Wednesday, April 26, 2006

Receptors - getting the message across

From Paul Greengard's Nobel Lecture in 2000:
It is estimated that there are about 100 billion nerve cells in the brain and that on average each of these nerve cells communicates with 1000 other nerve cells. A vigorous debate went on from the 1930s through the 1960s as to whether intercellular communication across the synapses between nerve cells was electrical or chemical in nature. The electrical school of thought held that the nerve impulse or action potential was propagated along the axon to the nerve ending, changed the electrical field across the postsynaptic plasma membrane, and thereby produced a physiological response. The chemical school believed that when the action potential came down the axon to the nerve terminal, it caused the fusion of neuro-transmitter-containing vesicles with the presynaptic plasma membrane, releasing a neurotransmitter, which then diffused across the synaptic cleft and, through activation of a (hypothetical) receptor, produced a physiological response. The chemical school won this debate: over 99% of all synapses in the brain use chemical transmission. (Paul Greengard won the 2000 Nobel Prize in Physiology or Medicine along with Arvid Carlsson and Eric R. Kandel "for their discoveries concerning signal transduction in the nervous system")

Life would be pretty dull if it wasn't for receptors. The senses of smell and vision, hormones, brain functioning - all rely on receptors to make them work. And the details of how they work are being discovered, down to the molecule-by-molecule layout of a single receptor. And the structures disclosed with these techniques are fascinating. No longer regarded as the passive “lock” of a “lock-and-key” mechanism, the receptors appear to work from a few simple elements and to achieve a wide range of effects. Robert Lefkowitz and his colleagues at the Howard Hughes Medical Institute at Duke University have been looking closely at epinephrine and norepinephrine receptor sites [since 1986] Lefkowitz and others succeeded in reading the full genetic sequence of the beta-2 receptor, which allowed them to clone the gene—in effect, to create thousands of copies of the beta-2 receptor in their laboratory. What they learned in the process was astonishing: a single receptor site spans the cell membrane, like a built-in tunnel, no fewer than seven times. The arrangement consists of seven recurring clusters of 20 to 25 amino acids, each crossing the membrane and all held together by loops of amino acids within the cell and just outside the membrane. This pattern appears to hold good for the other kinds of receptors as well. In a comparison of any two receptors, 40 to 50 percent of the sequence is identical—a high degree of conservation and an indication of how effective this structure must be. (From Sandra Ackerman's book Discovering the Brain)


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(A dopamine receptor is on the left, and a serotonin receptor is on the right


From Evolution of Signal Transduction: Animals are especially fond of these receptors. The genome of the nematode worm is now done and it contains 1049 G-protein coupled receptors. These may be chemosensory receptors.

From Richard Axel's 2004 Nobel Lecture: The completed sequence of both the [mouse] and human genome ultimately identified 1300 odorant receptors in the mouse and 500 in humans. If mice possess 20,000 genes, then as much as 5% of the genome, one in 20 genes encodes the odorant receptors. And all of these receptors that the sense of smell relies on are based on the same serpentine arrangement where the receptor protein passes through the membrane 7 times. The 3 "extra-cellular" loops of the protein that are outside of the cell membrane have a specific shape and composition that binds with 'ligands', i.e. molecules that can reversibly bind with the receptor protein (usually with some molecular surface features which 'fit' well with the shape of the receptor protein).

G protein-coupled receptor (GPCR)
For the receptors we've been discussing so far, there is a "G Protein" molecule inside of the cell membrane that gets activated when this change in shape occurs. It's called a "G Protein" because it is a glycoprotein that is anchored on the cytoplasmic cell membrane that binds the guanine nucleotide (one of the bases that is used to build DNA). When combined with the sugar ribose... guanine forms a derivative called guanosine (a nucleoside), which in turn can be phosphorylated with from one to three phosphoric acid groups, yielding the three nucleotides GMP (guanosine monophosphate), GDP (guanosine diphosphate), and GTP (guanosine triphosphate).(ref.).
Almost all members of this superfamily of proteins act as molecular switch, which is on when GTP is bound and off when GDP is bound. Binding is specific for the guanine base[ and is 6 orders of magnitude higher for GDP and GTP than it is for GMP and other nucleotide bases]. (ref.)



From Indiana State U.:
The link between neurotransmitters and intracellular signaling is carried out by association either with G-proteins (small GTP-binding and hydrolyzing proteins) or with protein kinases, or by the receptor itself in the form of a ligand-gated ion channel (for example, the acetylcholine receptor). One additional characteristic of neurotransmitter receptors is that they are subject to ligand-induced desensitization: That is, they can become unresponsive upon prolonged exposure to their neurotransmitter.


Bruce Jenks at Radboud University Nijmegen has put together some cool flash animations. From his site:
The G proteins associated with receptor transduction are the so-called "trimeric" G proteins because they are composed of three subunits, alpha, beta and gamma. (The trimeric designation distinguishes the receptor-associated G proteins from smaller intracellular "monomeric" G proteins that are involved in vesicular traffic and other processes within the cell.) When the neurotransmitter[or hormone or odour or whatever] binds with the receptor, it exerts molecular-level forces on the protein which cause the shape of segments inside of the cell membrane to change. Intracellular loops of the receptors are responsible for the activation of the G proteins. After activating a G protein the ligand-receptor complex remains active and can activate more G proteins. In this sense, one can think of the activated ligand-receptor complex as an enzyme.


Molecular tinkering of G protein-coupled receptors: an evolutionary success

GPCRs have a central common core made of seven transmembrane helices (TM-I to -VII) connected by three intracellular (i1, i2, i3) and three extracellular (e1, e2, e3) loops. The diversity of messages which activate those receptors is an illustration of their evolutionary success. The diagrams at left show the classification and diversity of GPCRs. Figure A: Three main families (1, 2 and 3) can be easily recognized when comparing their amino-acid sequences. Receptors from different families share no sequence similarity, suggesting that we are in the presence of a remarkable example of molecular convergence. Family 1 contains most GPCRs including receptors for odorants. Group 1a contains GPCRs for small ligands including rhodopsin and β-adrenergic receptors. The binding site is localized within the seven TMs. Group 1b contains receptors for peptides whose binding site includes the N-terminal, the extracellular loops and the superior parts of TMs. Group 1c contains GPCRs for glycoprotein hormones. It is characterized by a large extracellular domain and a binding site which is mostly extracellular but at least with contact with extracellular loops e1 and e3. Family 2 GPCRs have a similar morphology to group Ic GPCRs, but they do not share any sequence homology. Their ligands include high molecular weight hormones such as glucagon, secretine, VIP-PACAP and the Black widow spider toxin, α-latrotoxin. Family 3 contains mGluRs and the Ca2+ sensing receptors. Last year, however, GABAB receptors and a group of putative pheromone receptors coupled to the G protein Go (termed VRs and Go-VN) became new members of this family. Figure B: Family 4 comprises pheromone receptors (VNs) associated with Gi. Family 5 includes the 'frizzled' and the 'smoothened' (Smo) receptors involved in embryonic development and in particular in cell polarity and segmentation.(refs.)
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From the conclusion of Alfred G. Gilman's 1994 Nobel lecture:
Why G Proteins?
One might well ask why G proteins are included in signaling pathways and why the systems are so complex structurally. Transmembrane signaling is clearly accomplished with simpler [...] molecular assemblages, such as tyrosine kinases, ligand-gated ion channels, and receptor guanylyl cyclases. I believe there are several reasons for the evolution of complex signaling systems. At a relatively simple level, the existence of these molecular switches and timers permits enormous amplification in the signaling process. A single agonist-receptor complex can catalyze the activation of many G proteins during the time that a single G protein a subunit remains, active; delayed deactivation of the alpha subunit permits further amplification at the level of catalytic effector molecules. There is also the possibility of substantial regulatory complexity, with opportunities to modulate both the quantitative and qualitative aspects of signaling by alterations in rates of synthesis and degradation of many gene products, as well as more acute regulation by covalent modification of these molecules. Most importantly, perhaps, the tripartite nature of these signaling systems permits enormous diversity of outputs. G protein-regulated signaling pathways are characterized by both convergence and divergence at each step. Many different kinds of receptors can converge to activate a single type of G protein, while a single type of receptor can interact with more than one species of G protein to initiate several events. Similarly, different G proteins may converge on a single effector to alter its activity, either additively, synergistically, or antagonistically, while a single G protein may also interact with more than one effector. G proteins can also exert effects via either their alpha or beta gamma subunits. The complexity of the cellular switchboard thus appears sufficiently vast to permit each cell to design a highly customized signaling repertoire by expression of a relatively modest number of modular components.


Second messengers
From Brainfacts, from the Society for Neuroscience: Substances that trigger biochemical communication within cells, after the action of neurotransmitters at their receptors, are called second messengers; these intracellular effects may be responsible for long-term changes in the nervous system. They convey the chemical message of a neurotransmitter (the first messenger) from the cell membrane to the cell’s internal biochemical machinery. Second messenger effects may endure for a few milliseconds to as long as many minutes.

An example of the initial step in the activation of a second messenger system involves adenosine triphosphate (ATP), the chemical source of energy in cells. ATP is present throughout the cell. For example, when norepinephrine binds to its receptors on the surface of the neuron, the activated receptor binds G proteins on the inside of the membrane. The activated G protein causes the enzyme adenylyl cyclase to convert ATP to cyclic adenosine monophosphate (cAMP).
[Shockwave animation from Signal Transduction Cascades] The second messenger, cAMP, exerts a variety of influences on the cell, ranging from changes in the function of ion channels in the membrane to changes in the expression of genes in the nucleus, rather than acting as a messenger between one neuron and another. cAMP is called a second messenger because it acts after the first messenger, the transmitter chemical, has crossed the synaptic space and attached itself to a receptor. Second messengers also are thought to play a role in the manufacture and release of neurotransmitters, intracellular movements, carbohydrate metabolism in the cerebrum. Direct effects of these substances on the genetic material of cells may lead to long-term alterations of behavior.


OK, so when a ligand binds to a receptor it effectively turns on an enzyme reaction or an ion channel, which can cause quite significant changes of functionality within the cell. But a switch that can only be turned on once and can never be turned off is kinda useless. How do the receptors get 'turned off' once they've been activated? i.e. How do the ligands get removed from the receptors?

One of the ways this happens is with the help of clathrin (yes, our friend the super cool cellular transport molecule). In addition to having a binding site for a ligand molecule, receptors also have a binding site for a protein called adaptin. Once adaptin has bonded with the receptor, it tows it towards a clathrin coated pit, which is tethered to the cell membrane via the cell's cytoskeleton (ref.).

Once enough receptors have accumulated in the pit, the pit pinches off to form an enclosed bubble called an endosome which carries the receptors and their attached ligand molecules into the cell in a process called endocytosis. The pH within the endosome drops to 5.9-6.0, which is sometimes enough to strip the ligand from the receptor. The receptors can then be recyled, by moving back to the membrane.

Those endosomes carrying receptors with ligands that get past this stage are called 'late endosomes'. Their pH continues to drop to 5.0 to 6.0 range, and they can be given digestive enzymes that further help in removing the ligands from the receptors. If the ligand survives this stage, the endosome fuses with a lysosome within the cell, which drops the pH to the 5.0 to 5.5 range and is capable of breaking apart both the ligand and the receptor itself. Check out this cool website from the University of Texas for more information on this fascinating process.

Getting rid of the ligand by itself is not sufficient to turn off the process associated with the receptor - if there are more ligands (e.g. neurotransmitters or hormones) hanging around the cell membrane, they will simply bind with the recycled receptors and the process will continue. To truly turn off the signaling, the ligands have to be removed from the vicinity of the receptors. There are at least 4 ways that this can happen:

  • Diffusion: e.g. the ionic concentration in the extra-cellular fluid that the ligand is in can exert pressure on the molecule, causing it move to an area with a weaker ionic concentration.

  • Enzymatic degradation (deactivation): a specific enzyme changes the structure of the neurotransmitter so it is not recognized by the receptor. For example, acetylcholinesterase is the enzyme that breaks acetylcholine into choline and acetate.

  • Glial cells: astrocytes remove neurotransmitters from the synaptic cleft.

  • Reuptake: the whole neurotransmitter molecule is taken back into the axon terminal that released it. This is a common way the action of norepinephrine, dopamine and serotonin is stopped...these neurotransmitters are removed from the synaptic cleft so they cannot bind to receptors.
  • One of the major areas of pharmaceutical research is identifying 'uptake inhibitors' which can interfere with the uptake mechanism and prolong the length of time that the receptor is activated.
    (ref.)

    Within the brain, there are two broad categories of receptors - those that act slowly, like the GPCR receptors that trigger second messengers discussed earlier, and those that act quickly by controlling ion channels, for example.
    Back to Paul Greengard's Nobel Lecture:We know today that there are two categories of chemical transmission between nerve cells, which are referred to as fast and slow synaptic transmission. About half of the fast synaptic transmissions in the brain are excitatory, and most of these utilize glutamate as their neurotransmitter [which causes a conformation of the receptor it binds to, which opens up the ion channel and allows ions to rush into the cell, causing depolarization]. The other half of the fast synaptic transmissions are inhibitory and most use GABA [Gamma-AminoButyric Acid] as their neurotransmitter.

    Gary G. Matthews has created some cool animations that show how fast-acting neurotransmitters act directly as ligands which open ion channels for both excitatory and inhibitory synaptic transmissions, and slow-acting neurotransmitters act indirectly through G-protein coupled receptors.

    Wiki's entry on GABA:In vertebrates, GABA acts at inhibitory synapses in the central nervous system. GABA acts by binding to specific receptors in the plasma membrane of both pre- and postsynaptic cells. This binding causes the opening of ion channels to allow either the flow of negatively-charged chloride ions into or positively-charged potassium ions out of the cell. This will typically result in a negative change in the transmembrane potential.

    Three general classes of GABA receptor are known. These include GABAA and GABAC ionotropic receptors, which are ion channels themselves, and GABAB metabotropic receptors, which are G protein-coupled receptors that open ion channels via intermediaries


    Paul Greengard: Slow synaptic transmission, which occurs over periods of 10s of milliseconds, is enormously more complex than fast synaptic transmission. ... It is very likely that all of the biogenic amines, and all of the peptide neurotransmitters, produce their effects on the target cells through slow synaptic transmission. And even the fast acting neurotransmitters, including glutamate and GABA, produce many of their effects through slow synaptic transmission pathways. In fact, Liu et. al (Nature 403: 274-280) have shown that receptors can interact with each other in the cell membrane, even slow GPCR receptors like dopamine receptors and fast ion channel receptors like the GABA receptor. (check out Bruce Jenk's GPCR-ion channel interactions presentation). These type of interactions increase the range of responses a cell can produce in react to one or more neurotransmitters.

    And it's important to note that for each neurotransmitter there can be many different receptors, each capable of producing different effects in the cell when activated. In fact, the same neurotransmitter is frequently used in different areas for completely different purposes. This is one reason that therapies that involve artificially introducing neurotransmitters as a drug can have devastating side-effects.

    Another surprising thing is that just because a neurotransmitter gets released at one synapse that an axon terminates on doesn't necessarily mean that all of that axon's synapses will release that neurotransmitter. It's amazing how subtle and complex the brain really is.


    More...

  • HybridMedicalAnimation's extremely cool receptor and co-receptor agonist animation.

  • Linda Buck's excellent 2004 Nobel lecture Unraveling the Sense of Smell
  • Bruce Jenks website at Radboud University Nijmegen: Cell Signaling

  • Receptor Mediated Endocytosis - provides a lot of good info on how receptors are recycled within the cell

  • Orientations of Proteins in Membranes (OPM) database is quite mindblowing. 3D rotational views of proteins in membranes. Wow.
  • Evolution of Signal Transduction discusses other types of receptors not covered here, including nuclear receptors that drive gene transcription.

  • Thermodynamic properties and heterogeneity of membrane assemblies. - "It is assumed usually that the lipid membrane acts as an anchor for the proteins and has no independent function. However, membranes contain hundreds of different lipids which are different with respect to their physical properties (for example charge and size)."
  • Martin Rodbell's 1994 Nobel Lecture: SIGNAL TRANSDUCTION: EVOLUTION OF AN IDEA - points out interesting similarities between G-Proteins and Cytoskeletal proteins.
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