School of Life and Environmental Sciences

Signalling
[Phil Poronnik]

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[Beginning of material]
OK, so welcome back, let's think about signalling so when things bind to cells, how do the cells know
what to do? And it's really quite interesting and very, very elegant and complex. So, if you think about
it, how does insulin work, how does it talk to the cells, it must- it can't go through the membrane, as we
discussed, it's an amino acid-based structure, a peptide. So it has to interact with something on the
membrane, that has to tell the cell that insulin is present and to do something about it. OK, as you can
see from the cartoon there, something purple binds to some fingers on the membrane and something
inside the cells binds to the other side of the fingers and moves away. And that's, in fact, pretty much
how insulin actually works. So if you think about some terminology, insulin is a signalling molecule or a
hormone, it is a ligand that binds to a receptor in the membrane and the receptor has to have a higher
affinity interaction with the ligand which means it binds very, very tightly and also high specificity for
the ligand, which means it binds only to insulin and nothing else. OK, how you actually create specificity
within the body is only certain cells actually can express the insulin receptor to bind it. OK, and there's
many, many different types of cells all over the body, but the amount of receptor and distribution
governs how it works. And you can see from that picture down the bottom, you get the idea of a signal
molecule hormone binding to a receptor, that's reception phase. These are relay molecules in a
transduction pathway called transduction that actually leads to a cellular response. And the easiest
way to think about receptors and the ligands is that it's a lock and key, if you've got a key to your lock
at home, nobody else can get into your house except you. If you take that same lock and put it in
another house, they can go to the house as well. So it's really that simple. And it's a bit like a radio
signal. You have to have the receiver receptor to pick it up. So therefore, hormones target only those
organs or cells that can express those specific receptors. So when you think about signaling, it's really
this idea about cellular decision making. OK, so in response to external stimulus, the signal leads to a
cellular response and that in the context of hormones and everything else means they can secrete,
move, contract, divide, die, survive, catabolise, form tight junctions and any other kind of functions. OK,
so that's how the body communicates with all these different hormones and signals to keep itself in-
under homeostasis or whatever, or development. And once again, insulin is a polypeptide. It can't get
across the membrane. Therefore it requires some sort of a trans-membrane receptor that can bind the
insulin and then transduce the signal. OK, and we'll go into the the details of these more in second
year. But just so that you understand that there are these receptors with trans membrane domains that
can send signals. And just for those of you that really, really like biology, here's a beautiful picture
from about last year, I think, where they've actually done the crystal structure, the cryo-EM structure,
that's cryo electron microscopy structure of the insulin receptor. And you can see on the right hand side
are the images that they get from the microscope. And the left hand side is the actual model they can
build using those structures. It's very, very exciting science. In terms of things like insulin, these signal
pathways usually have these cascades. In the case of many receptors, it's this phosphorylation cascade
mediated by enzymes called kinases and these transfer phosphate from ATP and activate target
proteins. OK, so it's an ATP mediated thing and you can see here basically that at the top the
reception occurs. There's some sort of phosphorylation cascade that can activate proteins inside the
nucleus that can result in transcription of genes or whatever. OK, so it's a basic model where a receptor
binds and then you get phosphorylation cascades and some sort of activation response. And we've got
a couple of those in more detail. If you think about, in addition to a phosphorylation cascade, there's
this idea of second messengers, these are these key intermediates that are responsible for cell
signalling, OK? And a second messenger is something whose presence is a signal. OK, so it suddenly
appears in response to a hormone or a signal, it acts as an amplifier. It is either synthesized or
released from storage. And they act as intracellular ligands that can be made or released by effector
 Signalling
[Phil Poronnik]

proteins. So suddenly these these messengers suddenly appear like FedEx coming delivering parcels,
OK? And if you think about 'what does the second messenger need to be in order to be an effective
signalling or messenger', it needs to be in lower amounts in the resting state. So it has to be very low
when nothing's happening, when something happens, suddenly it increases by a lot. It has to be
somehow regulated in its appearance or synthesis. It has to be regulated or destruction removed and
be able to act through other proteins. OK, that's the basics behind all this. And the two classic second
messengers that we have we talk about in biology are cyclic AMP and calcium. OK, so what about
cyclic AMP? What is it? It's actually ATP turned into cyclic AMP. So for those of you, well you're all
studying chemistry so you can all enjoy this chemistry here. It starts off with ATP. There's an enzyme
called adenylate cyclase that then causes this cyclisation, the cyclic AMP. And when cyclic AMP- so
that's the synthesis of the cyclic AMP. When its job is done, it's degraded into AMP by this enzyme
called phosphodiesterase. OK, so you can see here the classic requirements of a signalling molecule. It's
non-existent or very low levels normally, it gets activated by adenylate cyclase. They get more and
more cyclic AMP and then it gets broken down when the signalling's finished. And we'll speak about
ATP more in later lectures, how it gets made. OK, so there we can see there what regulates cyclic AMP,
adenylate cyclase makes it from ATP. There's lots of ATP in the cytoplasm. And when adenylate
cyclase makes cyclic AMP, the presence then activates these kinases, and then that causes the cascade.
One question is 'what then regulates adenylate cyclase?' And this is an important, very important
concept in human biology, there's thousands of these G protein coupled receptors, but this is a simple
schematic, for the purposes of this lecture series you just need to remember that G proteins bind to
GTP. OK, so it's mediated by GTP. So you get the activation of the G protein-coupled receptor. So
these are these just like the insulin receptor, it's a specific kind of receptor that can bind G proteins. It's
activated by a molecule like epinephrine. The GTP activates the G protein. The G protein then
activates the adenylate cyclase. So adenylate cyclase is regulated by G proteins that are talking to
the receptors. OK, then you get the activation of a cyclic AMP which can then go and phosphorylate
different cascades. OK, so just remember there's a G protein linked receptor in the membrane that
binds the G protein, GTP activates it. And then adenylate cyclase activates ATP to make cyclic AMP
and the downstream cascade. It's very, very simple but also very, very elegant. And here's an example
here, we spoke about glucagon converting glycogen to glucose in the liver and glucagon binds to a G
protein-coupled receptor in the liver. And you can see here the hormone binds to the receptor. The G
protein is mobilized to activate adenylate cyclase, ATP is then then converted to cyclic AMP and the
cyclic AMP can go and activate all sorts of different kinases. OK. Simple as that. So from one- and the
important concept here is from one receptor, you can get the activation and production of a lot of
cyclic AMP. And so this is this concept of amplification, OK, so on the left hand side, you can see that
the binding of one molecule to a G protein coupled receptor can activate about 100 molecules of G
proteins, they can in turn activate adenylate cyclase, and you get this cascade till you get about the
glycogen being converted in huge amounts. On the right hand side you can sort of see a more
graphical representation of that. OK, so the activation of the receptor by a single hormone activates
adenylate cyclase, lots of cyclic AMP that can go and do lots and lots of things inside the cell. Here's a
nice example from Science, where you often- microscopy, where you can load cells with a dye, it turns
red as there's more cyclic AMP being produced and you can see both cells responding very much to
cyclic AMP. Over the course of a few minutes. OK, so moving on from this. Let's think about the other
second messenger, which is calcium. So once again, we learned in the first lecture that calcium in the
blood's probably ran about one millimolar or so, for pumping out calcium or getting out of the
cytoplasm. So the cytoplasmic calcium is actually very, very low. So just like the sodium-potassium
ATPase, there's a pump that pumps calcium, responds to ATP, so it can create this 10,000-fold gradient
for calcium. OK, and somehow along in the process of evolution, calcium was chosen as to be an ion
that could actually interact with all sorts of different structures on proteins and make them change their
behaviors. OK, so calcium is very, very low inside the cells and high outside. So you can imagine it can
come in very, very quickly and the concentration can go up quite a lot and affect a lot of proteins. And
you can remember this is that very important slide we saw in the first lecture where this is the first few
moments of each of our lives. With the sperm, penetrates the egg and sets off this wave of calcium. So
this increasing intracellular calcium in waves actually binds to all sorts of proteins inside the cell and
triggers all this activation and fertilisation events. That's a very, very powerful example. And just
because we've been speaking about intracellular organelles in previous lectures, there is certain areas
of concentration of calcium inside the cells, so the mitochondria and nucleus and ER have reasonably
high concentrations of sodium- calcium. But the concentration of calcium inside the cytoplasm itself

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 Signalling
[Phil Poronnik]

remains very, very, very low. And, of course, you learn more about these things in second and third
year. But just to remind yourselves that there is calcium inside the organelles, but the cytoplasmic
calcium is very, very low. So calcium, the second messenger, when you activate the receptors that you
saw from the previous video of the oocyte, calcium floods into the cell and changes cellular actions at
all levels. You can think about how fast calcium works in terms of releasing neurotransmitters and stuff
like that. Here's another example, here's a cardiac myocyte, so this is a few things we've been talking
about in the lecture so far, and this is a single cardiac myocyte that's beating all alone by itself quite
happily. OK, just a single rat myocyte, and it's been loaded with a dye that turns red when there's
calcium. And so you can see now as the muscle contracts, there's a calcium wave that passes along that
muscle. And I think you'll learn more about that with Peter Knight in the muscle section. OK, but once
again, to give you an example of how very, very powerful calcium is as a signalling messenger. OK,
the other thing is in the pancreas, which Peter will be talking about, this is the exocrine pancreas that
produces the digestive enzymes. You can see here there's about five or six pancreatic cells and you
can see a wave of calcium sweeping across them. So these cells using calcium to talk to each other
about what's going on in their environment and how to secrete digestive enzymes or stuff like that. So
once again, very, very lovely example of the power of calcium as a second messenger. The other thing
to think about is things like steroid hormone, like oestradiol or testosterone, because they're made up-
they're all basically derivatives of cholesterol, which is a normal part of a membrane. So these guys
can actually just go straight across the membrane. They don't need a membrane receptor, OK? And
oestradiol, for example, is secreted from ovaries, it acts on various target organs and causes the
growth of proteins, responsible for individual growth and involve menstruation as well. OK, so how do
these things get work inside the cells if they can cross the membrane? And in this case, although they
cross the membrane, they still need to find a receptor. OK, so the hormone moves across the cell
membrane, goes into the cytoplasm where it finds a receptor that it can bind to. And typically these
receptor hormone complexes will translocate into the nucleus where they can bind to DNA and
stimulate the transcription of various proteins. OK, so that's a very powerful, interesting mechanism. And
an example just to show you is here you can see how the hormone is tightly- this steroid hormone tightly
sits to the grooves within the DNA. OK. So this is how these things- each hormone can bind a specific
hormone receptor complex, as can be combined to specific grooves in the DNA to activate particular
transcription patterns and stuff. So it's a highly specific thing based on the structure of these proteins.
So in this little mini lecture, we've learned about how cells signal and communicate with each other and
particularly the roles of phosphorylation cascades, cyclic AMP and calcium and steroid hormones and
their modes of action. In the next lecture we'll have a look at a couple of examples, OK?
[End of recorded material]

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