Tag Archives: GPCRs

The uses and abuses of molarity — II

Just as the last post showed why a 1 Molar solution of a protein makes no sense at all, it is reasonable to ask what the highest concentration of a single protein in the cellular environment could be. Strangely, it was very hard for me to find an estimate of the percentage of protein mass inside a eukaryotic cell. There is one for the red blood cell, which is essentially a bag of hemoglobin. The amount is 33 grams/deciliter or 330 grams/liter. Hemoglobin (which is a tetramer) has a molecular mass of 64,000 Daltons.  So that’s 330/64000 = .5 x 10^-3 Molar.   So all proteins in our cells have a maximum concentration at most in the milliMolar range.

Before moving on, how do you think the red blood cell gets its energy?  Amazingly it is by anaerobic glycolysis, not using the oxygen carried by hemoglobin at all.  Why? If it used oxidative phosphorylation which runs on oxygen, it would burn up.  That’s why red cells do not contain mitochondria. 

On to Kd the dissociation constant.  At least 475 FDA approved drugs target G Protein Coupled Receptors (GPCRs), and our genome codes for some 826 of them.  Almost 500 of them code for smell receptors, and of the 300 or so not involved with smell 1/3 are orphans (as of 2019) with no known ligand.  There are GPCRs for all neurotransmitters which is why neurologists and psychiatrists are very interested in them. 

The Kd is defined as [ free ligand ][ free receptor ]/ [ ligand bound to receptor]  where all the  [  ]’s are concentrations in Moles/liter (e.g. Molar concentrations). 

There’s the rub.  Kd makes sense when ligand and receptor are swimming around in solution, but GPCRs never do this.  The working GPCR is embedded in our cell membrane which topologists tell us are 2 dimensional manifolds embedded in 3 dimensional space.  What does concentration mean in a situation like this?  Think of the entropy involved in getting all the GPCRs to lie in a single plane.  Obviously not so simple.  

People get around this by using radioactive ligands, and embedding GPCRs in membranes and measuring the time for ligands to bind and unbind (e.g. kinetics), but this is miles away from the physiologic situations — for details please see

2019 Apr 5; 485: 9–19.
The same is true for other proteins of interest — ion channels for the neurologist, hormone receptors for the endocrinologist, angiotension converting enzyme 2 (ACE2) for the pandemic virus.  
I think that all Kd’s of membrane embedded receptors do is give you an ordinal ordering (e.g. receptor A binds ligand B tighter than ligand C ) but not a quantitative one.
Next up, how a Nobel prizewinner totally misunderstood the nature and applicability of molarity and studies on a two dimensional gas (complete with Pressure * Area = n * Gas Constant * Temperature).


Cells are not bags of cytoplasm

How Ya Gonna Keep ’em Down on the Farm (After They’ve Seen Paree) is a song of 100+ years ago when World War I had just ended. In 1920, for the first time America was 50/50 urban/rural. Now it’s 82%.

What does this have to do with cellular biology? A lot. One of the first second messengers to be discovered was cyclic adenosine monophosphate (CAMP). It binds to an enzyme complex called protein kinase A (PKA), activating it, making it phosphorylate all sorts of proteins changing their activity. But PKA doesn’t float free in the cell. We have some 47 genes for proteins (called AKAPs for protein A Kinase Anchoring Protein) which bind PKA and localize it to various places in the cell. CAMP is made by an enzyme called adenyl cyclase of which we have 10 types, each localized to various places in the cell (because most of them are membrane embedded). We also have hundreds of G Protein Coupled Receptors (GPCRs) localized in various parts of the cell (apical, basal, primary cilia, adhesion structures etc. etc.) many of which when activated stimulate (by yet another complicated mechanism) adenyl cyclase to make CAMP.

So the cell tries to keep CAMP when it is formed relatively localized (down on the farm if you will). Why have all these ways of making it if its going to run all over the cell after all.

Actually the existence of localized signaling by CAMP is rather controversial, particularly when you can measure how fast it is moving around. All studies previous to Cell vol. 182 pp. 1379 – 1381, 1519 – 1530 ’20 found free diffusion of CAMP.

This study, found that CAMP (in low concentrations) was essentially immobile, remaining down on the farm where it was formed.

The authors used a fluorescent analog of CAMP which allowed them to use fluorescence fluctuation spectroscopy which gives the probability distribution function of an individual molecule occupying a given position in space and time (SpatioTemporal Image correlation Spectroscopy — STICS).

Fascinating as the study is, it is ligh tyears away from physiologic — the fluorescent CAMP analog was not formed by anything resembling a physiologic mechanism (e.g. by adenyl cyclase). A precursor to the fluorescent CAMP was injected into the cell and broken down by ‘intracellular esterases’ to form the fluorescent CAMP analog.

Then the authors constructed a protein made of three parts (1) a phosphodiesterase (PDE) which broke down the fluorescent CAMP analog and (2) another protein — the signaler — which fluoresced when it bound the CAMP analog. The two were connected by (3) a flexible protein linker e.g. the ‘ruler’ of the previous post. The ruler could be made of various lengths.

Then levels of fluorescent CAMP were obtained by injecting it into the cell, or stimulating a receptor.

If the sensor was 100 Angstroms away from the PDE, it never showed signs of CAMP, implying the the PDE was destroying it before it could get to the linker implying that diffusion was quite slow. This was at low concentrations of the fluorescent CAMP analog. At high injection concentrations the CAMP overcame the sites which were binding it in the cell and moved past the signaler.

It was a lot of work but it convincingly (to me) showed that CAMP doesn’t move freely in the cell unless it is of such high concentration that it overcomes the binding sites available to it.

They made another molecule containing (1) protein kinase A (2) a ruler (3) a phophodiesterase. If the kinase and phosphodiesterase were close enough together, CAMP never got to PKA at all.

Another proof that phosphodiesterase enzymes can create a zone where there is no free CAMP (although there is still some bound to proteins).

Hard stuff (to explain) but nonetheless impressive, and shows why we must consider the cell a bunch of tiny principalities jealously guarding their turf like medieval city states.


A molecular ruler

Time to cleanse your mind by leaving the contentious world of social issues and entering the realm of pure thought with some elegant chemistry. 

You are asked to construct a molecular ruler with a persistence length of 150 Angstroms. 

Hint #1: use a protein

Hint #2; use alpha helices

Spoiler alert — nature got there first. 

The ruler was constructed and used in an interesting paper on CAMP nanoDomains (about which more on the next post).

It’s been around since 2011 [ Proc. Natl. Acad. Sci. vol. 108 pp. 20467 – 20472 ’11 ] and I’m embarrassed to admit I’d never heard of it.

It’s basically a run of 4 negatively charged amino acids (glutamic acid or aspartic acid) followed by a run of 4 positively charged amino acids (lysine, arginine). This is a naturally occurring motif found in a variety of species. 

My initial (incorrect) thought was that this couldn’t work as the 4 positively charged amino acids would bend at the end and bind to the 4 negatively charged ones. This can’t work even if you make the peptide chain planar, as the positive charges would alternate sides on the planar peptide backbone.

Recall that there are 3.5 amino acids/turn of the alpha helix, meaning that between a run of 4 Glutamic acid/Aspartic acids and an adjacent run of 4 lysines/arginines, an ionic bond is certain to form between the side chains (and not between adjacent amino acids on the backbone, but probably one 3 or 4 amino acids away)

Since a complete turn of the alpha helix is only 5.4 Angstroms, a persistence length of 150 means about 28 turns of the helix using 28 * 3.5 = 98 amino acids or about 12 blocks of ++++—- charged amino acids. 

The beauty of the technique is that by starting with an 8 amino acid ++++—- block, you can add length to your ruler in 12 Angstrom increments. This is exactly what Cell vol. 182 pp. 1519 – 1530 ’20 did. But that’s for the next post. 

The perfect aphrodisiac ?

We’re off to London for a few weeks to celebrate our 50th Wedding Anniversary. As a parting gift to all you lovelorn organic chemists out there, here’s a drug target for a new aphrodisiac.

Yes, it’s yet another G Protein Coupled Receptor (GPCR) of which we have 800+ in our genome, and which some 30% of drugs usable in man target (but not this one).

You can read all about it in a leisurely review of “Affective Touch” in Neuron vol. 82 pp. 737 – 755 ’14, and Nature vol. 493 pp. 669 – 673 ’13. The receptor (if the physiological ligand is known the papers are silent about it) is found on a type of nerve going to hairy skin. It’s called MRGPRB4.

The following has been done in people. Needles were put in a cutaneous nerve, and skin was lightly stroked at rates between 1 and 10 centimeters/second. Some of the nerves respond at very high frequency 50 – 100 impulses/second (50 – 100 Hertz) to this stimulus. Individuals were asked to rate the pleasantness of the sensation produced. The most pleasant sensations produced the highest frequency responses of these nerves.

MRGPRB4 is found on nerves which respond like this (and almost nowhere else as far as is known), so a ligand for it should produce feelings of pleasure. The whole subject of proteins which produce effects when the cell carrying them is mechanically stimulated is fascinating. Much of the work has been done with the hair cells of the ear, which discharge when the hairs are displaced by sound waves. Proteins embedded in the hairs trigger an action potential when disturbed.

Perhaps there is no chemical stimulus for MRGPRB4, just as there isn’t for the hair cells, but even so it’s worth looking for some chemical which does turn on MRGPRB4. Perhaps a natural product already does this, and is in one of the many oils and lotions people apply to themselves. Think of the chemoattractants for bees and other insects.

If you’re the lucky soul who finds such a drug, fame and fortune (and perhaps more) is sure to be yours.

Happy hunting

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