W8L2 Flashcards
G protein-mediated signalling overview
- GPCRs are 7TM-spanning proteins; sometimes called metabotropic receptors
- Can function as monomers, but can also exist as homo-oligomers and heteromers (hetero-oligomers)
- mostly monomers
*The G-protein is a membrane-associated protein comprising three subunits (Gαβγ), the Gα-subunit can bind to GTP and GDP, and Gα-subunit possesses GTPase activity
*When the trimer binds to agonist-occupied receptor, the Gα-subunit is activated via binding of GTP, and is thought to dissociate and do downstream signalling by first activating an effector (a membrane enzyme or ion channel).
- “Activated” Gβγ-subunit can also contribute to signaling
*Activation of the effector is terminated when the bound GTP molecule is hydrolysed, which allows the Gα-subunit to recombine with Gβγ.
*There are multiple types of G-protein, which interact with different receptors and control different effectors.
*Effectors include adenylyl cyclase, phospholipase C, RhoGEFs, and ion channels
*Multiple intracellular components govern GPCR-initiated signals
G protein-coupled receptors
- 7 transmembrane spanning proteins
- largest family of receptor proteins (encoded by more than 3% of protein-coding genome)
- ~ 800 genes encode GPCRs in humans
- an estimated 30%-50% of all prescribed drugs target GPCRs
- interaction between receptor and target protein is mediated by G protein
- binding of ligand to receptor leads to alteration in :
(1) concentration of intracellular mediators, or
(2) ion permeability at plasma membrane - indirectly regulate activity of other membrane proteins (enzymes, ion channels) or intracellular proteins
GPCR STRUCTURE
Typically requires 20 aa to go through TM, but the TM domains can be more than 20 aa
Lots of TM domains have proline in them. Proline is a helix breaker, which explains the kinking
- a lot of the TM domains do not go straight through, they are kinked and go indirectly through the PM
It is very adaptable signalling mechanism; can respond to light, calcium, etc.; can respond to wide array of molecules
i3 is where the G protein binds to the receptor and the receptor activates the G protein
- extracellular N- terminus
- central core of 7 transmembrane helices (TM-I to –VII or H1-H7))
- 3 extracellular loops (e1, e2, e3)
- 3 intracellular loops (i1, i2, i3)
- intracellular C-terminus
– may also be alpha-helical in nature and be pinned back into the PM via a cysteine residue, and so it is sometimes counted as a seventh alpha-helical domain
GPCR - Tree of life
GPCR is important in many species of eukaryotes
Theres a few, if any, GPCR in plant kingdom.
- there are sometimes proteins that resemble, topologically, GPCRs
Everything from yeast, up to our cells, use GPCRs to sense their environment and for cells to communicate
Also important for researchers to understand species
- C. elegans
- D. rerio
- rats, mice
The total number of GPCRs in a species is variable
There is no correlation between how advanced a species is and the number of GPCRs
- as you go higher up in evolutionary scale, you sorta tend to lose GPCRs
The number of GPCRs within mammalians that are used for signalling between cells is very low (around 400 genes encoding those)
The difference in number of GPCRs between species comes from olfaction
- so mice need sense of smell, so that is why they have more than twice GPCR than humans do
Olfactory receptors vary greatly between species
Pseudogene - a gene that looks like it was once functional, but through evolution, it lost its function
Humans have 400 genes encoding olfactory GPCRs and 400 pseudogenes
Rats and mice each have over 1000 genes encoding olfactory GPCRs, with around 300-500 pseudogenes each
Human sense of smell is not well developed, but also not very useful because we use our eyes
Vomeronasal receptors
Vomeronasal receptors
- vestigial part within our nose, bone marrow nasal
- are called pheromone receptors; determines if another animal is mate, predator, or prey
Humans
- 400 genes encoding Vomeronasal GPCRs, and 400 pseudogenes
- much less than other species
- not sure if it is THAT important for pheromone detection; we have perfume now
Diversity and classification of GPCRs
The superfamily of G-protein-coupled receptors (GPCRs) is one of the largest families of proteins in the human genome
~800 GPCR genes have been identified in humans
– There are also many pseudogenes (encoding incomplete or nonfunctional proteins) as well as many full length “orphan” 7TM proteins whose endogenous ligands are unknown and which are potential new drug targets
– the 800 are split evenly between olfactory receptors and other receptors
Based on sequence similarities GPCRs can be divided into several classes, each with characteristic highly conserved residues distributed throughout the molecule
Three main GPCR classes (A, rhodopsin-like; B, secretin-like; C, glutamate-like) - easily recognized by comparing their amino-acid sequences – structures are highly conserved within GPCR classes.
There is also GRAS system of categorization. G stands for glutamate-like, R is rhodopsin-like, S is secretin-like.
Notwithstanding their similar topologies, receptors from different classes share little or no sequence similarity, a remarkable example of molecular convergence
GPCR Families in humans
- Class A (rhodopsin)
- largest number of gene products compared to other families
- 200 receptors with known ligands
- 90 orphan recepotrs
- 400 olfactory receptors
- 10 vision receptors
- 5 vomeronasal receptors - Class B (secretin)
- 15 total
- 15 receptors with known ligands - Adhesion
- 33 total
- 33 orphan receptors - Class C (glutamate)
- 23 total
- 12 are receptors with known ligands
- 8 are orphans
- 3 are for taste receptors - Frizzled
- 11 total
- 11 receptors with known ligands
- are important developmentally - Taste 2
- 25 total
- 25 for bitter taste specifically
Frizzled and Taste 2 are sometimes considered to be members of the same family
Adhesion was formerly considered a subset of Class B
Non-mammalian GPCRs and GPCR-like proteins include cAMP receptor family in dictyostelium (slime mould), pheromone receptors in yeast, and bacteriorhodopsin – a 7TM proton pump
Class A GPCRs
Class A - rhodopsin-like, all contain E/DRY motif at the cytoplasmic end of the third transmembrane domain and prolines at specific positions in helices 5, 6, and 7
Includes most GPCRs and can be subdivided into at least 3 groups.
Three main GPCR classes (1, 2 and 3) - easily recognized by comparing their amino-acid sequences – structures are highly conserved within GPCR classes.
- Group 1a - GPCRs for small ligands including adrenergic, muscarinic, serotonergic, and histamine receptors. The binding site is buried in between the transmembrane domains.
- Group 1b – GPCRs for peptides. The binding sites for these can include N-terminal, the extracellular loops and the transmembrane domains
- Group 1c – GPCRs for glycoprotein hormones. Characterized by large extracellular domains and mostly extracellular binding sites.
Class A GPCR (rhodopsin-like) phylogeny
Acetylcholine, dopamine, histamine, adrenaline, noradrenaline receptors are closely related
Receptors that are closely related and bind to lipid-based mediators: Lysophosphatidic acid, cannbinoid, leukotriene
Peptide activated receptors: opioid, endothelin, vasopressin, neurotensin, somatostatin, chemokine, angiotensin are closely related
P2Y purinergic and adenosine receptors are closely related
Beta 2 adrenergic receptor sequence
Is one of the prototypes of GPCR
Helps understand how drug targets work
Activation of the β2-adrenergic receptor involves disruption of an ionic lock between Asp130/Arg131 in TM3 and Glu268 in TM6
This “ionic lock” normally prevents movement in H6 relative to H3; mutation results in spontaneous activation
The two-state model of receptor activation
Receptors isomerize between resting (R) and active (R*) conformations
Agonist binding is not required to generate R* - spontaneous receptor activity
Agonists bind with higher affinity to and also promote isomerization to R*
This key initial step is defines the drug-receptor interaction, and is typically followed by a biochemical signaling cascade that leads to an ultimate cellular response
An antagonist will bind with equal affinity to both R and R*, while an inverse agonist will bind with higher affinity to the resting state. Either will competitively inhibit the effects of an endogenous agonist in vivo
Agonist binding promotes rearrangement of transmembrane helices – TM3 and TM6 move apart
This conformational change enables receptor to activate G protein
Inactive rhodopsin and active opsin
Image shows superposition of (inactive) rhodopsin and (active) opsin, viewed from the cytoplasmic side. The arrows indicate differences in the positions of TM5, TM6 and Tyr7.53 (from the NPXXY motif). GPCR activation includes a conformational change of the highly conserved NPXXY motif.
Activation by light is thought to open a cleft at the cytoplasmic end of the helix bundle, with separation of transmembrane helices III and VI and increased exposure of the inner faces of II, III, VI and VII.
Phylogenetic relationship between the GPCRs (TMI–TMVII) in the human genome.
ADHESION and SECRETIN classes have common ancestor
GLUTAMATE and FRIZZLED/TAS2 classes have common ancestor
All those 4 classes have an even older common ancestor with rhodopsin
Class B GPCRs
Class B - secretin receptor-like GPCRs
Significant homology between Class B
hormones
“Hot dog in a bun” binding mechanism
where the peptide agonist interacts with both the N-terminal extracellular domain and also the transmembrane domains.
All Class B GPCRs activate Gs/adenylyl cyclase, and some can couple to other pathways as well
Are not as easily drugable as Class A GPCRs because the binding pockets are not as tight. Class A’s have narrow pocket. Class B tends to be more broad pocket.
- small molecules can be more difficult to drug for Class B
