GPCRs

Question 1

Importance of understanding GPCRs

Answer 1

Ubiquitous in almost every cell

Understanding rhodopsin structure was important due to conserved structure in GPCRs allows us to develop improved ligands for others to treat disease. Ex. GPR84 has a fully conserved binding pocket across vertebrate orthologues. Can use a molecule that makes the same 5 H bonds as the natural agonist to activate. Drug tests on mice for human medicines will bind the same as the GPR84 in humans since conserved throughout evolution.

Question 2

Heterotrimeric proteins: why not direct interaction of GPCR to enzyme, number, examples and function

Answer 2

Guanidine nucleotide binding proteins

-Allows one G alpha protein (~20 in humans, less than no. of GPCRs) to associate with multiple signaling pathways (effector for one GPCR can be ex. ion channel, adenylyl cyclase, IP3, etc; versatile).
Some have a widespread role (ex. only 2 Gs isoforms since many cells need regulation of cAMP), others are distinctive and only expressed in one cell subtype
-One activated GPCR activating multiple G proteins, which can activate multiple effector proteins allows for signal amplification

The most diverse of G alpha proteins share 50% amino acid identity with a very similar tertiary structure due to all binding GTP (when active) and GDP (when inactive), and working in the same general way, similar to how kinases all have a similar structure due to binding ATP.

Some G proteins are ubiquitous in almost every cell (ex. only 2 Gs isoforms since many cells need regulation of cAMP) wheras others expressed in specfic tissues (ex. Golf is a specialised G protein in nasal epithelium to couple G proteins that control sense of odour perception to adenylyl cyclase)

Gs stimulates adenylyl cyclase, increasing cAMP levels.
Gi inhibits adenylyl cyclase, thereby reducing cAMP levels.
Gt1 (transducin alpha-T1) and Gt2 are specialized G proteins expressed in photoreceptor cells of the eye, where they mediate vision.
Gq activates phospholipase C-β, leading to increased intracellular calcium. Specialized versions of Gq are expressed in specific tissues and cell types

Question 3

First TM protein imaged, protein requirements for imaging and why said protein fit these. Information obtained from structure

Answer 3

Advances in biochemistry and related over last 20 years have changed how we think about how proteins and cell signalling works. Requirements for atomic protein imaging:
High purity
Stable, native conformation

Bovine rhodopsin is the most studied GPCR. The first transmembrane protein successfully purified (challenge to keep protein stable out native environment and detergent dissolves membrane) and imaged in 2000.

Rhodopsin is 50% of all protein in rod outer segment (couples to G alpha protein transducer (T1 for monochromatic recognition of light and T2 for colour vision)) so straightforward to obtain tissue and have sufficient protein from native source after purification (typically the amplification cascade in signalling means there’s low concentrations of receptor). Most GPCRs require 20,000-100,000 fold purification

7TM helices with kinks important in activation and deactivation

Covalently attached chromophore/ligand that isomerises when absorbs light on microsecond scale (very quickly): 11-cis retinal into all-trans retinal. This chemical change drives conformational change in protein

Question 4

Evolution of GPCRs in biology (how they arose, no. of genes, why different no. in other organisms, how we know function)

Answer 4

Typically via gene duplication the genes diverse over time to produce different physiological functions in different tissues. Still conserved enough to respond to the same ligand.

Sequence alignments and grouping similarities can see those that respond to the same ligand.

Groups in phylogenetic tree that indicate convergent evolution shows responding to the same ligand and having 7TM helices were structures that occurred multiple times in evolution (as well as divergence), highlighting that those traits must give an advantage.

~800 genes encode GPCRs (3% of coding genes). ~400 olfactory (which is why non-humans have more GPCRs since competitive advantage to have good sense of smell. In humans have pseudogenes; non-functional). ~200 don’t know natural ligand that regulates (may aid treating disease to understand). Done by KO studies and observe change in function/phenotype or DNA fingerprinting to reflect differences. UK biobank allows people to ask if they have SNP associated with susceptibility to a disease.

All organisms have GPCRs, highlighting importance (except parasites)

Question 5

Types of drug types based on activation

Answer 5

Full agonist: molecules that enrich the active state (R*)
In physiology, anything with less efficacy than a full agonist (partial agonist, antagonist, partial inverse agonist, inverse agonist) will partially block it’s effect

Full inverse agonist: molecules that enrich the inactive state (R)

Antagonist: molecule that doesn’t change balance between R and R*

All receptors (even rhodopsin which has extremely little) constitutive activity. Like how enzymes increase the rate of a reaction that would occur without it’s intervention, receptors can still become activated without their ligand. Different receptors have different levels of constitutive activity. All have it though since their activation must be energetically favourable to occur with ligand, and so must be able to activate alone.

Question 6

Information obtained through phylogenetic tree of GPCRs

Answer 6

Through informatics, aligning proteins and grouping by similarity, identified number of receptors that respond to a ligand

GPCRs frizzled, glutamate, or adhesion/secretin have no similarity to other GPCRs (other than having 7TM helices) that can all be grouped together (opiates, cannabinoid, etc)
This indicates they did not arise from gene divergence of a gene gaining mutations, instead convergent evolution.

Highlights use of GPCRs

Question 7

Direct Binding Experiments (method, information obtained)

Answer 7

• Use increasing concentrations of radiolabelled drug in the presence or
  absence of an excess of a second drug which is known to compete with the
  radioligand for the receptor
• Provides information on:
• The total number of receptors present
• The affinity of the radiolabelled ligand for the receptor
• In a similar fashion to linear transformation of the Michaelis-Menten
  equation to Lineweaver-Burk or Eadie-Hofstee plots, the same can be done
  with binding data. The most common form is the Scatchard plot.

Question 8

Criteria for specific, physiologically relevant binding

Answer 8

-Saturable (dose dependent so can regulate)
-Stereoselective (receptors are often chiral so will only bind one enantiomer)
-Suitable tissue profile (only picked up in target tissue; confirm expression of the target receptor in a tissue via qrt-PCR or protein levels)
-Binding should be competed for by pharmacological doses of
receptor selective drugs
-Binding should not be displaced by drugs of other classes
-Selective for the one receptor
-High affinity, low Kd (low dosage required)

Question 9

Initial studies on 2nd messengers showed and next steps

Answer 9

Addition of glucagon raised cAMP levels (measured with radioimmunoassay in which [3H]cAMP incubated with Ab and competitive antigen, and radioactivity measured and concentration calculated with calibration curve) in hepatocytes

Not obvious from these studies that GTP was required since ATP used was purified imperfectly from tissues and contaminated with sufficient GTP. Once chemically synthesised ATP was available in became clear that GTP
also had to be added for function
Conclusion: there is a step in the cascade from glucagon activating the GPCR to 2nd messenger cAMP production via AC that requires GTP

What was the molecular nature of this ‘transducer’?

Question 10

Experiment perform to prove drug target for a GPCR was a protein + next steps

Answer 10

Through covalent labelling

Example: Identification of muscarinic acetylcholine receptor

Modified antagonist propylbenzilylcholine to react via covalent bonds (propylbenzilylcholine mustard), comprised of radioactive hydrogens ([3H] propylbenzilylcholine mustard)

Knew muscarinic acetylcholine receptor is present in brain so incubated animal brain with antagonist, solbilised, denatured and added to SDS-PAGE

Added excess of another antagonist atropine. On SDS Page gel imaged with autoradiography, saw signal band without atropine but none with atropine, suggesting both antagonists bound the same receptor

Question 11

Experiment performed to purify a GPCR + next steps

Answer 11

Example: Purification of the delta-opioid receptor

Used neuroblastoma cell line grown in lab and knew delta-opioid receptor was present that has a fentanyl ligand

Modified fentanyl with radioligand and highly reactive chemical group to covalently bind to what fentanyl binds to ([3H] fentanyl isothiocyanate)

Solubilised membrane with detergent

Ran on SDS-PAGE which showed lots of bands (all proteins in cell solute) and autoradiograph showed multiple bands but less (only the few proteins bound to fentanyl since it has multiple targets)

Applied solution column of wheat germ agglutinin - a lectin so selective for carbohydrates (PTM for all PM; N-linked glycosylation) to purify fentanyl (with bound receptor). Saw only one band in autoradiograph so successfully purified out other proteins that bind fentanyl. SDPAGE showed fewer but lots of bands since purification isn’t perfect

Applied solution to another column of antibody to the ligand on sepharose beads.
30% purity of the opioid receptor was achieved.

Next steps:
Chopped up, sequenced, and designed primers based on cDNA library to be able to clone protein. However, sequencing doesn’t tell you which codons are used to encode that amino acid (except tryptophan which has has one codon that encodes for it since it was the last produced in evolution)

Question 12

Experiment performed to understand beta-2 adrenoreceptor + next steps

Answer 12

Took lung from guinea pig since they knew β-adrenoreceptors would be present there as drugs to treat asthma target lung and are selective for that receptor

Solubilised membrane (since it’s an intrinsic membrane protein) and did tested multiple detergents

Use affinity chromatography of sepharose column with aprenolol (treats heart failure), a high-affinity ligand for the receptor that doesn’t bind covalently (reversible attachment)

Purified 100,000 fold (typically need 20,000 - 100,000 fold for GPCRs) a single polypeptide of 64,000

Next steps: characterise and answer if the polypeptide is sufficient to produce functional characteristics of the receptor (are there other interacting proteins necessary)

Chopped up, sequenced, and obtain aa information to design primers based on cDNA library to be able to clone protein.
However, sequencing doesn’t tell you which codons are used to encode that amino acid (except tryptophan which has has one codon that encodes for it since it was the last produced in evolution)
Predicated MW based on amino acid sequence to be more than 64,000 (must have PTM). Using enzymes that cleave off carbohydrates measured MW to be 64,000 on gel

Discovered 7 segments rich in hydrophobic residues (20-24aa to pass PM), inferring these regions passed the PM. Since there were multiple proteins with this feature although in different tissues (muscarinic adrenoreceptor in muscle, and rhodopsin in eye), must be a protein family

Question 13

Example of GPCR with same ligand but different effects

Answer 13

Acetylcholine in brain (research for dementia), heart (parasympathetic slows heart rate), iris in eye, etc
Is this multiple genes that respond to acetylcholine, or the same one expressed in these tissues

Now know it’s different acetylcholine receptors

Drug Pirenzepine discovered that has different affinity to block muscarinic receptors in brain and heart, indicating almost certainly it’s a different protein

Question 14

How did we move from isolating individual GPCRs ‘one at a time’ to
identifying the full family in humans?

Answer 14

Linda Buck won Nobel prize for paper published in 1991

Noticed TMD3 and TMD6 tended to have high sequence similarity (now know highly conserved due to binding )
Intracellular loop 3 (links TMD5 and TMD6) varies in length

Developed primers for TMD3 going 3’ to 5’ and an opposite primer for TMD6 from 5’ to 3’ to amplify region inbetween that includes intermolecular loop 3

Run on agarose gel and obtained lots of bands of different sizes; fragment library of different lengths of 3rd loop, and so different GPCRs

Cut out band and sequenced. Compare with database to identify if it’s a GPCR that has been cloned before and matches known sequence. If no match then indicates it’s a novel GPCR.
Identified existence of over 400 novel GPCRs of odorant sensors in olfactory neurons of our nose

Question 15

Example of molecular basis of selective binding of a GPCR

Answer 15

GPCRs that bind catecholamine ligands

All have key residues conserved in the same position

Amine head group forms salt bridge with aspartic acid in TMD3

Hydroxyl groups in catechol ring make H bonds with pair of serines three amino acids away in primary sequence and so so adjacent in alpha helix (~3.6 aa per turn) in TMD5

Benzene core makes pi-pi stacking interactions with phenylalanine in TMDVI

Question 16

Structural alterations
associated with GPCR activation, experiments that show

Answer 16

Protein imaging based approach:

Bottom of TMD6 has glutamate
DRY domain in TMD3 (sometimes D is E) is the most conserved residues in family
Close in tertiary structure in inactive receptor state and interact, forming ionic lock

TMD6 moves outwards when ionic lock is broken, swinging out like a gate allowing space for G protein helix to enter bottom of receptor and induce next step in signal cascade (ex. initial experiments in x-ray crystallography imaged inactive rhodopsin to active opsin,TMD6 moves 12A when activated by isolating protein in dark/red light, and beta2 adrenoreceptor).

Disadvantages: static image of snapshot of a dynamic process

Fluorescence quenching based approach:

Modified Bimane so the closer to tryptophan, to more it quenches tryptophan signal (best fluorescent signal)

Added bimane to TMD6 (since moves a lot). Observed large change in fluorescent signal from inactive to active state.

Disadvanges: Needed to purify protein to perform

Hydrogen-deuterium exchange mass spectrometry (HDX-MS):
Used in early 2010s to study conformational changes in GPCR activation. Amide H on protein backbone can exchange with deuterons (2H) in D2O containing buffer, and the exchange rate increases with increased solvent accessibility and decreased H bonding.
With mass spectrometry, and rate of increase in mass over time for each peptide segment indicates flexibility and solvent exposure.
Performed on beta2 adrenoreceptor with isoproternol agonist and showed an increased in flexibility in ICL3 (for G protein binding) and TM6, and antagonists and inverse agonists caused more rigid structure.

Disadvantages: Information is low res.(5-15 aa peptides) on relative dynamics not specific aa movements, so coupled with imaging techniques and molecular dynamic simulations

Molecular dynamic simulations:
Computational technique to model movement of each aa over microsecond time scales
Uses data from experiments and mathmatical models based on Newton’s laws of motion to calculate movement under influence of physical forces (van der waals, etc)

Disadvantages: lots of computer power required

Question 17

Use of GPCRs in disease succeptibility

Answer 17

Splice and polymorphic variation (SNPs) can introduce further diversity
potentially modifying regulation and function
Some are associated with certain diseases. GPCRs are very prevalent in body (3% of coding genes) so high chance they contain SNPs

Ex. GPR65
I231L increases risk for Inflammatory Bowel Disease

Question 18

Mutations investigating activity of GPCRs

Answer 18

-Beta2-adrenoreceptor:

Mutated aa close to junction where TMD6 becomes the 3rd intracellular loop.

Modified beta2-adrenoreceptor with changes to look like alpha1-adrenoreceptor.
Thought since beta2 modifies cAMP but alpha1 calcium, thought they’d obtain receptor that responded to signals similar to alpha1, but with effects of beta2

Obtained constitutively active beta2-adrenoreceptor with higher agonist activity

R* agonists bind receptor more tightly than R, since it promotes binding of G protein to receptor

-Alpha2-adrenoreceptor:
Different amino acid substitutions of T348 changed balance of R to R* induced by agonist
WT had the lowest constitutive activity

-Alpha1-adrenoreceptor:
Changed A293 to all amino acids.
Alanine had the lowest basal activity

Question 19

Cholera toxin: mode of action in body, mechanism, activation

Answer 19

Cholera bacteria enters endothelial gut lining (sheds regularly so bacteria is excreted) and produces toxin
Causes efflux of water by modifying ion channel
Symptoms is watery diarrhea and persist if contaminated water is continually ingested

Activation:
Persistently increases cAMP levels in intestinal cells
ADP-ribosyltransferase: Covalently modifyes Galphas (takes NAD and adds ADP ribose)
Inhibits GTP hydrolysis, locking Galphas in active GTP bound state
Adenylyl cyclase continually activated and increases cAMP

Question 20

Method to identify components of GPCR signalling and what they found

Answer 20

S49 lymphoma cells WT and UV treated (allow cells to grow to clone out DNA damage), both have isoproterenol added.
In WT, cAMP levels increase (due to GPCR activation) leading to cell apoptosis

In UV treated cells survive, indicating the receptor is no longer expressed due to DNA damage to components of signalling: can’t produce cAMP or respond to it).

Multiple lines made (cyc-, unc, etc) since UV may have damaged multiple proteins so to validate results.

Termed cyc- cells for cyclic AMP deficient. Adenylyl cyclase originally thought to be the mutated protein

S49 cyc- cells were charecterised and showed the mutant protein linked the beta-adrenergic receptor to AC:

Receptor binding assay (radiolabelled agonist propranolol and measured binding affinity) showed the receptor was present and could bind ligand properly (not the mutant)

Adding a direct AC activator (forskolin) led to cell death (cAMP increase) in S49 cells, indicating the mutant protein in the GPCR signalling pathway acted upstream

Radioligand binding assay: incubate cell with radiolabelled non-hydrolysable GTP analogue [32S]GTPgammaS or [3H]GDP to measure guanidine nucleotide shift (change in affinity of GTP/GDP for G alpha once the GPCR is activated). No increase in GTP binding or GDP disassociation after receptor stimulation,

Cholera toxin:
Lack of incorporation of [32P]ADP ribose by cholera toxin to S49 cyc- cells and didn’t increase cAMP
In WT S49, using [32P]NAD+ with cholera toxin results in incorporation of [32P]ADP-ribose into a 45kDa polypeptide (G alpha s) to a key arginine

Indicates mutant in a protein that must link GPCR to AC
Found the specific mutation in cyc- cells was in promoter

Question 21

How to define molecular nature of Gs

Answer 21

Functional reconstitution assay

Membranes from S49 cyc- cells obtained (contains AC and beta-adrenergic receptor)

Add column fractions from detergent-extracted mouse liver membranes (relevant protein must be present since previous studies done with hepatocytes)

Optical density measured at 280nm, of the amount of protein present to monitor

If Gs is present in a fraction this should now allow isoproterenol stimulation of cAMP production

Further purification steps, keeping the fractions that reconstitutes the pathway
Ideally reach a single homogeneous polypeptide that displays the relevant activity

Found that no matter the fractionation scheme used, the active fraction always contained a 45 kDa protein (corresponds to protein cholera toxin modified), but also another protein at 35kDa and 8kDa

Presumably a hetero-trimeric complex

Question 22

Pertussis toxin: mode of action in body, mechanism, activation

Answer 22

Produced by bacteria that causes whooping cough

ADP-ribosyltransferase that in pancreatic islets with pertussis toxin, cAMP increases

Using [32P]NAD+ plus Pertussis Toxin
results in incorporation of [32P]ADP-ribose into a 41kDa polypeptide to a key cysteine residue
Toxin acts to prevent receptor interaction with Gi alpha protein by inhibiting a Gi mediated decrease of cAMP

Question 23

Mechanism of heterotrimeric G protein regulation

Answer 23

In the resting/inactive state, the G proteins are in a heterotrimeric complex with the G alpha subunit (guanine nucleotide binding protein) is bound to GDP

Receptor activation causes the receptor to form a complex with the G-proteins and promote the off-rate, allowing disassociation of GDP

In a healthy cell, the high energy charge (sufficient levels of ATP and GTP for energy, and GDP and ADP low) allows GTP concentrations to be sufficiently high it takes the place of GDP, promoting the disassociation of G alpha from the complex to activate downstream signals

To desensitise the signal, G alpha has GTPase activity to hydrolyse the terminal gamma phosphate of GTP to have GDP in the binding pocket. The G alpha protein recombines with G beta and gamma

Terms:
The receptor is a guanine exchange factor (GEF) - promotes the exchange of GDP for GTP

Regulators of G protein signalling (RGS) proteins - controls rate of GTPase rate
GTPase activating protein (GAPs) enhance the rate of GTPase activity

Question 24

Toxins effects on G proteins

Answer 24

Cholera toxin - ADP-ribosylation on a key arginine near the its GTPase domain blocks the GTPase. The Gs remains in active state, continually activating AC

Pertussis toxin - ADP-ribosylation on a key cysteine residue near the GTP binding site of Gi prevents release of GDP by preventing the interaction with the receptor. Locks in inactive state

Rare mutation that alters arginine cholera toxin modifies
Pituitary tumours occurs from increased cAMP production

Mutation

Question 25

Characterisation of S49 ‘Unc’ (uncoupled) cells

Answer 25

Another cell line with beta2-adrenoreceptors exhibiting ligand binding to receptor (radioactive ligand binding assay) so intact, and lack of guanine nucleotide shift receptor affininty ([32P]GTP or [3H]GTPgammaS binding assay) - indicates modified GPCR signalling Cholera toxin incorporates [32P]ADP ribose into a 45 kDa protein - indicates Gs is expressed In reconstitution experiment, addition of purified Gs restores cAMP production. Question: what is the mutation Single point mutation in the Gs c-terminal region (R into P of aa six aa from terminal). This region is an alpha helix that inserts into the cytoplasmic cavity (from movement of TMD6) at the bottom of the receptor when agonist bound Proline is a helix breaker (cyclic structure makes it rigid), so C-terminal can no longer engage with receptor

Question 26

Recent approaches to elucidate G protein roles

Answer 26

Previously: Addition of toxins and UV mutagens to S49 cells to produce cyc- and unc mutant lines Currently: CRISPR/Cas-9 to eliminate potential signalling pathways of FFA4 in HEK293 KO Gq/11 (involved in increasing intracellular Ca) Add agonist induced increase of Ca in WT, but no response in KO Reintroduce KO protein and confirm reconstitutes function (confirm another mutation didn't occur in experiment) IP concentration (relevant to Ca signalling) doesn't increase with agonist introduction but does in WT. Basal activity is higher in WT before agonist added compared to Gq - FFA4 has constitutive ability in absence of agonist for cell signalling (towards R*, FFA4 can engage with Gq without agonist) Can be measured with ex. radiolabeled inositol assay and fluorescent dye for Ca2+

Question 27

GPCRs engaging with multiple G proteins: mediating factors, examples, experiments to show, results

Answer 27

Rare for GPCR to only couple one G protein (influenced by ligand stabilising dif. conformation states, cell type, receptor density (high may simultaneous activate multiple), receptor phosphorylation or adaptor proteins alters preference) Different methods to give homeostatic information to regulate how metabolism is treated. Beta2 adrenoreceptor: Canonical signalling is Gs but under high agonist conc. or when phosphorylated can couple to Gi FFA4: Predominantly engages Gq: regulates release of hormones that regulate incretin secretion (ex. GLP1) Also engages with Gi/o: regulates satiety hormone release When C-terminal or ICL phosphorylated in immune cells: regulates anti-inflammatory effects Synthetic Protein Affinity Strength Modulation (SPASM) sensor: BRET to assess how different receptors engage different G proteins At the C terminal tail tether ER/K linker flanked by probes YFP and NanoLuc, then a specific G proteins. When the GPCR is active, BRET occurs. With the TUG-891 peptide, enhanced FFA4 activation. Found higher BRET response for Gq, then Gs, Gi, G13 - GPCR has preference for different G proteins, but can engage with all (at higher amounts of receptor or higher conc. of activating ligand) since all relatively similar structure - diversity in physiology Can also: KO different G protein with CRISPR/Cas9 and charecterise change in signallinng GTPgammaS binding assay

Question 28

Which regions of G protein alpha subunits control distinct functions

Answer 28

Map onto primary sequence regions of proteins for different functions Interact with receptor Interact with beta/gamma complex Interacts with effector proteins (ex. AC) Interacts with GTP/GDP Receptor - C terminal alpha5 helix Gs sequence in Unc mutant (6 aa from terminus) Pertussis toxin ADP-ribosylates the Cys 4 aa from terminus Both can't engage with receptor Crystal structure shows C-terminal forms alpha helix that engages with receptor G protein chimeras (possible since all are similar enough to fold in a similar way) of replacing the last 5aa, the receptors that interact switches selectivity for receptor GTP/GDP - G box motifs In 3D can identify area. In primary sequence can identify blocks (not next to one another) that form guanine nucleotide binding region 50% identity between all the G proteins is from the guanine nucleotide binding region (hard to make selective inhibitors targeting this) Screen mutants in [35S]GTPgammaS binding assay Beta/gamma - First 10-15 aa N-terminus Cleavage with trypsin of N-terminal 5kDa of alpha subunit eliminates beta/gamma interactions Mutagenesis of N terminal residues caused loss of complex when co-immunoprecipitated Atomic level structures

Question 29

Defining roles of beta/gamma complex

Answer 29

Initial experiments showing beta/gamma subunits do as much/if not more in signalling as G alpha Pertussis toxin in neutrophil: Many chemoattractant receptors increase IP3 via stimulating the Gq pathway When cells are pretreated with pertussis toxin (doesn't effect Gq), it blocks effect of ligands - Gi protein involved (unexpected) Suggested due to role of beta/gamma (pertussis toxin locks GDP bound state in a complex with beta/gamma, decreasing beta/gamma available in cell for other pathways; here, activation of PLCbeta) Decreasing beta/gamma available in neutrophil cells (via increasing GDP bound G alpha transducin (only in eye) in immune cells since it doesn't signal cAMP in white blood cells, so it's only effect will be to bind and sequester beta/gamma complex) Caused decrease in IP Proteins purified from native tissue so thought results may be from contamination of a sufficient amount of Gq to signal Instead, made recombinant G proteins (alpha, beta and gamma) since E coli (don't have GPCRs so no contamination of G proteins) - rGalphai Adding GDP bound rGalphai inhibited effect of hormone to regulate GIRK potassium channel in cardiac cells GTPgammaS analogue used to lock Galpha in active state with beta/gamma released and free to signal Microinjection: Purified G alpha had no effect on GIRK regulation Purified G beta/gamma regulated GIRK Beta/gamma often regulates ion channels through direct binding

Question 30

Diseases from GPCR

Answer 30

Note location of mutation is close to bottom of TMDVI (glutamate) that forms the ionic lock and need for TMDVI to move to accommodate the G protein alpha subunit Familial male precocious puberty (mutation in TMD5 of LH): males develop secondary sex characteristics at ~2 years old Luteinizing hormone receptor stimulates testosterone production (normally induced by surge of LH in puberty) Retinitis pigmentosa (mutation in TMD6 of rhodopsin): lose sensitivity to light and can cause decay of retina from overactivation; blindness Thyroid adenoma (mutation in intracellular loop 3 of TSH, ex. A623I): increase in cAMP induces division in thyroid. Dwarfism: mutation near TMD6 of Growth Hormone Releasing Hormone Receptor (GHRHR) causes misfolding and reduced cell surface expression, reducing GHRH response

Question 31

How are hetero-trimeric G proteins associated with the plasma membrane?

Answer 31

Post translational carboxylation - fatty acid buries into membrane 1 FA added directs it to the membrane, but 2 anchors it to the PM G alpha C16 palmitoyl and C14 Myristol (saturated) added as protein is made via consensus sequence Raft localised to stimulate AC G beta interacts with G gamma G gamma Farnesyl or geranylgeranyl groups to the C terminus (involved in cholesterol synthesis) After disassociation can relocalise to non-raft domains to interact with ion channels, etc

Question 32

Luteinizing hormone receptor mutation

Answer 32

Familial male precocious puberty (FMPP) is a gonadotropin-independent disorder that is inherited in an autosomal dominant, male-limited pattern Gain of function Affected males generally exhibit signs of puberty with testosterone production and Leydig cell hyperplasia by age 4 D578G in the 6th transmembrane helix of LH receptor found in the affected individuals from 8 different families Cells transfected to express the mutant LH receptor exhibit markedly increased cyclic AMP production in the absence of agonist This suggests that FMPP results from a constitutively activated LH receptor

Question 33

Vasopressin receptor mutations

Answer 33

One of the most common diseases associated with GPCR is Congenital nephrogenic diabetes insipidus (CNDI) with a mutation in Vasopressin (ADH) V2 receptor Loss of function (most common type of mutation in general) - mutation in promoter, coding region that result in poor/no expression, lack of function, have phenotype Different pedigrees of patients with this condition have a range of mutations in the coding region of this gene (range of genetic mutation in different families with disease) Common features: Inability of the kidney to concentrate urine in response to vasopressin This may lead to severe dehydration, and for newborns resulting to mental retardation and poor growth Sex linked inheritance -maps to q28 of x chromosome Cloning of a human vasopressin V2 receptor to cDNA allowed demonstration that the V2 vasopressin receptor also maps to this locus Take cDNA encoding with the V2 receptor and make the mutation (fluorescence hybridisation to map mutation) of G to A in codon 137. This results in R137H mutation in the protein (at DRH motif in ionic lock, expect it to be more active) Express either WT or mutant receptor in COS cells Destabilises the protein so quickly destroyed. Rate of degradation is faster than rate of synthesis

Question 34

Orphan GPCRs to develop medicine

Answer 34

Method: Expression profiling - For therapeutic intervention analyse where GPCR is expressed in body to indicate function Ex. GPR120 is an FFA1 receptor expressed in enteroendocrine cells (GLP-1 secretion), macrophages (anti-inflammation) and adipocytes (differentiation and glucose uptake) so targeted as a novel therapeutic for metabolic disorders (ex. reduces obesity to reduce risk of type II diabetes) Phenotype of KO mouse - Ex. GPR35 KO has higher systolic blood pressure Variant/SNP link to human disease - UK Biobank tot link SNPs in GPCRs (or any protein) and how it drives alteration disease susceptibility Ex. GWAS study found strong link of T108M with primary sclerosing cholangitis Link with other disciplines - With machine learning, found S294R in GPR35 to be highly correlated with developing coronary artery calcification. Also a TF associated with the receptor is a good marker of developing aspects of cardiovascular disease Example of GPCR targeted in therapeutics: Orexin receptor in CNS 2 that respond to neuropeptides Orexins have prominent role in stabilising wakefulness Since GPCRs are conserved, diseases associated in other vertebrates gives indication of effect in humans Ex Many dogs (especially Doberman pinscher) have genetic modification that causes them to sleep a lot (narcolepsy) and when mapped in dogs found genetic deficiency of OX2 Treat narcolepsy with orexin receptor activators Treat insomnia with orexin receptor blockers - Suvorexant/Belsorma antagonist for OX1 and OX2

Question 35

Example of use of diversity to treat disease

Answer 35

Melanocortin system is a complex network of signalling pathways involving melanocortin peptides and their receptors that regulate several physiological processes, primarily feeding behaviour and body weight 5 receptor subtypes MC1R, 3, and 4 have natural antagonist (agouti and agouti related protein; AgRP) as well as agonists (melanocyte stimulating hormone; MSH peptide) MC1R: role in pigmentation as expressed in melanocytes (in skin: blocker has yellow skin in mice, activator darker) MC2R - Involved in cortisol production in adrenal cortex MC3R and MC4R - Involved in feeding behaviour. Targets to treat obesity (in brain: blocker causes weight gain, activator weight loss) Mutation in MC4R mutation is the most strongly associated with obesity Transgenic mice models: AgRp (endogenous antagonist/inverse agonist for MC4R) ectopic overexpression induced obesity (higher body weight) Agouti (endogenous antagonist/inverse agonist for MC4R and MC1R) ectopic overexpression induced obesity and lighter coat Humans: Assessing genetics of those with extreme phenotype Here, morbidly obese people (BMI35-40) had mutations in MCR and none in control had Spread of mutations across protein, all which induced poor receptor expression/function Target to activate for obesity

Question 36

Viruses and GPCRs: HIV activation

Answer 36

Viruses have GPCR in genome and/or take advantage of human GPCRs for life strategy Ex. HIV to gain entry into white blood cells hijacks 2 chemokine receptors on some human white blood cell surface - CCR5 and CXCR4 GPCRs HIV can enter cell via CCR5 receptor entry - macrophagic tropic HIV go into macrophages and T cells (particularly CD4+) via CCR5 receptor on their surface or via a related receptor CXCR4 for entry -the T tropic HIV enter T cells and T cell lines via CXCR4 receptor on their surface

Question 37

Viruses and GPCRs: HIV resistance, origin, prevalence

Answer 37

Some people with a lifestyle suggesting they'd have HIV (used bathhouses, sexual preference) were resistant as carried CCR5delta32 deletion mutation (premature STOP codon, frameshift mutation) so virus can't enter. Homozygous for mutation has almost complete protection, heterozygotes have some protection and delayed AIDs progression Prevalence of mutation depends on population: around 10% of European Caucasians have mutation, increases going further North, particularly in Eastern Finland where it likely arose Mutation is not present in any other populations Given high prevalence suggested a relatively recent pandemic of HIV that resulted in few with mutation surviving and passing on to significant amount in population Viking invasions of northern Europeans likely spread the mutation to other regions Thought HIV mutation spread may be related to bubonic plague Large killer of medieval Europeans FPR1 R190W is the SNP that gives resistance Moved across world so mutation is present in a range of populations (unlike for HIV): Around 10% of Europeans, Africans, Americans descendant from either, and 20% of Asians have mutation

Question 38

Viruses and GPCRs: treating HIV with GPCR based therapy

Answer 38

Since CCR5delta32 provides resistance, induce in people to treat A Chinese researcher genetically edited baby twin girls to have the mutation Lots of ethical considerations (man was jailed) Shortly afterwards found CCR5delta32 may induce a shorter lifespan Analysis of UK Biobank wasn't consistent with that idea - no particularly negative component of overall physiology HIV-1 remission possible with stem cell transplant of hematopoietic SC from CCR5delta32 donor into a HIV infected patient CCR5 may become pseudogene since no competitive advantage, in populations with low prevalence of HIV

Question 39

Viruses and GPCRs: integration of GPCRs into genome

Answer 39

Viruses are small and efficient so don't keep gene if it doesn't give competitive advantage, and over time some has integrated human and animal GPCRs into genome Instead of also integrating protein ligand to activate, mutate to increase constitutive activity Ex. The human herpes virus 8 (HHV-8) encodes a viral GPCR acquired from the host that mimics CXCR1 and 2 to aid pathogenicity Causes Kaposi's sarcoma Mutations made highly constitutively active for continuous signalling Danger: Human herpes in many peoples genomes (regular cold sores) Transfecting ORF74 (a receptor that HHV-8 acquired) into NIH-3T3 cells and injecting cells into immune deficient mice (AIDs phenotype) Found constitutively active ORF74 is oncogenic, and mice develop Kaposi's sarcoma symptoms In early HIV infections, many AIDs patients developed cancer of Kaposi's sarcoma - immune suppression allowed constitutively active GPCR to cause and drive cancerous state