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Growth Hormone & its Receptor (Growth Hormone & its Receptor (extracellular extracellular domains)domains)

7.61 Eukaryotic CellBiology: Principles and

Practice2005

Lecture 2Receptors

7.61 Eukaryotic Cell7.61 Eukaryotic CellBiology: Principles andBiology: Principles and

PracticePractice20052005

Lecture 2Lecture 2ReceptorsReceptors

Detect and process extracellular physiologicand pathophysiologic molecules

Structure of Ligand determines structure ofreceptor and Mechanism of Action: Hydrophobic

vs. Hydrophilic

Hydrophobic: Nuclear Hormone ReceptorsCytoplasmic/Nuclear Localization

Complex with hsp90 and others to hold bindingsite open for the ligand

Olefsky JM.J Biol Chem. 2001 Oct 5;276(40):36863-4

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Nuclear Hormone Receptors:Four Mechanisms used by the Estrogen Receptor

Julie M. Hall, John F. Couse, and Kenneth S. Korach J. Biol. Chem., Vol. 276, Issue 40, 36869-36872, 2001

The retinoic acid receptor, in the absence of a hormone, is a repressor and it collects a series ofrepressor proteins like a magnet. Addition of the ligand reverses the polarity of the magnet,causing the repressors to fly off and the coactivators to bind, resulting in chromatin modification toactivate transcription. This is how the hormonal or physiological signals affect the recruitment of co-factors that modify the chromatin in target genes. Every cell has receptors and therefore canrespond, but each cell responds in its own unique way, and thus the same hormone can have adifferent effect in a neuron versus an epithelial skin cell versus a bone cell. Evans RM. 2003. PPARs andthe complex journey to obesity.Keio J Med. 2004 Jun;53(2):53-8.

48 HUMANNR GENES

Hydrophilic Ligand/Receptor SystemPlasma Membrane is Permeability Barrier

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WHAT ARE THE CHARACTERISTICPROPERTIES OF A LIGAND/RECEPTOR INTERACTION?

How do you define a receptor, how do you find a receptor,how do you study receptors?

MEASURE BINDING

Receptor-preparation:Intact Cells:

Cultured cells, tissue slices, perfused organs, whole organism

Ligand-preparation:Detection

Avoid artifacts of labeling(e.g. denaturation, crosslinking)

How to Label Ligands

Protein Ligands

Protein Labeling:

RadioisotopesFluorescent dyes

Epitope tagsOthers (biotin)

125I-Protein[3H]Protein

[35S]methionine

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BINDING Assays

Variables:Concentration, Time, Temperature

201510500

No AdditionsAcLDLSpecificControl

A. Saturation Kinetics

I-AcLDL (µg protein/ml)125

0

100

200

300

400

Rece

ptor

Act

ivity

“Hot + Cold”

Look out for bad data and beware of people who only show delta!

Alternative to Chemical Modification of Ligand

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Immunoreceptor vs Radioreceptor Assays

For immunorreceptor assay, nonspecific iscalculated by regression analysis

100%

time after binding

Reversible Bindingto Receptor

Off rates are criticalmust not loose signal during washing

Binding vs Cell Association:Inhibit uptake at 4°C

Quantitative Analysis:How many binding sites? How tight is the binding?

Rigorous analysis depends on system being in equilibrium

BINDING Assays

R + L RL

R = unbound receptorsL = unbound ligandRL = ligand/receptor complex

Rt = total # of receptors (binding sites) = R + RL

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Classic Scatchard Analysis

Bound/Free

Bound (RL)

slope = -1/K

intercept = R

d

t

[RL]bound

free

Kdiss

[RL][L]

[R ]Kd

tKd

[RL]=

Rt = R + RL

freebound =

=[R][L]

Simple Straight Line:Single Class of Binding

Sites

R = unbound receptorsL = unbound ligandRL = ligand/receptor complex

Rt = total # of receptors (binding sites) = R + RL

R + L RL

Complications!Error propagation

Bound

Bound/Free

0 2 4 6 8 100

2

4

6

Complications!Cannot always trust your eyes!

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Direct Plot:

ScatchardPlot:

-ive coop. +ive coop.

B/F

BBound (RL)

Bound/Free

fewer high affinitymore abundent lower affinity

Non-linear Scatchard Plots:Multiple Classes of Binding Sites

.

0 10 20 0 10 20 300

2

4

B (nM) B (nM)

0

2

4

Boun

d/Fr

eeBo

und/

Free

A. Correct Nonlinear Fit B. Correct Graphical Fit

C. Incorrect graphical fit D. Incorrect graphical fit

a

b

2

4

Boun

d/Fr

ee

00

Old FashionGraphicalAnalysis -

Not Good forQuantitativeDeterminations

2

4

Boun

d/Fr

ee

Correct Nonlinear Fit

0 10 20 30B (nM)

.

0 10 20 0 10 20 300

2

4

B (nM) B (nM)

0

2

4

Boun

d/Fr

eeBo

und/

Free

A. Correct Nonlinear Fit B. Correct Graphical Fit

C. Incorrect graphical fit D. Incorrect graphical fit

a

b

2

4

Boun

d/Fr

ee

00 10 20 30

B (nM)

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http://www.graphpad.com/http://www.graphpad.com/curvefit/introduction9e.htm

Use Non-linear Regression AnalysisGood Packaged Programs Available

E.g., GraphPad Prism

Plot Graph and Calculate!

Sources of Nonlinearity

Multiple Classes of Binding Sites

Bound (RL)

Bound/Free

fewer high affinitymore abundent lower affinity

-ive coop. +ive coop.

B/F

B

Complex Classes of Binding Sites- Negative and Positive Cooperativity- ensemble effect

Sources of NonlinearityMultiple Classes of Binding Sites

Complex Classes of Binding Sites -Negative and Positive

Cooperativity

Poor Data:Nonequilibrium Conditions

Instability of Ligand or ReceptorSite

Ligand Problems: Badmodifications, Dilution of ligand

(endogenous ligand)

Biological Relevance: LDL and Glass Beads

[inadequate ligand concentration range]

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Ligand Structure/Specificity β-Adrenergic Receptor System

R

C C NH CH 2

RHOHO

HO

H

CCH 3CH 3

= epinephrine

= isoproterenol

effects response effectsbinding

epinephrine + ßAR -> activation of adenylate cyclase -> increased cAMP

You can differentiate to some extent effects on binding andresponse, e.g.:

R = H (epinephrine); Kd = 5 x 10-6 MR = C3 (isoproterenol); Kd = 0.4 x 10-6 (higher affinity)

both epi. and isopro. stimulate cyclase(called ‘Agonists’)

R=C-C-C-C-phenyl-OH Kd = 0.06 x 10-6 M

R

C C NH CH 2

RHOHO

HO

H

CCH 3CH 3

= epinephrine

= isoproterenol

effects response effectsbinding

C C NH CH 2

RHO

CCH 3CH 3

CH 2O

CH 2

R =CH 2

CH 3

Alprenolol, Kd = 3 x 10-9 M

C C NH CH 2

RHO

CCH 3CH 3

CH 2O

R =

Propranolol, Kd = 5 x 10-9 M

Bind Tightly!

Don’t Activate Cyclase!

R

C C NH CH 2

RHOHO

HO

H

CCH 3CH 3

= epinephrine

= isoproterenol

effects response effectsbinding

Antagonists(vs Agonists and Inverse Agonists)

R R*(active)

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How to isolate receptors?

A) Need an assay to follow purification radioligand binding, immunochemical detect. (Ligand blot)

Filter binding in reconstituted liposomes

Tricks to help purify:1) Affinity Labeling (crosslinking)2) Ligand affinity chromatography:

a) nonionic detergent solubilize protein - detergentmicelles

b) run on ligand-crosslinked column c) elute with competitor or altered salt or pH

3) Immunoaffinity chromatography4) Clone by functional expression (e.g., cellular response)

SR-AI (innate immunity pattern recognition receptor)Ligand affinity, ion exchange chromatography, preparativeSDS-PAGE-> monoclonal antibodyImmunoaffinity -> 240,000-fold purification from lung

SR-BI – epitope tag, express, immunoaffinity – gel

ßAR - 125I-cyanopindolol (CYP) binding assay; alprenolol-sepharose in digitonin, reconstitution requires proper lipids

Examples:

Purified BINDING ACTIVITY, but how do you know that you'veactually purified the biologically relevant receptor?

Number of indirect and some direct tests Indirect:

1) exhibits binding affinity and specificity seen in cells2) protein exhibits cell type and tissue type distribution ofactivity

3) expressed on cell surface - proteolysis, surface labelingreagents4) glycoprotein5) regulation makes sense

Direct:1) Reconstitution of activity in vitro

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cell

βAR/liposome

fuse with polyethylene glycol

e.g. Fusion of ßAR (60 kD)/liposomes to XenopusRBC and assay for cAMP production

1 20

2

4

6

8

no addition+ agonist isopro.agonist + antagonistprost. E1 or NaF

Reconstitution of purified ß-Adrenergic Receptor into Xenopus RBC

Xenopus + empty liposome Xenopus + ß-AR/liposomeagonist: isoproterenol;antagonist: propanolol

Purified BINDING ACTIVITY, but how do you know that you'veactually purified the biologically relevant receptor?

Direct:2) Express cloned gene in cells or tissues and measurebio activity3) Inhibit activity in appropriate system (blockingantibody, siRNA, KO animal)

STRUCTURE OF THE ß-ADRENERGIC RECEPTOR

Out

In

Heptahelical (7-Transmembrane) domain receptor

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7-TM or G-Protein Coupled Receptors (GPCR)

large family (>2000) - ~5% of worm genome codingsequences, perhaps 3% of mammals

α2-AR, ß1 and ß2-AR, dopamine receptors, muscarinicacetyl choline receptors (4 subclasses), sight(Rhodopsin), taste, smell, angiotensin receptor (masoncogene), serotonin receptor, a- and α- factor yeastreceptors, LH receptor, PAR (protease activated receptor)

many intronless genes!

all operate using similar signal transduction systems (G-proteins)

Major Drug Targets for Pharmaceutical Industry…

Family of structurally and functionally similar proteinsprovides special opportunity to explore structure/function

method: insert cDNA into expression vector, transfect into Lcells because they are ßAR -ive, look for binding and correctpharmacologic specificity, affinity, etc.-G-protein and cyclaserequired to work!

For example:Delete N- or C-terminus -> binding okDelete 5-6 loop (C3=i3) -> binding ok Conclusion -> not needed for binding

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For example:Co-express SR(1-5, aa1-262) +SR(6-7, aa263-end) -> still works! Conclusion -> self assemble

Delete 6-7 loop (E4) or helices 6&7 -> No binding! Conclusion -> ???????????

What can you do?

Similar structures permittesting of chimeras ofGPCRs for structuraldeterminants of ligandspecificity and downstreamtarget (G-protein) specificity

α2-AR -> decrease inadenylate cyclase activityß2-AR -> increase in adenylatecyclase

different ligand specificities

Major determinant of specificbinding in 7th membranespanning domain

B3-AR from outside of cell bindingto norepinepherine

Combined Use of Chemistry (Pharmacology - alter structureof the ligand) and Molecular Biology (mutagenesis- modify

putative ligand binding sites) to define Ligand/ReceptorInteractions - ‘complementary mutations’

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Side chain interactions within the transmembrane region of the β2-adrenergic receptor with agonist isoproterenol (A) and antagonistsalprenolol (B), as derived from mutagenesis and/or modelling studies.For clarity of pictorial representation the interaction of Phe-289 is notshown.

Agonist:Isoproterenol

Antagonist:Alprenolol

Scheme 3. Schematicdrawing of theinteraction of ATP withthe P2Y1 receptor asderived frommutagenesis andmodeling studies. Forreasons of clarity theinteraction withArg310(TM7) is notshown.

Palczewski K, Kumasaka T, Hori T, Behnke CA, Motoshima H, Fox BA, Le Trong I, Teller DC, Okada T, Stenkamp RE,Yamamoto M, Miyano M. Crystal structure of rhodopsin: A G protein-coupled receptor. Science. 2000 Aug 4;289(5480):739-45. Henry R. Bourne and Elaine C. Meng Rhodopsin Sees the Light Science 2000 289: 733-734

Figure 2. Ribbon drawing of rhodopsin (A) viewed parallel to the plane of the membrane and (B)seen from the extracellular (intradiscal) side of the membrane revealing a counterclockwisearrangement of the 7-TM helices.[8] The 11-cis-retinal molecule is shown in red. The figureshave been prepared using the programs Molscript[102] and Raster3d.[103]

11-cis-retinal

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Figure 4. Model ofRS-102895 boundto the MCP-1receptor. RS-102895 is thespace-fillingmolecule in thecenter of the bundleof helices, indicatedby the ribbons.[33]Receptor residueGlu291 is shown inthe ball-and-stickpresentation; it isshown interactingwith the basicnitrogen atom of thespiropiperidinestructure. Pleasenote that in contrastto Figure 2 B theview from theintracellular side isdisplayed

Figure 3. Three-ligand binding sites ofthe 5-HT1A receptor in a rhodopsin-based 3D model according to Jacobyand co-workers.[24] Left: extracellularview; right: side view with extracellularside at the top. The three ligands areserotonin or 5-HT (yellow),propranolol (cyan), and 8-OH-DPAT(green). Residues identified bymutagenesis data are indicated. The 5-HT site is located between TM3 (TM III;with Asp116 as the key recognitionsite) and TM5 (TM V; providing Ser199and Thr200 to interact with the 5-OHgroup of serotonin). A second site, thepropranolol binding site, is locatedbetween TM3 and TM7 (contributing,for example, Asn386 to hydrogen bondthe oxygen atoms of the oxy-propanolamine fragment in beta -blockers). The third binding site, the 8-OH-DPAT binding site is also locatedbetween TM3 and TM7. Ligandsaddressing this site, like 8-OH-DPAT,are thought to be oriented parallel tothe helices (interactions by 8-OH toSer393 and Asn396 and by aminogroup to Asp116).

Desensitization: tendency of biological responses to wane overtime despite continuous presence of stimulus of constant intensity.How- inactivate, destroy, or sequestration

Homologous desensitization : ß-adrenergic receptor kinase(ßARK) phosphorylates receptor and lowers activity somewhat,after phosphorylation, ß-arrestin binds and really drops activity.NB at high ligand occupancy

Heterologous desensitization: general reduction on stimulation ofother receptors, protein kinase A (PKA) plays a role here, PKA isabout 6x slower than ßARK at phosphorylating the receptor also,ßARK mediated desensitization is t1/2<15 sec while PKA is 3.5min (>14 fold) see Roth et al, PNAS 88:6201-6204 (91)), arrestinindependent.

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beta-Arrestin, a promiscuous mediator of receptorendocytosis. (A) Binding of ligand to its GPCR activatesheterotrimeric G proteins, causing dissociation of thealpha subunit from the beta/gamma dimer (not shown).The betagamma dimer, anchored to the inner surface ofthe plasma membrane by the prenylated gammasubunit, facilitates translocation of GRK to the plasmamembrane. This enables GRK to phosphorylate theserine/threonine motif in the GPCR carboxyl terminus,resulting in beta-arrestin translocation to the GPCR.beta-Arrestin binds to adaptor proteins in clathrin-coated pits, resulting in endocytosis of the GPCR, whichis then either recycled or degraded with concomitantactivation of G protein-independent signaling pathways.(B) On binding to its ligand, the Fz4 receptor inducestranslocation of Dvl2 to the plasma membrane.Phosphorylation of Dvl2 by protein kinase C (PKC) leadsto binding of beta-arrestin 2. Binding of Wnt5A to thecysteine-rich domain (CRD) in the extracellular aminoterminus of Fz4 leads to endocytosis of the Fz4complex. (C) Transforming growth factor-beta type IIreceptor (TbetaR-II) binds to, and its intracellular kinasedomain phosphorylates, the intracellular carboxylterminus of TbetaR-III. This leads to translocation ofbeta-arrestin 2 to the plasma membrane, followed byendocytosis of the TbetaR-II/TbetaR-III complex. Thisprocess does not require binding of TGF-beta to itsreceptor, hence the TbetaR-II extracellular binding site(triangle) is depicted as empty.

end