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Signal Transduction
March 2009March 2009
Advanced Biochemistry CourseAdvanced Biochemistry Course
First lectureFirst lecture
Plasma Membrane Structure and FunctionPlasma Membrane Structure and Function
The plasma membraneplasma membrane separates the separates the internal environment of the cell from its internal environment of the cell from its surroundingssurroundings
The plasma membrane is aThe plasma membrane is a phospholipid phospholipid bilayerbilayer withwith embedded proteinsembedded proteins.
The plasma membrane has a fluidfluid consistency and aconsistency and a mosaicmosaic pattern of pattern of embedded proteins.embedded proteins.
Protein dynamics in the lipid bilayerProtein dynamics in the lipid bilayer
1) Proteins can move laterally in the plane of the membrane (capping)2) Proteins can rotate around an axis vertical to the plan of the membrane (channels)3) Proteins cannot tumble through the plan of the membrane
LateralLateral
RotationalRotational
ThumblingThumbling
Signal Transduction
Endocrinic- “ (Insulin; adrenalin)
Paracrinic -transduction (histamin;prostaglandins)
Autocrinic - “ (TGF/ IGF)
Synaptic - transmission (neurotransmitters)
Models of Cell-Cell Models of Cell-Cell signalingsignaling
(D)
Receptors
Membrane receptors Cytosolic receptors
1. G-coupled receptors2. Channel/receptors3. Enzyme/receptors
1. Steroid receptors2. Vitamin D3. Retinoic acid
Cell surface receptorsCell surface receptors
Gramicidin A in lipid bilayer and water
Antibiotic peptide
Forms a pore in the cell wall of a bacteria and lets out monovalent cations (K+, Na+).
[Membrane potential disappears and bacteria dies!]
15 amino acids, helicalChannel is formed by a head- to-head dimer
3.5 nm3.5 nm3.5 nm
4 nm
Glycophorin
The Free Energy for Transferring a Helix of 20Residues from the Membrane to Water
Hydropathy plotHydropathy plot
-adrenergic receptor
Rhodopsin
Membrane topology
One of the largest families of membrane proteins Common structural architecture
Extracellular N-terminal domainExtracellular N-terminal domainGlycosylated
Ligand recognition
Intracellular C-terminal domainIntracellular C-terminal domainContains several putative phosphorylation sites
Involved in desensitisation/internalisation
7 membrane spanning domains7 membrane spanning domains
Couple to G-proteins signal transduction
Divided into subfamilies based on sequence homology
definitiondefinition
G-protein-coupled receptorsG-protein-coupled receptors
Current estimation ~1000 GPCRs in human genomeCurrent estimation ~1000 GPCRs in human genome
How many GPCRs are encoded by the human genome?How many GPCRs are encoded by the human genome?
Orphan GPCRsOrphan GPCRs
First estimation based on comparison with First estimation based on comparison with C. elegansC. elegans
19.100 genes19.100 genes ~1000 GPCRs~1000 GPCRs ~5% of genome~5% of genome
HuGoHuGo ~1800 GPCRs~1800 GPCRs
~700 olfactory, gustatory and chemokinine receptors~700 olfactory, gustatory and chemokinine receptors
~300-400 transmitter GPCRs300-400 transmitter GPCRs
27.000 genes27.000 genes
210 GPCRs bind un known natural ligands
160 orphan GPCRs160 orphan GPCRs remain to be characterised
~400 transmitter GPCRs400 transmitter GPCRs
Orphan GPCRsOrphan GPCRs
Rationale for oGPCR characterisationRationale for oGPCR characterisation
A. GPCRs are good drug targets
50% of subscription drugs interact with 50% of subscription drugs interact with GPCRsGPCRs
Why is the pharmaceutical industry interested in oGPCRs?
• Hypertension• Stomach ulcers• Migraine• Allergies
B. GPCRs in disease states
Disease states associated with GPCR Disease states associated with GPCR mutationsmutations•Rhodopsin
receptor retinitis pigmentosa•Vasopressin
V2nephrogenic diabetes•Glucago
ndiabetes, hypertensionGRF-receptor dwarfism Asp(60) Gly (60)
GPCR subfamiliesGPCR subfamilies
•Largest family•Conserved DRY motif (i2)•Conserved cysteines -S-S-
Family A: Rhodopsine-like
Family B: Secretine-like
•Large N-terminal domain•Several well conserved
cysteine residues•High Mr hormone ligands
Family C: Metabotropic glutamate
•Long N-terminal domain•N-terminus sufficient for
ligand binding
R R*
S-F-L-L-R-N
Protease activated receptor (PAR)Protease activated receptor (PAR)
Thrombin (LARGE GREEN SPHERE) recognizes the N terminal of the thrombin receptor called PAR1. The c-terminal (RED oval) and the N-terminal (Blue oval) are involved in the binding. This is similar to the sequence of the thrombin hyrudin inhibitor. Thrombin cleaves the peptide bond between Arg 41 and Ser42. This unmasks a sequence S-F-L-L-R-N. This is sequence that the receptor recognizes and is activated. The synthetic S-F-L-L-R-N activates the PAR-1 receptor independent of the cleavage
Lipid head groupsPolar/negatively charged
Chains hydrophobic
Lipid head groups
The positioning of the the 7TMR in the membraneThe positioning of the the 7TMR in the membrane
Extracellular
Cytoplasmic
COOH-
-NH2
i1
i2
i3
e1e2 e3
TM1 TM2 TM3 TM4 TM5 TM6 TM7
DRY
G-protein-coupled receptorsG-protein-coupled receptors
-S-S-
How does the ligand activates the How does the ligand activates the receptor in a selective way? receptor in a selective way? A two state model is commonly used to A two state model is commonly used to characterize this activationcharacterize this activation
Fluorescence spectroscopy was used to Fluorescence spectroscopy was used to characterize the diversity of characterize the diversity of conformational states of the conformational states of the 2AR and its 2AR and its mode of activationmode of activation
AGONIST-INVERSE AGONIST - AGONIST-INVERSE AGONIST - ANTAGONISTANTAGONIST
● Drug effects can be classified into three major phenotypes: agonistagonist, , antagonistantagonist and inverse and inverse agonistagonist. .
● AgonistAgonist and inverse agonistinverse agonist effects are associated with receptor activation and inactivation, respectively
● AntagonismAntagonism implies that a drug produces no effect when administered alone but blocks the effects of agonists and inverse agonists.
Inverse agonist - a ligand that prefers the inactive form of the receptor
Agonist- a ligand that activates the receptor
Antagonist- a ligand that inhibits the receptor
Partial agonist- a low affinity agonist
r R*Inactive formInactive form Active formActive form
Two- State Model
r R
Agonist
rA R*A
InverseAgonist
activeinactive
Partial agonists and antagonistsbind to both r and R states
Receptor states and inverse agonists
Activation in the absence of an agonist; over-expression
(Two-State Model)
Energy landscape diagram describing a possible mechanism of GPCR activation by an agonist
1) Inverse agonistInverse agonist (propranolol) binds to the rr form of the receptor and the activity of the system is suppressed below its normal spontaneous state2) In between fullfull and and inverse agonistsinverse agonists are those agonists that bind to
both rr and RR states. These are partial agonistspartial agonists. These are unable to achieve maximal stimulation even if all receptor binding sites are occupiedInverse agonism offers a potential of developing new drugs that attenuate the effect of mutant receptors that are constitutively active
3) The neutral antagonistsneutral antagonists (-blockers such as pindolol) bind to both rr
and RR conformations and are better regarded as passive antagonistspassive antagonists They impede the binding of both agonists and inverse agonists.Therefore, pindolol affects the heart only during exercise and stress while propranolol also suppresses the resting heart rate
Activation of G-protein-coupled receptors
Ligand Efficacy: The effect of different classes of drugs on a GPCRthat has some detectable basal activity
FULL AGONISTFULL AGONIST PATRIAL AGONISTPATRIAL AGONIST
ANTAGONISTANTAGONIST INVERSE AGONISTINVERSE AGONIST
RhodopsinRhodopsin
DopamineDopamine
How can we determine the How can we determine the mechanism of activation?mechanism of activation?
Three different methods are used, which Three different methods are used, which could be applied to explore the could be applied to explore the
mechanism of activation of 7TMR or any mechanism of activation of 7TMR or any other receptors other receptors
The focus will be on the adrenergic The focus will be on the adrenergic receptorsreceptors
F R E T fluorescence resonance energy transfer
A donor chromophore, in its electronic excited state, may transfer energy to an acceptor chromophore (in
close proximity < 10nm) through Non-radiative dipole-dipole coupling
When both chromophores are fluorescent, the term
"fluorescence resonance energy transfer (FRET)" is often used instead, although the energy is not
actually transferred by fluorescence
Fluorescence resonance energy transfer (FRET)Fluorescence resonance energy transfer (FRET) The two fluorescent probes report in real time through a fastdecrease in FRET the intra-molecular conformational rearrangements associated with receptor activation
Norepinephrine (NE)Norepinephrine (NE)AgonistAgonist
Inverse agonistInverse agonistYohimbineYohimbine
Swaminath, G. et al. J. Biol. Chem. 2004;279:686-691
Binding site for norepinephrine in the 2AR
Top-down view of hormone receptor with an adrenalin molecule
A multi-step agonist bindingA multi-step agonist binding
good partial Agonist
selective Agonist
selective Agonist
selective Agonist
inverse Agonist
antagonist
weak partial agonist
Gether et al., J. Biol. Chem. 1998;273:17979
Arrangement of transmembrane domains of a prototypical G protein-coupled receptor as viewed from the extracellular surface of the membrane (based on the projection maps
from two-dimensional crystals of rhodopsin)
The Asp3-Arg3 pair at the cytoplasmic end of transmembrane domain 3 (TM3) is part of the highly conserved (D/E)RY motif found in beta2-AR and other rhodopsin-family GPCRs, whereas the Glu6at the cytoplasmic end of TM6 is highly conserved in amine and opsin receptors. The ionic link between the Asp3-Arg3pair and Glu6 is known as the ionic lock
Break ionic lock
Activate rotamer toggle switch
The Ionic LinkThe Ionic Link
The Asp3-Arg3 (D/R)Asp3-Arg3 (D/R) pair at the cytoplasmic end of
transmembrane domain 3 (TM3TM3) is part of the highly conserved (D/E)RY(D/E)RY motif found in 2AR and other rhodopsin-family GPCRs, whereas the
Glu6Glu6 at the cytoplasmic end of TM6TM6 is highly conserved in amine and
opsin receptors. The ionic link between the Asp3-Arg3Asp3-Arg3 pair and Glu6 Glu6 is known
as the ionic lockionic lock
How is the receptor activated ?
● Previous biophysical studies on the 2-AR suggest that agonist
binding and activation occurs through at least one
conformational intermediate, implying that at least one
molecular switch is involved.
● These studies also show that structurally different agonistsdifferent agonists and
partial agonistspartial agonists differ in their ability to induceinduce specific
conformational transitions.
Swaminath, G. et al. J. Biol. Chem. 2004;279:686-691
Cellular responses to catecholamines
cAMP accumulation in HEK cells expressing 2AR
The state that stabilizesthe binding of the catechol ring alone (R1) is not sufficient toactivate the G proteins
Fluorescence spectroscopy Fluorescence spectroscopy to monitor disruptionto monitor disruptionof the ionic lockof the ionic lockat the at the AR AR
Fluorescence spectroscopy Fluorescence spectroscopy to monitor disruptionto monitor disruptionof the ionic lockof the ionic lockat the at the AR AR
Mutated A271C and binding of mBrBimaneMutated I135WRelies on the quenching of of bimane fluorescence by Trp at near contact distance in the 5-15 Å range
The bimane-tryptophan technique
Quenching during agonist binding to the site
TM 6TM 6TM 3TM 3
TM6TM6 E 268E 268
TM3TM3 D R YAsp-Arg-Tyr
A271CIle135W
-9 -8 -7 -6 -5 -4 -3 -2-10
0102030405060708090
100SAL
NE
DOP
HAL
EPI
CAT
ISO+ascorlg [Ligands] (M)
IsoIso
EpiEpi
SalSal
NorepiNorepi
DopDop
CatCat
HalHalLog[ligand]Log[ligand]
% m
axim
al q
uen
chin
g%
max
imal
qu
ench
ing
Effect of ligand structure Effect of ligand structure on the ionic lockon the ionic lock
D130
R131E268
Ionic Lock
A135W
H271C-Bimane
InactiveActive
•DRYDRY
Ballesteros, J. A. et al. J. Biol. Chem. 2001;276:29171-29177
Molecular three-dimensional representations of the interaction of TM3 and TM6 at their cytoplasmic ends and the effects of 6.30
mutations
Jensen, A. D. et al. J. Biol. Chem. 2001;276:9279-9290
Proposed conformations of the inactive and active states of the 2AR
Fluorescence studies of receptor activation
These studies show that 2ARs labeled at Cys265Cys265 on the cytoplasmic end
of TM6, adjacent to the G protein–coupling domain,
is able to report conformational changes in the G protein–coupling domain.
These modifications alter the molecular environment around the
fluorophorefluorophore, which is translated to changes in fluorescence intensity and
fluorescence lifetime. In these experiments, fluorescence lifetime analysis
can detect discrete conformational states in a population of molecules,
while fluorescence intensity measurements reflect their weighted average
To monitor agonist induced conformational changes purified receptors were labeled at Cys 265 (at the third intracellular loop) with tetramethyl rhodamine tetramethyl rhodamine maleimidemaleimide -Fluorescence intensity was followed as a function of time
Norepi induced conformational changes are biphasic.
The rapid change is similar for both dopamine and norepi
Fluorescence Life-time spectroscopyFluorescence Life-time spectroscopy ex 541nm and em at 571nmex 541nm and em at 571nm
Swaminath, G. et al. J. Biol. Chem. 2005;280:22165-22171
Norepinephrine induces a biphasic conformational change in TMR-2AR
Swaminath, G. et al. J. Biol. Chem. 2004;279:686-691
Conformational changes in TMR-2AR in response to a panel of catecholamine-related ligands reveal the structural features of catecholamine ligands responsible for the rapid
and slow components of the biphasic conformational change
Swaminath, G. et al. J. Biol. Chem. 2004;279:686
Norepinephrine induces a biphasic conformational change in TMR-2AR
Differences between the L- and D- enantiomers
Swaminath, G. et al. J. Biol. Chem. 2005;280:22165-22171
Catechol-induces conformational changes in TMR-2AR in the presence of a saturating concentration of salbutamol
Swaminath, G. et al. J. Biol. Chem. 2005;280:22165-22171
Catechol (CAT) competes with isoproterenol, but not with salbutamol (SAL) or alprenolol, for binding to the
2AR
Break ionic lock
Activate rotamer toggle switch
Swaminath, G. et al. J. Biol. Chem. 2005;280:22165-22171
Molecular model of the agonist binding pocket of the 2AR
The model was generated using the crystal structure of rhodopsin as a template. TM segments involved in agonist binding are colored as follows: red, TM3; green, TM5; blue, TM6. A, the proposed binding site for isoproterenol.B, salbutamol docked into the upper (extracellular) region of the binding pocket. The aromatic ring of salbutamol interacts with Tyr-174, Phe-193, and Tyr-1995.38.
3D structure of the adrenergic receptorHigh-resolution structural information is essential for understanding
molecular mechanisms of protein function; however, some limitations of crystallography must be recognized.
In forming a crystal, a protein becomes locked in a single conformational state. This is a significant drawback when one considers the body of functional and biophysical evidence that GPCRs are conformationally
complex and dynamic proteins.
They do not behave as simple bimodal switches but adopt conformations that are specific for the bound ligand and the associated signaling partner
(e.g. G proteins, arrestins). In the reported crystal structures, the conformation of the 2AR bound to carazolol is close to an inactive state. Carazolol is an inverse agonist, but suppresses not, very similar 50% of
basal activity in the 2AR.
Therefore, the 2AR structure might not represent a fully inactive receptor and could differ significantly from the unliganded receptor or one of the
potential active states.
Nature 450 (2007), pp. 383–387
BA
BRAIN regions affected by Parkinson’s diseaseBRAIN regions affected by Parkinson’s disease
Pars compacta region of the substantia nigra in the
neuronal brain appears dark
Normal Parkinsonian
Dopaminergic Neurons
Na+
TyrosineTyrosine
Ca++
Receptor
MAOMAO
DopamineDopamine
DopaDopa
Dopamine is converted toepinephrine
TyrosineTyrosineHydroxylaseHydroxylase
Tyrosine Hydroxylase
Dopa decarboxylase
Dopamine- -hydroxylase
Norepinephrine- transmethylase
Pathway for the synthesis of catecholamines
The receptor and effector are independent entities
GG
L
Effector
G-proteinsG-proteins
Signal
Swaminath, G. et al. J. Biol. Chem. 2004;279:686-691
Norepinephrine (Norepi) induces a biphasic conformational change in TMR-2AR
Swaminath, G. et al. J. Biol. Chem. 2004;279:686-691
Norepinephrine induces a biphasic conformational change in TMR-2AR