GPCR

Question 1

GPCR structure

Answer 1

• 7 TM domain receptor protein
• 800 human GPCRs
• 33% of small molecule drugs are GPCR targeting
• activates trimeric G protein via GTP exchangve
• leads to activation of further enzymes

Question 2

GPCR signals

Answer 2

• photons (vision)
• exogenous small molecules (smell, taste)
• neurotransmitters (serotonin)
• peptide/small molecule hormones
• proteins

Question 3

Pharmacological terms

Answer 3

• all GPCRs have small level of basal/constitutive activity
• full agonist: native ligand gives 100% full activity
• partial agonist: close to full activity
• neutral agonist: suppress activity back to basal levels
• inverse agonist: inhibit to 0% (even basal levels inhibited)

Question 4

B2-adrenergic receptor

Answer 4

• class A GPCR rhodopsin-like

- two conformations (active vs inactive)

Question 5

GPCR Families

Answer 5

Class A: normal GPCR structure
Class B: larger extracellular domain/peptide hormones
Class C: orthosteric binding site/allosteric binding site/ intracellular dimerisation of receptors
Class D: frizzled and smoothened receptors/larger extracellular domains

Question 6

G Protein Activation

Answer 6

• heterotrimeric protein activated by ligand bound GPCR

(1) Agonist binding leads to a conformational change in the GPCR, which stimulates GDP/GTP exchange in the G protein.
(2) Active, GTP-bound, G protein dissociates into α subunit and βγ dimer (both are membrane-bound due to lipid modifications).
(3) The α subunit and βγ dimer regulate effector proteins (AC, adenylyl cyclase).
- AC converts ATP to cAMP as secondary messenger
(4) GTP hydrolysis deactivates the α subunit, which reassembles with the βγ dimer.
- By dimer can inactivate effectors (like Ca ion transporter)

Question 7

Models of GPCR activation

Answer 7

• memorise models *
  Simple binding and activation:
• equilibrium of inactive/active form relating to the protein a-helical change

Simple ternary complex model:
- describes G protein association

Extended ternary complex model:

• describes the constitutive activity levels (can have signalling without input)
• agonist drives equilibrium to a state which is more effective in coupling (increased activity)

Question 8

Adenylyl Cyclase Structure

Answer 8

• G alpha protein effector
• membrane spanning protein with 12 TM helices
• two TM domains with both termini inside cell
• two intracellular loops (C1a/C2a) with enzymatic activity

Question 9

Adenylyl Cyclase Activity

Answer 9

• Ga unit binds to AC protein
• removes inorganic diphosphate from ATP to form cyclic AMP
• cAMP phosphodiesterase hydrolyses cAMP to AMP

Question 10

Cyclic AMP-dependent protein kinase A

Answer 10

• Mediate cAMP effects/Activated by cAMP
• Kinase phosphorylates specific serine/threonines on target proteins to regulate activity
• Inactivate state: PKA has two catalytic units and two regulatory units. cAMP binding to regulatory units alters conformation causing dissociation
• regulatory unit penetrates active site cleft to block activity
• Released catalytic units activated to phosphorylate target proteins
• In unstimulated cells, a phosphodiesterase keeps cAMP conc. Low to inactivate PKA
• PKA phosphorylates a target protein that is a phosphodiesterase to lower the cAMP concentration and make a pulse of PKA activity

Question 11

Somatostatin

Answer 11

• inhibitory hormone
• in digestive system: decreases gastric emptying and release of pancreatic hormones
• in nervous system: decreases pituitary hormone release
• expression induced by cAMP via PKA dependent pathway
• slow response mediated by PKA: transcription factors
• cAMP activates the gene that encodes this hormone. The regulatory region of the somatostatin gene contains a short cis-regulatory sequence, called the cyclic AMP response element (CRE), which is also found in the regulatory region of many other genes activated by cAMP.
• A specific transcription regulator called CRE-binding (CREB) protein recognizes this sequence. When PKA is activated by cAMP, it phosphorylates CREB on a single serine; phosphorylated CREB then recruits a transcriptional coactivator called CREB-binding protein (CBP), which stimulates the transcription of the target genes
• Thus, CREB can transform a short cAMP signal into a long-term change in a cell, a process that, in the brain, is thought to play an important part in some forms of learning and memory.

Question 12

Fight or Flight Response

Answer 12

• fast response mediated by PKA: ion channel
• B-adrenergic receptor activated by adrenaline
• cAMP production increases to activate PKA
• phosphorylates B0subunit to release from the Ca ion channel
• releases inhibitory effect to allow Ca signalling

Question 13

Phospholipase C-B

Answer 13

• Ga protein effector
• CT domain in membrane linked to PLC-B in intracellular leaflet
• GPCRs can use G proteins to activate the membrane bound enzyme phospholipase C-B
• PLC-B has X/Y linker blocking active site when inactivated
• PLC-B in the active state has X/Y linker moved out (allosteric activation)

Question 14

Phospholipase C-B activity

Answer 14

• Phospholipase acts on a phosphorylated ionositol phosphoplipid (phosphoinositide) called PIP2.
• Receptors that activate this inositol phospholipid signaling pathway mainly do so via a G protein called Gq, which activates phospholipase C-β in much the same way that Gs activates adenylyl cyclase. The activated phospholipase then cleaves the PI(4,5)P2 to generate two products: inositol 1,4,5-trisphosphate (IP3) and diacylglycerol. At this step, the signaling pathway splits into two branches.
  1) IP3 binds to IP3 gated calcium channels in the ER to release ions. Increase in cytosolic calcium propagates the signal by influencing activity of calcium sensitive proteins.
  2) Diaglycerol is in the membrane where it activates protein kinase C (calcium dependent). Initial rise in Ca alters PKC to translocate to the cytoplasmic face of the membrane where it is activated.
• ER membrane contains another type of Ca channel opening in response to rising Ca levels to amplify the signal. Calcium induced calcium release results in positive feedback loops. The receptors are inhibited after some delay by the high Ca concentrations lowering the signal.

Question 15

PIP2 hydrolysis

Answer 15

• learn structure*

- PLC-B hydrolyses phosphatidylinositol 4,5-bisphosphate into diacylglycerol and inositol 1,4,5-trisphosphate

Question 16

IP3

Answer 16

• secondary messenger generated by PLC-B
• diffuses to ER and opens IP3 gated Ca ion release channel
• Ca ions then activate protein kinase C

Question 17

Calcium waves

Answer 17

• positive and negative feedback produces ion waves
• based on IP3 and ryanodine receptors
• IP3 gated receptors open to release Ca ions
• Ca induced Ca ion release from neighboring receptors
• inhibition caused by high ion concentration
• Ca pumps in ER membrane restores low Ca cytosolic concentration

Question 18

Vasopressin

Answer 18

• induces Ca ion oscillations in liver cells
• stress response causes vasopressin to release glucose from glycogen
• vasopressin receptor is a class A GPCR with downstream signalling via the PLC-B-IP3 pathway
• frequency of Ca bursts related to concentration of hormone applied to cells

Question 19

Calmodulin

Answer 19

• relays Ca ion increase to other proteins
• Single chain with 4 Ca binding sites. When activated it undergoes a conformational change. Calmodulin binds and activates other proteins.
• Ca ion binds to Calmodulin to change conformation and give a middle flexible linker
• flexible linker can wrap protein around CaM kinase to form CaM kinase peptide
• As one example, Ca2+/calmodulin binds to and activates the plasma membrane Ca2+-pump that uses ATP hydrolysis to pump Ca2+ out of cells. Thus, whenever the concentration of Ca2+ in the cytosol rises, the pump is activated, which helps to return the cytosolic Ca2+ level to resting levels.

Question 20

Calmodulin dependent kinases structure

Answer 20

• dodecamer with two rings of 6 subunits each
• subunits have kinase domain and a non catalytic hub domain which are connected by a linker containing the CaM binding site and an autophosphorylation site
• twelve copies of the enzyme are assembled into a stacked pair of rings, with kinase domains on the outside linked to a central hub.
• This structure helps the enzyme function as a molecular memory device, switching to an active state when exposed to Ca2+/calmodulin and then remaining active even after the Ca2+ signal has decayed.

Question 21

CaMK II Activity

Answer 21

• In the inactive form, a kinase domain pops out and is activated by Ca/calmodulin binding
• binding stabilises the active conformation
• Adjacent kinase subunits can phosphorylate each other (a process called autophosphorylation) when Ca2+/calmodulin activates them. Once a kinase subunit is autophosphorylated, it remains active even in the absence of Ca2+, thereby prolonging the duration of the kinase activity beyond that of the initial activating Ca2+ signal. The enzyme maintains this activity until a protein phosphatase removes the autophosphorylation and shuts the kinase off. CaM-kinase II activation can thereby serve as a memory trace of a prior Ca2+ pulse, and it seems to have a role in some types of memory and learning in the vertebrate nervous system. Mutant mice that lack a brain-specific form of the enzyme have specific defects in their ability to remember where things are.

Question 22

Calcium Oscillations

Answer 22

Low frequency oscillation:
- time between Ca spikes is long enough for phosphatase to dephosphorylate CaMKII

High frequency oscillation:

• more frequenct spikes doesn’t give time for removal of phosphate groups
• system’s activity is saturated and activity increases

Question 23

Olfactory System Structure

Answer 23

1. olfactory cilia detect odor molecules
2. olfactory sensory neuron
3. subtentacular cell (support cells in between neurons) + stem cells
4. olfactory bulb connects to neurons and leads to the brain

Question 24

Principles of Olfactory System

Answer 24

• each olfactory sensory neuron expresses only one type of odorant receptor (can sense many different molecules)
• OR are the largest GPCR family in mammals
• neurons expressing the same receptor connect to the same glomerul region
• each odorant can activate a set of receptors and different receptor combinations give a scent

Question 25

Odorant Signalling

Answer 25

- activated OR couples to Golf and the a unit activates adenylyl cyclase - cAMP increase binds and activates PKA and opens cAMP-gated Na/Ca channels - depolarisation is amplified by Cl efflux through Ca dependent Cl channels - expression of all other OR is silenced by the GB/y units - Study on zebrafish showed that OR gene silencing was dependent on OR activity: signalling through G protein By units was both necessary and sufficient to suppress the expression of OR genes

Question 26

OR Desensitisation

Answer 26

- Negative feedback response: Influx of Ca binds to calmodulin to form CaM; binding to the ion channel and closing it. CamKII is activated phosphorylating AC and reducing cAMP concentration. It also activates phosphodiesterase’s hydrolyzing cAMP - Main mechanism is not phosphorylation of AC but it is the Ca rise due to influx of ions inhibiting the gate channel

Question 27

Photoreception

Answer 27

1. retinal pigment epithelium sense light (but is farthest away) 2. photoreceptor cells: rod (sensitive to light) and cone (sensitive to color) cells 3. bipolar cells connect photoreceptors to ganglion cells 4. ganglion cells connect to axons of optic nerve

Question 28

Rod Photosensor Cells

Answer 28

- outer segment: light sensitive - inner segment - nuclear region - synaptic region: signals to neurons in retina - photoreceptors hyperpolarise in response to light - two types of bipolar cells for cone cells: ON bipolar cells depolarise in response to light/OFF bipolar cells hyperpolarise in response to light - ON/OFF pathways are important for the detection of light increments and decrements

Question 29

Rhodopsin

Answer 29

- light-sensitive receptor protein involved in visual phototransduction - found in rods and is a GPCR - Rhodopsin consists of two components, a protein molecule also called scotopsin and a covalently-bound cofactor called retinal. - Scotopsin is an opsin, a light-sensitive G protein coupled receptor that embeds in the lipid bilayer of cell membranes using seven protein transmembrane domains. These domains form a pocket where the photoreactive chromophore, retinal, lies horizontally to the cell membrane, linked to a lysine residue in the seventh transmembrane domain of the protein

Question 30

Vision Mechanism

Answer 30

- Vision uses cyclic GMP by guanylyl cyclase - Receptor activation causes a fall in cyclic nucleotide levels - The plasma membrane surrounding the outer segment contains cyclic-GMP-gated cation channels. Cyclic GMP bound to these channels keeps them open in the dark. Paradoxically, light causes a hyperpolarization (which inhibits synaptic signaling) rather than a depolarization of the plasma membrane (which would stimulate synaptic signaling). - Hyperpolarization results because the light-induced activation of rhodopsin molecules in the disc membrane decreases the cyclic GMP concentration and closes the cation channels in the surrounding plasma membrane

Question 31

Rhodopsin Mechanism (Activated)

Answer 31

- the aldehyde group of retinal is covalently linked to the amino group of a lysine residue on the protein in a protonated Schiff base - When rhodopsin absorbs light, its retinal cofactor isomerizes from the 11-cis to the all-trans configuration - this conformational change activates transducin (G protein) via GDP -> GTP exchange - transducin (a unit) activates phosphodiesterase, which results in the breakdown of cGMP - Decrease in cGMP concentration leads to closing of cation channels and hyperpolarization of the membrane potential

Question 32

Rhodopsin Mechanism (Inactivated)

Answer 32

- transducin bound to GDP and inactive - phosphodiesterase inactive - guanylyl cyclase converts GTP to cGMP - cGMP binds to cGMP gated cation channels to allow Na/Ca entry

Question 33

Vision Desensitisation

Answer 33

- GAP binds to transducin a subunit and stimulates GTP hydrolysis - closing of cGMP gated channels increases cytosolic Ca - stimulates guanylyl cyclase to make more cGMP - falling Ca activates rhodopsin kinase by causing dissociation of inhibitory EF-hand protein = recoverin - kinase phosphorylates cytosolic tail of rhodopsin to partially inhibit transducin activation - arrestin binds to phos. rhodopsin to further inhibit transducin activation

Question 34

Arrestin

Answer 34

- binds to the phos. C terminal tail (done by GRK1) | - binds at same face as G protein to prevent further transducin activation

Question 35

Full Cycle of Rhodopsin

Answer 35

* memorise cycle * | - need this

Question 36

Biased System

Answer 36

System can be biased towards arrestin or G protein (both bind to same place on GPCR - arrestin: internalisation, MAPK signalling, TF - G protein: B/y unit activates ion channels, phospholipase, receptor kinases vs a unit activates cAMP/Ca/IP3/Rho

Question 37

Biased Signalling

Answer 37

1. biased ligand: different ligand cause different conformational changes in GPCR 2. biased receptor: absence of phos. site in C tail is biased to G protein 3. biased system: differential expression of transducer elements (ie. higher GRK1 levels = more likely to use arrestin)