily produce hybrids that come out in the same place because again, we would not expect these genes to rearrange. Now, we're going to hybridize to with the DNA from these six different T cell lineages and whoa! The hybrids come out in different places and they come out in different places depending on the length of the relevant gene that's left behind after rearrangement in these various T cell lineages. So, there we have, we have identified number two as being one of our receptors. When the scientists went through all 10 of these, they found happily that two of them produced a pattern like this, the other eight produced a pattern that indicated no rearrangement. So, they had identified two messages that allowed them to identify two genes that were rearranged in the T cell and once they had this DNA or RNA, they could then hybridize it with the chromosomes and locate those genes on the chromosome, and of course naming them Alpha or Beta is strictly arbitrary. But basically, they found the genes and that was a good first step into studying them further.
