Tag Archives: gpcr

Proline rides again !

Proline is a kinky amino acid.  Kinky in the sense that it is only one of the twenty with a fixed configuration of its alpha carbon because of the ring (which may be why there is more of it in organisms living at high temperature) and kinky in the sense that when present in alpha helices it produces a kink.  The previous post shows how it is used to schlep the body weight’s worth of ATP we make each day out of our mitochondria — https://luysii.wordpress.com/2019/01/30/3939/.

Well here it is in one of the marijuana receptors (CB1).  Binding of delta9 THC in the 7 transmembrane alpha helix bundles of the G Protein Coupled Receptor (GPCR) causes an alteration in the kink allowing transmembrane helix 6 (TM6) to move outward toward the cytoplasm, creating a cavity on the intracellular side, where the G protein trimer can bind.

You can read much more about this in an exquisite paper [ Cell vol. 176 pp. 448 – 458 `19 ] describing the CB1 receptor bound to a synthetic ligand 20 times more potent that delta-9 tetrahydrocannabinol (delta9 THC).  It is a cryoEM study which used 177,000 projections to come up with a 3 Angstrom resolution structure of CB1 bound to MBDB-FUBINACA in complex with its G protein trimer.  They had to use a single chain variable fragment (scFv6) along with a positive allosteric modulator (PAM) called ZCZ-011 to stabilize the complex.

MBDB-FUBINACA is a story in itself.  It is presently the fentanyl of synthetic cannabinoids, which “has been linked to thousands of hospitalizations and numerous fatalities”  [ New England Journal of Medicine vol. 376 pp. 235 – 242 ’17 ].  I’m surprised I’ve never heard of it — have you? But then I’ve been retired from clinical practice for some time. Perhaps the mainstream press, pushing marihuana legalization as it has been, kept it quiet, or more likely there have been no further episodes of mass intoxication from the AMB-FUBINACA (aka the zombie drug) since 2017.

I’ve never knowingly used marihuana.  Frankly it scares me — for why please see — https://luysii.wordpress.com/2014/05/13/why-marihuana-scares-me/.

There are 4 molecular switches buried in GPCRs [ Current Med. Chem. vol. 19 pp. 1090 – 1109 ’12 ]

1. The ionic lock switch between the D/E R Y sequence at the cytoplasmic end of TM3 and E286 at the cytoplasmic end of TM6 (single letter amino acid code used) –

2. TM3 – TM7 lock switch.  In rhodopsin it is between the protonated Schiff base of lysine and a glutamic acid and it broken on light activation,.=

3. Toggle switch linked with the n P x x Y motif in TM7 (x stands for any amino acid) — much more about this later in the post.

4. Transmission switch — produced by agonist binding, the outward movement of TM6 to to ligand binding creating a hole fo the G protein to bind to the receptor on the cytoplasmic side.

So why did I call the Cell paper exquisite?  Because of the molecular detail it provides about just how MDMB FUBINACA activates CB1.  Here’s the structure of AB-FUBINACA — https://en.wikipedia.org/wiki/AB-FUBINACA.   Both look like drugs designed by a committee.  They both have a para-iodophenyl group, an amide, and a fused indole ring with an extra nitrogen (imidazole ring — I never could keep heterocyclic nomenclature straight).    MDMB has a methyl ester (in place of the amide) and a tertiary butyl group (in place of the isoPropyl group).

I don’t have time to look up how Pfizer came up with it.  The FUBINACAs do not resemble delta9 THC at all — https://en.wikipedia.org/wiki/Tetrahydrocannabinol.

The pictures in the paper show how the hydrophobic aromatic side chains of FIVE phenylalanines and 2 tryptophans create a nice oily space for delta9 THC and MBDB-FUBINACA to bind.

F200 (phenylAlanine 200) and W356 are the toggle twin switch which stabilize the inactive conformation of CB1.  The rotation of F200 to interact with the imidazole of FUBINACA, allows W356 to rotate outward, changing the kink produced the the proline #358  in TM6 allowing the helix to straighten and rotate outward toward the cytoplasm, creating a cavity for the G protein to bind to.

Definitely a tour de force for the blind watchman.


Time for drug chemists to go to the Multiplex

30 – 40% of all the drugs currently in clinical use are thought to target G Protein Coupled Receptors (GPCRs). Just how many GPCRs inhabit our genome isn’t clear. The latest estimate is 850 which is 4.2% of the 20,077 annotated protein genes we have. That being the case, it behooves drug chemists to know everything about them and how they work.

A recent paper [ Cell vol. 166 pp. 907 – 919 ’16 ] shows that a lot of the old thinking about GPCRs is wrong. Binding of a ligand to a GCPR results in a conformational change in its 7 transmembrane segments, so that the parts inside the cell bind to a heterotrimer of proteins which bind (and hydrolyze) GTP — this is the G protein. So far so good. The trimer splits up into its 3 constituents, unimaginatively called alpha, beta and gamma, each of which can act as a messenger that a ligand from outside the cell has landed on a GPCR, binding to other proteins causing all sorts of effects (e.g. can act as a second messenger)

All good things must end, and termination of GPCR signaling was thought to involve phosphorylation of the intracellular segment of the GPCR, binding of another protein (betaArrestin), removal from the cell membrane (so it can no longer bind its extracellular ligand) and then either destruction or recycling back to the cell membrane. So the old paradigm was betaArrestin binding equals the end of signaling.

It was thought that betaArrestin and the G protein competed for binding to the same intracellular amino acids of the GPCR. Not so says this paper. For some GPCRs both can bind, and signaling can continue, even though the complex of GPCR, G protein and betaArrestin is now inside the cell in an endosome. The complex is called the Multiplex. The examples given are GPCRs for parathyroid hormone (PTH) and Thyroid Stimulating Hormone (TSH). Blurry pictures are given of the complex. GPCRs have been divided into several classes and GPCRs for TSH and PTH are class B GPCRs — which contain a long phosphorylatable tail in the cytoplasm. The G protein binds to these GPCRs by its core region, while betaArrestin binds to the tail. Signaling continues apace.

A probably original (and probably wrong) idea

This post is not for the pharmacologically or chemically faint of heart.  Drug chemists should have no problem with it. You’ll need to bring your own background to the party, older posts on this blog don’t provide it.

It involves a particularly well studied G protein coupled receptor (GPCR), the beta2 adrenergic receptor.  Our genome codes for over 800 GPCRs, and at least 30% of all the drugs we currently use bind to them.

So here are my notes on  the paper that set me to thinking.  The idea follows the **** at the end.

       [ Science vol. 335 pp. 1055 – 1056, 1106 – 1110 ’12 ]  Conformation changes on ligand binding follow a common theme in the 3 activated GPCRs (including the beta2 adrenergic receptor) that have so far been crystallized.  Agonist binding results in an outward displacement of transmembrane segments 5 and 6 (TM5 and TM6) which opens a pocket on the intracellular surface of the receptor accepting the G protein.  Binding of beta-arrestin to GPCRs requires phosphorylation of sites on the cytoplasmic surface on TM7 toward the carboxy terminus of the GPCRs.

        This work attached 19F probes to cysteines at the intracellular ends of TM6 and TM7 of the beta2 adrenergic receptor for NMR spectroscopy.  They used 2,2,2 trifluoroethanethol to label 3 native cysteine residues (#265, #327, #341).  With no bound ligand two conformations in the ligand free state are in equilibrium with each other.  Then different ligands were added.  Carvedilol induced a shift to the TM7 active state, but had little effect on the conformational equilibrium at TM6.  Carvedilol is an antagonist which blocks G protein signaling and induces receptor desensitization and internalization.  

      Older work shows that the weakest agonists loosen interactions (the ionic lock and toggle), that juxtaposeTM3 and TM6 in the inactive state.  More potent agonists perturb the Asn Pro X X Y sequence of TM7 which permits phosphorylation and beta-arrestin binding.  Substituents on the hydroxylamine tail of the ligand are important — large nonpolar groups are particularly effective.  

       Agonist induced receptor bias for G protein vs. beta-arrestin signaling is one of many instances of the plastic and multistate behavior characteristic of GPCRs. 


So what’s the idea?  Drug chemists spend a lot of their time designing and making slightly different GPCR ligands to have slightly different effects.  On p. 1109 of the paper, the structures of 8 are given.  Probably hundreds exist.  The work here describes two different conformations of the beta2 GPCR, each of which triggers a different cellular response,  because each conformation binds a different set of proteins.  The drugs (particularly carvedilol which is in clinical use) cause the GPCR to favor one conformation over another.

Based on what we’ve been taught about neurotransmitters and neurotransmission, this makes no evolutionary sense.  The ‘natureal’ ligand for beta2 adrenergic GPCR is norepinephrine.  What selective advantage can there be to having the GPCR flopping between 2 (or more) conformations?  Well there might be if other neurotransmitters bound to it producing different conformations.

Back in the day there was a lot of talk about false neurotransmitters.  These were small molecules which somehow got into the synaptic vesicle and were released into the synaptic cleft when the vesicle was exocytosed, but which didn’t bind to the receptor.  One such false neurotransmitter was octopamine (norepinephrine without the meta-hydroxyl group).  It was thought to be formed in excess in liver failure, enter into synaptic vesicles and do nothing useful when released, causing hepatic encephalopathy.

A less fanciful example is available [ Neuron vol. 46 pp. 1 – 2, 65 – 74 ’05 ] Serotonin, a true neurotransmitter we all know and love, can enter dopamine neurons, when reuptake is blocked (by a tricyclic antidepressant, or by a SSRI), where it accumulates in synaptic vesicles.

Back in the day when I was in medical school, there was something called the Dale Feldberg law, which said that an identical chemical transmitter is liberated at all terminals of a single neuron.  We now know that this is wrong — neurons release two classes of chemical transmitters (1)  biogenic amines such as norepinephrine, dopamine and serotonin and (2) neuropeptides, and many release one from each class.  Whether or not a single neuron releases more than one biogenic amine isn’t known (despite the example above).

So perhaps the body is a better drug chemist than we think, and the same GPCR can bind different neurotransmitters with different effects, explaining the multiple conformations they adopt.  If true, this means that there are probably other transmitters out there that we don’t know about.  In the case of the beta2 adrenergic receptor, they are probably small molecules, as the binding site is deep within the transmembrane region of the GPCR.

So is this sort of thing why GPCRs have multiple conformations?