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?

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  • Curious Wavefunction  On March 16, 2012 at 9:22 am

    This is a fascinating topic about which I have occasionally blogged. Yes, it’s possible that there could be other neurotransmitters which bind to it. But could it really be otherwise? It’s obviously not possible for a protein to stay in one rigid conformation, so I interpret your question to mean why GPCRs have different *functionally relevant* conformations.

    I think it could be a simple requirement of multiple regulation. Think about what would happen if the beta-AR had only one productive conformation that bound to adrenaline and activated a single circuit. This would increase the risk of that circuit getting activated all the time and going haywire. You would need an inhibitory circuit – exemplified in this case by beta-arrestin recruitment leading to downregulation of the beta-AR – to counter its effects. I think of the multiple functional conformations of GPCRs as introducing a failsafe mechanism into the whole process. I think the same goes for the unliganded basal activity of most GPCRs (which can be shut down by inverse agonists, but not by antagonists), another failsafe in case there’s no adrenaline around.

    To me, this kind of built in safeguard actually makes a lot of evolutionary sense and only illustrates evolution’s remarkable ability to design homeostatic systems.

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