Part 3: Protein domains Flashcards
1
Q
Domain: SH2
A
Src-homology2~100AA
2 alpha helices flanking beta sheet (antiparallel)
2
Q
SH2 binding affinity
A
phosphorylated tyrosines
3
Q
Domain: PTB
A
phsophotyrosine binding: ~100-150AA
beta barrel (antiparallel) followed by c-term alpha helix
binds in cleft between helix strands
4
Q
PTB binding affinity
A
phophorylated tyrosines
5
Q
Domain: SH3
A
src-homology 3: 60AA
Beta-barrel fold (2 antiparallel beta sheets)
binds in shallow hydrophobic pocket
6
Q
SH3 binding affinity
A
prolines (-X-P-p-X-P)
7
Q
PH domain
A
pleckstrin homolgy: ~120 AA
2 perpindicular beta sheets (antiparallel) followed by C term ampipathic alpha helix
binds in cleft between loops connecting strands
8
Q
PH binding affinity
A
phosphorylated inositol phospholipids
9
Q
molecular switches
A
kinase
phosphatase
Guanine nucleotide binding
10
Q
protein kinases
A
protein phosphorylation
phosphate groups added to proteins using adenosine triphosphate
11
Q
protein phosphotases
A
dephosphorylation
phosphate groups removed from proteins by hydrolysis
12
Q
Guanine nucleotide binding
A
G protein cycle
- Input signal Exchanges GDP for GTP
- Exchange assisted by activated receptor for trimeric G, Guanine nucleotide exchange factors (GEFs) for monomeric G
- Output signal–>hydrolysis (catalyzed by intrinsic GTPase for trimerG & w/ help from GTPase-activating proteins (GAPS) for monomeric G
13
Q
Criteria of receptors
A
display specificity by detecting only those signal molecules the cell wants to perceive
- appropriate binding affinity (Kd) for the signaling molecule in order to detect it at the likely concentration in the vicinity of the cell
- transmit the message of the signaling molecule by modulation of further component in the signaling cascade
14
Q
Receptor classes
A
intracellular
cell-surface
15
Q
cell surface receptors
A
Ligand gated ion channel
G-protein coupled
Enzyme linked
cytokine
16
Q
Ligand gated ion channel action
A
Binding of ligand changes ion permeability of plasma membrane and allows passage of specific ions
17
Q
Ligand gated ion channel system
A
synaptic
18
Q
Ligand gated ion channel ligands
A
neurotransmitters
19
Q
Ligand gated ion channel binding
A
Kd=10^-6 to 10^-3 (very low affinity)
20
Q
Ligand gated ion channel control
A
acute regulation (release of NT-containing vesicles from neurons; contraction of muscle cells) long lasting activation of Ca-sensitive gene expression
21
Q
Ligand gated ion channel examples
A
cation-selective: excitatory (nicotinic ACh, glutamate)
anion selective: inhibitory (gly, GABA)
22
Q
Ligand gated ion channel Drugs
A
psychotropics, anesthetics, anticonvulsants, drug abuse
23
Q
Ligand gated ion channel: termination
A
Ligan removal occurs rapidly by:
- diffusion away from receptor and synaptic gap
- degradation by enzymes on cell surface (acetylcholinesterase)
- reuptake into pre synaptic neuron
Formation of an inactive ligand bound state ensures brief periods of transduction
24
Q
G-Protein coupled receptor action
A
binding of ligand activates heterotrimeric G protein which conveys signal to next component in pathway
25
G-Protein coupled receptor system
synaptic, endocrine, paracrine, autocrine
26
G-Protein coupled receptor ligands
NT, hormoes, cytokines (chemokines)
27
G-Protein coupled receptor binding
Kd= 10^-9 to 10^-6M
28
G-Protein coupled receptor control
mediation of sensory sytems (vision, taste, smell)
| Acute regulation of critical physiological responses (cardiac contractility, metabolism, complex behavior)
29
G-Protein coupled receptor examples
Muscarinic ACh, beta adrenergic, rhodopsi
30
G-Protein coupled receptor drugs
antihistamines, anticholinergics, beta blockers, opiates
31
G-Protein coupled receptor structure
Transmembrane alpha helices
large ligands bind to extracellular loops, small bind in pocket
Extracellular subject to glycosylation
intracellular subject to phosphorylation
32
heterotrimeric g proteins
coupling proteins
alpha subunit - ;argest (39-46kDa)
-hydrophilic; covalently attached to membrane
-many different forms
-guanine nucleotide-binding site and GTPase activity
-domains that interact with effector proteins
33
beta-gama complex
dimeric complex of smaller subunits (35, 10kDa)
- hydrophobic, covalently attached to membrane
- similar form for different G-protein subtypes
- some interaction with effector proteins
34
G protein coupled receptor mechanism
1. ligand binds
2. Conformational change, recognition site exposure for G protein binding
3. GDP/GTP exchange, G protein alpha dissociates from beta-gamma
4. Subunit alpha binding to enzyme (release second messengers)
5. Intrinsic GTPase activation, hydrolysis of GTP to GDP, release enzyme
6. G-protein reformation with GDP, returns to receptor
35
G protein coupled receptor termination
extracellular enzymes metabolize or inactivate many of the small ligands
- Receptor mediated endocytosis accounts for some desensitization
- receptor phosphorylation by protein kinases is the major mechanism of sensitization
- protein kinase A--> receptor +/- ligand
- GPCR specific protein kinases (GRKs)->receptor +ligand
36
Caffeinated alcohol drinks: ethanol
1. ethanol binds to allosteric binding site on GABA bound receptor
2. Allows receptor to stay open longer
3. causes membrane potential to become more negative
4. Increases GABAs suppression of neural activity
5. Increases dopamine release
37
Caffeinated alcohol drinks: caffeine
1. Caffeine blocks adenosine receptor on its G-protein (is an antagonist)
2. cancels adenosines effect (suppresion of neural activity, increase blood flow)
3. Allows increased neural activity
4. Leads to blood vessel constriction, epinephrine release, increased alertness
5. increases dopamine release
38
Enzyme linked receptor action
Binding of ligand activates intrinsic enzymatic activity of cytoplasmic domain
39
Enzyme linked receptor system
endocrine, paracrine
40
Enzyme linked receptor ligands
hormones, growth factors
41
Enzyme linked receptor binding
Kd= 10^-12 to 10^-9
42
Enzyme linked receptor control
long-lasting changes in gene expression (cell division, programmed cell death, cell differentiation)
43
Enzyme linked receptor examples
receptor tyrosine kinase (EGF,FGF,PDGF, insulin
| receptor serin/threonine kinase: TGF-beta, BMP
44
Enzyme linked receptor drugs
Cancer, type 2 diabetes
45
Enzyme linked receptor structure
superfamily of more than 80 proteins
-each subunit is a single polypeptide chain consisting of: large extracellular n terminal for ligan binding, single transmembrane domain, intracellular C-terminal catalytic domain
46
Functional Enzyme linked receptors
```
mainly dimers (RTKS) and tetramers (serine/threonine kinases)
Variations: insulin receptor
```
47
Receptor tyrosine kinase (RTK)
1. inactive RTK
2. Ligan binds->dimerization, kinase activation
3. Active RTK-> autophosphorylation of tyrosine residues (cross phosph)
4. Binding/activation of signaling proteins->initiation of cascade
48
MAP kinase cascade
Mitogen-activated protein kinase cascade
1. adaptor protein
2. Ras activation protein
3. active Ras
4. activated MAPKKK (serine/threonine kinase)
5. activate to MAPKK, threonine/tyrosine)
6. activate to MAPK - effector protein (serine/threonine kinase)
7. Phosphorphylates cystolic membrane proteins of nuclear gene regulatory protein
8. Change in cystolic/membrane proteins or change in gene expression
49
Enzyme linked receptor Serine/threonine kinase
1. inactive
2. ligand binding to type II, dimerization with type I, kinase activation and cross phosph of type I
3. SMAD binding and phosphorylation , SMAD unfolding and activation
4. SMAD dissociation, dimerization with different SMAD subtype, exposure of nuclear localization signal (NLS)
5. Translocation to nucleus, altered gene expression.
50
Enzyme linked receptor termination
Endocytosis down regulation
1. Binding of ADAPTIN to exposed intracellular ligand receptor complex, binding of clatherin to adaptin, both cluster at invagination site
2. clathrin polymerization, forms vacuole with coated pit
3. Release of clatherin-coated vesicle into cytoplasm, shedding of clathrin coat, fucion of vesicle with endosome, dissociation of ligand-receptor complexes
4. Potential recycling or transfer remains to lysosome for degredation
51
Cytokine receptor action
binding of ligand facilitates association and activation of cytoplasmic enzymes, particularly tyrosine kinases, receptor lacks intrinsic enzymatic activity
52
Cytokine receptor system
paracrine, autocrine
53
Cytokine receptor ligands
cytokines, some GFs
54
Cytokine receptor binding
Kd=10^-9 to 10^-6 M
55
Cytokine receptor control
long lasting changes in gene expression (cell growth/differentiation)
56
Cytokine receptor examples
Class I- interleukin: IL-2 dimers
Class II: interferon: IFN multimers
Tumor necrosis factor : trimers
57
Cytokine receptor drugs
cancer, antivirals, immunosuppressives
58
Cytokine receptor structure
great diversity, recruit broad range of intracellular signaling proteins
each subunit is a single polypeptide consisting of: extracellular N terminal ligand binding domain, single transmembrane, intracellular c terminal with different protein-protein motifs but no intrinsic enzymatic activity
59
Functional Cytokine receptor
multimeric complexes (two or more)
60
Cytokine receptor mechanism
1. Inactive
2. Cytokine binds--> dimerization of JAK
3. JAK cross phosph, subunit phosphorylation
4. STATs bind to subunit
5. Phosphorylation of STATs, activated
6. STATS dissociate and dimerize
7. Translocation to nucleus--> altered gene expression
61
Cytokine receptor termination
phosphatases remove tyrosine phosphates from receptor/STATS
SOCS (suppressor of cytokine signaling) protein inhibit STAT phsophorylation by binding/inhibiting JAKs or competing with STATs for phosphotyrosine binding sites on receptor
-multimeric formation of receptor after ligan binding triggers endocytosis of ligand receptor complex
62
Normal class II cytokine receptor mechanism (without ebola)
1. IFN-gamma binds to JAK, activates it
1b. Release of phosphorylated STAT1s and subsequent dimerization
2. Binding of STAT1 dimer to importin alpha5 subunit of importin alpha5beta complex
3. This is transported through nuclear por
4. where it dissociates by Ran-GTP
5. so STAT1 can bind to DNA targets (GAS)
6. This leads to expression of antiviral respons.
63
Ebola virus and Cytokine receptor pathway
EBOV VP24 protein competes with STAT dimer on the import complex
-Transport of infected complex thru nuclear pore
-which gets dissociated by Ran-GTP complex
causing a suppression of antiviral response
64
Rate of formation Ligand receptor
k(on)[L][R]
65
Rate of LR dissociation
k(off)[LR]
66
equilibrium dissociation constant
k(on)[L][R]=k(off)[LR]
so
Kd=K(off)/k(on)
=[L][R]/[LR]
67
small Kd
high affinity for ligand
68
large Kd
low affinity for ligand
69
Bmax
=total # of receptors=[R]o at t=0
| [LR]=0
70
At t(equilibrium)
Free receptor =[R]-[LR]
Free ;igand = [L]-[LR]=approx [L]o
bound ligand= [LR]
71
Saturation binding relation
[LR]=[R][L]/(Kd+[L])
Bound= Bmax (free/(Kd+Free)
if free is >> Kd, then bound=Bmax
if free =Kd, then bound =.5Bmax
nonlinear regression used to measure
72
Saturation plot assumptions
equilibrium conditions
homogeneous, monovalent (1:1) populations of lgand and receptor
negligible ligand depletion (bound<10% of free)
Negligible inactivation of ligand and receptor
Negligible cell surface interactions
73
Stachard plot
linearization of saturation binding equation
slope=-1/Kd
y int= Bmax/Kd
x int= Bmax
Advantage: visual evaluation is easy
Disadvantage: bound on both axes magnifies experimental error, saturation plot gives more accurate estimate of Kd, Bmax
74
Dose response
Half Maximal Effective concentration = EC50
| EC50
75
Saliva stimulation in diabetes
Observation of reduced saliva in diabetic rats
Parotid gland: Kd=Kd for control vs diabetic but Bmax>Bmax
Submandibular: Kd control
