Nobel Prizes Flashcards
1
Q
Who did Gilman work with initially?
A
Rall (Sutherland’s collaborator)
2
Q
What did Gilman work with initially?
A
Cyclic nucleotides (cAMP)
3
Q
What research question was Gilman studying?
A
How are receptors coupled to adenylyl cyclase
4
Q
What was the issue with Rodbell’s experiments of a transducer?
A
Other researchers could not replicate his results
5
Q
How come other researchers could not replicate Rodbell’s results?
A
They did not have access to the purified membranes that he did
6
Q
What was the turning point of Gilman’s research?
A
Experiments with cyc- mutants
7
Q
What was the most interesting cyc- mutant that Gilman studied?
A
Uncoupled receptor and AC, had both components working, but no cAMP response
8
Q
What did the blots that Gilman did reveal?
A
Purification of aGs and bGs
9
Q
What is special about fluoride activation of aG?
A
Requires a cofactor (Al3+)
10
Q
Why does fluoride need an aluminium cofactor to activate aG?
A
AlF4 mimics GTP for activation of aGs
11
Q
What does ARF stand for?
A
ADP-ribosylation factor
12
Q
What does ARF do?
A
Required for cholera to bind NAD to aGs
13
Q
What was the moral of Gilman’s experiments with Gi?
A
Never throw anything away (found Gi through leftover samples)
14
Q
How were different families of G-proteins discovered?
A
Parallels between vision transduction and adrenaline were discovered
15
Q
What was the evolutionary relationship between the alpha subunits of G-proteins?
A
They were distinct but related
16
Q
What was the evolutionary relationship between the beta subunits of G-proteins?
A
They were very similar or identical
17
Q
What are the groups of G-proteins, and their size?
A
Small Gs group, large Gi group, Gq, and G12
18
Q
What is the general function of G-proteins?
A
Function as switches and timers
19
Q
What is the speed of GTPase activity?
A
Fairly slow catalysis - sec to min
20
Q
If there is no GTP, what complex of G-protein is stable?
A
Hormone-receptor-aG complex
21
Q
What does aG have a high affinity for?
A
GTP
22
Q
True or false: many G-proteins undergo lipid covalent modifications
A
True: these modifications help for affinity of a subunit to beta/gamma complex
23
Q
What big biochemical breakthrow allowed for studying the properties of G-protein subunits?
A
In transfecto (adding DNA to cells for expression)
24
Q
What was the advantage of using In transfecto to study G-protein subunits?
A
Could see their function without other G-protein subunits present
25
What is gusducin?
A G-protein found selectively in taste buds
26
What protein is gusducin similar to?
Transducin
27
What does the beta/gamma subunit function as?
Noise suppressor
28
How does the beta/gamma subunit function as a noise suppresor?
Helps prevent random GDP dissociation
29
What is the functional advantage of utilizing G-proteins?
Allows for large amplification, and convergence or divergence
30
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
31
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
32
What is the practical advantage of utilizing G-proteins signaling?
Highly customized signaling repertoire with relatively few components
33
What are the most versatile chemical sensors?
GPCRs
34
What is the general pathway of a GPCR?
GPCR detects extracellular agonist, activates G-protein, modulates downstream effectors
35
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
36
What is ligand efficacy?
The effect of a ligand on the structure and biophysical properties of a receptor
37
What is the largest group of targets for drug discovery?
GPCRs
38
Where did the first structural insights of GPCRs come from?
Rhodopsin and bets 2 AR
39
What is the signature of a GPCR?
7 transmembrane topology
40
How were the domains for ligand binding and G-proteins identified?
Using beta 2 AR and alpha 2 AR
41
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
42
What was the problem of doing biophysical studies on beta 2 AR?
Expression and purification of unstable membrane proteins
43
How was beta 2 AR purified and expressed for biophysical studies?
Inserted a cleaveable signal sequence
44
What was used to characterize mechanisms of activation of beta 2 AR?
Fluorescence spectroscopy
45
What is a rheostat?
A device that adjusts a value along a range
46
How are GPCRs molecular rheostats?
They have a range of conformations to allow for specific ligand binding
47
What is meant by a "ligand specific conformation" for GPCRs?
A specific ligand causes a specific conformation (out of a range) of the GPCR
48
Why did rhodopsin give the first insight into GPCRs?
Relatively high stability, and natural abundance
49
What were the 2 impediments to crystallography of beta 2 AR?
Dynamic character, and small polar surface area for crystal lattice contact
50
How were the problems with crystallography of beta 2 AR overcome?
Used antibodies and protein engineering to crystallize the inactive state
51
What was the challenge of crystallography of active beta 2 AR?
Agonists alone do not fully stabilize beta 2 AR
52
What is needed to fully stabilize beta 2 AR?
Gs
53
What was used to stabilize active beta 2 AR for crystallography?
Nanobodies
54
True or false: there is one active state of beta 2 AR
False: conformational changes are not consistent with a single active state (sequential)
55
Which transmembrane unit undergoes the largest changes in response to agonists?
TM6
56
Why does beta 2 AR have little polar surface?
It is largely a transmembrane protein, and thus needs large hydrophobic regions
57
What maintains the inactive state of beta 2 AR?
Packing of conserved amino acids
58
What is required to accommodate agonist binding in beta 2 AR?
Rearrangement of conserved amino acids
59
What were the two postulates of receptors?
They interact with stimuli, and act on effectors to alter function
60
True or false: the existence of receptors was generally accepted
False: there was a lot of skepticism surrounding receptors
61
What was radioligand binding used for (in terms of studying GPCRs)?
Regulation, subtypes, and theories of mechanisms
62
What was the "Rosetta Stone" of GPCR families?
Purification of the first four (rhodopsin, adrenaline, etc.)
63
How could Gi be converted into Gs?
Replacing the third cytoplasmic loop
64
How is homeostasis present in GPCR function?
Desensitization (stop reacting to signal)
65
What is BARK?
beta-adrenaline receptor kinase, now called GRK2
66
How many enzymes are in the GRK family?
7
67
How many groups are there in the GRK family?
3
68
What are the groups in the GRK family?
1. 1,7
2. 2,3 (BARK 1,2)
3. 4,5,6
69
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
70
What type of kinases are GRKs?
Serine/threonine kinases
71
How are GRKs translocated to the plasma membrane?
Interactions with beta/gamma G
72
How is arrestin binding similar and different in different GPCRs?
Same mechanisms, but with great specificity for the particular receptor
73
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
74
What is the structure of arrestin?
Many antiparallel beta sheets with a polar core
75
True or false: PKA and PKC are receptor specific
False: they are fairly general, and not receptor specific
76
How does desensitization of GPCRs occur?
Receptor phosphorylation, and arrestin binding
77
What motif can function as a signal transducing system (in the GPCR system)?
Arrestin-GRK system
78
What are the three functions of beta-arrestin?
Desensitization, internalization (endocytosis), and signaling
79
What does beta-arrestin binding link to?
MAP kinase pathway, or clathrin coated pit endocytosis
80
What do biased agonists do?
Stabilize particular active form, stimulating some responses and not others
81
What pathways can be biased in the GPCR system?
G-protein pathway, or beta-arrestin pathway
82
What are the characteristics of beta-arrestin signaling?
Diverse, uses similar pathways to G-proteins, and it has distinct cellular effects
83
What governs the fraction of receptors with bound ligand?
Ligand concentration
84
What is another name for GPCRs?
7TMs
85
What does 7TM stand for?
7 transmembrane (regions)
86
What was the classical receptor theory based on?
Law of mass action and dose-response data
87
What was used to prove the existence of receptors?
Radioactively labelled agonists
88
Why are receptors that bind to diffuseable ligands harder to purify?
They are found in smaller amounts
89
What is the same across GPCRs?
Same manner of signal transmission
90
How many conformations do GPCRs have?
A large number, ranging from inactive to active
91
What shape do the helices resemble?
Rods
92
Where are the helices found in a GPCR?
In the plasma membrane (7 transmembrane regions)
93
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
94
How big is the structural change on the ligand side of the GPCR?
Small change
95
How big is the structural change on the intracellular side of the GPCR?
Large change
96
What is the holy grail of GPCR research?
High resolution picture of active ternary complex (receptor, ligand, and G-protein)
97
How stable is the ternary complex (GPCRs)?
Not very stable
98
Why is there inherent instability of the ternary complex (GPCRs)?
Able to activate many G-proteins
99
What 5 biochemical strategies were used to visualize GPCRs?
1. Apyrase treatment (ATP -> ADP -> AMP)
2. Special detergent
3. Nanobody stabilization (instead of G-protein)
4. Covalent agonist binding (keep agonist)
5. Protein engineering (reduce mobility of helices)
100
What is an agonist (in terms of GPCRs)?
A molecule that binds to GPCR and stabilizes a conformation that activates a G-protein
101
What is an inverse agonist (in terms of GPCRs)?
A molecule that binds to and stabilizes an inactive conformation of the GPCR
102
What is an antagonist (in terms of GPCRs)?
A molecule that competes with agonists to bind to and block binding sites on GPCR
103
How does an antagonist block GPCR signaling?
Prevents conformational change that activates G-protein
104
What are the design criteria of developing a new drug?
High specificity, high efficacy, and few side effects
105
What is the importance of the biochemical techniques used to purify AR?
Can be used to purify other 7TM receptors
106
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
107
Why can a GPCR signal through different intracellular pathways depending on the ligand?
Relative flexibility of receptor in membrane (different active and inactive forms)
108
True or false: the same 7TM receptor can be both G-protein dependent and G-protein independent
True: this is done through biased signaling
109
How does nature and nurture interact?
Mutually and synergistically
110
What broad issues did Rodbell study?
Broad issues of biological communication
111
What laid the foundation for the transducer theory?
AC
112
What was the critical lesson by Rodbell?
Check sources, and never interpret a hyperbolic curve (describes behavior of entire universe)
113
What was needed to measure the pharmacology of hormone receptors (in Rodbell's time)?
Indirect tests
114
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?
115
What was Rodbell's argument regarding receptors and enzymes?
Multiple receptors interacted with a common catalytic unit
116
What was the common structural element of the transducers?
A structure that converted MgATP into cAMP
117
How was it discovered that lipids were somehow involved in structural interactions?
Receptors were sensitive to agents that affected membrane structure
118
What is meant by "too many angels on a pinhead"?
Hard to believe that several hormones were structurally annealed to the same enzyme
119
What influenced transducer theory?
Informational processing (cybernetic theory)
120
What does a transducer do (in cybernetic theory)?
Couples discriminator and amplifier
121
What does a discriminator do (in cybernetic theory)?
Decides a state
122
What is the discriminator in biology?
Receptor
123
What does an amplifier do (in cybernetic theory)?
Amplify a response
124
What is the amplifier in biology?
AC (makes cAMP to amplify signal)
125
What system do adipocytes have?
Multiple receptor-AC system
126
Why did Rodbell move from a fat system to a liver system?
Simpler system (multiple receptor AC-system to just glucagon specific)
127
What do guanine nucleotides do?
Lower affinity of receptor for hormone
128
How long was the system active with non-hydrolyzable GTP?
Three days at room temperature
129
What must the transducer do (in a biological sense after Rodbell's experiments)?
Have capacity to hydrolyze GTP
130
What is the limiting step in G-protein signaling?
Release of Pi (not hydrolysis)
131
What is the difficult problem in membrane biology?
Understand how components are organized in the membrane
132
What questions are central to the difficult problem in membrane biology?
How are proteins distributed to obtain efficiency and rapidity
133
What did the irridation studies suggest?
Disaggregation theory (based on lower functional size)
134
What components are in excess: G-proteins or receptors?
G-proteins (10:1)
135
What helps separate aG from beta/gamma G?
Interactions of enzyme, Mg, and GTPase
136
What are the similarities between G-proteins and cytoskeletal proteins?
Similar functions, substrates, activators, regulation, etc.
137
What do G-proteins sense?
Chemical signals
138
What do tubulin / actin sense?
Mechanical signals
139
What was the prevailing theory in Sutherland's time regarding hormone action?
Hormone action could not be studied in the absence of cell structure
140
What was the inspiration for Sutherland's studies?
Biosynthesis of urea
141
What is necessary for scientific progress?
Young / talented investigators, with free exchange of ideas
142
Why was glycogen breakdown Sutherland's model of interest?
It was rapid, large, and reproducible
143
What is the rate-limiting step in glycogen breakdown?
Phosphorylase
144
How did hormones improve the rate-limiting step in glycogen breakdown?
Increased phosphorylase activity
145
What was the inactivating enzyme in glycogen breakdown?
Phosphatase
146
What was the heat stable factor that activated phosphorylase?
cAMP
147
What was found about AC in Sutherland's experiments?
Converted ATP > cAMP, widely distributed
148
What was found about phosphodiesterase in Sutherland's experiments?
Converted cAMP -> AMP, widespread occurance of CAMP
149
What is the mechanism of action for glucagon and epinephrine?
Increased cAMP by stimulated AC (not inhibiting PDE)
150
Where was AC originally thought to be located?
In the nucleus
151
How was the idea that AC was in the nucleus disproved?
Fragmentation of the membrane also fragmented AC
152
Why do cells have different responses to cAMP?
Different cells have different enzymes
153
What was Sutherland's 4 criteria for his studies involving cAMP?
1. AC should be stimulated
2. cAMP levels should change
3. PDE inhibitors should add synergistically
4. Effect should be mimicked with external cAMP
154
What was done to make cAMP able to enter the membrane?
Make it more lipid soluble
155
Which cAMP derivative gave a higher response in Sutherland's studies?
Lipid-soluble (perhaps due to penetration of cell membrane)
156
What was the significance of Sutherland's rat heart studies?
Measured cAMP levels and a mechanical / functional response (not chemical)
157
What is interesting about slime molds?
cAMP is used as a first messenger, not a second messenger
158
What are the two naturally occurring cyclic nucleotides?
cAMP and cGMP
159
What are the differences between AC and GC?
GC is more soluble, requires Mn2+, and is not stimulated by fluoride
160
True or false: the biological role of cGMP is the same as cAMP
False: they have different roles in biology
