Term 2 Lecture 15: G Protein Coupled Receptors Flashcards
Transmembrane proteins span up to 3 domains in the lipid bilayer
Exoplasmic and cytosolic transmembrane proteins have a hydrophilic exterior surface that interacts with the aqueous environment of the cytoplasm or ECM.
Membrane spanning proteins are hydrophobic, their AA side chains interact with the hydrocarbon core of the bilayer and they usually consist of alpha helices or beta strands
Lipid anchored membrane proteins
Are bound covalently to one or more lipid molecules.
The hydrophobic tail of the attached lipid is embedded in one leaflet of the membrane and anchors the protein to the membrane.
The polypeptide chain itself does not enter the phospholipid bilayer.
Covalently attached lipids anchor some otherwise soluble proteins to one or the other plasma membrane leaflet in eukaryotic cells
Lipid anchored membrane proteins
Are bound covalently to one or more lipid molecules.
The hydrophobic tail of the attached lipid is embedded in one leaflet of the membrane and anchors the protein to the membrane.
The polypeptide chain itself does not enter the phospholipid bilayer.
Covalently attached lipids anchor some otherwise soluble proteins to one or the other plasma membrane leaflet in eukaryotic cells
Acylation, prenylation and GPI anchors
Acylation:
cytosolic proteins anchored by a single fatty acid chain attached to an N terminal of glycine
Common acyl anchors are myristate(C14) and palmitate (C16)
Prenylation:
Cytosolic proteins anchored to the membrane by a prenyl group thioether bond to one or two c termini of Cys sulphyl acyl groups.
Common anchor geranylgeranyl (C20)
GPI anchor:
Anchors extracellular protein to the exoplasmic surface of the plasma membrane.
a) phosphatidyl inositol anchor - inserts 2 fatty acyl chains into the bilayer
b) phospho-ethanolomine unit links protein to anchor
The attached sugar units vary in number.
They can form lipid rafts
Sometimes a second geranylgeranyl or fatty acyl palmitate group is linked to a nearby cysteine residue (usually myristate)
The additional hydrocarbon anchor is thought to reinforce the attachment of the protein to the membrane.
Mechanistically phenylation that occurs after the myristillation. An N-myristyl-transferase enzyme that chops off the first methionine (the start of the aa) exposing the glycine (motif) and palmityl transferase comes in later and recognises the motif.
It happens in this order as the association with the membrane is necessary for palmitylation.
Double acylation causes lipid raft formation.
G protein coupled receptors (GPCRs)
Perhaps the most numerous class of receptors they are used to detect and respond to many kinds of signal e.g. neurotransmitters, hormones involved in glycogen and fat metabolism and even photons.
(See a table of human G protein coupled receptors of pharmaceutical importance e.g. Table 15.01 molecular cell biology 2016)
GPCRs are of great medical importance ~30% of drugs are agonists or antagonists of specific GPCRs or closely related groups of GPCRs
E.g. claritin is an antagonist of histamine and thus reduces pollen allergy symptoms
Despite diversity all GPCR signal transduction pathways have these common elements
1) a receptor that contains 7 membrane spanning alpha helices
2) a heterotrimeric G protein, a receptor activated switch by cycling between active/inactive forms
3) a membrane bound effector protein
4) proteins that participate in desensitisation often signalling pathways (that end or reduce the signal)
Second messengers are part of many GPCR pathways
GPCR pathways usually have short-term effects in the cell by quickly modifying existing proteins, either enzymes or ion channels.
They allow cells to respond rapidly to a variety of signals e.g. light or hormone stimuli.
G protein linked receptors have a common structuse
7 units (H1-7)
Linked by 4 internal (C1-4)
and 4 external (To 1-4) bonds
The largest family of cell surface receptors (7 TM receptors) with a huge variety of signalling ligands (hormones, neurotransmitters and local mediators)
The receptors mediate processes as diverse as metabolism, learning and memory, olfaction and vision.
Common structure of 7 membrane passes, extracellular ligand binding domain (first messenger external) and intracellular G-protein binding domain (second messenger internal)
The ligand for the receptor is known as the first messenger, thought to have evolved from non G-protein linked structurally similar receptors in lower organisms
(Lefkowitz and Kobilka Nobel prize 2012)
G protein structure
Understanding the structure allows us to think about how the ligand binds and how drugs can be designed to integrate or promote the process
Gilman and Rodbell Nobel prize 1994. Found at the cytoplasmic face of the plasma membrane the message is relayed from activated 7-TM receptor to a number of target molecules 3 subunits (alpha, beta and gamma)
Alpha subunit is bound to guanosine diphosphate (GDP) in inactive state and GTP in active state.
The alpha and gamma subunits are attached to the plasma membrane by covalently attached lipids alpha myristate and/or palmitate.
The beta gamma dimer is prenylated.
There are many types of G protein that perform distinct functions within the cell
The G protein activation cycle
See diagram start of notebook 3
1) binding of hormones induces confirmational change in receptor activating it
2) activated receptor binds to the G alpha subunit which is at this point bound to the beta gamma dimer and a molecule of GDP
3) activated receptor causes confirmational changes in the g alpha subunit triggering dissociation (release) of GDP
4) binding of GTP to G alpha subunit triggers dissociation of G alpha from the receptor and G - beta gamma dimer.
↓
GTP takin in
↓
5) hormone dissociates from the receptor. G alpha with bound GTP now binds to the activated receptor
6) hydrolysis of GTP→GDP causes G alpha subunit to dissociate from the effector and reassociate with G-beta,gamma dimer
7) return to resting state
Major classes of mammalian heterotrimeric G proteins and their effectors
Different g proteins are activated by different GPCRs and regulate different effector proteins
Humans have:
21 types of G alpha subunit encoded by 16 genes (several generated by alternate splicing) they have specific functions
6 G beta subunits
and 12 G gamma subunits
G beta gamma dimers have interchangeable activities.
(See a table of major classes of mammalian heterotrimeric G proteins and their effectors e.g. table 15-2 in molecular cell biology 2016)
G alpha 0 and g alpha Q increase IP3 and DAG (deacycl glycerol) by removing the head group of lipids. They have different receptors but both are associated with phospholipase C affector
Cellular functions dependent on cAMP
3’5’ cyclic AMP (adenosine monophosphate) the cyclic part is cyclised between carbons 3,5 and 4.
Important in all eukaryotes. Activator of protein kinase a (that phosphorylates proteins)
Various target tissues involved:
Target tissue/hormone/major response
Ovary/luteinising hormone/ progesterone secretion
Muscle and heart/ adrenaline/ glycogen breakdown and increased heart rate for fight or flight response
Liver/glucagon/ glycogen breakdown
Kidney/ vasopressin/ water resorption
G proteins regulate cAMP by regulating adenyl cyclase
cAMP is synthesised by the enzyme adenyl cyclase (AC.) AC is activated by G alpha s protein and inhibited by g alpha i.
AC synthesises cAMP from ATP, cAMP is degraded by cAMP phosphodiesterase to end the signal. cAMP is a ‘second messenger.’ There are 10 isoforms of AC in mammals 9 are regulated by G proteins. For added complexity G proteins regulated by ACs can also be regulated by G beta gamma dimers and second messenger Ca²+
(Sutherland Nobel prize 1971)
G proteins regulate adenyl cyclase
ATP
→active adenyl cyclase→
cyclic AMP and 2Pi
→phosphodiesterase and H2O→
AMP
G proteins regulate adenyl cyclase different hormones bind to either stimulatory or inhibitory receptors that act to stimulate or inhibit AC
The end result is cAMP upregulation or blocking.
Stimulatory examples are epinephrine, glucagon,l and ACTH (bind to G alpha s to promote cAMP)
Inhibitory examples are PGE1 and adenosine (bind to G alpha i to block cAMP production)
Together they regulate cAMP production
