G Protein Coupled Receptors Flashcards
What is signal transduction?
- For cells to respond to extracellular signalling molecules (e.g. hormones, neurotransmitters, growth factors, etc…) they must possess the appropriate “receptor”.
- Receptors can be intracellular (e.g. receptors for steroid and thyroid hormones, etc…), but the majority of extracellular signalling molecules do not readily cross the plasma membrane and therefore their receptors are located at the cell-surface.
- Although some receptors can directly alter cellular activity, many require “transduction” of the initial ligand binding event via other intracellular signalling components to generate a response, e.g. contraction, secretion, proliferation, differentiation, etc.
What are the three superfamilies of cell-surface receptor?
- Ligand-gated (receptor-operated) ion channels (e.g. nicotinic acetylcholine receptors)
- Receptors with intrinsic enzymatic activity (receptor tyrosine kinases (e.g. insulin receptor)
- G protein-coupled (7TM) receptors (e.g. muscarinic acetylcholine receptors)
What is the difference between an agonist and and an antagonist?
Agonists: bind to the receptor and activate it
(leading to intracellular signal transduction events)
Antagonists: bind to the receptor but do not activate it (block the effects of agonists at the receptor)
What stimuli do GCPR’s respond to?
Sensory GPCRs sense light (e.g. rhodopsin), odours and tastes
Different GPCRs respond to:
• Ions (H+, Ca2+) • Neurotransmitters (e.g. acetylcholine, glutamate) • Peptide and non-peptide hormones (e.g. glucagon, adrenaline) • Large glycoproteins (e.g. thyroid-stimulating hormone (TSH))
What is the basic structure of GPCR?
- Single polypeptide chain
- 7-transmembrane (7TM)- spanning regions
- Extracellular N-terminal
- Intracellular C-terminal
Two regions of GPCRs can be responsible for ligand binding:
- For some receptors the ligand binding site is formed by (2-3 of) the transmembrane (TM) domains
- In other cases the N-terminal region (and other extracellular domains) form the ligand binding site
How is GPCR signalling initiated?
- G-Proteins are made up of three distinct subunits; alpha, beta, gamma. The beta and gamma subunits bind tightly to each other and function as a single unit. The alpha subunit has a guanine nucleotide-binding site, which binds GTP and slowly hydrolyses it to GDP (GTPase activity).
- Under basal conditions, the G-Protein is present at the inner face of the plasma membrane predominantly in its heterotrimeric form, with GDP bound to the alpha-subunit.
- Activated receptor (Agonist bound) has a high affinity for this form of the G protein and a protein-protein interaction occurs, which leads to GDP being released by the alpha-subunit and binding GTP in it’s place (i.e. the receptor act as a guanine nucleotide exchange factor (GEF))
- Once GTP has bound to the alpha-subunit, the affinity of the receptor for both alpha-GTP and the beta-gamma subunits is decreased. They are both subsequently released and are each able to interact with effectors (secondary messengers).
How is G-protein signalling terminated?
The effector interaction is terminated by the intrinsic GTPase activity of the alpha-subunit hydrolysing GTP. Once this occurs, the affinity of the alpha-subunit for the beta-gamma subunit increases, and the heterotrimer is reformed and awaits reactivation by an agonist-activated receptor to reinitiate the cycle.
G proteins can be thought of as on/off switches and timers. The on switch is receptor-facilitated GDP/GTP exchange and the timer/off switch is governed by the length of time taken for GTP hydrolysis.
Describe GPCR diversity
The human genome encodes 20 Gα (alpha), 5 Gβ (beta) and 12+ Gγ (gamma) proteins. Therefore, there are >1000 possible Gα-βγ protein combinations
What governs Receptor-G protein selection?
Activated GPCRs preferentially interact with specific types of G protein. The Gα subunit is a primary determinant.
In turn, Gα subunits and Gβγ subunits interact with specific effector proteins.
In this way, an extracellular signal, working via a specific GPCR, will activate a single, or small sub-population of G proteins and effectors in the cell to bring about a specific cellular response.
Describe the GPCR interactions of different ligands
see lecture slide
Describe the actions of the cholera toxin (CTx) and pertussis toxin (PTx)
CTx eliminates the GTPase activity of Gs. This leads Gs to become irreversibly activated.
PTx interferes with the GDP/GTP exchange on Gi. This leads Gi to become irreversibly inactivated.
Describe some examples of effectors that g proteins can interact with
ADENYLYL CYCLASE:
ATP to cyclic AMP
PHOSPHOLIPASE C:
PIP2 to IP3 + DAG
PHOSPHOINOSITIDE 3 KINASE (PI3K):
PIP2 to PIP3
cGMP PHOSPHODIESTERASE: cyclic GMP to 5’-GMP
Give examples of GPCR’s that have an effect on adenyl cyclase
Gs-coupled receptors:
β-adrenoceptors
D1- dopamine receptors
H2- histamine receptors
Gi coupled receptors:
α2-adrenoceptors
D2-dopamine receptors
μ-opioid receptors
Describe the agonist stimulated regulation of adenyl cyclase
Adenylyl Cyclase is an integral plasma membrane protein that can be either activated (via Gs) or inhibited via (Gi) by activation of different receptors.
This enzyme hydrolyses cellular ATP to generate cyclic AMP. Cyclic AMP interacts with a specific protein kinase (Cyclic AMP-dependent Protein Kinase, or PKA), which in turn phosphorylates a variety of other proteins within the cell to affect activity. (cAMP binds to regulatory subunits of PKA, which in turn allows catalytic subunit to disassociate and phosphorylate substrates)
Describe the agonist stimulated regulation of phospholipase C
Phospholipase C is an enzyme that hydrolyses the membrane phospholipid (PIP2) to IP3 and DAG. It is activated by Gq. DAG stays in membrane.
IP3 exerts its effects by interacting with specific intracellular receptors on the endoplasmic reticulum (ER) to allow Ca2+ to leave the lumen of the ER and enter the cytoplasm.
