Biology 304: April 6, 2000

Lecture notes:

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When I spoke about the differences between neuropeptides and small molecule transmitters, one of the points I made was that the post-synaptic response to neuropeptides tended to be slower and longer lasting than the classical response to small molecule transmitters -- like ACh at the nmj.

Partly lack of inactivation; partly nature of the post-synaptic receptor. Unlike the nicotinic ACh receptor at the neuromuscular junction, in which the actual receptor for the neurotransmitter is part of the same large macromolecule that contains the ion channel whereby the neurotransmitter signal is transduced to the post-synaptic cell, the receptor molecules that neuropeptides bind to are distinct from the molecules that mediate the observed post-synaptic response. So rather than the receptor actually mediating the final event of interest -- like changing membrane permeability -- the receptor is coupled to some sort of transducer that conveys the signal to an effector protein

In many cases the effector molecule that mediates the post-synaptic response can be an ion channel but sometimes the post-synaptic activation of a receptor can effect  other molecules in the post- synaptic cell that have nothing to do with ion flux across the cell membrane -- they might, for instance,  effect  the post-synaptic cytoskeleton, or enzyme activity and they frequently involve the action of second messengers such as cAMP or arachidonic acid.

With this type of indirectly acting receptor it turns out that the interaction between the receptor itself and the effector protein --whatever that might be -- is mediated by interaction of the receptor with a regulatory protein

Two classes of indirectly acting receptors mediating the effects of neuropeptides:
 

G-protein-coupled receptors; in which the receptor interacts directly with a group of regulatory proteins that bind GTP and are known as G-proteins.

Receptor tyrosine kinases

Receptor tyrosine kinases involve the action of G-proteins, too, but they're a different class of G-proteins and the receptor doesn't interact with them as directly

They are also sometimes called metabotropic receptors, to indicate that they can produce a variety of metabolic effects, rather than simply fluxes through ion channels.

G-protein-coupled receptors

Compared to directly-activated ligand-binding ion channels, the action of G-protein coupled receptors is both slower and more complicated. However it is also more sensitive: activation of one receptor can lead to activation of many G-proteins:

Up to about 37,000 in some cases (rhodopsin in the eye; a bit of an extreme case)

and hence amplification of the signal, permits a much greater diversity of response than a simple depolarization or hyperpolarization of the cell membrane, and provides far more flexibility of response.

The whole area of G-protein coupled receptors and the various G-protein activated second messenger pathways is a huge field that's not only important in neurobiology but in mechanisms of communication between cells in general.

G-protein coupled receptors form a large family of proteins:

The G-protein-coupled receptors form a large family of proteins: over a hundred of them have now been cloned. Some small-molecule transmitters use G-protein coupled receptors: for example most of the effects of the catecholamines are mediated through G-protein-coupled receptors, as well as the effects of acetylcholine that are mediated through the muscarinic ACh receptor, a subset of glutamate receptors, rhodopsin in the photoreceptors of the retina functions as a G-protein coupled receptor, and odorants that you smell act by binding to G-protein-coupled receptors.

Only peptide messengers utilize tyrosine kinases

I.Directly-gated ionophoric receptors vs G-protein coupled receptors:

Structure:

Directly-gated ionophoric receptors are typically comprised of several very similar subunits which assemble to form one large macromolecular complex which both binds the transmitter and contains the channel through which the ions flow.

In contrast, the actual receptor -- or the neurotransmitter binding moiety -- of the G-protein coupled receptors consists of a single polypeptide chain, that has seven membrane spanning regions.

Serpentine

Hydrophobicity mapping

of the amino acid sequences of G-protein coupled receptors has led to a model of the typical G-protein-coupled receptor as containing 7 hydrophobic membrane-spanning regions with the amino terminal on the extracellular side of the membrane and the carboxy terminal on the intracellular side of the membrane.

Many of these receptors have been cloned and site-directed mutagenesis has given some information about which parts of the receptor are involved in which of its functions. The seven transmembrane regions tend to be well conserved from one receptor to the next.

Helpful in the cloning strategy for a number of these receptors.

Parts of the third cytoplasmic loop and of the carboxyl tail are believed to be involved in interactions with the G-protein. Variations in these domains influence which specific G-proteins the receptor is able to interact with. You can make a chimeric receptor, where you splice in the G-protein binding regions from one receptor with the rest of another receptor -- in this case they activate the G-protein characteristic of the donor receptor.

For ligands like the catecholamines, the ligand-binding domains are believed to reside just inside the lipid bilayer towards the extracellular surface of the cell. Variations in this domain affect the specificity of a receptor for its given ligand. Its important to remember that while these structures are pictured as a flat chain that loops back and forth the plasma membrane, in reality, the 7 transmembrane domains probably form more of a cluster so that the whole protein is sort of globular. Most small ligands fit into a sort of pocket-like structure and are thereby able to alter the conformation of the protein.
Movie of 3-D conformation

Interestingly enough, receptors that bind the same ligand don't necessarily seem to be the most closely related in terms of overall homology.

The binding of large ligands -- like peptides -- that won’t fit into such a pocket is less clearly understood. Receptors for such ligands have a long amino terminal extension that is believed to bind to part of the ligand and perhaps help orient the rest of the peptide so that a piece of it extends into some sort of cleft between the different transmembrane domains.

Just as the ionophoric glutamate receptors are considered to belong to a different gene family from the other ionotropic receptors, the G-protein-coupled glutamate receptors also appear to belong to a receptor family distinct from that of the other G-protein coupled receptors.

The G-protein coupled glutamate receptors, like those for the various neuropeptides, have a large amino terminus extension to which glutamate appears to bind.

II. Signal transduction via G-proteins

A. What are G-proteins anyway?

As the name implies, G-protein-coupled receptors transduce their signals to their effector or target molecules by means of a class of proteins known as G-proteins

G-proteins are a group of proteins that are able to activate various other target proteins in the cell. The G in G-protein stands for guanine nucleotide binding protein.

G-proteins are associated with the plasma membrane although they are NOT integral membrane-spanning proteins like the receptors or ion channels. They can exist in two different conformations: an inactive form which is bound to guanosine diphosphate (GDP) and an active form which is bound to guanosine triphosphate (GTP).

G-proteins exist as trimers of three distinct subunits: a, b and g. The a subunit is the one that binds GDP or GTP and also is the one that interacts with the receptor. Initially, a was thought to be the only subunit that directly affected the effector proteins as well; effects of b and g have been controversial, but it now appears they do play some direct role as well.  b and g subunits, which are very closely associated with each other also act to anchor the a subunit in the plasma membrane and are also required to enable the receptor to activate the a subunit.

B. Activation and inactivation:

In unstimulated cells, G-proteins exist overwhelmingly in the inactive, trimeric form bound tightly to GDP.

(purified G-proteins retain bound GDP after weeks in storage)

Occupancy of a G-protein-coupled receptor by its transmitter enables the receptor to interact with the G-protein. This interaction enables GTP to bind to the G-protein instead of GDP.

This in turn leads to dissociation of the a,b,g complex

The freed a subunit then diffuses through the membrane to and activates its effector or target protein.

The free a subunit itself has inherent GTPase activity. This limits the lifetime of the activated form
 

C. Effector (target) proteins and specificity:

Direct actions of G-proteins on ion channels

G-proteins have many effector -- or target -- proteins within the cell. One class of targets: ion channels. Either  by direct interaction of the G-protein with the ion channel or indirectly by turning on a second messenger pathway.

Direct activation of a K⁺ channel by the free activated a subunit of a G-protein is believed to underlie the slowing of the heart rate upon activating a muscarinic receptor with Acetylcholine.
 

Patch clamp:

Could do inside out patches:

See if you need GTP

G-proteins were involved in the response at all: knew that this was the case because GTP had to be present to see any effect of ACh on the channels. Furthermore, the effect was enhanced by inclusion of Gpp (NH)p, a non-hydrolyzable analog of GTP, and it was inhibited by pertussis toxin, which inhibits a variety of G-proteins.

Looking at the effect of acetylcholine on these K channels while doing a cell-attached patch clamp. They found that ACh only affected channels if it was applied inside the patch electrode: not to the rest of the cell membrane. This meant that there was some sort of barrier preventing the signal initiated by ACh’s binding to its receptor from reaching the K channel.

Most second messengers are small substances that readily diffuse through cytoplasm. The blocking of the effect by the walls of the patch pipet suggested that the K channels were activated by something that was restricted to the plasma membrane -- like the a subunit of the G-protein. This idea was further substantiated by the observation that the channels could also be activated by addition of pre-activated a subunits to the cytoplasmic surface of inside-out patches.

Direct action of a G-protein on a channel is also believed to underlie the autoregulation of release by norepinephrine on sympathetic neurons. In this case, released norepinephrine feeds back on presynaptic a-adrenergic autoreceptors. This leads to inhibition of a calcium channel and a reduction in the subsequent release of norepinephrine.

Indirect actions mediated through 2nd messengers: amplification and multiple effects

The targets of G-proteins include not only ion channels, but also membrane transporters, and perhaps most importantly, enzymes involved in the synthesis of various second messengers. Thus G-proteins can have indirect actions on a target molecule mediated through activation of a second messenger pathway.

While these responses are even slower than those mediated by the G protein directly, they permit the response to be amplified many many times (each receptor activates a number of G-proteins; each activated G-protein can lead to the creation of many second messenger molecules). Since 2nd messengers themselves may have many target proteins, this also permits a single event -- occupancy of a receptor by a transmitter -- to have many different simultaneous sequelae.

This possibility of divergence of signals; together with the possible convergence of different neurotransmitters on the same G protein and G protein mediated signal transduction pathways -- for instance the M current in sympathetic ganglia is inhibited following activation of G proteins by both acetylcholine and by LHRH -- allows for a great deal of flexibility in G protein-mediated signaling

Specificity G_s, G_i, G_o:
Most of the specificity of the G-protein is determined by the a subunit. The b and g subunits appear to be far more interchangeable.

G_s stimulates adenylyl cyclase, the enzyme responsible for synthesis of cAMP. Events mediated by G_s can often be recognized pharmacologically because they are mimicked by cholera toxin, which irreversibly activates a_s.

G_i  inhibits adenylate cyclase and hence the production of cAMP. While the mechanism of action of G_i in inhibiting adenylate cyclase is incompletely understood, at least part of its mechanism of action appears to result from the liberation of a large number of free b,g complexes, which act to grab up the free a s subunits and hence limit their activation of adenylate cyclase.

While there are other classes of G proteins (about 20 distinct G proteins have mow been cloned) that are similarly named by the effector proteins they turn on, for instance G_K, which turns on K channels, it's common to lump all of the G-proteins that act on other target proteins besides adenylyl cyclase as G_o for G_other.  Many of the G_i and G_o proteins are inhibited by pertussis toxin.

RECEPTOR TYROSINE KINASES

Many neurotrophins; also neurite outgrowth and long-term signaling events

A. Single pass through the membrane

B. Ligand-binding leads to dimerization and autophosphorylation

C. Autophosphorylation activates and permits the recruitment and phosphorylation of their own cascade of cytoplasmic proteins

D. Interestingly enough, small monomeric G-proteins in the Ras family play a pivotal role in some receptor tyrosine kinase pathways
 

Ras themselves involved in differentiation and proliferation

Rho affects actin cytoskeleton

Rab involved in intracellular trafficking

Slides Selected References and Further Reading

Ross EM (1992) "G proteins and receptors in neuronal signaling" in An Introduction to Molecular Neurobiology, Z Hall (ed), Sinauer, Sunderland, MA, p 181 - 206.

Zigmond MJ et al. (1998) "Neurotransmitter receptors" in Fundamental Neuroscience, Zigmond MJ, Bloom FE, Landis SC, Roberts JL and Squire LR (eds), Academic Press, NY.

Schwartz JH and Kandel ER (1992) "Synaptic transmission mediated by second messengers" in Principles of Neuroscience, ER Kandel, JH Schwartz and TM Jessell (eds), Elsevier, NY, p 173-192.

Hille B (1994) "Modulation of ion channel function by G-protein coupled receptors" TINS 17:531-535.

Ibáñez CF (1995) "Structure-function relationships in the neurotrophins: a review" http://cajal.mbb.ki.se/Structure-Function-Review