1Growth Hormone & its Receptor (Growth Hormone & its Receptor (extracellular extracellular domains)domains)7.61 Eukaryotic CellBiology: Principles andPractice2005Lecture 2Receptors7.61 Eukaryotic Cell7.61 Eukaryotic CellBiology: Principles andBiology: Principles andPracticePractice20052005Lecture 2Lecture 2ReceptorsReceptorsDetect and process extracellular physiologicand pathophysiologic moleculesStructure of Ligand determines structure ofreceptor and Mechanism of Action: Hydrophobicvs. HydrophilicHydrophobic: Nuclear Hormone ReceptorsCytoplasmic/Nuclear LocalizationComplex with hsp90 and others to hold bindingsite open for the ligandOlefsky JM.J Biol Chem. 2001 Oct 5;276(40):36863-42Nuclear Hormone Receptors:Four Mechanisms used by the Estrogen ReceptorJulie M. Hall, John F. Couse, and Kenneth S. Korach J. Biol . Chem ., Vol. 276, Issue 40, 36869-36872, 2001The 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 GENESHydrophilic Ligand/Receptor SystemPlasma Membrane is Permeability Barrier3WHAT 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 BINDINGReceptor-preparation:Intact Cells:Cultured cells, tissue slices, perfused organs, whole organismLigand-preparation:DetectionAvoid artifacts of labeling(e.g. denaturation, crosslinking)How to Label LigandsProtein LigandsProtein Labeling:RadioisotopesFluorescent dyesEpitope tagsOthers (biotin)125I-Protein[3H]Protein[35S]methionine4BINDING AssaysVariables:Concentration, Time, Temperature201510500No AdditionsAcLDLSpecificControlA. Saturation KineticsI-AcLDL (µg protein/ml)1250100200300400Receptor Activity“Hot + Cold”Look out for bad data and beware of people who only show delta!Alternative to Chemical Modification of Ligand5Immunoreceptor vs Radioreceptor AssaysFor immunorreceptor assay, nonspecific iscalculated by regression analysis100%time after bindingReversible Bindingto ReceptorOff rates are criticalmust not loose signal during washingBinding vs Cell Association:Inhibit uptake at 4°CQuantitative Analysis:How many binding sites? How tight is the binding?Rigorous analysis depends on system being in equilibriumBINDING AssaysR + L RLR = unbound receptorsL = unbound ligandRL = ligand/receptor complex Rt = total # of receptors (binding sites) = R + RL6Classic Scatchard AnalysisBound/FreeBound (RL)slope = -1/Kintercept = Rdt[RL]boundfreeKdiss[RL][L][R ]KdtKd[RL]=Rt = R + RLfreebound==[R][L]Simple Straight Line:Single Class of BindingSitesR = unbound receptorsL = unbound ligandRL = ligand/receptor complex Rt = total # of receptors (binding sites) = R + RLR + L RLComplications!Error propagationBoundBound/Free0 2 4 6 8 100246Complications!Cannot always trust your eyes!7Direct Plot:ScatchardPlot:-ive coop.+ive coop.B/FBBound (RL)Bound/Freefewer high affinitymore abundent lower affinityNon-linear Scatchard Plots:Multiple Classes of Binding Sites.0 10 20 0 10 20 30024B (nM)B (nM)024Bound/FreeBound/FreeA. Correct Nonlinear Fit B. Correct Graphical FitC. Incorrect graphical fit D. Incorrect graphical fitab24Bound/Free00Old FashionGraphicalAnalysis -Not Good forQuantitativeDeterminations24Bound/FreeCorrect Nonlinear Fit0 10 20 30B (nM ).0 10 20 0 10 20 30024B (nM)B (nM)024Bound/FreeBound/FreeA. Correct Nonlinear Fit B. Correct Graphical FitC. Incorrect graphical fit D. Incorrect graphical fitab24Bound/Free00 10 20 30B (nM )8http://www.graphpad.com/http://www.graphpad.com/curvefit/introduction9e.htmUse Non-linear Regression AnalysisGood Packaged Programs AvailableE.g., GraphPad PrismPlot Graph and Calculate!Sources of NonlinearityMultiple Classes of Binding SitesBound (RL)Bound/Freefewer high affinitymore abundent lower affinity-ive coop.+ive coop.B/FBComplex Classes of Binding Sites- Negative and Positive Cooperativity- ensemble effectSources of NonlinearityMultiple Classes of Binding SitesComplex Classes of Binding Sites -Negative and PositiveCooperativityPoor Data:Nonequilibrium ConditionsInstability of Ligand or ReceptorSiteLigand Problems: Badmodifications, Dilution of ligand(endogenous ligand)Biological Relevance: LDL and Glass Beads[inadequate ligand concentration range]9Ligand Structure/Specificity β-Adrenergic Receptor SystemRC C NH CH2RHOHOHOHCCH3CH3= epinephrine= isoproterenoleffects responseeffectsbindingepinephrine + ßAR -> activation of adenylate cyclase -> increased cAMPYou 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 MRC C NH CH2RHOHOHOHCCH3CH3= epinephrine= isoproterenoleffects responseeffectsbindingC C NH CH2RHOCCH3CH3CH2OCH2R =CH2CH3Alprenolol, Kd = 3 x 10-9 MC C NH CH2RHOCCH3CH3CH2OR =Propranolol, Kd = 5 x 10-9 MBind Tightly!Don’t Activate Cyclase!RC C NH CH2RHOHOHOHCCH3CH3= epinephrine= isoproterenoleffects responseeffectsbindingAntagonists(vs Agonists and Inverse Agonists) R R*(active)10How to isolate receptors?A) Need an assay to follow purification radioligand binding, immunochemical detect. (Ligand blot) Filter binding in reconstituted liposomesTricks to help purify:1) Affinity Labeling (crosslinking)2) Ligand affinity chromatography:a) nonionic detergent solubilize protein - detergentmicellesb) run on ligand-crosslinked column c) elute with competitor or altered salt or pH3) 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