Part 3: Protein domains Flashcards
Domain: SH2
Src-homology2~100AA
2 alpha helices flanking beta sheet (antiparallel)
SH2 binding affinity
phosphorylated tyrosines
Domain: PTB
phsophotyrosine binding: ~100-150AA
beta barrel (antiparallel) followed by c-term alpha helix
binds in cleft between helix strands
PTB binding affinity
phophorylated tyrosines
Domain: SH3
src-homology 3: 60AA
Beta-barrel fold (2 antiparallel beta sheets)
binds in shallow hydrophobic pocket
SH3 binding affinity
prolines (-X-P-p-X-P)
PH domain
pleckstrin homolgy: ~120 AA
2 perpindicular beta sheets (antiparallel) followed by C term ampipathic alpha helix
binds in cleft between loops connecting strands
PH binding affinity
phosphorylated inositol phospholipids
molecular switches
kinase
phosphatase
Guanine nucleotide binding
protein kinases
protein phosphorylation
phosphate groups added to proteins using adenosine triphosphate
protein phosphotases
dephosphorylation
phosphate groups removed from proteins by hydrolysis
Guanine nucleotide binding
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
Criteria of receptors
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
Receptor classes
intracellular
cell-surface
cell surface receptors
Ligand gated ion channel
G-protein coupled
Enzyme linked
cytokine
Ligand gated ion channel action
Binding of ligand changes ion permeability of plasma membrane and allows passage of specific ions
Ligand gated ion channel system
synaptic
Ligand gated ion channel ligands
neurotransmitters
Ligand gated ion channel binding
Kd=10^-6 to 10^-3 (very low affinity)
Ligand gated ion channel control
acute regulation (release of NT-containing vesicles from neurons; contraction of muscle cells) long lasting activation of Ca-sensitive gene expression
Ligand gated ion channel examples
cation-selective: excitatory (nicotinic ACh, glutamate)
anion selective: inhibitory (gly, GABA)
Ligand gated ion channel Drugs
psychotropics, anesthetics, anticonvulsants, drug abuse
Ligand gated ion channel: termination
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
G-Protein coupled receptor action
binding of ligand activates heterotrimeric G protein which conveys signal to next component in pathway
G-Protein coupled receptor system
synaptic, endocrine, paracrine, autocrine
G-Protein coupled receptor ligands
NT, hormoes, cytokines (chemokines)
G-Protein coupled receptor binding
Kd= 10^-9 to 10^-6M
G-Protein coupled receptor control
mediation of sensory sytems (vision, taste, smell)
Acute regulation of critical physiological responses (cardiac contractility, metabolism, complex behavior)
G-Protein coupled receptor examples
Muscarinic ACh, beta adrenergic, rhodopsi
G-Protein coupled receptor drugs
antihistamines, anticholinergics, beta blockers, opiates
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
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
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
G protein coupled receptor mechanism
- ligand binds
- Conformational change, recognition site exposure for G protein binding
- GDP/GTP exchange, G protein alpha dissociates from beta-gamma
- Subunit alpha binding to enzyme (release second messengers)
- Intrinsic GTPase activation, hydrolysis of GTP to GDP, release enzyme
- G-protein reformation with GDP, returns to receptor
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
Caffeinated alcohol drinks: ethanol
- ethanol binds to allosteric binding site on GABA bound receptor
- Allows receptor to stay open longer
- causes membrane potential to become more negative
- Increases GABAs suppression of neural activity
- Increases dopamine release
Caffeinated alcohol drinks: caffeine
- Caffeine blocks adenosine receptor on its G-protein (is an antagonist)
- cancels adenosines effect (suppresion of neural activity, increase blood flow)
- Allows increased neural activity
- Leads to blood vessel constriction, epinephrine release, increased alertness
- increases dopamine release
Enzyme linked receptor action
Binding of ligand activates intrinsic enzymatic activity of cytoplasmic domain
Enzyme linked receptor system
endocrine, paracrine
Enzyme linked receptor ligands
hormones, growth factors
Enzyme linked receptor binding
Kd= 10^-12 to 10^-9
Enzyme linked receptor control
long-lasting changes in gene expression (cell division, programmed cell death, cell differentiation)
Enzyme linked receptor examples
receptor tyrosine kinase (EGF,FGF,PDGF, insulin
receptor serin/threonine kinase: TGF-beta, BMP
Enzyme linked receptor drugs
Cancer, type 2 diabetes
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
Functional Enzyme linked receptors
mainly dimers (RTKS) and tetramers (serine/threonine kinases) Variations: insulin receptor
Receptor tyrosine kinase (RTK)
- inactive RTK
- Ligan binds->dimerization, kinase activation
- Active RTK-> autophosphorylation of tyrosine residues (cross phosph)
- Binding/activation of signaling proteins->initiation of cascade
MAP kinase cascade
Mitogen-activated protein kinase cascade
- adaptor protein
- Ras activation protein
- active Ras
- activated MAPKKK (serine/threonine kinase)
- activate to MAPKK, threonine/tyrosine)
- activate to MAPK - effector protein (serine/threonine kinase)
- Phosphorphylates cystolic membrane proteins of nuclear gene regulatory protein
- Change in cystolic/membrane proteins or change in gene expression
Enzyme linked receptor Serine/threonine kinase
- inactive
- ligand binding to type II, dimerization with type I, kinase activation and cross phosph of type I
- SMAD binding and phosphorylation , SMAD unfolding and activation
- SMAD dissociation, dimerization with different SMAD subtype, exposure of nuclear localization signal (NLS)
- Translocation to nucleus, altered gene expression.
Enzyme linked receptor termination
Endocytosis down regulation
- Binding of ADAPTIN to exposed intracellular ligand receptor complex, binding of clatherin to adaptin, both cluster at invagination site
- clathrin polymerization, forms vacuole with coated pit
- Release of clatherin-coated vesicle into cytoplasm, shedding of clathrin coat, fucion of vesicle with endosome, dissociation of ligand-receptor complexes
- Potential recycling or transfer remains to lysosome for degredation
Cytokine receptor action
binding of ligand facilitates association and activation of cytoplasmic enzymes, particularly tyrosine kinases, receptor lacks intrinsic enzymatic activity
Cytokine receptor system
paracrine, autocrine
Cytokine receptor ligands
cytokines, some GFs
Cytokine receptor binding
Kd=10^-9 to 10^-6 M
Cytokine receptor control
long lasting changes in gene expression (cell growth/differentiation)
Cytokine receptor examples
Class I- interleukin: IL-2 dimers
Class II: interferon: IFN multimers
Tumor necrosis factor : trimers
Cytokine receptor drugs
cancer, antivirals, immunosuppressives
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
Functional Cytokine receptor
multimeric complexes (two or more)
Cytokine receptor mechanism
- Inactive
- Cytokine binds–> dimerization of JAK
- JAK cross phosph, subunit phosphorylation
- STATs bind to subunit
- Phosphorylation of STATs, activated
- STATS dissociate and dimerize
- Translocation to nucleus–> altered gene expression
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
Normal class II cytokine receptor mechanism (without ebola)
- IFN-gamma binds to JAK, activates it
1b. Release of phosphorylated STAT1s and subsequent dimerization - Binding of STAT1 dimer to importin alpha5 subunit of importin alpha5beta complex
- This is transported through nuclear por
- where it dissociates by Ran-GTP
- so STAT1 can bind to DNA targets (GAS)
- This leads to expression of antiviral respons.
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
Rate of formation Ligand receptor
k(on)[L][R]
Rate of LR dissociation
k(off)[LR]
equilibrium dissociation constant
k(on)[L][R]=k(off)[LR]
so
Kd=K(off)/k(on)
=[L][R]/[LR]
small Kd
high affinity for ligand
large Kd
low affinity for ligand
Bmax
=total # of receptors=[R]o at t=0
[LR]=0
At t(equilibrium)
Free receptor =[R]-[LR]
Free ;igand = [L]-[LR]=approx [L]o
bound ligand= [LR]
Saturation binding relation
[LR]=[R][L]/(Kd+[L])
Bound= Bmax (free/(Kd+Free)
if free is»_space; Kd, then bound=Bmax
if free =Kd, then bound =.5Bmax
nonlinear regression used to measure
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
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
Dose response
Half Maximal Effective concentration = EC50
EC50
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
