Extracellular to Intracellular Signalling Mechanisms

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G-protein Related Signalling - The Movies

click picture to play movie
opens in a new window. file - 17MB

g-protein cascade movie

protein kinase movie

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Review of the Basic Schema

In eukaryotes - plant and animal

Mutations of the proteins anywhere in the pathway
can constituitively turn on or turn off the process

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G-Protein Coupled Receptors -GPCRs

The GPCR superfamily of related receptors make up 1-3% of a typical mammalian genome.

The generic GPCR has an extracellular binding region, 7 transmembrane helices and an intracellular binding region.

Rhodopsin, the photopigment of retinal rods, is the first (and as of October 2002 the only) completely solved GPCR high-resolution crystal structure.

Here is a representation of its actual geometry in the membrane

Rhodopsin is unique because its agonist, 11-cis retinal, is actually bound to it in an inactive state shown in red above, and isomerizes to the active all-trans configuration upon absorbing a photon.

The "bleached rhodopsin changes shape to a form known as metarhodopsin II, which fits its G-protein target, transducin. If the rhodopsin was upside down in its membrane it would still capture a photon, but the surface that changes shape would not have access to the transducin and no signal would be sent.

More about this in a later lecture.

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Why GPCR crystallization is hard

Different substructures need different solvents.

Here is a brief summary of the process for rhodopsin (Okada et al.) (all in dim red light)

• Get membranes of photoreceptors into suspension by disruption techniques using Nonyl glucoside (NG)
• Concentrate and resuspend in
  • 0.5% (plus w/w NG and rhodopsin) Nonyl glucoside (NG) (detergentlike substance)
  • 30 mM 2-(N-morpholino)ethanesulfonic acid (MES)
  • 5 mM beta-mercaptoethanol
  • 65 mM Zn(OAc)2
  • 0.6 mM heptane-1,2,3-triol (HPTO) (a critical component)
  • 0.85M Ammonium sulfate (AS)
• Hang 10 microliter drops on a coverslip sealed over a well containing MES buffered AS for at least 45 DAYS.

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The rhodopsin structure has already been used to model parts of other GPCRs, such as the muscarinic acetylcholine receptor. The next figure shows how binding of acetylcholine to the extracellular face of the receptor causes a configuration change.

In the unbound state on the left the amino acids in green and in blue (spacefill) associate via weak bonds to hold the helices in a closed configuration. Note how binding of acetylcholine (CPK colors) disrupts the weak bonds and brings about movement of the helices.

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The movement caused by ligand binding on the extracellular face causes shifting of the transmembrane helices an that in turn causes movement of the intracellular loops of the GPCR. This disrupts the association of the GPCR with the g-protein, allowing the g-protein to dissociate from the GPRC. This picture shows a hypothetical model of the binding of rhodopsin to the g-protein transducin.

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click the above picture for a Chime-based presentation
about g-protein structure (appears in a new window)

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Four main G-alpha-chain families

Some GPCRs can activate more than one g-protein in different families.

Some g-proteins can be activates by non-gpcr pathways.

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GTP binding to the alpha-subunit causes dissociation from the GPCR and of alpha-subunit from the beta-gamma pair. Here is GTP bound in the alpha subunit binding pocket.

The space-fill version of the above view when seen from the left, shows the narrow entry cleft

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When associated with the beta-gamma pair, the amino-terminal residues of the alpha subunit form a helix that is stabilized by its interaction with the side of the beta-gamma pair. When the alpha subunit dissociated from the beta-gamma pair, its terminal helix assumes more of a random coil configuration and tucks back against the bottom of the alpha-subunit. this contributes to the shape change of the subunit that allows it to bind to the cyclase.

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Interaction of alpha-subunit with adenylate cyclase.
Note the intercalation that changes cyclase shape

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The resultant cAMP activates a kinase

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Activation of the Kinase

"In the absence of cAMP, the two regulatory (R) and two catalytic (C) subunits of cAPK [cyclic A Protein Kinase] form a catalytically inactive, tetrameric holoenzyme complex. R subunits possess a conserved domain structure, which is comprised of a dimerization domain at the N terminus, two tandem cyclic-nucleotide binding domains at the C terminus, and a variable, interconnecting linker region. The linker region contains a substrate-like inhibitor sequence that docks to the active site cleft of the C subunit."

"Each R subunit cooperatively binds two cAMP molecules and releases concomitantly the C subunit as an active protein kinase." This picture shows the region that binds the two cAMP molecules on one of the two regulatory subunits. The region has two similar domains, A and B, that bind a cAMP molecule (stick figure).

"The solvated interface with its A-site cAMP enables dynamic transmission of conformational change from the A site through the B domain to the B site, and this transmission is critical for the cooperative binding and release of four cAMP molecules per RII dimer upon holoenzyme formation."

The region that binds the cAMP is highly conserved in most cAMP binding proteins and is a motif called the Phosphate Binding Cassette.

Note the hydrogen bonds of the side chains (purple) that anchor the cAMP, and other hydrogen bonds of residues (blue) that help position the side chains that interact with the cAMP.

Different isoforms of the Regulatory subunit RIalpha and RIIalpha have been identified. They vary enough in overall shape and function that they make potentially good targets for drug interventions.

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Kinase Targets - The alpha-s pathway

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Termination of G-protein signalling

For example, "There is strong evidence implicating RGS proteins in the subsecond kinetics of G i- and G o-mediated ion channel activation and deactivation in neurons. . . . One other intriguing aspect of RGS proteins is that they undergo rather profound regulation of expression by signal transduction events. This makes them uniquely well-suited to play a role in the cellular changes that underlie tolerance and/or dependence that are the hallmarks of drugs of abuse. . . . amphetamine-induces upregulation of RGS2 and -3 with chronic administration . . . stimulation of both mu and delta opioid receptors induced RGS4 upregulation in PC12 cells expressing those receptors." (Neubig)

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Below is an actual example

• "Organization of a synaptic neurotransmitter receptor complex by interactions of PSD-95, GKAP/SAPAP, shank/ProSAP and homer proteins. Shank/ProSAP and its domains are depicted in the centre."
• "Several receptors, both ionotropic (the NMDA receptor) and G-protein-coupled (sst2, CIRL and mGluR), are shown which have the potential to bind to this complex via interactions with PDZ domains or ena/VASP homology domains."
• "Note the dual anchorage of this complex to the cytoskeleton by the actin-binding proteins fodrin and cortactin."

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An example of kinase localization

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Another localization example

"A Schematic Representation of the Membrane with the Associated Components of the GIRK Channel Signaling Complex with its Opposing Modulatory Pathways

(Viewed from the intracellular side of the membrane.) GIRK channels are gated following the activation of GPCRs associated with pertussis toxin-sensitive G proteins (G i/o ) that release G?? dimers to directly gate the channel (blue). The phospholipid PIP 2is closely associated with the channel to stabilize its functional integrity. Reduction of channel activity can be mediated by GPCRs that are associated with Gq-type G proteins (green), to activate phospholipase C (PLC), which breaks down PIP 2. Increase in channel activity can be mediated by the activation of protein kinase A (PKA) following the activation of GPCR linked to G?s-type G proteins (yellow). Both PLC and PKA may be soluble and thus do not have to directly associate with the GIRK channel activation complex." (Sadja et al.)

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Second messanger localization can lead to specificity

In some cases the g-protein opens a Ca++ channel. The calcium that enters is then a second messanger. However, localized pumps and vesicles that take up the Ca++ can keep it from spreading throughout the cell, thus localizing its action.

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Some g-protein pathways are inhibitory

When activated by bound GTP, the G-alpha type-i subunit binds to adenylate cyclese and inhibits cAMP production.

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Agonist induced desensitization - An example

"When agonists continuously or sequentially stimulate a receptor, a progressively reduced response is usually observed. . . . [This] frequently involves receptor phosphorylation by G protein-coupled receptor kinases (GRKs). . . that phosphorylate GPCRs only in the agonist-bound state. According to most recent models, GPCRs occupied by agonists activate heterotrimeric G proteins, releasing G complexes. Membrane-bound G heterodimers and phosphatidylinositol bisphosphate bind to the carboxyl terminal domain of soluble GRKs, targeting the kinases to the receptors. These enzymes phosphorylate GPCRs, increasing their affinity for arrestin molecules. The binding of arrestin proteins to the receptors sterically interdicts the receptor–G protein interaction, stabilizing the uncoupled state of the receptor." (Prado et al.)

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Some Disorders

Adrenergic receptors have noradrenalin or adrenalin as their ligands. There are at least 6 different alpha-type and 3 different beta-type adrenergic receptors. These receptors are targets for drugs that deal with various disease types.

• Beta1 receptor activation causes stronger contraction of heart muscle. The beta1-blockers can be used to decrease heart muscle contraction and thus reduce high blood pressure. These blockers are also used to decrease the load on heart muscle that has been damaged by heart attacks.
• Beta2 receptor activation (and multiple alpha subtype activation) causes smooth muscle contraction. Thus, inhalation of beta2 blocker substances is used to relax constricted lung bronchioles in asthma.
• Beta1 and beta 3 receptors are found in fat tissue and their activation causes the breakdown of fat for its ultimate use in energy production. Recent studies suggest malfunction of these receptors in obesity.

Disorders of the g-protein signalling pathway are implicated in numerous types of cancers. This is not surprising given the major role of these pathways in the cell cycle and cell differentiation.

Old-age disorders of the heart are associated with the decline in beta1-adrenergic receptors (a GPCR) and in G-alpha-s as well as the increase in G-alpha-i. Reasons for these changes are still being sought.

A mutation in the human gene GNAT2 causes a truncation defect in cone-photoreceptor transducin that leads to a total lack of color discrimination (achromatopsia).

References and the sources of the pictures and information on this page

Disease References