GPCRs Flashcards
Ligand binding to GPCRs
Activated a heterotrimeric G protein
Comprise 60% of all TM receptors
350 hormones, 500 visual and smell, 150 orphans
Pharmaceutical interest
Around 30% of all small drug molecules are targeted at GPCRs
Antihistamines, b-adrenergic, opiates
Muscarinic receptors for ACH
M1, M3, M5 = Gq
M2, M4 = Gi
Adrenergic receptors and G protein
A1- phospholipase C Gq
A2- adenylate cyclase Gi
B1- adenylate cyclase Gs
B2
Target and effect caused by B adrenergic receptors
Hepatic- glycogenolysis Adipose- lipolysis Heart- contraction rate Smooth muscle intestine- relaxation Bronchial smooth muscle- dilation
Target and effect caused by a2 adrenergic receptors
Gi cAMP
Smooth muscle blood vessels- constriction
Intestine- constriction
Skin- constriction
Kidney-
Affects blood supply to peripheral organs
Structure of GPCRs
1100 residues
7 TM helices, 21-23 residues named H1-H7
4 extra cellular loops E1-E4. E1 is N terminus
4 cytosolic loops C1-4, C4 is the c terminus
N terminus outside, c terminus inside
Ligand binding determined by conserved residues in the TM domains
Chimeric experiment, C3 loop for G protein interaction
Xenopus ooycytes
Injected with mRNA for either a2,b2 or chimeric.
They don’t normally express GPCRs but do express G proteins
In chimeric receptors- depended if the receptor had a b2 or a2 loop as to whether it bound Gi or Gs
GPCR family A
Rhodopsin/b adrenergic like
Disulphide bridge between E2 and E3
Diffusion of ligand to binding pocket for b-adrenergic receptor
May cap covalently bound retinal of rhodopsin
DRY motif at C2
20 conserved residues in TM regions- mainly N and P
Binding sites for ligands between the helices
Biogenic amine receptors
GPCR family B
Secretin/glucagon/calcitonin like
Large n terminal E1 domain with 6 Cys residues to form disulphide bonds
Disulphide between E2-E3
Can form heterodimers which modifies ligand binding and signalling
GPCR family C
Metabotropic neurotransmitter/ Ca sensing receptor
Very long N terminus E1
Disulphide between E2-E3
Very small C3
20 TM conserved residues, high conservation in C3
Homo and heterodimers
Taste, glutamate, GABA, Ca receptors
Frizzled receptors
Unique amoung 7 TM
Signalling through Wnt
Interact with other plasma membrane receptors
Olfactory and gustatory receptors
Largest GPCR family in vertebrates
Human-400 mouse 1200
Disulphide bridge extra cellular
Between end of Tm3 and middle of ECL2
Folds ECL2 in half into ECL2a and ECL2b
Thought to stabilise the receptor in the membrane
Binding of ligands to GPCR
Depends on ligand size and n terminus size
Most small diffusable ligands bind inside hydrophobic core, e.g. Those derived from amino acids
Peptide hormones bind to the N terminus segment and on exposed loops e.g. ATCH and glucagon
4 general components of GPCRs
Receptor with 7 TM domains
Coupled trimeric G protein
Membrane bound effector or ion channel that is regulated by the G protein
Proteins involved in feedback and desensitisation of the pathway
Epinephrine binding to B2 model
The AA interior of different GPCRs is diverse
Can allow hydrophilic or hydrophobic binding
Helices 3,5,6 involved in epinephrine binding
Binds to Ser, Asp, Asn residues
Nh2+ forms ionic bond with the carboxylate side chain of D113 in H3
Catechol ring forms hydrophobic interactions to F290 in H6
Hydrogen bonding between catechol OH and S203,204,207 in H5
Ligand binding gives a conformational change in TM5 and 6 and C3 loop
GPCR toggle switch overview
Helix 5 extends into cell 2 helical turns
Extra cellular segment of helix 6 tilts inwards, intracellular segment tilts outwards by 1.4nm
Allows entry of G protein
Inactive conformation- indole interacts with water hydrogen bond network
In active rotamer, indole reacts with F208 at bottom of binding pocket
Y306 of the NpxxY motif in TM7 interacts with F313 when inactive and in a. Hydrophobic cluster when active
3 micros witches- W265, Y306, R135
Role of GPCR extra cellular loops
Critical role in GPCR function- crystal structure
Different between even closely related GPCRs
Development for receptor specific drugs
GPCRs exist as dimers and oligomers instead of monomers
Studies that suggest GPCR dimers
Heterotetramers- heteromers of homodimers coupled to G protein
Canonical antagonistic interaction between Gs and Gi which simultaneously bind to receptors and AC
A2a-D2 heteromers- the A2A agonists decrease affinity and efficacy of D2 agonists. This means that A2A antagonists have been used to treat Parkinson’s (act as dopamine agonists)
Discovery of the role of GTP
Glucagon + GTP raised cAMP levels 3x more than glucagon alone
G protein cycle
Activation by nucleotide exchange
Deactivation by intrinsic GTPase activity in Ga, activated when Ga binds to effector
‘Land-ladies switch’
Regulators of the GTPase switch
GEFS- GDP dissociation rate, activated GPCRs act as GEFs
GAP- increase rate of hydrolysis
RGS- regulator of G protein signalling
GDI- Guanine dissociation inhibitor ( stop dissociation of either GTP or GDP, can lead to prolonged off or on)
Composition of Ga
N-myristoylation of amino terminal glycine gives attachment to membrane
GTP binding site
BY binding site
Receptor recognition at extreme c terminus
Effector binding site
Allows discrimination between multiple receptors
Humans have 21 a chains which interact with 6 b subunit and 12 y subunit
Tissue specific combinations
ON state of G proteins
Switch I and II bound to YP of GTP
Switch III stabilises II in GTP bound form
Y phosphate also binds to Lys (followed by Ser) in P loop. The switches and Lys hydrogen bond to the oxygens of the phosphate
Ser of P loop - bonds to Mg2+ cofactor and bonds to the oxygen of the Y phosphate from its amide
Lys of P loop- the NH3+ of lysine bonds to the oxygens on the B and Y phosphates
Gas- Thr204 (I), Gly226 (II)
Gai- Thr181 (I), Gly203 (II)
OFF state of G proteins
Switch II unwinds and binds BY rather than effector enzymes
GTPase activity causes loss of h bonding to thr and gly residues
Ga switch I
Thr (204 Gsa, 181 Gai) amide bonds with Y phosphate
Role in catalysis
Arg 201 in Gas can be modified by cholera toxin giving inactivated GTPase activity.
Also site of spontaneous point mutations in some cancers
Ga switch II
The NH of glycine hydrogen bonds to O of Y phosphate in GTP
Role in catalysis
(S 226, i 203)
Gln residues have a role in catalysis- mutations prevent GTP hydrolysis
Switch II is the primary effector binding surface
Ga switch III
Mobile loop with close proximity to GTPase site
Nucleotide responsive communication
Stabilises switch II in GTP bound form
Allows effector recognition, e.g. AC
Conserved ED (negative) binds to R231 (s) or R208 (i) to stabilise
P loop of Ga
Ser47 (ai) or Ser54 (as) - it’s oxygen binds to Mg and its amide bonds to Mg
Lys46 (ai) or Lys53 (as)- it’s positive group takes over from Arg and binds between the B and Y phosphates
Subfamilies of Ga subunits
Overall 40% homology, with 60-90% homology within families
Ga- ubiquitous
Gi- brain and eyes
Gq- B lymphocytes, T cells, blood vessels PLC
G12/13- ubiquitous, direct effect in enzymes I.e. RhoGEF
16 genes, but 20 splice variants
Gas class
Adenylate cyclase
Increases camp
B adrenergic, glucagon,my serotonin, vasopressin
Gai class
Adnenylyl cyclase
The BY acts on the K+ channel
Decrease in cAMP and change in membrane potential
a2 adrenergic, mACH
Gaolf
Adenylate cyclase
Camp increase
Odour receptors
Gaq
Phospholipase C
Increase in DAG and IP3
a1 adrenergic receptor
Gao
Phospholipase C
Increase in IP3 and DAG
ACH receptor in endothelial cells
Gat
cGMP phosphodiesterase (hydrolyses cGMP) cGMP reduced Rhodopsin receptor in rod cells
1- bind of hormone gives conformational change
TM5 extended by 2 helical turns
TM6 moved outward by 1.4nm
C2 extended loop forms an alpha helix when in a B2ARGaBY complex
2- Activated receptor binds to Ga
Interface of TM 5 and 6 and C2/3 loop of GPCR and alpha helices in Ga
Receptor acts as a GEF, disturbs switch stabilising effect of receptor (GDI) so GDP is released
GPCR-Ga has a similar affinity for GDP and GTP- but additional gamma phosphate stabilises the new switch formation
3- binding induces change in Ga, GDP dissociates and Ga dissociates from BY
Stable GaGTP is formed with no affinity for receptor or BY but high affinity for GTP and effector
Additional Y phosphate stabilises switch III which stabilises switch II in GTP bound form
4- hormone dissociates from receptor, Ga binds to effector and activates it
Activated receptor acts as a GAP and hydrolyses GTP
Interaction is due to reconfigure switch II that rotates in the presence of Y phosphate of GTP
5- hydrolysis of GTP to GDP causes Ga to dissociate from effector and reassociate with GBY
Complexing of Ga with effector activates GTPase activity
Rate of GTPase increased by GAP (effector)
RGS- stabilises the G protein in the transition state
In some pathways the BY can activate the effector
Hormone induced activation and inhibition of AC in adipose cells
Inhibitory- PGE1 and adenosine
Activating- epinephrine, glucagon, ACTH
In a lot of tissues, PGE1 stimulates camp but in adipose it is linked to Gai instead of Gas
In a2 epinephrine activates Gai, but in adipose it is linked to Gas
Shows that different effectors can have opposite effects in different tissues
Breakdown of cAMP
cAMP phosphodiesterase
cAMP -> 5’AMP
Two routes for inhibition of AC by Gi protein
1- direct action of Gi on AC
2- Subunit exchange. Gia is less inhibitory than its BY. The BY is more inhibitory in the presence of activated Gsa
4 pieces of evidence for the G protein activation model
1 non hydrolysable GTP
2 amplification
3 modifications of G proteins alter signals
4 FRET
Non hydrolysable GTP analogues
GTPYS-Binds to Ga but can’t be hydrolysed. The =O on the gamma phosphate is replaced by a Sulphur
Methylene GTP (P-CH2-P) Imino GTP (P-NH-P)
These lead to permanent activation, proving that GTP is needed for activated G protein
Amplification of hormone signal
Activation of 100 Gs before the hormone dissociates from the receptor
Each Gsa acts on one AC
100 ACs produce about 10^4 cAMP
Shows that the levels of cAMP produced are much higher than those if the hormone acted directed on AC- there are 100 Ga activated per H:R
Cholera toxin
Mechanism
How causes disease
Ribosylates switch I residues
Arg201 in Gs, Arg174 in Gt. Doesn’t affect Gi.
Inhibits GTPase activity, low affinity for BY
Means that this is constitutively active
Increased camp in gut-> PKA active -> phosphorylates CFTR channel Cl- channel and an Na:H exchanger
NA and Cl leave, water loss to compensate osmotically
Pertussis toxin
Mechanism
Disease
Bordetella pertussis
Ribose latest a Cys residue in the a subunit when in inactive heteromers
Cys351 or 347 in Gt
Affects Gi, Go and Gt
Block g coupling to receptor and prevents Gi activation
Means that AC is active, loss of fluids electrolytes and music in airway epithelium
In Gt, this instead activates cGMP phosphodiesterase which breaks down cGMP
Bastia destroys epithelial giving cough
What does Gz do?
355 residue lacks Cys for PTX
Different AA sequence in highly conserved regions in Ga
Highly expressed in neural tissues
,dedicate signal transduction in PTX insensitive systems
FRET
Excited molecule can either fluoresce at a slightly lower wavelength or can do FRET
GFP - has got absorbing S65, Y66,G67 centre. Emits 400-500nm
Fluorophores such as GFP-YFP or CFP-YFP
Emit at a much lower wavelength when in close proximity
FRET for evidence of model
Amoeba transfected with 2 fusion proteins
Ga with CFP, 490nm
Gb with YFP, 527nm
In the inactive complex, yellow light emitted
When activated, cyan light emitted
Measure light change when a ligand is added
4 Functions of BY subunits
Binding to the inactive GaGDP. Allows it to interact with the receptor again
Has an isoprenylated Y subunit that anchors the complex to membrane
Acts on some effectors- mACH in heart, acts directly on K+ channels
AC regulation- stimulates isoforms 2 and 4, inhibits type 1
Regulation of receptor phosphorylation- such as B-ARK
Role of BY in the heart
mACH receptor, Gai GPCRs Acts on K+ channel Increases efflux from cytosol Increases negative potential inside Reduction of muscle contraction Termination by reassociation with a
Clinical- increase in Ga
Neoplasia
Pituitary tumours, defective GTPase in Gsa and overstimulation of AC
Ovarian tumours have defective Gia
Clinical- decrease in Ga
Osteodystrophy
Pseudohypoparathyroidism not because of hormone deficiency but because hormone can’t activate Gsa (expression or function)
Leads to decreased camp
Expression is influenced by thyroid conditions, glucocorticoids, insulin deficiency and heart failure
Mutations in Gta cause night blindness- Gta can’t effect PDE
Sequence variation in gene encoding GB subunit associated with hypertension, obesity and atherosclerosis
Examples of second messengers
camp -> PKA
cGMP -> PKG and opens cation channels
Ca2+ - other kinases
DAG -> PKC, is lipophillic and retained in membrane
IP3-> opens Ca channels in endoplasmic reticulum
2 main groups of protein kinases
Ser/thr- includes PKA and PKC, phosphorylase kinase
Tyr- RTKs and JAKs. Adds a negative charge to the enzyme, can disrupt and form electrostatic interactions.
Why is phosphorylation a favoured regulation mechanism?
Reversible- kinases and phosphorylases
Amplification and quick response
Enzymes can show cross talk between pathways
Stimulation of camp
Tropic hormones Melanocytes stimulating hormone B adrenergic agonist Glucagon Vasopressin Parathormone Calcitonin Some prostaglandins e.g. PGE1
Inhibition of camp
Somatostatin A2 agonists Insulin mACH agonist Opiates Prostaglandins e.g. PGE2
Topology of AC
Two repeats which both have 6 TM helices
Two catalytic sites C1 and C2 on the cytosolic face
Intracellular N and C termini
Both sites are required for full activity- mutations inhibitory
9 isoforms in humans,
Activation of AC by Ga
Interaction of switch II with AC
Needs GTP bound conformation
Ca/calmodulin AC
1,3,8 Present in brain All activated by Ca bound to calmodulin AC1 is reduced by BY AC1 and AC3 activated by PKC
GBY stimulated AC family
2,4,7
Lungs, 7 is ubiquitous
a1 receptors activate Gq which generate BY. When in the presence of the Gsa created by B adrenergic, this stimulates AC.
In 2 PKC activates, in 4 it inhibits
Ca2+and Gai AC family
5 and 6
Ubiquitous
Inhibited by BY and Gai and Ca
5 is activated by PKC, 6 is inhibited
Equation of AC
MgATP -> cAMP + MgPPi2- + H+
Pyrophosphatase breaks down PPi from ATP, energy used for cyclisation
Breakdown- broken to 5’AMP using water, Mg2+ and PDEs
Forskolin
Binds directly between split catalytic AC site
Distinct from Gsa site
Ligand induced activation of PKA
Inactive tetrameric complex
Binds 2 camp at each side (CNB-A and CNB-B)
Binding of camp to R subunits releases the C subunits
Each R contains a pseudo sequence that binds active site
R subunits are joined by AKAP protein
Camp changes pseudo substrate domain
Positive cooperative binding- binding at one lowers the Kd for camp binding to other site
Structure of PKA regulatory units
Glycogen breakdown
With no camp the CNB-A loop bonds C subunit
Glutamate and Arginine residues bind camp
Binding of AKAP to the regulatory tails allows localisation
PKA regulates phosphorylase kinase B by phosphorylation Ser to regulate glycogen breakdown
Use of FRET to measure camp in cells
Attach BFP to regulatory and GFP to catalytic
Inactive with give green light
Active will give blue
FRET to measure PKA activity
Protein with link between CFP and YFP
In linker is the serine target and a 14-3-3 phodphoserine binding domain
When Ser-p, this folds the protein over as it binds to the 14-3-3
This brings the CFP and YFP close
Inactive blue light, active yellow light (FRET to longer wavelength)
How much camp for PKA activation?
Normal 0.3 uM
Only 2-3 fold increase needed
Effect of PKA on glycogen
B Epinephrine -> cAMP -> PKA
PKA -> phosphorylase kinase -> phosphorylase -> increase in glycogen break down
PKA -> glycogen synthase -> decrease in glycogen synthesis
Also activates inhibitor of phosphoprotein phosphatase
This stops the phosphatase from dephosphroylating activated enzymes
When PKA inactive, phosphoprotein phosphatase then dephosphrylates -> glycogen synthesis
Effect of epinephrine on adipose cells
Ovarian cells
Epinephrine ->
PKA activation
Phosphorylates and activates phospholipase
Release of FA (lipolysis) into blood
Allows energy for heart, kidney and muscle
Ovarian cells -> PKA -> oestrogen and progesterone synthesis
Glycogen phosphorylase kinase substrates
GP can be activated by GPK activated by PKA
GPK acts on GP and GS
PKA cannot act on GP because it has an Arg near the Ser-14 site
Signal amplification in glycogen synthesis
One epinephrine receptor -> 10x Gsa
1:1 Gsa to AC, 10^4 fold
Camp -> PKA
PKA -> activates phosphorylase b kinase (x10 more)
PBK -> activates glycogen phosphorylase a (x10 more)
GPA -> converts glycogen to G-1-P (x10 more)
3 last steps give another 10^4 amplification, total 10^8
