Lecture 5 - GPCRs & G-proteins Flashcards
What is another name for GPCRs (G-protein coupled receptors)?
Metabotropic receptors
Do GPCRs have ion channels?
no - however G-proteins can be activated
What type of binding site do GPCRs have?
Neurotransmitter binding site
What 3 G-subunits are attached to GPCRs?
Alpha, beta & gamma G-proteins
What leads to the G-protein (inactive trimer) being activated?
GDP is bound till activation following binding of GTP. This leads to a signalling cascade.
What are G-protein coupled receptors?
Cells use approximately 25 different families of receptor proteins to detect and respond to a myriad of chemical and physical stimuli - GPCRs are the most diverse.
What are the structural features of GPCRs?
- 7 transmembrane (a helices) of all GPCRs are packed in a similar way
- TM3 centrally located next to binding pocket, crucial for ‘transduction’ of ligand binding
- other transmembranes and extracellular N+ terminus also contribute to ligand binding
- N terminal on extracellular side and where the ligand binds. C terminal where G-protein is bound on the intracellular side
What terminal is the G-protein bound to?
C terminal
What determines GPCR classes?
- structural features of the extracellular domains defining the ‘ligand’ binding site
- linked directly to the huge diversity of the stimuli GPCRs can detect.
What are different types of ligands?
Amino acids, hormones, proteins etc.
What determines what ligand will bind?
Differences in the binding pocket. Different cell types express different amounts and types of receptors
What is an example of a protease-activated receptors (PAR) in platelets?
Receptor activated by cleavage of the N terminal which in turn acts as a tethered ligand - part of the receptor itself acts as the agonist.
- Receptors work together to elicit a response - 3 independent stimuli activate platelets
- N-terminal acts as its own ligand, as it is cleaved by thrombin
- The resting platelet expresses 3 types of receptors - binding of collagen receptors leads to activation of ADP, leading to that receptor becoming activated. Meanwhile the thrombin receptors are activated
What 3 receptors are found on platelets?
- collagen receptors
- ADP receptors
- thrombin receptors
Which side of the GPCR is the N & C terminus found?
N-terminus at extracellular - ligand binding
C terminus at intracellular - G-protein binding
What are G-proteins?
- Guanine nucleotide-binding proteins - belong to the GTPase family
- Act as molecular switches inside cell to transmit signals from extracellular stimuli
- Regulated by ability to bind and hydrolyse GTP (‘on’) to GDP (‘off’)
- Exist as heterotrimeric complexes made up of a, B and y subunits
What occurs when the GDP is bound to the alpha subunit?
The G-protein is in an inactive state
How is G-protein signalling controlled?
- G-proteins are timers
- Duration of signalling by activated trimeric G protein is regulated by rate of GDP hydrolysis by Ga(alpha)
- RGS (regulators of G-protein signalling) proteins stimulate GTPase activity in the a subunit
What is RGS?
Regulators of G-protein signalling
How many different families of G-proteins exist?
6:
Gs
Gi
Gq
G12/13
Gti (Transducin)
Gz
What does Gs do?
Stimulates adenylyl cyclase (increase cAMP)
What does Gi do?
Inhibits adenylyl cyclase (decrease cAMP) and activates phospholipase CB
What does Gq do?
Activates phospholipase CB, increasing IP3, DAG and intracellular calcium
What does G12/13?
Modulates Rho GTPases, affecting the cytoskeleton and cell migration
What does Gti (Transducin) do?
Involved in visual signaling through rhodopsin and phosphodiesterase activation
What does Gz do?
Regulates ion channels and modulates neurotransmitter release and platelet aggregation
What does G(olf)a subunit do?
Subunit & cell type specificity to detect smell
How do organisms respond specifically to particular stimuli?
Selective expression of certain receptors and the molecules involved in signal transduction allow cells to respond specifically to particular stimuli
The cell type and where it is can also create diversity.
It is the specific subunit and cell type that determine the response using the same signalling as the other cell type
Describe how effectors are determined by the class of Ga (alpha) subunit
Effectors include enzymes that create 2nd messengers and ion channels whose gating is regulated either directly (By subunits), or indirectly by 2nd messengers and their effectors
Describe the process of direct activation of an ion channel
- Similar mechanism as ligand-gated channels
- Slow to open or close
- Stay open or closed for longer - minutes rather than milliseconds
Describe how activated G proteins regulate the activities of enzymes that control the levels of second messengers
Second messengers are small molecules that carry signals inside the cells
These include:
- hydrophobic lipids confined to the membrane in which they are generated
- small soluble molecules that diffuse through the cytoplasm (cAMP, cGMP)
- calcium ions
Why do we need these second messenger system?
A single ligand binding to a single GPCR results in the phosphorylation (activation) of millions of proteins.
Describe how cholera toxin leads to disease?
Vibrio Cholera - Gs a subunit (stimulatory). The second messenger activates adenylate cyclase = catalyses cAMP = activation protein kinases. The effect on the cell is an increase in Cl- ion secretion = increase in Na+ ion secretion + H2O. The physiological effect is excess fluid and electrolytes in the lumen of the small intestine, leading to Diarrhoea & extreme dehydration.
Describe how a whooping cough leads to disease?
Bordatella pertussis - Gi a subunit (inhibitory). Secondary messenger inactivates inhibitory G protein = increase in cAMP. This leads to too much intracellular cAMP. The physiological effect is that it leads to erosion of the respiratory epithelium and large quantities of mucus-containing fluid. This triggers coughing fits
What is an example of mutations in GPCRs?
Uveal melanoma - GNAQ and GNA11
- over 90% of uveal melanoma have mutations in Gq (a) subunit
- leads to blocking of GTP hydrolysis so subunits always active causing permanent signal transmission (constitutively active growth pathways)
- thought to occur early on in tumour development
- not yet targeted for therapy but can be used for diagnosis
