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