Nobel Prizes Flashcards
Who did Gilman work with initially?
Rall (Sutherland’s collaborator)
What did Gilman work with initially?
Cyclic nucleotides (cAMP)
What research question was Gilman studying?
How are receptors coupled to adenylyl cyclase
What was the issue with Rodbell’s experiments of a transducer?
Other researchers could not replicate his results
How come other researchers could not replicate Rodbell’s results?
They did not have access to the purified membranes that he did
What was the turning point of Gilman’s research?
Experiments with cyc- mutants
What was the most interesting cyc- mutant that Gilman studied?
Uncoupled receptor and AC, had both components working, but no cAMP response
What did the blots that Gilman did reveal?
Purification of aGs and bGs
What is special about fluoride activation of aG?
Requires a cofactor (Al3+)
Why does fluoride need an aluminium cofactor to activate aG?
AlF4 mimics GTP for activation of aGs
What does ARF stand for?
ADP-ribosylation factor
What does ARF do?
Required for cholera to bind NAD to aGs
What was the moral of Gilman’s experiments with Gi?
Never throw anything away (found Gi through leftover samples)
How were different families of G-proteins discovered?
Parallels between vision transduction and adrenaline were discovered
What was the evolutionary relationship between the alpha subunits of G-proteins?
They were distinct but related
What was the evolutionary relationship between the beta subunits of G-proteins?
They were very similar or identical
What are the groups of G-proteins, and their size?
Small Gs group, large Gi group, Gq, and G12
What is the general function of G-proteins?
Function as switches and timers
What is the speed of GTPase activity?
Fairly slow catalysis - sec to min
If there is no GTP, what complex of G-protein is stable?
Hormone-receptor-aG complex
What does aG have a high affinity for?
GTP
True or false: many G-proteins undergo lipid covalent modifications
True: these modifications help for affinity of a subunit to beta/gamma complex
What big biochemical breakthrow allowed for studying the properties of G-protein subunits?
In transfecto (adding DNA to cells for expression)
What was the advantage of using In transfecto to study G-protein subunits?
Could see their function without other G-protein subunits present
What is gusducin?
A G-protein found selectively in taste buds
What protein is gusducin similar to?
Transducin
What does the beta/gamma subunit function as?
Noise suppressor
How does the beta/gamma subunit function as a noise suppresor?
Helps prevent random GDP dissociation
What is the functional advantage of utilizing G-proteins?
Allows for large amplification, and convergence or divergence
How is convergence seen in G-protein signaling?
Many receptors can converge on one G-protein, and many G-proteins can converge on one effector
How is divergence seen in G-protein signaling?
A single receptor can activate many G-proteins, and one G-protein can activate more than one effector
What is the practical advantage of utilizing G-proteins signaling?
Highly customized signaling repertoire with relatively few components
What are the most versatile chemical sensors?
GPCRs
What is the general pathway of a GPCR?
GPCR detects extracellular agonist, activates G-protein, modulates downstream effectors
True or false: one receptor can only couple to one G-protein
False: Beta 2 AR can couple to both aGs and aGi in cardiac myocytes
What is ligand efficacy?
The effect of a ligand on the structure and biophysical properties of a receptor
What is the largest group of targets for drug discovery?
GPCRs
Where did the first structural insights of GPCRs come from?
Rhodopsin and bets 2 AR
What is the signature of a GPCR?
7 transmembrane topology
How were the domains for ligand binding and G-proteins identified?
Using beta 2 AR and alpha 2 AR
Why was beta 2 AR a good model system?
Had many structural studies through mutagenesis, and there were a diversity of ligands present for different effects
What was the problem of doing biophysical studies on beta 2 AR?
Expression and purification of unstable membrane proteins
How was beta 2 AR purified and expressed for biophysical studies?
Inserted a cleaveable signal sequence
What was used to characterize mechanisms of activation of beta 2 AR?
Fluorescence spectroscopy
What is a rheostat?
A device that adjusts a value along a range
How are GPCRs molecular rheostats?
They have a range of conformations to allow for specific ligand binding
What is meant by a “ligand specific conformation” for GPCRs?
A specific ligand causes a specific conformation (out of a range) of the GPCR
Why did rhodopsin give the first insight into GPCRs?
Relatively high stability, and natural abundance
What were the 2 impediments to crystallography of beta 2 AR?
Dynamic character, and small polar surface area for crystal lattice contact
How were the problems with crystallography of beta 2 AR overcome?
Used antibodies and protein engineering to crystallize the inactive state
What was the challenge of crystallography of active beta 2 AR?
Agonists alone do not fully stabilize beta 2 AR
What is needed to fully stabilize beta 2 AR?
Gs
What was used to stabilize active beta 2 AR for crystallography?
Nanobodies
True or false: there is one active state of beta 2 AR
False: conformational changes are not consistent with a single active state (sequential)
Which transmembrane unit undergoes the largest changes in response to agonists?
TM6
Why does beta 2 AR have little polar surface?
It is largely a transmembrane protein, and thus needs large hydrophobic regions
What maintains the inactive state of beta 2 AR?
Packing of conserved amino acids
What is required to accommodate agonist binding in beta 2 AR?
Rearrangement of conserved amino acids
What were the two postulates of receptors?
They interact with stimuli, and act on effectors to alter function
True or false: the existence of receptors was generally accepted
False: there was a lot of skepticism surrounding receptors
What was radioligand binding used for (in terms of studying GPCRs)?
Regulation, subtypes, and theories of mechanisms
What was the “Rosetta Stone” of GPCR families?
Purification of the first four (rhodopsin, adrenaline, etc.)
How could Gi be converted into Gs?
Replacing the third cytoplasmic loop
How is homeostasis present in GPCR function?
Desensitization (stop reacting to signal)
What is BARK?
beta-adrenaline receptor kinase, now called GRK2
How many enzymes are in the GRK family?
7
How many groups are there in the GRK family?
3
What are the groups in the GRK family?
- 1,7
- 2,3 (BARK 1,2)
- 4,5,6
How are the GRKs distrubuted in the body?
GRK 1 and 7 are found only in the retina; GRK 2, 3, 5, 6 and ubiquitously expressed
What type of kinases are GRKs?
Serine/threonine kinases
How are GRKs translocated to the plasma membrane?
Interactions with beta/gamma G
How is arrestin binding similar and different in different GPCRs?
Same mechanisms, but with great specificity for the particular receptor
How are the arrestins distributed in the body?
Arrestin 1 and X arrestin are in the retina, beta-arrestin 1 and beta-arrestin 2 are ubiquitously expressed
What is the structure of arrestin?
Many antiparallel beta sheets with a polar core
True or false: PKA and PKC are receptor specific
False: they are fairly general, and not receptor specific
How does desensitization of GPCRs occur?
Receptor phosphorylation, and arrestin binding
What motif can function as a signal transducing system (in the GPCR system)?
Arrestin-GRK system
What are the three functions of beta-arrestin?
Desensitization, internalization (endocytosis), and signaling
What does beta-arrestin binding link to?
MAP kinase pathway, or clathrin coated pit endocytosis
What do biased agonists do?
Stabilize particular active form, stimulating some responses and not others
What pathways can be biased in the GPCR system?
G-protein pathway, or beta-arrestin pathway
What are the characteristics of beta-arrestin signaling?
Diverse, uses similar pathways to G-proteins, and it has distinct cellular effects
What governs the fraction of receptors with bound ligand?
Ligand concentration
What is another name for GPCRs?
7TMs
What does 7TM stand for?
7 transmembrane (regions)
What was the classical receptor theory based on?
Law of mass action and dose-response data
What was used to prove the existence of receptors?
Radioactively labelled agonists
Why are receptors that bind to diffuseable ligands harder to purify?
They are found in smaller amounts
What is the same across GPCRs?
Same manner of signal transmission
How many conformations do GPCRs have?
A large number, ranging from inactive to active
What shape do the helices resemble?
Rods
Where are the helices found in a GPCR?
In the plasma membrane (7 transmembrane regions)
What is the mechanical motion of the GPCR when an agonist binds?
Grips bundle at one end, opens up like a rose bouquet at the other end
How big is the structural change on the ligand side of the GPCR?
Small change
How big is the structural change on the intracellular side of the GPCR?
Large change
What is the holy grail of GPCR research?
High resolution picture of active ternary complex (receptor, ligand, and G-protein)
How stable is the ternary complex (GPCRs)?
Not very stable
Why is there inherent instability of the ternary complex (GPCRs)?
Able to activate many G-proteins
What 5 biochemical strategies were used to visualize GPCRs?
- Apyrase treatment (ATP -> ADP -> AMP)
- Special detergent
- Nanobody stabilization (instead of G-protein)
- Covalent agonist binding (keep agonist)
- Protein engineering (reduce mobility of helices)
What is an agonist (in terms of GPCRs)?
A molecule that binds to GPCR and stabilizes a conformation that activates a G-protein
What is an inverse agonist (in terms of GPCRs)?
A molecule that binds to and stabilizes an inactive conformation of the GPCR
What is an antagonist (in terms of GPCRs)?
A molecule that competes with agonists to bind to and block binding sites on GPCR
How does an antagonist block GPCR signaling?
Prevents conformational change that activates G-protein
What are the design criteria of developing a new drug?
High specificity, high efficacy, and few side effects
What is the importance of the biochemical techniques used to purify AR?
Can be used to purify other 7TM receptors
True or false: the same GPCR can signal through different intracellular pathways depending on the ligand
True: there are different active forms based on the ligand
Why can a GPCR signal through different intracellular pathways depending on the ligand?
Relative flexibility of receptor in membrane (different active and inactive forms)
True or false: the same 7TM receptor can be both G-protein dependent and G-protein independent
True: this is done through biased signaling
How does nature and nurture interact?
Mutually and synergistically
What broad issues did Rodbell study?
Broad issues of biological communication
What laid the foundation for the transducer theory?
AC
What was the critical lesson by Rodbell?
Check sources, and never interpret a hyperbolic curve (describes behavior of entire universe)
What was needed to measure the pharmacology of hormone receptors (in Rodbell’s time)?
Indirect tests
What was the central question studied by Rodbell (with fat cells)?
Do all hormones operate on the same enzyme, or is it coupled to separate cyclases?
What was Rodbell’s argument regarding receptors and enzymes?
Multiple receptors interacted with a common catalytic unit
What was the common structural element of the transducers?
A structure that converted MgATP into cAMP
How was it discovered that lipids were somehow involved in structural interactions?
Receptors were sensitive to agents that affected membrane structure
What is meant by “too many angels on a pinhead”?
Hard to believe that several hormones were structurally annealed to the same enzyme
What influenced transducer theory?
Informational processing (cybernetic theory)
What does a transducer do (in cybernetic theory)?
Couples discriminator and amplifier
What does a discriminator do (in cybernetic theory)?
Decides a state
What is the discriminator in biology?
Receptor
What does an amplifier do (in cybernetic theory)?
Amplify a response
What is the amplifier in biology?
AC (makes cAMP to amplify signal)
What system do adipocytes have?
Multiple receptor-AC system
Why did Rodbell move from a fat system to a liver system?
Simpler system (multiple receptor AC-system to just glucagon specific)
What do guanine nucleotides do?
Lower affinity of receptor for hormone
How long was the system active with non-hydrolyzable GTP?
Three days at room temperature
What must the transducer do (in a biological sense after Rodbell’s experiments)?
Have capacity to hydrolyze GTP
What is the limiting step in G-protein signaling?
Release of Pi (not hydrolysis)
What is the difficult problem in membrane biology?
Understand how components are organized in the membrane
What questions are central to the difficult problem in membrane biology?
How are proteins distributed to obtain efficiency and rapidity
What did the irridation studies suggest?
Disaggregation theory (based on lower functional size)
What components are in excess: G-proteins or receptors?
G-proteins (10:1)
What helps separate aG from beta/gamma G?
Interactions of enzyme, Mg, and GTPase
What are the similarities between G-proteins and cytoskeletal proteins?
Similar functions, substrates, activators, regulation, etc.
What do G-proteins sense?
Chemical signals
What do tubulin / actin sense?
Mechanical signals
What was the prevailing theory in Sutherland’s time regarding hormone action?
Hormone action could not be studied in the absence of cell structure
What was the inspiration for Sutherland’s studies?
Biosynthesis of urea
What is necessary for scientific progress?
Young / talented investigators, with free exchange of ideas
Why was glycogen breakdown Sutherland’s model of interest?
It was rapid, large, and reproducible
What is the rate-limiting step in glycogen breakdown?
Phosphorylase
How did hormones improve the rate-limiting step in glycogen breakdown?
Increased phosphorylase activity
What was the inactivating enzyme in glycogen breakdown?
Phosphatase
What was the heat stable factor that activated phosphorylase?
cAMP
What was found about AC in Sutherland’s experiments?
Converted ATP > cAMP, widely distributed
What was found about phosphodiesterase in Sutherland’s experiments?
Converted cAMP -> AMP, widespread occurance of CAMP
What is the mechanism of action for glucagon and epinephrine?
Increased cAMP by stimulated AC (not inhibiting PDE)
Where was AC originally thought to be located?
In the nucleus
How was the idea that AC was in the nucleus disproved?
Fragmentation of the membrane also fragmented AC
Why do cells have different responses to cAMP?
Different cells have different enzymes
What was Sutherland’s 4 criteria for his studies involving cAMP?
- AC should be stimulated
- cAMP levels should change
- PDE inhibitors should add synergistically
- Effect should be mimicked with external cAMP
What was done to make cAMP able to enter the membrane?
Make it more lipid soluble
Which cAMP derivative gave a higher response in Sutherland’s studies?
Lipid-soluble (perhaps due to penetration of cell membrane)
What was the significance of Sutherland’s rat heart studies?
Measured cAMP levels and a mechanical / functional response (not chemical)
What is interesting about slime molds?
cAMP is used as a first messenger, not a second messenger
What are the two naturally occurring cyclic nucleotides?
cAMP and cGMP
What are the differences between AC and GC?
GC is more soluble, requires Mn2+, and is not stimulated by fluoride
True or false: the biological role of cGMP is the same as cAMP
False: they have different roles in biology
