Week 4 MPW Lecture 13 Flashcards
How do G-protein coupled receptors usually work?
A ligand binds to the outer face of the receptor, and this leads to a conformational switch on the inner face, which is detected and carried on by a G protein that binds to the inner face – hence G-protein coupled.
a) Conformational switch occurs by rotation of helices. Which produces a conformational change. G protein binds and is activated by the GPCR, which is acting as a GEF
What is the general structure of G-protein coupled receptors?
GPCRs all have the same basic structure: 7 transmembrane helices, starting at the outside of the cell and ending on the inside. The third intracellular loop is where the G protein binds
What is the biological importance of a tryptophan ring inside the protein?
the tryptophan ring creates a switch because of its size.
a) so when the helices move, it moves while taking a lot of space and therefore moving lots of areas in the GPCR
Why can GPCR work as an on/off switch?
because the switch involves the rotation of a rigid helix
How is the signal detected on the other side of the membrane?
Binding of ligand moves the tryptophan, which makes the bent helix rotate. This changes the conformation of loop 3, which opens up a groove for the Ga protein. A helix from the Ga fits into the groove and opens up the binding site on Ga to allow GDP to leave
How does the G-protein interact?
The G-protein that interacts with the receptor forms a complex with two other proteins. This is usually described as a heterotrimeric complex, comprising Ga (the G protein), plus Gb and Gg, which always go around together. Lipid anchors that keep the G protein components attached to the membrane
a) This shows Ga with GDP bound, and with a little bump on top. This bump is showing the bit that interacts with the GPCR. The idea is that when the GPCR is bound to its ligand, the intracellular loop 3 changes shape, the bump then fits into a corresponding groove in the GPCR, and this opens up the space where the GDP binds, allowing GDP to dissociate and GTP to bind
Where does the alphaG-GTP targets?
A common target of Ga-GTP is adenylyl cyclase, which converts ATP to cAMP, but only when bound to Ga-GTP. The cAMP then acts as a second messenger elsewhere in the cell.
a) Bound GTP is rapidly hydrolysed to GDP by Ga, turning off the signal (ie Ga is its own GAP). Adenylyl cyclase also acts as a GAP.
A complete signalling pathway for a typical GPCR acting via cAMP:
Pathway:
a) Signalling molecules (1st messenger) binds to GPCR
b) GPCR activates G-proteib which activates adenylyl cyclase
c) Adenylyl cyclase synthesises cAMP
d) cAMP activates protein kindaA
e) Activated protein kinase A subunits enter nucleus (diffuses)
f) PKA phosphorylate CREB
g) Activated CREB recruits Creb binding protein (CBP)
h) CREB-CBP activates genes
What other targets can be targeted by activated G(alpha)?
Some G proteins indirectly open or close ion channels.
Smell and vision work in this way
Binding of odorants to specific receptors activates a Ga, which activates adenylyl cyclase. The cAMP opens Na+ channels, which initiates neuron depolarisation.
Light leads to alteration in concentration of cGMP, which works in a similar way.
a) An important class of GPCRs work by the Ga activating phospholipase C (PLC), usually the isoform PLCb.
What is IP3?
Inositol 1,4,5-triphosphate (IP3) which is made from an inositol sugar that is phosphorylated at several positions.
What is PIP2?
IP3 + Diacylglycerol (DAG) = phosphatidyl inositol 4,5-bisphosphate, or PIP2
What are the uses of inositol phosphates?
Different inositol phosphates are recognised by different proteins. They are found mainly on the inner leaflet of the cell membrane. In particular, PIP2 binds to proteins involved in the actin cytoskeleton, and in endocytosis.
Explain the activation of PLC (phospholipase C)
Activation of PLC leads to cleavage of PIP2 into IP3 and DAG:
a) IP3 is used to open Ca2+ gated channels
b) Ca2+ bind to protein kinase C
c) PKC binds to DAG to phosphorylate substrates.
Describe the structure of GPCRs
They are 7-TM proteins. The ligand binding site is buried fairly deeply on the external face. The third intracellular
loop comprises a binding site for Ga of a heterotrimeric complex.
How does rhodopsin differ from typical GPCRs?
for most GPCRs, it is the binding of the ligand that turns on the signal. For rhodopsin, the ligand (retinal) is covalently bound to the GPCR. Retinal is activated by light which converts a double bond in retinal from cis to trans (I think I got it the wrong way round in the lecture). This changes its shape and activates the GPCR. Resetting of the rhodopsin GPCR requires removal of the trans retinal and its replacement by a trans form.
