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Binding of Sap102 and Usp4 to the A2a

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Binding of Sap102 and Usp4 to the A2a Diplomarbeit BINDING OF SAP102 AND USP4 TO THE A2A ADENOSINE RECEPTOR CARBOXYTERMINUS Zur Erlangung des akademischen Grades Doktorin der Zahnheilkunde (Dr. med. dent.) an der Medizinischen Universität Wien ausgeführt am Institut für Pharmakologie unter der Anleitung von Prof. Dr. Christian Nanoff eingereicht von Ivana Ostrouska Matr.Nr.: n0347690 Gallmeyergasse 6/1/5, A-1190 Wien Wien, 31.März 2009 Diploma Thesis BINDING OF SAP102 AND USP4 TO THE A2A ADENOSINE RECEPTOR CARBOXYTERMINUS Obtainment of the academic degree Doctor of Dentistry (Dr. med. dent.) at the Medical University Vienna performed at the Institute of Pharmacology supervised by Prof. Dr. Christian Nanoff submitted by Ivana Ostrouska Matr.No.: n0347690 Gallmeyergasse 6/1/5, A-1190 Vienna Vienna, 31.March 2009 My very best thanks to Christian Nanoff, Oliver Kudlacek, Ingrid Gsandtner and to the Institute of Pharmacology. 3 Content 1 Summary ................................................................................................................ 11 2 Introduction ........................................................................................................... 12 2.1 The A2A adenosine receptor..................................................................................... 12 2.1.1 A2A receptor signaling.......................................................................................... 15 2.2 Synapse Associated Proteins and SAP102 .............................................................. 17 2.2.1 SAP Structure...................................................................................................... 17 2.2.2 Regulation, interactions and functions ................................................................ 20 2.3 USP4........................................................................................................................ 22 3 Experimental Procedures ..................................................................................... 25 3.1 Cell culture and transfection.................................................................................... 25 3.2 DNA Constructs...................................................................................................... 25 3.3 Yeast-Two-Hybrid Interaction test.......................................................................... 27 3.4 Cyclic AMP Accumulation Assays – Determination of cAMP formation ............. 30 3.5 Radioligand Binding Experiments .......................................................................... 31 3.6 Protein Determination – Bradford Assay ................................................................ 32 3.7 Mitogen-activated Protein (MAP) Kinase Assay .................................................... 32 3.8 Fluorescence Microscopy........................................................................................ 33 3.9 FACS (Fluorescence activated cell sorting) ............................................................ 34 4 Results..................................................................................................................... 36 4.1 Binding of SAP102 to the carboxyl terminus of the A2A adenosine receptor ......... 36 4.2 Characteristics of the A2A receptor with the DVELL to RVRAA mutation ........... 39 4.3 Activation of cAMP-formation by wild-type and mutant receptor ......................... 40 4.4 Identification of stable HEK293 cell clones by radioligand binding ...................... 41 4.5 Cyclic AMP-formation in stable cell lines .............................................................. 43 4.6 Receptor-dependent phosphorylation of ERK1/2.................................................... 43 4.7 Mapping the USP4 recognition site on the A2A receptor c-tail ............................... 45 4.8 Time course of the effect of USP4 on the expression of functional A2A receptors 47 4.9 FACS analysis of A2A receptor surface expression................................................. 49 4 5 Discussion and Conclusions.................................................................................. 51 5.1 Mapping the binding-site for SAP102 on the A2A adenosine receptor.................... 51 5.2 The binding site of USP4 on the A2A adenosine receptor ....................................... 53 5.3 Conclusion............................................................................................................... 54 i. Abbreviations........................................................................................................... 6 ii. Index of Figures and Tables ................................................................................... 8 iii. Index of Appendix ................................................................................................. 10 iv. References .............................................................................................................. 55 v. Appendix ................................................................................................................ 60 5 i. Abbreviations A2AR A2A adenosine receptor AD activation domain ADA adenosine deaminase ADP adenosine diphosphate AMPA-receptor α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptor ATP adenosine triphosphate BCA bicinchonic acid BDNF brain-derived neurotrophic factor BRCA2 breast cancer gene 2 cAMP cyclic AMP, cyclic adenosine monophosphate CNT concentrative nucleoside transporters CREB cAMP response element-binding protein c-tail carboxyl terminal tail Dlg product of the homologous drosophila gene, Disc Large DUBs deubiquitinating enzymes DUSP domain present in USP ERK1/2 extracellular signal-regulated kinase FACS Fluorescence activated cell sorting GFP green fluorescent protein GKAP guanylate-kinase associated protein GPCR G-protein coupled receptor GTP guanosine triphosphate GUK guanylate kinase MAGUK membrane-associated guanylate kinase-like domain MAP-kinase mitogen-activated protein kinase NMDA-receptor N-methyl-D-aspartic acid receptor PCR polymerase chain reaction PDZ domains letters of PSD95, DlgA and zonula occludens-1 protein PKA protein kinase A PSD post synaptic density protein SAP102 synapse associated protein of 102 kDa 6 SH3 Src homology domain TrkB tyrosine kinase B UAS upstream activating sequence Ub ubiquitin USP4 ubiquitin-specific protease UTR untranslated region 7 ii. Index of Figures and Tables Introduction Figure I- 1: Interspecies similarity of the A2A adenosine receptor gene. ............................ 14 Figure I- 2: SAP family – modified from (Fujita and Kurachi, 2000, Kim and Sheng, 2004) ..................................................................................................................................... 18 Figure I- 3: Ribbon diagram representing the structure of an isolated PDZ domain (red - - helix, yellow - -sheet)................................................................................................ 19 Figure I- 4: Ribbon structure of an SH3 domain................................................................. 19 Figure I- 5: Proposed model for the role of SAP102 (Sans, et al., 2005)............................ 20 Table 1: Overview of PSD/SAP interactions ...................................................................... 21 Figure I- 6: The ubiquitin-conjugation machinery (Ravid and Hochstrasser, 2008)........... 23 Figure I- 7: Substrate targeting to the 26S proteasom (Ravid and Hochstrasser, 2008) ..... 24 Experimental Procedures Table 2: List of vectors used in yeast-two-hybrid interaction assays................................. 27 Table 3: Supplements of the synthetic drop-out media for cultivating transformed yeast.. 29 Figure M- 1: Principles of yeast-two-hybrid assay ............................................................ 30 Figure M- 2: FACS example .............................................................................................. 35 Results Figure 1 - Interaction of SAP102 with the A2AR c-terminus; Importance of the DVELL (382-386) sequence...................................................................................................... 38 Figure 2 - Epifluorescence microscopy. Imaging the fluorescent A2A receptor in HEK293 cells. (left) wild-type receptor; (right) mutant receptor.............................................. 40 Figure 3 - Activation of cAMP-formation by the wild-type and mutant receptor. ............. 43 8 Figure 4 – (A) A2A receptor dependent ERK-phosphorylation; (B) Summary of experiments performed after transient transfection of HEK293 cells with the wild-type and mutant A2A receptor. ............................................................................................. 45 Figure 5 - Mapping the USP4 recognition site on the A2A receptor c-tail; Interaction of USP4-DUSP and truncated variants of the receptor c-tail in yeast. ............................ 47 Figure 6 - Radioligand binding to membranes from HEK293 cells transfected with the A2A adenosine receptors in the presence and absence of USP4. ........................................ 48 Figure 7 - A2A receptor surface expression in cells co-transfected with USP4................... 49 9 iii. Index of Appendix v.1 Zusammenfassung (deutsche Übersetzung der „Summary“) 10 1 Summary The A2A adenosine receptor has an extended cytoplasmic carboxyl terminal tail (c-tail) that contains putative docking sites for accessory cellular proteins. The binding of two candidate interaction partners to the receptor c-tail, these are (i) the MAGUK (membrane- associated guanylate
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