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JOANNE STUBBE: --that Brown and Goldstein carried out,

which in conjunction with many other experiments

and experiments by other investigators

have led to the model that you see here.

And so we'll just briefly go through this model, which,

again, was the basis for thinking

about the function of PCSK9 that you learned

about recitation last week, as well as providing

the foundation for thinking about the recitation.

This week, we really care how you sense cholesterol levels

in membranes, which is not an easy thing

to do given that it's lipophilic and so are many other things.

OK.

So the LDL receptor--

that was their model, that there is a receptor--

is generated in the endoplasmic reticulum.

If you looked at the handout, you'll

see that it has a single transmembrane-spanning region,

which means it's inserted into a membrane.

And the membrane where it functions, at least

at the start of its life, is in the plasma membrane.

So somehow, it has to get from the ER to the plasma membrane.

And this happens by forming coated vesicles.

We'll see a little bit of that, but we're not

going to talk about this methodology in any detail.

But Schekman's lab won the Nobel Prize

for this work, either last year or the year

before, of how do you take proteins

that are not very soluble and get them to the right membrane.

And they do this through coated vesicles

that, then, move through the Golgi stacks

that we talked about at the very beginning.

And then, eventually, they arrive at the plasma

membrane and become inserted.

So these little flags are the LDL receptor.

OK.

So that's the first thing that has to happen.

And I just know that this whole process is extremely complex.

And patient mutants are observed in almost every step

in this overall process.

It's not limited to the one set of types of experiments,

where something binds and doesn't bind to LDL receptor

that we talked about last time.

So the next thing that has to happen-- again,

and we haven't talked about the data for this at all,

but not only do these receptors have to arrive at the surface,

but they, in some way, need to cluster.

And it's only when they cluster that they

form the right kind of a structure that, then,

can be recognized by the LDL particles

that we've talked about.

And so they bind in some way.

And that's the first step in the overall process.

And then, this receptor, bound to its cargo, its nutrients--

and, again, this is going to be a generic way

of bringing any kinds of nutrients into cells.

It's not limited to cholesterol--

undergoes what's now been called receptor-mediated endocytosis.

And so when the LDL binds to the receptor,

again, there's a complex sequence

of events that leads to coding of the part that's

going to bud off, by a protein called clathrin.

Again, this is a universal process.

We know quite a bit about that.

And it buds off.

And it gives you a vesicle.

And these little lines along the outside are the clathrin coat.

I'll show you a picture.

I'm not going to talk about it in any detail,

but I'll show you a picture of it.

So the LDL binding, we talked about.

We talked about binding in internalization.

Those are the experiments we talked about last time

in class that led, in part, to this working hypothesis.

And so we have clathrin-coated pits.

And it turns out that there's a zip code.

And we'll see zip codes throughout-- we'll

see zip codes again, in a few minutes,

but we'll see zip codes which are simply

short sequences of amino acids that signal to some protein

that they're going to bind.

So how do you target clathrin to form these coated pits?

How do you form a pit, anyhow, in a circle?

And how does it bud off?

And where do you get the curvature from?

Many people study these processes.

All of these are interesting machines

that we're not going to cover in class.

So you form this coated pit, and then it's removed.

So once it's formed, and you've got a little vesicle,

it's removed.

And then it can go on and do another step.

And another step that it does is that it

fuses with another organelle called an endosome, which

is acidic pH.

How it does that, how it's recognized,

why does it go to the endosome and not directly

to the lysosome-- all of these things, questions,

that should be raised in your mind

if you're thinking about the details of how

this thing works, none of which we're going to discuss.

But it gets into the endosome, and then what you want to do

is separate the receptor from its cargo, the LDL.

And we know quite a bit about that.

If you read-- I'm not going to talk about that either,

but if you read the end of the PowerPoint presentation,

there's a model for actually how this can happen.

And you can separate the receptor from the cargo.

And the receptors bud off, and they

are recycled in little vesicles to the surface, where

they can be reused.

The LDL particles can also, then-- and what's left here

can then fuse with the lysosome.

And that's, again-- we've talked about this--

it's a bag of proteases and a bag of esterases, hydrolysis,

lipids.

That's what we have in the LDL particle-- hydrolysis.

We talked about ApoB being degraded

with iodinated tyrosine, last time.

That's where this happens and gives you amino acids

and gives you cholesterol.

OK.

And then, again, depending on what's

going on in the environment of the cell,

the cholesterol would then be shuttled, somehow,

to the appropriate membranes.

OK.

So you can see the complexity of all of this.

If the cholesterol is present, and we don't need anymore

in the membranes, then it can become esterified

with long-chain fatty acids.

Those become really insoluble, and they

form these little globules inside the cell.

And then the process can repeat itself.

And the question we're going to focus on in lectures 4 and 5,

really, are how do you control all of this.

OK.

So this is the model.

And so I think what's interesting

about it is people have studied this in a lot of detail.

It was the first example of receptor-mediated endocytosis.

So we know something about the lifetime of the receptor.

We know it can make round trip from surface inside, back

to the surface in 10 minutes.

We also know it doesn't even have to be

loaded to make that round trip.

It could be one of the ones that isn't

the clustering of the receptors, which

is required for clathrin-coated vesicles to form.

And so you can tell how many trips it makes in its lifetime.

And so the question, then, what controls all of this?

But before we go on and do that, I just

want to briefly talk about, again,

mutations that have been found in the LDL receptor processing.

And they're really, basically, at every step in the pathway.

So the initial ones we found, that we talked about,

we'll come to in a minute.

But we had some patients with no LDL receptor express at all.

So somehow, it never makes it to the surface.

OK?

There are other examples-- and these have all been studied

by many people over the decades--

that it takes a long time to go through this processing.

And it gets stuck somewhere in the processing.

That may or may not be surprising,

in that you have transmembrane insoluble regions.

And if the processing goes a little astray or some mutation

changes, then you might be in trouble.

So we talked about this last time.

We talked about that they had just looked at 22 patients.

Some of the patients had no binding of LDL

to the surface of the fibroblast that they

were using as a model, at all.

Some have defective binding.

So if they compared it to a normal,

they had a range of dissociation constants.

And we'll talk quite a bit about dissociation constants,

not this week but next week, in recitation.

It's not so easy to measure dissociation constants

when things bind tightly.

And thinking about how to measure them correctly,

I think, is really important.

And I would say, probably, I could pull out

10 papers out of current journals, really good journals,

where people haven't measured dissociation

constant correctly, when you have tight binding.

So this is something that we put in

because I think it's important that people need to know how

to think about this problem.

So anyhow, let's assume that Brown and Goldstein

did these experiments correctly, which I'm sure they did.

And they got a range of binding.

And we also saw that the patient we looked at,

JD, had normal binding.

That indicates he was the same as normal patients,

but something else was problematic.

And that something else wasn't that it

failed to form coated pits, but that it failed

to bring this into the cell.

So it failed to internalize the LDL.

That was JD's defect.

We also, in recitation last week--

hopefully, you've had time, now, to go back and look

at the paper a little bit.

But LDL, in the model we were just looking at, gets recycled.

It goes in and gets back to the surface.

But what happens if, on occasion, instead

of budding off into vesicles and returning to the surface, it,

with the LDL cargo, goes to the lysosome and gets degraded?

Well, that was the working hypothesis for what PCKS did.

It targeted to the wrong place and degraded it.

And the phenotypes of those patients were interesting,

and that's why it was pursued.

So there are many, many defects.

And despite the fact that we have these statins,

people are still spending a large amount

of time thinking about this because of the prevalence

of coronary disease.

So I'm not going to talk about this,

but I'm just going to show you two slides.

And you can go back and think about this yourself.

But this is the LDL receptor.

We know quite a bit about it now.

And one of the questions you can ask yourself,

which is an interesting question we're not

going to describe-- but you have LDL

particles that are different sizes.

How do you recognize all these different sizes?

And how does the clustering do that?

And so that's done up here.

And there's calcium binding.

We know quite a bit about that, but I don't think we really

understand the details.

You have a single transmembrane helix in the plasma membrane.

And this is the part--