Medizinische Fakultät der Charité - Universitätsmedizin Berlin Campus Benjamin Franklin aus dem Institut für Pharmakologie Direktor: Univ. Prof. Dr. med. Walter Rosenthal

Ligand-induced N-terminal proteolysis of the human endothelin B receptor

Inaugural-Dissertation zur Erlangung der medizinischen Doktorwürde der Charité - Universitätsmedizin Berlin Campus Benjamin Franklin

vorgelegt von Evelina Grantcharova-Angelova aus Sofia, Bulgarien

Referent: Priv.-Doz. Dr. med. Alexander Oksche Korreferent: Priv.-Doz. Dr. med. H.-D. Orzechowski

Gedruckt mit Genehmigung der Charité – Universitätsmedizin Berlin Campus Benjamin Franklin

Promoviert am: 02.12.2005

to my parents and my husband

Table of contents

1 INTRODUCTION...... 1

1.1 G protein-coupled receptors ...... 1 1.1.1 Signaling of the G protein-coupled receptors ...... 3 1.1.2 Regulation of G protein-coupled receptor activity...... 4 1.2 Endothelins...... 6 1.3 Endothelin receptors...... 10 1.3.1 Signal transduction of endothelin A and endothelin B receptors...... 12 1.3.2 Internalization and down-regulation of the endothelin receptors...... 14 1.4 Physiological role of the endothelin system ...... 15 1.5 The role of the endothelin system in disease ...... 17 1.6 The aim of this study ...... 20

2 MATERIALS AND METHODS ...... 21

2.1 Materials...... 21 2.1.1 Chemicals, kits, cells and antibodies ...... 21 2.1.2 Technical equipment and software ...... 25 2.2 Methods...... 27 2.2.1 Cell culture, transient transfection and nucleofection...... 27 2.2.2 Protein analysis...... 29 2.2.3 Saturation and displacement binding experiments with 125I-ET-1 ...... 37 2.2.4 Inositol phosphate assay ...... 38 2.2.5 Determination of cAMP content of intact HEK 293 cells by radioimmunoassay...... 39 2.2.6 Laser Scanning Microscopy...... 40

3 RESULTS ...... 41

3.1 Detection of the wild-type and mutant ETB receptors in immunoblots...... 41

3.2 The ETB receptor is expressed as a full-length receptor at the cell surface of HEK 293 cells and vascular smooth muscle cells...... 44

3.3 The N-terminal cleavage of the ETB receptor occurs at the plasma membrane ...... 46

3.4 The N-terminal cleavage of the ETB receptor is ligand-dependent...... 47

3.5 Time-course of N- and C-terminal proteolysis of the ETB receptor...... 49 3.6 The N-terminal cleavage is reduced by metal chelators but not by inhibitors of lysosomal proteases or the proteasome...... 51

3.7 The N-terminal cleavage of the ETB receptor is prevented by metalloprotease inhibitors ...... 52 3.8 Substitutions of amino acids in the cleavage site do not prevent N-terminal proteolysis ...... 54

I 3.9 The N-terminally truncated ∆2-64 ETB receptor shows a dramatically reduced cell surface expression when compared to the wild-type...... 55

3.10 The ∆2-64 ETB receptor retains its ability to stimulate inositol phosphate formation and to inhibit forskolin-induced cAMP formation...... 57

3.11 Wild-type and N-terminally truncated ETB receptors show differences in ERK1/2 activation .....59

3.12 The second phase of ERK1/2 activation depends on βγ subunits released from Gi proteins...... 61 3.13 Matrix metalloproteases are involved in the second phase of ERK1/2 activation...... 63 3.14 N–linked glycosylation is essential for the late phase of ERK1/2 activation...... 64

3.15 The ∆2-64 ETB receptor shows normal internalization upon ET-1 stimulation...... 65

4 DISCUSSION ...... 67

5 SUMMARY...... 74

6 REFERENCES...... 76

7 PUBLICATIONS LIST ...... 91

8 ACKNOWLEDGEMENTS...... 94

9 CURRICULUM VITAE...... 95

II Abbreviations

AC adenylyl cyclase

Ang II angiotensin II APS ammonium persulfate ATP adenosine triphosphate

Bmax maximal binding BSA bovine serum albumin cAMP cyclic adenosine-monophosphate CBB Coomassie Brilliant blue G250 cDNA complementary DNA cpm counts per minute DMEM Dulbecco’ s modified Eagle’ s medium DNA desoxyribonucleic acid dpm desintegration per minute DSMZ Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH DTT dithiothreitol ECE-1 endothelin-converting-enzyme-1 EDTA ethylendiamine-tetraacetate EGF epidermal growth factor ER endoplasmic reticulum ERK1/2 extracellular-signal regulated kinase 1/2 pERK1/2 phosphorylated extracellular-signal regulated kinase 1/2 ET endothelin

ETB receptor endothelin B receptor FCS fetal calf serum FMP Berlin Forschungsinstitut für Molekulare Pharmakologie, Berlin GDP guanosine diphosphate GFP green fluorescent protein GPCR G protein-coupled receptor

III GRK G protein-coupled receptor kinase GTP guanosine triphosphate HEK 293 cells human embryonic kidney 293 cells HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid

IRL1038 ETB receptor-selective antagonist

IRL1620 ETB receptor-selective agonist

KD dissociation constant

Ki inhibitory constant LSM Laser Scanning Microscopy LT low temperature MAPK mitogen activated protein kinase MMP matrix metalloprotease NP-40 ethylphenyl-polyethylene glycol OD optical density PAGE polyacrylamide gel electrophoresis PAR protease-activated receptor PBS phosphate buffered saline PGE1 prostaglandin E1 PKA protein kinase A PKC protein kinase C PLC phospholipase C PMSF phenylmethylsulfonyl fluoride PTX pertussis-toxin QM quiescent medium

RES-701-1 ETB receptor-selective antagonist RNA ribonucleic acid rpm rotations per minute RT room temperature SDS sodiumdodecyle sulfate TACE tumor necrose factor α-convertase TBS Tris-buffered saline TEMED N,N,N´,N´-Tetramethylethylene diamine

IV TM transmembrane Tris Tris (hydroxymethyl)-aminomethan ¡ Triton X-100 Octylphenylpoly(ethylene glycol ether)n TSH receptor thyrotrophic stimulating hormone receptor

V2 receptor vasopressin 2 receptor VSMCs vascular smooth muscle cells

V Summary

5 Summary

The human endothelin B (ETB) receptor comprises 442 amino acids, of which the first 26 function as a signal peptide. The signal peptide, which is essential for cell surface transport is cleaved off by a signal peptidase in the ER lumen during receptor biosynthesis. In addition, a second protheolytic cleavage within the extracellular N terminus (at R64↓S65) has been identified, which results in an N-terminally truncated receptor, lacking amino acids 26 to 64. The regulation and the physiological significance of this proteolysis were not known when this study was started. To gain more insight into the process of N-terminal proteolysis, ETB receptor or ETB·GFP fusion protein stably or transiently expressed in human embryonic kidney 293 (HEK 293) cells and in vascular smooth muscle cells (VSMCs) were analyzed. After incubation of cells with 125I-ET-1 at 4°C, only the full- length ETB receptor was detected at the cell surface. When cells were incubated at 37°C in the presence of endothelin-1, N-terminal cleavage was observed. The cleavage was not prevented by inhibitors of internalization (sucrose, phenylarsine oxide) or of serine and cysteinyl proteases. However, when cells were incubated with internalization and metalloprotease inhibitors (batimastat, inhibitor of TNFα-converting enzyme Ro32-7315) or metal chelators (EDTA, phenanthroline), the cleavage was blocked. The data show that metalloproteases mediate an agonist-dependent cleavage of the ETB receptor at the cell surface.

Functional analysis of a mutant ETB receptor lacking the first 64 amino acids (∆2-64 ETB) revealed normal ligand binding properties and preserved G protein-signaling (increase of inositol phosphate formation and inhibition of forskolin-induced cAMP-formation) when compared to the wild-type receptor. However, the ∆2-64 ETB receptor showed a 15-fold reduced cell surface expression and an altered ability to activate ERK1/2. Although the wild-type and the ∆2-64 ETB receptor elevated an early phase of ERK1/2 phosphorylation (within 5 min), only the wild-type receptor induced a second phase of ERK1/2 activation

(starting after 80 min). The second phase was mediated via βγ subunit of Gi proteins and was abolished by inhibitors of matrix metalloproteases (batimastat and an inhibitor of TNFα-converting enzyme Ro32-7315).

74 Summary

The data presented in this study strongly suggest, that the N-terminal proteolysis of the human ETB receptor is mediated by a metalloprotease in an agonist-dependent manner.

Removal of the ETB receptor’ s N terminus yields a receptor with a dramatically reduced cell surface expression and an altered ability to stimulate ERK1/2 activation. The data suggest, that the N-terminal cleavage of the ETB receptor could be involved in the regulation of cell surface expression and of ERK1/2 activation. The functional role of the observed biphasic ERK1/2 activation via the full-length ETB receptor and the monophasic

ERK1/2 activation via the ∆2-64 ETB receptor requires further characterization.

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