7.61 Eukaryotic Cell Biology: Principles and Practice Lecture 2 Receptors. (extracellular domains) Growth Hormone & its Receptor (extracellular

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1 Growth Hormone & its Receptor (extracellular (extracellular domains) 7.61 Eukaryotic Cell Biology: Principles and Practice 005 Lecture Receptors Detect and process extracellular physiologic and pathophysiologic molecules Structure of Ligand determines structure of receptor and Mechanism of Action: Hydrophobic vs. Hydrophilic Hydrophobic: Nuclear Hormone Receptors Cytoplasmic/Nuclear Localization Complex with hsp90 and others to hold binding site open for the ligand Olefsky JM.J Biol Chem. 001 Oct 5;76(40):

2 Nuclear Hormone Receptors: Four Mechanisms used by the Estrogen Receptor Julie M. Hall, John F. Couse, and Kenneth S. Korach J. Biol. Chem., Vol. 76, Issue 40, , HUMAN NR GENES The retinoic acid receptor, in the absence of a hormone, is a repressor and it collects a series of repressor 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 to activate transcription. This is how the hormonal or physiological signals affect the recruitment of cofactors that modify the chromatin in target genes. Every cell has receptors and therefore can respond, but each cell responds in its own unique way, and thus the same hormone can have a different effect in a neuron versus an epithelial skin cell versus a bone cell. Evans RM PPARs and the complex journey to obesity.keio J Med. 004 Jun;53():53-8. Hydrophilic Ligand/Receptor System Plasma Membrane is Permeability Barrier

3 WHAT ARE THE CHARACTERISTIC PROPERTIES 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: Radioisotopes Fluorescent dyes Epitope tags Others (biotin) 15 I-Protein [ 3 H]Protein [ 35 S]methionine 3

4 BINDING Assays Variables: Concentration, Time, Temperature Receptor Activity A. Saturation Kinetics No Additions AcLDL Specific Control 0 0 Hot + Cold I-AcLDL (µg protein/ml) 0 Look out for bad data and beware of people who only show delta! Alternative to Chemical Modification of Ligand 4

5 Immunoreceptor vs Radioreceptor Assays For immunorreceptor assay, nonspecific is calculated by regression analysis 100% Reversible Binding to Receptor time after binding Off rates are critical must not loose signal during washing Binding vs Cell Association: Inhibit uptake at 4 C BINDING Assays Quantitative Analysis: How many binding sites? How tight is the binding? Rigorous analysis depends on system being in equilibrium R + L RL R = unbound receptors L = unbound ligand RL = ligand/receptor complex R t = total # of receptors (binding sites) = R + RL 5

6 bound free Bound/Free Classic Scatchard Analysis R = unbound receptors R + L L = unbound ligand RL = ligand/receptor complex R t = total # of receptors (binding sites) = R + RL bound Rt = R + RL K diss = [R][L] [RL] = free [RL] [L] = [R ] K d t slope = -1/K [RL] K d d RL Simple Straight Line: Single Class of Binding Sites intercept = R t Bound (RL) Complications! Error propagation 6 Bound/Free Bound Complications! Cannot always trust your eyes! 6

7 Direct Plot: Scatchard Plot: Non-linear Scatchard Plots: Multiple Classes of Binding Sites Bound/Free Bound (RL) fewer high affinity more abundent lower affinity Boun Old Fashion Graphical Analysis 0 - C. Incorrect graphical fit D. Incorrect graphical fit a b Correct Nonlinear Fit Bound/Free 4 Not Good for Quantitative Determinations Bound/Free 4 Bound/Free B (nm) B (nm) B (nm) Bound/Free 7

8 Use Non-linear Regression Analysis Good Packaged Programs Available E.g., GraphPad Prism Plot Graph and Calculate! Sources of Nonlinearity Multiple Classes of Binding Sites Bound/Free Complex Classes of Binding Sites - Negative and Positive Cooperativity - ensemble effect -ive coop. +ive coop. B/F Bound (RL) B fewer high affinity more abundent lower affinity Sources of Nonlinearity Multiple Classes of Binding Sites Complex Classes of Binding Sites - Negative and Positive Cooperativity Poor Data: Nonequilibrium Conditions Instability of Ligand or Receptor Site Ligand Problems: Bad modifications, Dilution of ligand (endogenous ligand) [inadequate ligand concentration range] Biological Relevance: LDL and Glass Beads 8

9 Ligand Structure/Specificity β-adrenergic Receptor System effects response HO H O effects binding R C C NH CH HO R H = epinephrine C CH 3 = isoproterenol CH 3 epinephrine + ßAR -> activation of adenylate cyclase -> increased camp You can differentiate to some extent effects on binding and response, e.g.: R = H (epinephrine); Kd = 5 x 10-6 M R = C 3 (isoproterenol); Kd = 0.4 x 10-6 (higher affinity) both epi. and isopro. stimulate cyclase (called Agonists ) R=C-C-C-C-phenyl-OH effects response effects binding H HO O R Kd = 0.06 x 10-6 M C C NH CH HO R H = epinephrine C CH 3 = isoproterenol CH 3 CH 3 CH R = CH H O C CH 3 CH 3 R O CH C C NH CH Alprenolol, Kd = 3 x 10-9 M H O R = C CH 3 CH 3 R O CH C C NH CH Propranolol, Kd = 5 x 10-9 M effects response H HO O effects binding R Bind Tightly! Don t Activate Cyclase! C C NH CH HO R H = epinephrine C CH 3 = isoproterenol CH 3 Antagonists (vs Agonists and Inverse Agonists) R R*(active) 9

10 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) ) Ligand affinity chromatography: a) nonionic detergent solubilize protein - detergent micelles b) run on ligand-crosslinked column c) elute with competitor or altered salt or ph 3) Immunoaffinity chromatography 4) Clone by functional expression (e.g., cellular response) Examples: SR-AI (innate immunity pattern recognition receptor) Ligand affinity, ion exchange chromatography, preparative SDS-PAGE-> monoclonal antibody Immunoaffinity -> 40,000-fold purification from lung SR-BI epitope tag, express, immunoaffinity gel ßAR - 15 I-cyanopindolol (CYP) binding assay; alprenololsepharose in digitonin, reconstitution requires proper lipids Purified BINDING ACTIVITY, but how do you know that you've actually purified the biologically relevant receptor? Number of indirect and some direct tests Indirect: 1) exhibits binding affinity and specificity seen in cells ) protein exhibits cell type and tissue type distribution of activity 3) expressed on cell surface - proteolysis, surface labeling reagents 4) glycoprotein 5) regulation makes sense Direct: 1) Reconstitution of activity in vitro 10

11 1 e.g. Fusion of ßAR (60 kd)/liposomes to Xenopus RBC and assay for camp production βar/liposome cell fuse with polyethylene glycol Reconstitution of purified ß-Adrenergic Receptor into Xenopus RBC 8 no addition + agonist isopro. agonist + antagonist prost. E1 or NaF 6 4 agonist: isoproterenol; antagonist: propanolol 0 Xenopus + empty liposome Xenopus + ß-AR/liposome Purified BINDING ACTIVITY, but how do you know that you've actually purified the biologically relevant receptor? Direct: ) Express cloned gene in cells or tissues and measure bio activity 3) Inhibit activity in appropriate system (blocking antibody, sirna, KO animal) STRUCTURE OF THE ß-ADRENERGIC RECEPTOR Out In Heptahelical (7-Transmembrane) domain receptor 11

12 7-TM or G-Protein Coupled Receptors (GPCR) large family (>000) - ~5% of worm genome coding sequences, perhaps 3% of mammals α -AR, ß 1 and ß -AR, dopamine receptors, muscarinic acetyl choline receptors (4 subclasses), sight (Rhodopsin), taste, smell, angiotensin receptor (mas oncogene), serotonin receptor, a- and α- factor yeast receptors, LH receptor, PAR (protease activated receptor) many intronless genes! all operate using similar signal transduction systems (Gproteins) Major Drug Targets for Pharmaceutical Industry Family of structurally and functionally similar proteins provides special opportunity to explore structure/function method: insert cdna into expression vector, transfect into L cells because they are ßAR -ive, look for binding and correct pharmacologic specificity, affinity, etc.-g-protein and cyclase required to work! For example: Delete N- or C-terminus -> binding ok Delete 5-6 loop (C3=i3) -> binding ok Conclusion -> not needed for binding 1

13 For example: Co-express SR(1-5, aa1-6) +SR(6-7, aa63-end) -> still works! Conclusion -> self assemble Delete 6-7 loop (E4) or helices 6&7 -> No binding! Conclusion ->??????????? What can you do? Similar structures permit testing of chimeras of GPCRs for structural determinants of ligand specificity and downstream target (G-protein) specificity α -AR -> decrease in adenylate cyclase activity ß -AR -> increase in adenylate cyclase different ligand specificities Major determinant of specific binding in 7th membrane spanning domain Combined Use of Chemistry (Pharmacology - alter structure of the ligand) and Molecular Biology (mutagenesis- modify putative ligand binding sites) to define Ligand/Receptor Interactions - complementary mutations B3-AR from outside of cell binding to norepinepherine 13

14 Agonist: Isoproterenol Antagonist: Alprenolol Side chain interactions within the transmembrane region of the β- adrenergic receptor with agonist isoproterenol (A) and antagonists alprenolol (B), as derived from mutagenesis and/or modelling studies. For clarity of pictorial representation the interaction of Phe-89 is not shown. Scheme 3. Schematic drawing of the interaction of ATP with the PY1 receptor as derived from mutagenesis and modeling studies. For reasons of clarity the interaction with Arg310(TM7) is not shown. 11-cis-retinal 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. 000 Aug 4;89(5480): Henry R. Bourne and Elaine C. Meng Rhodopsin Sees the Light Science :

15 Figure 4. Model of RS bound to the MCP-1 receptor. RS is the space-filling molecule in the center of the bundle of helices, indicated by the ribbons.[33] Receptor residue Glu91 is shown in the ball-and-stick presentation; it is shown interacting with the basic nitrogen atom of the spiropiperidine structure. Please note that in contrast to Figure B the view from the intracellular side is displayed Figure 3. Three-ligand binding sites of the 5-HT1A receptor in a rhodopsinbased 3D model according to Jacoby and co-workers.[4] Left: extracellular view; right: side view with extracellular side at the top. The three ligands are serotonin or 5-HT (yellow), propranolol (cyan), and 8-OH-DPAT (green). Residues identified by mutagenesis data are indicated. The 5- HT site is located between TM3 (TM III; with Asp116 as the key recognition site) and TM5 (TM V; providing Ser199 and Thr00 to interact with the 5-OH group of serotonin). A second site, the propranolol binding site, is located between TM3 and TM7 (contributing, for example, Asn386 to hydrogen bond the oxygen atoms of the oxypropanolamine fragment in beta - blockers). The third binding site, the 8- OH-DPAT binding site is also located between TM3 and TM7. Ligands addressing this site, like 8-OH-DPAT, are thought to be oriented parallel to the helices (interactions by 8-OH to Ser393 and Asn396 and by amino group to Asp116). Desensitization: tendency of biological responses to wane over time 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 of other receptors, protein kinase A (PKA) plays a role here, PKA is about 6x slower than ßARK at phosphorylating the receptor also, ßARK mediated desensitization is t1/<15 sec while PKA is 3.5 min (>14 fold) see Roth et al, PNAS 88: (91)), arrestin independent. 15

16 beta-arrestin, a promiscuous mediator of receptor endocytosis. (A) Binding of ligand to its GPCR activates heterotrimeric G proteins, causing dissociation of the alpha subunit from the beta/gamma dimer (not shown). The betagamma dimer, anchored to the inner surface of the plasma membrane by the prenylated gamma subunit, facilitates translocation of GRK to the plasma membrane. This enables GRK to phosphorylate the serine/threonine motif in the GPCR carboxyl terminus, resulting in beta-arrestin translocation to the GPCR. beta-arrestin binds to adaptor proteins in clathrincoated pits, resulting in endocytosis of the GPCR, which is then either recycled or degraded with concomitant activation of G protein-independent signaling pathways. (B) On binding to its ligand, the Fz4 receptor induces translocation of Dvl to the plasma membrane. Phosphorylation of Dvl by protein kinase C (PKC) leads to binding of beta-arrestin. Binding of Wnt5A to the cysteine-rich domain (CRD) in the extracellular amino terminus of Fz4 leads to endocytosis of the Fz4 complex. (C) Transforming growth factor-beta type II receptor (TbetaR-II) binds to, and its intracellular kinase domain phosphorylates, the intracellular carboxyl terminus of TbetaR-III. This leads to translocation of beta-arrestin to the plasma membrane, followed by endocytosis of the TbetaR-II/TbetaR-III complex. This process does not require binding of TGF-beta to its receptor, hence the TbetaR-II extracellular binding site (triangle) is depicted as empty. end 16