GPCRs Flashcards
Importance of understanding GPCRs
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.
Why have heterotrimeric proteins link receptor to an enzyme, instead of direct interation
Allows one G alpha protein (~20 in humans, less than no. of GPCRs) are to associate with multiple signalling pathways. Some have a widespread role, others are distinctive and only expressed in one cell subtype
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)
First TM protein imaged, protein requirements for imaging and why said protein fit these. Information obtained from structure
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
Evolution of GPCRs in biology (how they arose, no. of genes, why different no. in other organisms, how we know function)
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)
Types of drug types based on activation
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.
Information obtained through phylogenetic tree of GPCRs
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
Diseases from GPCR
Note location of mutation is close to bottom of TMDVI 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.
Direct Binding Experiments (method, information obtained)
- 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.
Criteria for specific, physiologically relevant binding
-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)
Initial studies on 2nd messengers showed and next steps
Addition of glucagon raised cAMP levels in hepatocytes (specifically alpha cells of pancreatic islets)
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 receptor to 2nd messenger
production that requires GTP
What was the molecular nature of this ‘transducer’?
Method to identify components of GPCR signalling
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 (can’t produce cAMP) or components of signal cascade are mutated (can’t respond to it)
Experiment perform to prove drug target for a GPCR was a protein + next steps
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
Experiment performed to purify a GPCR + next steps
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 selective for carbohydrates (PTM that receptor will have since on 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)
Experiment performed to understand beta-2 adrenoreceptor + next steps
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, 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 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)
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
Example of GPCR with same ligand but different effects
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
How did we move from isolating individual GPCRs ‘one at a time’ to
identifying the full family in humans?
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
Example of molecular basis of selective binding of a GPCR
GPCRs that bind catecholamine ligands
All have key residues conserved in the same position
Amine head group interacts 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
Structural alterations
associated with GPCR activation
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. in inactive rhodopsin to active opsin,TMD6 moves 12A when activated by isolating protein in dark/red light)
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
Molecular dynamic simulations:
Observe movement of each aa over microsecond time scales
Disadvantages: lots of computer power required
Use of GPCRs in disease succeptibility
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. GPR35
S294R changes levels of calcification build up in arteries
T108M associated with likelihood of developing inflammatory bowel disease
T253M was found to be associated to anthracycline-induced cardiotoxicity (ACT), severe drug reactions for children treated with anthracycline in chemotherapy
Mutations investigating activity of GPCRs
-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
