• Release of Signal

• Binding of Ligand toReceptor

• Signal Transductionand Amplification

• Cellular Response

• Response of Organ and Organism

Communication in the Organism

Principles in intercellular communication:Release of Signals

GAP-Junction Receptor mediated

Secreted molecules Membrane boundligand

Hormone

granulocytes/endothel. cells via selectineT-cell receptor/MHC complex

autocrineparacrineendocrineEicosanoideNeurotrans-mitter, NO

Interleukine

A B

3

Model ofGap-Junction:

6 TM-Proteins/cellform Connexon

GAP-Junctions

4

Cell‐Cell Contact

5

Principles in intercellular communication

Endocrine pathway

Cell of Gland

Hormone receptor

Targetcell

HormoneBlood stream

paracrine

autocrine

Principles in intercellular communication

…synapse

Receptor Binding

7

Describes how and where the ligand interacts with the receptor!

8

Signal Transduction

Affinity and Activity

[c4,C7] Chem9 derivates

-12 -10 -8 -6

0

50

100

150

Chemerin-9

cp5 cp7

cp2log c [M], peptide

Res

pons

e [%

]concentration/response

versusdose/response

• Frequently no linear correlation: semilogarithmic scale

• Effect correlates with occupied receptors, but frequently more, because of second messenger: maximal effect achieved before all receptors are occupied.

Dose/Concentration-Response Curves

11

Emax

Emax2

EC50

Efficacy = Maximal effect, correlates with intrinisic activity

EC50 = concentration of half maximal activity

EC80, EC20 = concentration with 80% or 20 % max. effectPotency = pEC50 = negative Log of molar EC50-concentration

Intrinsic Activity = Efficacy

Definition

Conc. Ligand

Effe

ct

12

Important Values

13

Full/Partial Agonist

• A und B full agonists with different affinity• A is a partial agonist• Different slope, different binding mechanism

Efficacy:C<A=B

EC50:C<A<B

Potency:C>A>B

14

Antagonist Effect

Competitive antagonist• Competes with the agonist • Shifts concetration-response curve of agonist• Has no influence on maximal activity of agonist• Effect can be reverted with high concentrations of agonist

Receptor Antagonist Complex

noagonistbinding

15

Antagonist Effect

Receptor Agonist Complex

noeffect

Non competitive antagonist•Different binding site of agonist and antagonist •Lowers maximal effect of agonist, EC50 is maintained•Curve gets less steep•Effect cannot be reverted with high concentrations of agonist

Antagonist

16

Inverse Agonists

17

Introduction in Receptor Theory

Induced Fit: Receptor conformation is changed by the bindingof a ligand (agonist), which leads tosignal transduction. Antagonists bind, but do not lead to a conformational change

Conformational Selection: There exists an equilibrium ofdifferent receptor conformations, agonistsshift the equilibrium to the active conformation,inverse agonists to the inactive,antagonists don‘t change the equilibrium.

18

R

R

Agonist:Stabilizes active conformation

EffectorInverse Agonist:Stabilizes inactiveconformation

Conformational Selection

Antagonist: stabilisation of equilibrium

Constitutively activereceptors

R*

R*

L

KAKA*

R*

19

Advanced Pharmacology

Methods to Study Receptors

Cell System

Ligand-Receptor InteractionAgonist-Antagonist-inverse Agonist

BindingSignal Transduction

Receptor Mutagenesis

Protein-Protein InteractionReceptor DimerisationInteracting MoleculesReceptor Trafficking

Cell Systems

• Tissue, which is rich of endogenously expressedreceptors, e. g. homogenized brain, rat liver, rabbit kidney….

• Cell lines that endogenously express the receptorImmortilized cells (tumor or induced immortality)

ATCC (SK-NM-C, MCF7, etc.)Primary cells

• Transfected cell lines (stabile or transcient)

Cell line Origin Properties

CHO hamster Stable, transcient

BHK hamster Stable, transcient

HEK 293 human Stable, transcient

COS monkey SV40 antigene,Only transcient

Expression vector(plasmid)

Origin of replication

Beta-Lactamase (resistance gene E. coli)

Eukaryotic transcription unit:

Receptor

Selection marker

mutant cell

Defect is complemented by plasmid

Cells die as theyMiss something

Minimal media

transfectionRezessiv marker

normal cell

detoxification by plasmid

Cells die in toxicmilieu

toxic media

transfectiondominant marker

Frequently used genes for selection marker

Lipofection

defect cell

endocytosis

Mix Lipid solution

slow degradation offoreign DNA

Assays to test receptors

Affinity: Binding assays: Kd-Value, BmaxAutoradiography: Receptor distribution

Activity: functional assaysAgonistsAntagonistsSuperagonistsinverse Agonists

Activity without ligand: constitutive activity

30

Receptor Binding Assay

Receptor Binding

Cell lineCell line

centrifugationorfiltration

32

Radioactive labelling of hormones

Why?• Tracer for binding studies and autoradiography• Biodistribution and pharmacokinetics (stability)

How?• Direct iodination of tyrosine• Bolton‐Hunter (like) reaction of lysine

33

Direct iodination of tyrosine

Autoradiography of brain slices with125I nociceptin

Classical procedure

separation

Chloramine T

34

Bolton‐Hunter (like) reaction of lysine

125I

Lys

ON

O

O

O3H3H

3H

3H 3HTritiation by propionic acid NHS

NHS N‐hydroxy succinimide, Lys lysine, SFB = Succinimidyl‐fluorobenzoate

18F

O

ON

O

O

[18F]‐SFB

[18F]‐Fluoride

18F

NH

O

Peptid

[18F]‐Fluorobenzoyl‐Peptide

(Cyclotron)

+ Peptid

Affinity: specific binding at receptor

Specific bindingBinding of compound to receptor,saturable

Non specific binding1. Binding of ligand to other binding sites (same receptor, other

enzyme, transporter, etc. ), saturable2. Binding to non-receptor components of the tissue, membrane,

uptake in cells, or vesicle, non-saturable3. Unbound ligand that could not be separated from the bound ligand,

non-saturable

RBC3 - 36

total binding: Variation of concentration of radioligand

non-specific binding: Variation of concentration of radioligand in the presence of unlabelled ligand (c > 100-1000-fold Kd)

Kd represents affinity

Bmax receptor number per cell

Saturation Experiment, Kd-Value

Kd

Bmax

[L] Occupation of receptor

0 0%

1 x Kd 50%

4 x Kd 80%

9 x Kd 90%

99 x Kd 99%

Bmax – all receptors are occupied with ligand

Kd – conentration of ligand required for 50 % occupied receptors

• Specific binding of ligand and receptor is saturable and goes along classic kinetics

L + R LRkon

koff

LR

LRRLLR

RL max

on

offd

kkK• equilibrium:

bindingspec.L

LR[LR]d

max

K

kon: constant of association velocitykoff: constant of dissociation velocity

• Kd : equilibirum dissociation constant for a specific ligand L

… direct test to test binding of a radioligand to the specific receptor, determination of Kd and Bmax

max

Saturation Experiment, Kd-Value

Receptor Binding: Melatonin

RBC3 - 39

Typical values Bmax 10-1000 fmol binding sites per milligram of protein, Kd between 10 pM and 100 nM.Determination of Bmax and Kd:Past: Scatchard plot, today:fit data to the equation using nonlinear regression.

This analysis is based on these assumptions:• Binding follows the law of mass action and has equilibrated.• There is only one population of receptors.• Only a small fraction of the radioligand binds so that the free

concentration is essentially identical to the concentration added.• There is no cooperativity. Binding of a ligand to one binding site does

not alter the affinity of another binding site. In other words,the Kd is constant during the experiment.

Receptor Binding

RBC3 - 40

max

Receptor Analysis of Scatchard

-1/Kd

41

Binding Assays

Radiolabelled ligand; Saturation curve3H oxytocine, 125I insuline

Competition Binding

Constant amount oftracerVarying concentrationof unlabeled competitor

RBC3 - 43

Competition Binding , KI- and IC50-Values

Relevant to determine dissociation constants of unlabelled ligands that compete with the radiotracer for the same binding site

• Constant amount of radiotracer L ([L]), usually lower than KD• Variation of concentration of unlabelled ligand/inhibitor

IC50 : Concentration of Inhibitor/Ligand, required to displace 50 % of specific binding of tracer

Log IC50

0,5B0+NS

B0+NS

NS[L][I]1

[L]

ID

maxI

KK

BB

• Specific binding in absence of the inhibitors B0:

• if [L] = IC50, then BI = 0,5B0

• After rearrangement Cheng-Prusoff-Gleichung

[L][L]

Dmax0

K

BB

[L][L]0,5

[L]IC1

[L]D

max

I

50D

maxIC50

K

B

KK

BB

DK

K[L]1

IC50I

Competition Binding , KI- and IC50-Values

Concentration of tracerand KD of tracer

Competition binding [3H]DHA and β2-adrenergic ligands for human β2-adrenoceptors expressed in the CHO cells

David A. Sykes et al. Mol Pharmacol 2014;85:608-617

DHA:[3H]-dihydroalprenololantagonist

46

Competition  Binding Curve

Fixed concentration of tracer: 1 nM 3H cannabinoid

Cannabinoid receptor

47

Competition  Binding Curve

More advanced….two binding sites and Hill slope

• Heterogeneous receptors. The receptors do not all bind the unlabeled drug with the same affinity. This can be due to the presence of different receptor subtypes, or due to hetero-geneity in receptor coupling to other molecules such as G proteins.

• Negative cooperativity. Binding sites are clustered (perhapsseveral binding sites per molecule) and binding of the unlabeled ligand to one site causes the remaining site(s) to bind the unlabeled ligand withlower affinity.

• Curve fitting problems.

48

Radiobinding versus Fluorescence

49

Novel attempts: Fluorescently labelled ligands+ no radioctivity‐ High unspecific binding‐ Autofluorescence of cells‐ Sensitivity‐ Fluorescence bleaching‐ Effect affinity due to size

Fluorescene polarisation

Radiobinding versus Fluorescence

+ Solubile receptors (TF)+ Extracellular domain (RTK)- Membrane bound receptors

(GPCR, ion channel)

Tamra(tetramethylrhodamin)

50

51

Tb-labeled SNAP-CB2R monitored by HTRFScheme of the homogenous HTRF-based binding technique.

Eva Martínez-Pinilla et al. J Pharmacol Exp Ther 2016;358:580-587

Binding ligandNon binding ligand