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 kinase-like domain) protein SAP102 (synapse associated protein of 102 kDa) and (ii) the ubiquitin-specific protease USP4 was evaluated. The yeast-two- hybrid test was employed to assess interaction with the receptor c-tail at its original length and with several variants that had been truncated from the distal end. Various subdomains (PDZ, SH and GUK) were isolated from the multi-domain protein SAP102 and tested separately; the substrate recognition domain (DUSP) was used in case of USP4.
For SAP102 a series of interaction tests indicated that the receptor c-tail binds - by virtue of a five-residue motif located at position 382-386 (that is 22 amino acids removed from the c-terminal end) - to the GUK domain. Signal transduction was examined by a receptor mutant where the original five-residue motif (DVELL) had been supplanted by an alternative sequence (RVRAA) such that the receptor could not bind to SAP102. In HEK293 cells neither expression of the receptor nor receptor-dependent formation of cAMP or activation of the MAP-kinase ERK1/2 differed between mutant and wild-type receptor.
In contrast to SAP102, the binding site of the USP4-DUSP domain could not be unambiguously mapped on the receptor c-tail. Progressive truncation of the human c-tail identified a candidate interaction site that however was not detected in the highly homologous sequence of the mouse species orthologue. Thus, the conclusion is that (i) the yeast-two-hybrid interaction test may have limitations requiring the results to be re- evaluated by an independent experimental approach and that (ii) the consequence of mutating the SAP102 interaction site in the receptor should be revealed when the mutant receptor is expressed in a neuronal cell.
11 2 Introduction
Receptors for purines are comprised in two families, the P1- and P2-purinergic receptors. P2 receptors bind adenine and uridine nucleotides whereas P1 is the alternative prefix denoting the receptors for adenosine. Adenosine is formed in the extracellular space from released adenine nucleotides. Alternatively it is formed within the cell and extruded by transporters. Regardless of the underlying mechanisms, extracellular increase of adenosine reflects cellular activity (e.g., neuronal release of ATP) or cellular distress and cell damage. Accordingly, the most fruitful concept to understand the biological action of adenosine is the concept of adenosine as the retaliatory metabolite. In many instances adenosine can be thought of as a messenger that elicits both short term and long term effects that allow the tissue to recover. This response typically results from the concerted action of and graded recruitment of adenosine receptors. Four subtypes of G-protein coupled receptors have been characterized and are thought to mediate all biological actions of extracellular adenosine: the A1AR, A2AAR, A2BAR and A3AR (Fredholm, et al., 2001).
A1- and A3-adenosine receptors couple to Gi/o proteins while A2A and A2B–adenosine receptors couple to Gs.
Adenosine is unstable and its lifetime in the circulation is only a few seconds: the elimination of extracellular adenosine is by rapid cellular uptake, most often through specific nucleoside concentrative nucleoside transporters (CNT), which utilize the Na+- gradient for concentrative transport (Cass, et al., 1999). In addition, adenosine must also be eliminated enzymatically, because excess adenosine is toxic. Intracellular adenosine is metabolised enzymatically either by adenosine deaminase to inosine or by adenosine kinase, forming 5’-AMP (Poulsen and Quinn, 1998). This rapid degradation limits the action of adenosine to the immediate vicinity of the site from where it is released. If adenosine is not degraded the overflow of deoxadenosine or of adenosine itself is detrimental for T-cells and T-cell precursors (Hershfield, 2005). Accordingly, genetic deficiency in adenosine deaminase causes a severe combined immunodeficiency.
2.1 The A2A adenosine receptor
The A2A adenosine receptor is a G-protein coupled receptor that is expressed to high density in diencephalic nuclei, corpus striatum and nucleus accumbens, and the olfactory
12 tubercles. The A2A receptor is expressed in many neurons and glial cells and at lesser densities can be found in many brain regions (Moreau and Huber, 1999). In the vasculature, the A2A receptor is on endothelial and smooth muscle cells, where it mediates vasodilation and blood pressure control. A2A receptors on platelets inhibit aggregation, in T-lymphocytes they reduce the growth stimulus elicited by T-cell receptor activation. Due to the expression of the A2A receptor on T-cells as well as on monocytes/macrophages, dendritic cells, mast cells, neutrophils, endothelial cells adenosine is considered to exert a strong anti-inflammatory response (Haskó and Pacher, 2008).
The genomic organization of the A2A receptor gene includes two invariant exons. The transcripts identified contain the same coding sequence and a common 3′-untranslated region (UTR) but differ in the 5′-UTR. The role of the distinct 5′-UTR that is derived from multiple A2A promoters is unresolved. There a four tentative promoters (P1A, P1B, P2 and
P3; Fig. I-1) governing gene transcription. For the two downstream A2A promoters (P2 and
P3), the corresponding 5′UTRs suppress the expression of the A2A receptor at the translational level via an upstream open reading frame (Fredholm, et al., 2007). By contrast, a rat DNA fragment containing the three downstream promoters (P1A, P2, and
P3) was found sufficient to direct transgenic expression in many brain areas where the A2A receptor is expressed, but not in five peripheral tissues (heart, lung, liver, kidney, and lymph nodes) which also express significant levels of the A2A receptors (Fredholm, et al.,
2007, Lee and Chao, 2001). While the three A2A promoter regions (P1A, P2, and P3) appear to contain only the cis element(s) important for CNS expression they are insufficient to produce enrichment of receptor expression in the striatum. Alternative promoter usage hence may be the primary mechanism for the generation of the A2AR transcript diversity and tissue-specific expression. The 5′UTR of the A2A receptor gene possesses strong interspecific homology; therefore the translational regulation of the rat
A2A receptor gene likely is a general mechanism.
13
Figure I- 1: Interspecies similarity of the A2A adenosine receptor gene.
The most spectacular consequences of inactivating the A2A receptor gene are on the behavioral level (locomotion, anxiety and aggressiveness) as well as in pain perception.
The A2A receptor regulates wakefulness and sleep; diminished receptor activation through chronic caffeine consumption (= repeated receptor blockade) affords a protective effect against neurodegeneration. The role in degenerative disorders of the CNS and in modulating inflammation has renewed interest in the pharmacology of the A2A receptor.
The A2A receptor is a G-protein coupled receptor with the common architecture including seven -helices that are embedded in the membrane lipid bilayer. They are linked by three extracellular and three intracellular loops which are variable in size. The N-terminus is rather short (consisting of four amino acids); compared to other GPCRs with extended N- termini, the structure of the extracellular face therefore is mainly composed by the extracellular loops. Their structure is stabilized by four disulphide bonds which connect between loops and buttress the structure of loop 3. The extensive disulphide bond network stabilizes a rigid crevice exposing the ligand binding cavity to solvent, possibly allowing free access for small molecule ligands (Jaakola, et al., 2008).
14 The ligand binding pocket has been located by site-directed mutagenesis to the outward facing region of -helices 5, 6 and 7 and this was confirmed by analysis of the crystal structure of the receptor. In the receptor crystal, the bound antagonist ligand is in contact with side chains belonging to each of the three transmembrane helices. The orientation of the ligand (ZM241385) is almost perpendicular to the membrane plane (Jaakola, et al., 2008). The portion of the ligand directed toward the more-solvent-exposed extracellular region (ECL2 and ECL3) tolerates different chemical substitutions. In contrast, the moiety (which typically is a furan ring in antagonist ligands) pointing to the receptor protein core is constrained by at least one amino acid residue indispensible for ligand binding (His250). Its position is close to a tryptophan residue in helix 6, an important residue in receptor activation (the rotamer toggle).
2.1.1 A2A receptor signaling
Activation of the A2A adenosine receptor leads to stimulation of adenylyl cyclase. Earlier studies did not distinguish the A2A receptor from the other stimulatory adenosine receptor subtype (the A2B receptor). To date a subtype selective agonist (e.g. CGS21680) and antagonist (SCH 58261) ligands have become available. Thus, CGS 21680 leads to an increase in cAMP, mediated by the stimulatory G-proteins G s or G olf (e.g in striatal nerve cells).
When CGS21680 is applied to explanted nerve cells (brain slice or dissociated nerve cells in culture) receptor activation results in facilitated release of neurotransmitters (such as acetylcholine, noradrenaline, glutamate and GABA) an action most likely mediated by the canonical second messenger, cAMP (Seino and Shibasaki, 2005). In other neurons, the A2A receptor elicits postsynaptic effects either via cAMP-regulated effectors (through protein kinase A and possibly Epac, the nucleotide exchange factor of Rap and other small GTP- binding proteins). PKA transmits receptor activation to the cell nucleus by phosphorylating the transcription factor CREB (cAMP-responsive binding protein). In addition, A2A signaling has been shown to lower the activation threshold of the ionotropic NMDA-type glutamate receptor (Rebola, et al., 2008) to transactivate the BDNF (brain-derived neurotrophic factor) receptor TrkB (Lee and Chao, 2001) and to antagonize activation of the D2-dopamine receptor (Ferré, et al., 2008). It is however not clear if the communication between the A2A receptor and these neuronal receptors does require cAMP.
15
Signaling that occurs independently of cAMP has been demonstrated for the A2A adenosine receptor and another prototypical Gs-coupled receptor, the 2-adrenergic receptor. To account for the cAMP independent effect (on the activation of MAP-kinase) by the 2- receptor a model has been proposed that relies on receptor endocytosis (Maudsley, et al.,
2000). By contrast with the 2-receptor, the A2A receptor internalizes poorly and with substantial delay when occupied by an agonist (Brand, et al., 2008, Klinger, et al., 2002, Vidi, et al., 2008). Therefore a cAMP-independent signal must emanate from the surface- bound receptor.
In the receptor G-protein complex, the cytoplasmic peptide loops of a GPCR are believed to be covered up by the cognate heterotrimeric G protein. Ancillary signaling proteins may dock to the c-terminal segment of the receptor protein which is extraordinarily long in the
A2A receptor (122 peptide bonds). Truncation of the receptor c-tail indeed provided evidence for an accessory docking site. While (surprisingly) activation of MAP-kinase was preserved c-terminal truncation affected the constitutive activity of the A2A receptor (Klinger, et al., 2002). Thus, truncated receptors were less active than the wild-type receptor but the difference was only observed in intact cells (but not broken cells or fractionated membranes), indicating a role for a component that is lost upon cell lysis.
In search for ancillary protein components a brain cDNA library has been screened using the receptor c-tail as bait. The cDNA library was inserted in a yeast expression vector and yeast was transformed together with a vector encoding the c-tail. Both the brain proteins encoded by the cDNA library and the c-tail were expressed as fusion proteins merged to the split domains of a yeast transcription factor; if the two domains come in close contact due to the interaction of the c-tail with a protein partner (bait-prey interaction) transcription of a specific target gene becomes active. A metabolically deficient yeast strain is used for transformation where the deficiency is corrected by inducing the target gene; the target gene is under the control of the promoter recognized by the split transcription factor (see Methods).
This work has confirmed the interaction of two putative ancillary proteins (USP4 and SAP102 see below) with the receptor c-tail and continued the analysis using shortened
16 versions of the c-tail. Employing a “salami-strategy” where the c-tail is shortened (like slicing salami sausage) the aim was mapping within the length of the c-terminal polypeptide the docking regions for USP4 and SAP102. Apart from positively confirming the interaction the goal was to create receptors that would not bind either SAP102 or USP4, hence be impervious to their respective action. These receptor variants should eventually have a full-length c-tail carrying specific mutations that negate interaction with
SAP102 or USP4; in theory they should be capable of signaling via GS and may be used to investigate regulation by SAP102 and USP4 of the A2A adenosine receptor.
In the search for the amino acids, constituting the docking sites for two putative interactors
(SAP102 and USP4) of the A2A adenosine receptor, a human cDNA library has been used to identify possible interactors in a yeast complementation assay by the isolated carboxy- terminal receptor fragment (the c-tail) as bait. It has substantiated the findings of the interactor hunt with truncated forms of the receptor c-tail; truncated c-tail constructs were used to determine the minimal peptide length capable of binding the interacting proteins. Thus, it was possible to exclude distal c-tail segments as irrelevant for the interaction with either USP4 or SAP102.
2.2 Synapse Associated Proteins and SAP102
Synapse associated proteins (SAP) are known to regulate trafficking and localization of membrane proteins in synapses. These properties of SAP102 make it a logical candidate for A2A receptor regulation. The A2A receptor is present in the CNS and many of its actions are commensurate with synaptic localization; actions both pre- and postsynaptic have been described for the A2A receptor (in nerve cells of different origin). For example, in hippocampal nerve cells A2A receptors appear to enhance signaling by NMDA-glutamate- receptors (Rebola, et al., 2008, Tebano, et al., 2005); SAP102 may be relevant for juxtaposing the A2A- and NMDA-receptor within the extensions of the postsynaptic density.
2.2.1 SAP Structure
To date five members of the SAP family have been identified: SAP102, SAP97/hDlg, chapsin-110/PSD-93, PSD-95/SAP90 and Dlg (named after the product of the homologous
17 drosophila gene, Disc Large). Another name for SAP is membrane associated guanylate kinase (MAGUK). MAGUK family proteins consist of three n-terminal PDZ domains, one Src homology (SH3) domain and one guanylate kinase (GUK) domain at their c-terminal region.
Figure I- 2: SAP family – modified from (Fujita and Kurachi, 2000, Kim and Sheng, 2004)
The PDZ domain is a common structural domain of 80-90 amino-acids present in components of signaling pathways. PDZ is an acronym combining the initial letters of three proteins — post synaptic density protein (PSD95), Drosophila disc large tumor suppressor (DlgA), and zonula occludens-1 protein (zo-1) — which were first discovered to share the domain. PDZ domains represent the main binding site to interact and build large complexes with other proteins. The PDZ domain is composed of two -helices and six -sheets with which it connects to the PDZ-binding motif. Because SAPs have three PDZ domains they are able to bind more than one protein at a time bringing them close together.
In the c-terminus of SAP binding partner proteins a conserved binding sequence has been identified, the tSXV-motif which is present in glutamate receptor and potassium channels subtypes (Fujita and Kurachi, 2000, Kim and Sheng, 2004). However, neither this nor other PDZ-binding sequences form the A2A receptor c-terminus (Tonikian, et al., 2008).
18
Figure I- 3: Ribbon diagram representing the structure of an isolated PDZ domain (red - -helix, yellow - -sheet)
The Src homology 3 domain (or SH3 domain) is another protein domain [~60 amino acids] and has been identified in a number of signal-protein components (receptors, GTPases, kinases and adapter proteins). SH3 is able to bind target proteins due to a characteristic beta-barrel fold which consists of five or six β-strands (the latter arranged as two tightly packed anti-parallel β sheets); SH3 recognizes in binding partners proline-rich peptides where a series of prolines is flanked by hydrophobic residues. Although rich in proline residues the A2A receptor c-tail lacks the precise motif.
Figure I- 4: Ribbon structure of an SH3 domain
The GUK domain in MAGUK-proteins shares high sequence similarity with the guanylate kinase from yeast, a nucleoside monophosphate kinase that converts GMP to GDP using ATP as a phosphate donor. However, catalytic activity is absent from the GUK domain in MAGUK family members (which include the ZO1-like and Dlg-related proteins). Instead, the GUK domain is involved in an intramolecular interaction with the MAGUK’s own SH3 domain; this intramolecular interaction for example regulates the PSD-95/SAP-90 clustering of ion channels (Kim, et al., 1997, Shin, et al., 2000) or binding to guanylate- kinase associated protein (GKAP) (Wu, et al., 2000).
19
2.2.2 Regulation, interactions and functions
Sans et al. have identified a protein that modulates the SAP102-interaction with cognate partners. The mammalian homologue of “Drosophila melanogaster partner of inscuteable” (mPin) contains TPR (tetratricopeptide) repeats and a G-protein recognition motif (GoLoco) at the N-terminus. Pins-like proteins (such as LGN or MuNA) control the polarity in cell division of neuroblasts, a process that apparently requires the -subunit of the Gi protein. Sans et al. suggested a postmitotic function of Pins in the morphogenesis of neurites. In addition, the interaction of mPins with the SH3/GUK domain of SAP102 has been found a prerequisite for the formation of a complex between the NMDA-receptor NR2-subunit and the SAP102 MAGUK domain; mPins-binding is suggested to convert SAP102 from a closed to an open state capable of binding NMDA-receptors (Sans, et al., 2005).
Figure I- 5: Proposed model for the role of SAP102 (Sans, et al., 2005)
SAPs are renowned for clustering membrane proteins that would not cluster if expressed alone. The table below lists examples of PSD/SAP interaction with channel and receptor proteins. It is important to note that each SAP-family member can be found expressed in a tissue-specific pattern; expression appears to be regulated during stages of development (see Table 1). For instance, SAP102 mediates synaptic trafficking of NMDA and AMPA glutamergic receptors during synaptogenesis. When a synapse has been formed PSD-95 sustains the receptor complement by increasing the amount of AMPA receptors; in addition it causes a switch from NR2B- to NR2A-receptor subtypes, a role that is not
20 substituted for by SAP102 when the gene for PSD95 has been deleted (Elias, et al., 2008). The role of SAP102 apparently is in positioning NMDA-receptors. Only NMDA-receptors, bound to SAP102, are linked to the activation of kinases which correlates to the transcriptional regulation of a typical set of genes (Coba, et al., 2008).
NMDA and AMPA glutamate receptors have eminent importance in synaptic neurotransmission and long-term potentiation. Disorders in the organization of the postsynaptic membrane therefore are expected to impair cognitive function. In fact, individuals with a disorder of the SAP102 gene have a mental handicap, both mouse (Marks and Fadool, 2007) and human (Tarpey, et al., 2004).
Table 1: Overview of PSD/SAP interactions Type Interaction partner PDZ-binding Consequence of interaction Ref. motif SAP Shaker-type K+-channels TDV-motif Heteromultimeric channel (Kim, et al., (Kv1.1, Kv1.2, Kv1.4) complexes in specific brain 1995), regions (Gomperts, 1996) SAP Inwardly rectifying K+- ESEI/ESRI- Localization (Fujita and channels (Kir2.1, Kir2.3, motif Kurachi, Kir4.1) 2000, Kim and Sheng, 2004) SAP G-protein gated K+-channels ESKV-motif (Fujita and (Kir3.1(2)/GIRK1(2) Kurachi, 2000, Kim and Sheng, 2004) SAP NR1-3, NR1-4, NR2A, E-T/SXV motif Links NMDA-receptors to (Cuthbert, NR2B, NR2C and NR2D-2 ERK, important for LTP et al., 2007) (long-term potentiation) PSD-95 Kv1.3 channels & Relieves IR-dependent (Marks and Insulin receptor (IR) inhibition of Kv1.3- currents Fadool, 2007) PSD-95 Kv1.4, Kir2.3 Blocks channel (Cohen, et internalization al., 1996, Jugloff, et al., 2000) SAP102 NrCAM (Neuron-related cell SFV motif Neurite sprouting (Davey, et adhesion molecule) (c-terminal) al., 2005)
PSD 95 1-adrenergic- receptor E-T/SXV Decreases receptor (Hu, et al., (c-terminal) internalization 2000) SAP102/ 5-HT2A- and 5-HT2C-receptor SXV Decreases receptor (Backstrom, PSD-95/ (c-terminal) internalization et al., 2000, SAP97 Xia, et al., 2003) PSD 95 D1 dopamine receptor c-terminal tail Decreases signaling (Yao, et al., 2004)
21 2.3 USP4
The interaction between USP4 (ubiquitin-specific protease) and the A2A receptor is interesting because it is related to a fundamental question in biology, that is substrate recognition by deubiquitinating enzymes (DUBs). The posttranslational attachment of ubiquitin, a 76-amino-acid protein is considered a key step in the specific destruction of proteins and DUBs counteract the degradation initiated by ubiquitin attachment. This is the proposed mechanism by which USP4 increases functional A2A receptors in a heterologous expression system: USP4 binds to the c-terminus and deubiquitinates the A2A receptor.
This weakens the quality control for improperly folded A2A receptors and leads to an increase in their surface expression (Milojevic, et al., 2006).
Ubiquitin modification requires three different ligases (E1-E3). E1 activates ubiquitin through an ATP-dependent reaction leading to a thioester bond with an active-site cysteine. Ubiquitin is transferred to the active site cysteine of an E2 conjugating enzyme and eventually is conjugated to its substrate by an E3 ligase, which confers substrate specificity. The targeted amino acid is a lysine residue. A protein thus modified is destined for degradation by the 26S-proteasome. Alternatively, ubiquitination can be interpreted by the cell as a regulatory signal, e.g. for a protein to be endocytosed.
The counter-regulation achieved by deubiquitination is carried out by a enzymes (DUBs) encoded by five groups of genes encoded in metazoan genomes (ubiquitin C-terminal hydrolases (UCHs), ubiquitin-specific peptidases (USPs/UBPs), ovarian tumor domain proteins/otubain proteases (OTUs), Machado-Joseph disease protein domain proteases and JAMM motif (zinc metallo) proteases (Ravid and Hochstrasser, 2008, Singhal, et al., 2008)). Because USPs regulate the degradation of proteins they are an important part of the cellular quality control determining the turnover of proteins. Thus, USPs have been identified in cancers or diseases caused by mutated genes. For example, USP6 mutations are linked to aneurysmal bone cysts (altered USP6-expression), USP7 binds to the tumor suppressor protein p53; USP11 interacts with BRCA2, mutated BRCA2 predisposes breast cancer; USP4 has been implicated in lung cancer, retinoblastoma and Sjögren’s syndrome (associated with the autoantigen Ro52) (Singhal, et al., 2008, Soboleva, et al., 2005, Wada and Kamitani, 2006).
22 Embedded between an amino- and a carboxy-terminal domain, three specific domains are at the core of ubiquitin-specific peptidases: a finger domain, where Ub binds and induces a conformational change, a palm domain and a thumb domain, which purportedly are required for catalysis (Ha and Kim, 2008). Presumably, the ancillary N- and c-terminal regions may be necessary to recognize the protein substrate independent of the ubiquitin modification.
The number of putative ubiquitin-splitting enzymes encoded in the human genome is estimated to be around 100. DUBs recognize a substrate when a ubiquitin protein is attached. To ensure specific substrate targeting DUBs must be capable of distinguishing between different proteins with ubiquitin attachement. DUBs cleave a glycine-glycine peptide bond and release the substrate protein where a lysine side chain is bonded to the residual glycine. Analysis of the USP4 recognition sequence in the A2A receptor c-tail should provide information on how a deubiquitinating enzyme, the ubiquitin-specific protease 4, recognizes its substrate, i.e. the A2A receptor c-tail.
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 3 Experimental Procedures
3.1 Cell culture and transfection
The human embryonic kidney (HEK293) cells were cultured in DMEM (Dulbecco’s modified Eagle medium) supplemented with 10% fetal calf serum, 2mM L-glutamine, - mercaptoethanol, non-essential amino acids, at 5% CO2 and 37°C. Additionally, 0.2 mg/ml Geneticin (G418) was added for selection-pressure of cells transfected with plasmids driving the expression of a neomycin resistance gene.
HEK293 cells were transfected by CaPO4-precipitation of the vector DNA. Cells were grown to ~50% confluence. DNA (10 µg for cells seeded on a 10 cm dish) was dissolved in HEBS buffer pH 7.2, gently mixed with an equal volume of CaCl2-solution (10 mM) before it was added to the cells. After 3 hours medium was aspirated and cells exposed to a brief glycerol shock.
3.2 DNA Constructs
For the yeast-two-hybrid assay plasmids provided with the matchmaker lexA two-hybrid- system (Clontech) were used. Various versions (full length and truncated) of the human and mouse A2A adenosine receptor were cloned into the pLexA vector (bait). Proteins tested for interaction with the A2A receptor c-tail were expressed from the pB42-AD vector (prey).
Receptor c-tail constructs were amplified with the use of specific oligonucleotide primers and the human or mouse cDNA as template. In the PCR products the receptor coding sequence was flanked by EcoRI and XhoI restriction sites; these restriction sites were also present in the pLexA vector and allowed for insertion of the PCR product into the plasmid. A construct was thus generated with the LexA sequence preceding the receptor sequence; the expression resulted in a fusion protein provided that the oligonucleotide primers were positioned to give an uninterrupted open reading frame. Preparation of these vectors followed the same principle:
25 pB42 vectors expressing SAP subdomains pB42 vector expressing the USP4-DUSP domain
pEYFP-N1 vector (Clontech) expressing the human A2A receptor (cDNA cloned via HindIII/EcoRI)
The composition of each vector construct was verified by DNA sequencing.
The DVELL-RVRAA mutation was performed using the QuikChange ® mutagenesis Kit (Stratagene, La Jolla, CA). QuikChange can be applied to circular double strand cDNA. Using a pair of complementary oligonucleotides the DNA is amplified by PCR. Through specific substitution of bases the wild-type sequence has been replaced by the mutant sequence in the oligonucleotides; in the resulting PCR product the desired (RVRAA) mutation replaces the original sequence. In order to separate PCR product from –template the template is degraded by an endonuclease (DpnI) specific for methylated bases (base methylation is a consequence of bacterial DNA synthesis; PCR-produced cDNA is free of methylation).
After digestion with DpnI only the intact vector DNA can be amplified in bacteria. Positive clones are selected for antibiotics-resistance. The correct insert (i.e. receptor with appropriate mutation) needs to be identified by DNA sequencing.
GFP- and FLAG-tagged mouse homolog USP4 was kindly provided by D. Gray (Ottawa Regional Cancer Center, Ottawa, ON, Canada)
26
Table 2: List of vectors used in yeast-two-hybrid interaction assays
Vector Insert 1(A2A C-tail) Amino Acids pEG202 Mouse full-length R286-S410 Mouse 360 R286-G359 Mouse 385 R286 – L376 Mouse 391 R286-Q381 Mouse 395 R286-Q384 Human 311 R291-E311 Human 345 R291-V345 Human 360 R291-G360 Human 370 R291-S370 Human 385 R291-K391 Human 391 R291-K391 Human 395 R291-P395 Human 401 R291-D401 Human full-length R291-S411 Human full-length DVELL D482-L486 mutated to RVRAA pB42-AD SAP 3PDZ (rat) E148-Q482 SAP 2PDZ (rat) E148-K329 SAP PDZ-SH (rat) K404-V585 SAP-GUK (rat) I656-L849 DUSP-USP4 (human) Y57-T201
3.3 Yeast-Two-Hybrid Interaction test
The MATCHMAKER LexA two-hybrid system is a LexA-based interaction trap for detecting specific protein-protein interactions in yeast. The LexA System can be used to test for interaction between two previously cloned proteins or to screen a library for a gene encoding a novel protein that interacts with a known target (bait).
27 The yeast-two-hybrid interaction is based on a strong growth selection to detect and select transformations for interacting proteins. The LexA-based two-hybrid-system utilizes a powerful growth selection due to the conditional expression of a nutritional reporter gene.
The Matchmaker ® system kit provides a yeast strain (EGY48) carrying a reporter plasmid (LEU2); strain EGY48 has the genotype MATα trp1 his3 leu2 LexAop-LEU2; this means that the medium has to be supplemented with the amino acids tryptophan, histidine, leucin for the strain to proliferate (see below for a list of deficient genes). EGY48 can be distinguished from its parent strain by independence of uracil supplement:
HIS3 Imidazole glycerol-phosphate dehydratase (IGPD) catalyzes the sixth step of histidine biosynthesis. URA3 Orotidine-5'-phosphate (OMP) decarboxylase, catalyzes the sixth enzymatic step in the de novo biosynthesis of pyrimidines, converting OMP into uridine monophosphate (UMP) TRP1 Phosphoribosylanthranilate isomerase that catalyzes the third step in tryptophan biosynthesis LEU2 -isopropylmalate dehydrogenase
Strain EGY was used for the transformation of “bait” and “prey” pLexA and pB42. They were grown at 30°C in medium supplemented to select transformants (see Table below; if for example the EGY48 strain was transformed with the bait vector (pLexA) the synthetic drop-out (SD) medium contained tryptophan, leucin and glucose).
Yeast cells were transformed with the use of lithium acetate (LiAc). In this method, yeast was made competent to take up foreign DNA and was transformed in a single procedure. Yeast cells were grown in suspension, collected by centrifugation, washed and resuspended in 1.5 ml LiAc solution (containing 0.1µg of plasmid DNA along with 0.1 mg carrier DNA). Polyethylene glycol (PEG, 600µl 40%) was added and the mixture was incubated at 30°C for 30 minutes for an osmotic shock. Yeast cells were heat shocked (42°C in the presence of 3% DMSO) allowing DNA to enter. After cooling and centrifugation yeast was plated on the appropriate medium to select for transformants.
28 Table 3: Supplements of the synthetic drop-out media for cultivating transformed yeast
Plasmid Uracil Histidin Tryptophan Leucin Glucose Galactose
EGY48[p6op-LEU2]a - + + + + -
pLexA - - + + + -
pB42AD - + - + + -
pLexA and pB42AD - - - + + -
Interaction test - - - - - +
The yeast-two-hybrid assay is based on the fact that eukaryotic trans-acting transcriptional regulators are composed of independent domains, i.e. they can be physically separated but retain their function. (i) The DNA-binding domain (DNA-BD) binds to a specific promoter sequence, an upstream activating sequence, UAS; (ii) the activation domain (AD) directs the RNA polymerase II complex to transcribe the gene downstream of the DNA-binding site. Both domains are required to activate a gene and, normally, the two domains are part of the same protein. If physically separated the DNA-BD and AD peptides do not directly interact with each other and thus cannot activate the responsive genes. However, if the DNA-BD and AD can be brought into close physical proximity in the promoter region, the transcriptional activation function will be restored (Matchmaker® manual see Figure M-1).
29
A Prey + transactivation domain (AD)
B Bait + DNA-Binding domain (DB)
C Transactivation
Figure M- 1: Principles of yeast-two-hybrid assay
3.4 Cyclic AMP Accumulation Assays – Determination of cAMP- formation
Activated GS-protein coupled receptors stimulate adenylylcyclase followed by intracellular cAMP increase. Measurement of cAMP formation was according to the method developed by Salomon that involves incubating cells with [3H]adenine to label intracellular pools of adenine nucleotides. Accumulation of [3H]cAMP is then used as an index of the formation of cAMP; when cellular activity of phosphodiesterase is inhibited, this index is related to adenylyl cyclase activity. Separation of radioactively labeled cAMP from other components of the reaction mixture is efficiently accomplished utilizing sequential chromatography on Dowex cation-exchange (Dowex 50W-X4, Bio-Rad) and alumina columns (Salomon, 1991). The method was used to measure the degree of stimulation of
A2A receptor activated adenylylcyclase.
HEK293 cells seeded in 6-well plates were grown until 60% confluence and then transfected with plasmids driving the expression of the A2 adenosine receptor (wild-type or mutant) using CaPO4-precipitation. 48 hours later cells were incubated overnight with
30 [3H]adenine (1 µCi/ml); on the next day, the labelling medium was removed and substituted by 2 ml of serum-free DMEM medium containing adenosine deaminase (0.8 units/mL) and the phosphodiesterase inhibitor Ro 20-1724 (100 μM). The cells were incubated for 30 minutes at 37ºC in a 5% CO2 atmosphere. To initiate adenylyl cyclase a selective A2A receptor agonist CGS21680 was added, followed by 20min at 37°C. The reaction was terminated by aspiration of the reaction medium and addition of 1 mL ice- cold perchloric acid (PCA, 2.5%) containing 100 μM cAMP. 0.9 ml mL of each sample was neutralized with 0.1 mL of 4.2 M KOH. Separation of cAMP was performed by sequential chromatography on Dowex and Alumina columns. First, a large amount of contaminating ATP is eluted from the Dowex with water because negatively charged ATP does not bind to but is repelled by the anionic resin. Cyclic AMP can be eluted in the following fractions with water (repulsion of cAMP is weaker than of ATP). The eluted cAMP fractions are transferred to alumina columns from which [3H]cAMP is eluted with 3 ml 10 mM Imidazol plus 200 mM NaCl. After elution, the levels of [3H]cAMP are determined by liquid-scintillation counting: the vials are filled with 5 ml of scintillation cocktail, vortexed and counted in a -szinitillation counter (Packard 1900CA TRI-CARB). The assay was routinely prepared in triplicates.
3.5 Radioligand Binding Experiments
3 Equilibrium binding of the antagonist radioligand [ H]ZM241385 to the A2A adenosine receptor was measured in a final volume of 50 to 100 µL containing cell membranes (10 to
30 µg), HME buffer (25 mM HEPES-Na0H (pH 7.5), 1 mM EDTA, and 2 mM MgCl2), the indicated concentrations of [3H]ZM241385 and adenosine deaminase (0.8 units/mL). Adenosine deaminase (ADA) is an indispensable ingredient in binding and functional experiments (in membranes and intact cells) because it degrades contaminating adenosine. The binding was allowed to proceed to equilibrium at 30°C and was terminated after 60 minutes by filtration and washing over glass fiber filters using a cell harvester (Skatron). Filters were transferred to scintillation vials and 2 mL of scintillation cocktail were added. Radioactivity was measured using a liquid -szinitillation counter (Packard 1900CA TRI- CARB). Unspecific binding was determined in the presence of 10 µM xanthine amine congener (XAC) and amounted to ~5-10% of total binding in the KD concentration range. Data were fitted using the Sigma Plot software. In experiments using membranes from
31 clones with a high receptor expression level (1.5-8 pmol/mg of membrane protein) or low radioligand concentrations, the amount of membrane protein added and the assay volume was adjusted to avoid depletion of the radioligand (bound <10% of total).
Radioligand binding was used to quantitate the expression of the A2A adenosine receptor and the mutant A2A adenosine receptor (DVELL to RVRAA). For cell homogenization and the preparation of membranes, the culture medium was removed from the cells and these were scraped off in 2 ml PBS. After centrifugation at 9,000 g for 10 minutes, the cell pellet was resuspended in 1 ml HME-buffer (25 mM HEPES-NaOH pH 7.5, 2 mM MgCl2 and 1 mM EDTA) supplemented with 0.1 mM PMSF, an inhibitor of serine proteases. Samples were then subjected to a freeze/thaw cycle in liquid nitrogen, further homogenized by sonication with a sonication probe (4 pulses, 40%) and centrifuged again at 36,000 g for 15 minutes at 4ºC. After centrifugation, the supernatant was discarded and the pellet resuspended in a variable volume of 0.1 to 0.5 mL HME-buffer. Samples were frozen in liquid nitrogen and stored at –80ºC.
3.6 Protein Determination – Bradford Assay
To determine the concentration of protein in the samples, the Bradford Assay was used with the dye concentrate provided by Bio-Rad. The assay is based on the observation that the absorbance maximum for an acidic solution of Coomassie Brilliant Blue G-250 shifts from 465 nm to 595 nm upon binding to a protein. Both hydrophobic and ionic interactions stabilize the anionic form of the dye, causing a visible color change. The assay is useful since the extinction coefficient of a dye-albumin complex solution is linear over a 10-fold concentration range (i.e. from 1 to 10 μg of protein). Protein standards were prepared in the same buffer as the samples to be assayed. The standard curve was generated using bovine serum albumin (BSA) with concentrations of 0, 0.2, 0.4, 0.6, 0.8 and 1 mg/mL for the standard assay; the absorbance was measured at 595 nm.
3.7 Mitogen-activated Protein (MAP) Kinase Assay
Activation of the A2A receptor causes phosphorylation and activation of the MAP-kinase ERK1/2 (extracellular signal-regulated kinase) in many cell types. Receptor-mediated ERK-phosphorylation was followed over time. Stable transfected HEK293 cells were
32 seeded on 20 mm-dishes, grown to near-confluence and kept in serum-free medium overnight. Before the assay fresh medium with 1 µl ADA (0.8 U/ml) was added. After 20 minutes cells were stimulated with the selective A2A receptor agonist CGS21680 (0.5 µM) for the indicated time periods (0-60 min). The reaction was terminated by aspiration, addition of 80 µl lysis buffer (mM: 50 Tris.HCl, pH 7.4, 40 -glycerolphosphate, 100 NaCl, 10 EDTA, 10 p-nitrophenol phosphate, 1 PMSF, 1 Na3VO4, 10 NaF; 1% Nonidet P40, 0.1% SDS, 250 U/ml aprotinin, 40 µg/ml leupetin) and freezing in liquid nitrogen. After thawing, the cell debris was separated by centrifugation at 13,000g, the supernatant was transferred and the protein content was determined using bicinchonic acid (BCA). The BCA-method offers the advantage of being non-susceptible to detergents. The principle is in the reduction of bivalent copper ions by protein (due to cystein, tryptophan- and tyrosine residues); BCA chelates monovalent copper ion resulting in an absorption maximum at 562 nm, which may be measured photometrically.
Aliquots of the cellular lysate (15µg of protein) were applied to an SDS-polyacrylamide gel and were separated electrophoretically. The separated proteins were electro-transferred to nitrocellulose. Phosphorylated ERK was detected with a polyclonal rabbit antibody (1:1000; UnitedBiotech Inc., USA) and a horseraddish peroxidase-conjugated anti-rabbit antibody. The signal was recorded upon incubation with a peroxidase substrate on X-ray film. To demonstrate that equal amounts of p42 and p44 MAP-kinase had been loaded prebound antibody was removed by acid-strip (glycine-adetate, pH 3.0) and an incubation in potassium thiocyanate. After the blot was washed it was re-probed with an antibody directed against ERK independent of its phosphorylation status (1:1000; UnitedBiotech Inc., USA) to determine the total. The QuantiScan program (Biosoft, Leicester, UK) was used for densitometric quantification of the immunoreactive bands.
3.8 Fluorescence Microscopy
The first fluorescent protein GFP was found in the jellyfish Aequoria victoria. Through mutating the sequence proteins with different excitation/emission maxima could be generated inaddition to green emission, i.e. cyan, yellow and red. To detect the fluorescence the light-microscopes are equipped with at least two wavelength filter systems. The first is the excitation filter which is specific for a small wavelength (e.g. 420 nm) that excites fluorescence. The second filter is specific for the emitted wavelength by
33 the fluorescent protein and excludes light of different wavelength, e.g. the excitation beam. The filtered wavelengths are captured by a CCD-camera and the images can be viewed, stored and manipulated on a computer. In molecular biology, fluorescent proteins are used to follow proteins within the cell interior, to evaluate expression levels, to detect interaction by two proteins or to watch proteins moving in cells. Modern microscopes are able to differentiate - in real-time – between light in distinct wavelengths. The method has been used to record the receptor position using CFP-tagged A2A receptor (wild-type) and
CFP-tagged A2A RVRAA (mutant receptor); the receptor proteins were extended with cyan fluorescent protein on its c-terminus. In addition, GFP-tagged USP4 was used to identify co-expression with the A2A receptor.
For epifluorescence microscopy HEK293 cells were seeded on coated coverslips. Images could be recorded from live cells as coverslips were mounted in a 2 ml chamber filled with Krebs-Hepes-Buffer (pH 7.3). Images were taken on a inverted epifluorescence microscope (Zeiss Axiovert) using 63x oil immersion objective, a CFP/YFP filter set (Chroma Technology Corp., USA; Ludl-Filter-Wheel by Visitron GmbH, Germany) and a cooled charge-coupled camera (CoolSnap fix, Roper Scientific, USA). Images were stored and analyzed with Metamorph (Universal Imaging) and ImageJ (W. Rasband, T. Collins, National Institute of Mental Health, Bethesda, Maryland, USA).
3.9 FACS (Fluorescence activated cell sorting)
The method has been derived from the principle of flow cytometry. Cells labelled with either fluorescent antibodies transfected with proteins tagged to GFP are fed - through a low-diameter pipe - into the FACS machine where an optical system detects the emitted wavelengths. This method was used to quantify surface expression of wild-type and truncated A2A receptors with or without USP4.
HEK293 cells were seeded in a 6-well dish and were transfected with plasmids endcoding either wild-type or truncated A2A receptors. Simultaneously, cells were transfected with a second type of plasmid encoding either unfused GFP or GFP fused to USP4. After 23 hours at 37°C cells were detached using buffered EDTA-solution. Cells were suspended in a volume of 1 ml PBS and were counted. About 100,000 cells were centrifuged at 1,500g at 4°C for 8 minutes. Cells were resuspended in 100µl PBS plus 0.5 % BSA and incubated
34 with an antibody directed to the FLAG-epitope on the N-terminus of the A2A receptor (monoclonal M5 antibody from Sigma, working dilution 1:100) at 4°C for 30 minutes, followed by centrifugation to remove unbound antibody. After incubation with the secondary antibody (anti-mouse PE-Cy7-labelled rabbit IgG from BD Biosciences, 1:200) cells were washed a final time and resuspended in 100µl PBS plus BSA (0.5 %). Finally, the entire sample was analyzed by a FACScanto II (BD Biosciences, USA). Due to the incubation in the cold it was highly unlikely that antibody would be taken up by endocytosis which is very slow at low temperatures.
The specificity of the antibody labelling was assessed on control-transfected HEK293 cells
(that underwent a CaPO4-transfection with an empty plasmid vector, pcDNA3.1). After transfection cells were labelled with antibody as described above and submitted to FACS. Figure M-2 shows the labeling intensities; the x-axis represents the light intensity emitted by the PE-Cy7 fluorophore, the y-axis gives the emission of the fluorescent protein. The diagram demonstrates that cells that neither expressed GFP nor the FLAG epitope scored low on either fluorescence axis, all recorded events (blue dots) were gathered in the bottom left corner (the low intensity emission apparently represents cell-endogenous fluorescence). Thus, emission had to exceed the boundary of Q3 to score a cell as positively expressing GFP and epitope-tagged receptor (Fig. M-2). Consequently, events recorded in the right upper corner (Q2) represented cells double-positive in both GFP and FLAG-expression.
Figure M- 2: FACS example
35 4 Results
4.1 Binding of SAP102 to the carboxyl terminus of the A2A adenosine receptor
The extended intracellular carboxyl terminus, the c-tail of the A2A receptor is thought to present a docking site for components that regulate the expression and function of the receptor. Binding of SAP102 to the receptor c-tail was examined by expressing the isolated domains (these are the GUK, the SH3, and the PDZ domains) in yeast and putting each of the domains to an interaction test.
The EGY 48 yeast strain contains the LEU2 reporter plasmid (pSH 18-34). The plasmid expresses a reporter gene that enables yeast to grow on leucine-deficient agar. Expression of the LEU2 gene is contingent on the binding of LexA (the DNA-binding domain) to the DNA and of B42 (the activation peptide) to LexA (pEG202).
The coding sequence (open reading frame from human cDNA) for the protein subdomains was cloned into the pLexA vector, generating five plasmids (PDZ1-2, SH3, PDZ3, GUK, and SH3/GUK). The EGY48 strain was transformed with each of the plasmids (the empty vector was used as negative control); histidin-independent colonies were raised and transformed with the pB42-AD encoding the A2AR c-tail. Co-transformed yeast was plated on the combined synthetic drop-out medium that also excludes leucine; colonies grew to visible size four days after plating.
Figure 1A demonstrates colony growth of yeast transformed with the A2A receptor c-tail and the SAP102 GUK domain whereas none of the other transformants had a growth advantage over the control. The direct comparison suggested that SAP102 binds to the A2A receptor via its GUK domain.
In order to map the SAP102 docking site on the receptor c-tail truncated forms of the A2AR c-tail consisting of 106 (“395”; pEG202-A2AR 395), 102 (“391”), 91 (“380”,) and 71 amino acids (“360”) were constructed. Due to a restriction site common in the variant reverse primer the cDNA products were uniformly inserted into the multicloning site of the
36 pEG202 vector. Each of the plasmids encoding c-tails of different length was co- transformed with the “prey vector”, the SAP102 GUK domain.
Figure 1B demonstrates the effect of receptor truncation. Three individual co-transformed yeast clones were plated on the selective drop-out dish to test for the bait-prey interaction. The white hue that covers the plate segments becomes more intense with increasing growth. It is apparent that growth decreased when the receptor c-tail was shortened. A significant decline occurred with the constructs 380 and 360 suggesting that peptide sequence peripheral to position 380 was required for productive interaction. Since there was little difference between 391 and the full-length c-tail (and between 380 and 360) it was concluded that the GUK domain may recognize a peptide stretch flanked by residues 380 and 391 (381 GLPDVELLSHE 391).
The sequence comprises an acidic dileucin motif (E/D-xx-LL) known to mediate protein- protein interactions (which represents a sorting signal). A DxxLL motif is known to mediate binding of transmembrane proteins to GGA proteins, a family of ADP-ribosylation factor dependent clathrin-adaptors (trans-Golgi to lysosome sorting (Bonifacino and Traub, 2003). In addition a DXXLL motif was discovered in the vasopressin V2 receptor (membrane-proximal c-tail) where the sequence is required for cell surface targeting of the receptor (Schülein, et al., 1998).
The DVELL sequence of the A2A receptor carboxyl terminus has been mutated to RVRAA and inserted into pEG202. Transformed yeast was used to assess the interaction with the SAP102 GUK domain; the result is shown in Fig. 1C. The experiment reproduced the results from Fig. 1B; in yeast GUK binding required an extended c-tail and the interaction was lost with a shortened c-tail (380). Substituting the DVELL with RVRAA similarly resulted in poor growth, indistinguishable from that of the empty vector. This indicated that the interaction indeed relied on the presence of the DVELL sequence. Figure 1C thus led to the conclusion that the mutation abolishes the binding of the SAP102 and affirms that the interaction between A2A receptor and SAP102 GUK domain requires a receptor sequence flanked by residues 382 and 386; the results shown are representative of seven experiments.
37 SH SH 3 A GUK Yeast-Two-Hybrid: GUK PDZ 3 • Bait: human A2A receptor c- terminus (full length) • Prey: as indicated EMPTY PDZ 1-2 VECTOR
B A2A A2A 380 391 Yeast-Two-Hybrid:
A2A •Bait: as indicated •Prey: SAP102 GUK domain A2A FL 360 EMPTY VECTOR
C A2A A360 2A 380 A2A 391 391 Yeast-Two-Hybrid: RVRAA •Bait: as indicated A2A 395 •Prey: SAP102 GUK EMPTY domain A2A A2A 401 FL
Figure 1 - Interaction of SAP102 with the A2AR c-terminus; Importance of the DVELL (382-386) sequence.
38 4.2 Characteristics of the A2A receptor with the DVELL to RVRAA mutation
Since DVELL represents a renowned sorting signal it has been assessed the importance of the DVELL sequence in localization and signaling of the A2A receptor. Mutating DVELL to RVRAA in the receptor could lead to various consequences. They may be due to a loss of the sorting signal or due to severing the interaction with SAP102. To appreciate the mutation induced consequences it has been assessed GS dependent receptor signaling, that is agonist-dependent stimulation of cellular cAMP production. The A2A receptor is thought to be tightly coupled to the effector adenylyl cyclase and does not generate spare receptors (all the receptors populating the cell surface transmit the regulatory impulse to adenylyl cyclase). Hence, the degree of receptor-dependent cAMP production reflects the receptor density on the cell (Armstrong, et al., 2001).
First it was made sure that in HEK293 cells the mutant receptor reached the cell surface. Figure 2 shows HEK293 cells transiently transfected with plasmids encoding the CFP- tagged A2A receptor (wild-type sequence) and the CFP-tagged mutant receptor. An image of representative cells was captured one day after transfection on an inverted epifluorescence microscope (63-fold oil immersion objective; images captured with a cooled charge-coupled device camera).
As can be seen in Fig. 2, the A2A adenosine receptor was found at the cell surface and the distribution was similar for the mutant and wild-type receptor. Some receptor fluorescence was located in the cell interior and presumably represented unprocessed, immature receptor protein; this was also similar in cells with wild-type or mutant receptor. The experiment has been repeated several times, indicating that mutation of the DVELL motif to RVRAA shows no difference in the subcellular localization of the wild-type and mutated A2A receptor in HEK293 cells. Thus, the DVELL sequence is not a prominent sorting signal in transfected cells and its mutation did not abolish surface expression.
.
39
A2A-CFP A2A-RVRAA-CFP
Figure 2 - Epifluorescence microscopy.
Imaging the fluorescent A2A receptor in HEK293 cells. (Left) wild-type receptor; (right) mutant receptor.
4.3 Activation of cAMP-formation by wild-type and mutant receptor
HEK293 cells were transiently transfected to gauge the ability of the mutant receptor to stimulate the cellular formation of cAMP. The receptor was activated by the selective agonist CGS21680 which binds to the receptor with high affinity (EC50 ~ 8 nM). Transiently transfected cells were seeded in 6-well dishes and the cellular adenine nucleotide pool was metabolically pre-labeled by incubation with [3H]adenine (1µCi/ml). After 16 hours the medium was aspirated and cells were preincubated in fresh serum-free medium containing adenosine deaminase (1 U/ml) and the inhibitor of phosphodiesterase activity Ro20-1724 (100 µM). CGS21680 was added at the indicated concentrations; after a 20 minute-incubation the reaction was stopped by aspiration of the supernatant and by lysis of the cells with ice-cold perchloric acid. The supernatant (0.9 ml) was adjusted to neutral pH and cAMP was isolated by sequential column chromatography.
As can be seen in Figure 3A, transiently transfected HEK293 responded poorly to the receptor agonist. In cells that expressed either the wild-type or the mutant receptor a significant increase in cAMP occurred only at concentrations 10 nM; the half-maximal
40 concentration (EC50) was ~50 nM which is relatively low for an effect mediated by the A2A receptor. In order to circumvent the experimental problem it was generated stable cell lines with the wild-type and the mutant receptor. Stable lines were generated by selecting transfected cells for growth in the presence of the aminoglycoside antibiotic G418 (exploiting a kanamycin resistance gene encoded by the transfection plasmid). Thus the number of the cells that expressed receptor was expected to be markedly higher than in transiently transfected cells.
4.4 Identification of stable HEK293 cell clones by radioligand binding
After expanding each of the seven colonies they were transferred to a 10 cm dish; cells were grown to confluence and harvested. Cells were washed, homogenized in hypotonic buffer and membranes were prepared by differential centrifugation. A pellet obtained at 18,000g was resuspended to homogeneity in HME buffer. The membrane protein concentration was measured using a Bradford reagent. Membranes (30 µg/tube) were subjected to equilibrium radioligand binding with the selective antagonist [3H]ZM241385. Radioligand binding was used to assess the receptor expression level.
Figure 3B shows a saturation binding experiment using membranes of clone 2 expressing 3 the mutant version of the A2A receptor. Equilibrium binding of [ H]ZM241385 at increasing concentrations demonstrated that binding was saturated (Bmax = 1069±71 fmol/mg) with a KD of 1,6 (± 0,4) nM; the KD-value was compatible with the affinity value obtained for the wild-type A2A receptor.
The remaining cell clones were examined in a binding experiment carried out with a single saturating concentration of radioligand. Binding was performed on membranes obtained 48 hours after transfection with the mutant receptor. Only two of 7 clones (# 6 and # 8) expressed a significant amount of receptor. Specific binding values (i.e. the difference between total und unspecific binding) using [3H]ZM241385 at a concentration of 25 nM (and an excess of the non-selective unlabeled antagonist XAC to define unspecific [3H]ZM241385 binding) amounted to 2.5 pmol/mg membrane protein (clone 6) and 3.9 pmol/mg (clone 8) which is higher than the level obtained after transient transfection (700 – 1200 fmol/mg). I also obtained a stable clone expressing the wild-type receptor at a somewhat lower level (~ 500 fmol/mg).
41
42 Figure 3 - Activation of cAMP-formation by the wild-type and mutant receptor. (A) Formation of [3H]cAMP in the presence of increasing concentrations of the receptor agonist CGS21680 in HEK293 cells following transient transfection with the wild-type and 3 mutant A2A receptor and (C) in stable cell lines. (B) [ H]ZM241385 saturation binding curve for the wild-type A2A receptor; shown is specific binding in fmol/mg of membrane protein.
4.5 Cyclic AMP-formation in stable cell lines
Figure 3C shows cAMP-formation in the two stable cell lines (6 and 8) with the mutant receptor. Compared to cells that had been transiently transfected, the agonist response was much more robust, both in the amount of cAMP formed and the concentration-dependence of the effect. Clone 6 and clone 8 differed in the maximal response (~4000 vs. ~6500 cpm) and the apparent agonist potency (2.8 (±0.4) nmol/l vs. 4.5 (±0.9) nmol/l). While the agonist concentration response curves obtained the wild-type receptor and the mutant receptor in clone 6 were superimposable, the curve from clone 8 was shifted to the left.
Thus, there was no evidence that the mutation impaired the ability to activate GS and stimulate adenylyl cyclase. Thus, the c-terminal region harbouring the DVELL sequence (aa 382-386) is unlikely involved in G-protein coupling. The difference between the mutant clones may be related to receptor density.
4.6 Receptor-dependent phosphorylation of ERK1/2
ERK activation due to the A2A receptor stimulates cell proliferation for example in endothelial cells (Klinger, et al., 2002). As opposed to endothelial cells, in HEK293 cells the A2A receptor-dependent phosphorylation and activation of ERK1/2 (ERK) is dependent on cAMP (Seidel, et al., 1999). Receptor-dependent ERK-phosphorylation was found to be biphasic; while only the immediate effect was driven by cAMP a second delayed wave in ERK-phosphorylation was not due to cAMP (Gsandtner, et al., 2005). Therefore, ERK- phosphorylation has been investigated in HEK293 cell clones with either the wild-type or the mutant A2A adenosine receptor.
43 Receptor expressing cells were starved under serum-free medium overnight; upon exchanging the medium cells were incubated with the A2A adenosine receptor agonist CGS21680 (1µM) for increasing intervals. The reaction was stopped by harvesting the cells with ice-cold lysis buffer containing phosphatise inhibitors. Aliquots of the cellular lysates (15 µg of protein) were applied to SDS-polyacrylamide gels. After electrophoretic resolution and transfer to nitrocellulose, the level of active MAP-kinase was evaluated by immunoblotting with an antiserum recognizing the dually phosphorylated, active enzyme (Figure 4A upper panels, P-ERK). The loading control (Figure 4A, lower panels, holo ERK) was made by determining the total level of p42 and p44 MAP-kinase.
As can be seen from Figure 4A, the wild-type and the mutant A2A receptor similarly increased phosphorylation of ERK. The initial rise in phosphorylation was similar occurring within five minutes. The second protracted rise could be identified in the wild- type receptor clone where phosphorylated bands were detected at 20 and 30 minutes (after the addition of CGS21680) following a decline at 10 minutes. In the mutant receptor cell clone, however, the second phosphorylation phase had no peak; ERK phosphorylation continued to occur constantly but at a lower level. The increase in phosphorylation was not due to altered expression of ERK, the ERK1/2 levels were invariable over the time course (Fig. 4A, right hand panels).
Figure 4B shows a summary of several experiments studying the time course of agonist- mediated ERK-phosphorylation shown in Fig. 4A. The bar diagram represents the densitometric quantification of the phospho-ERK immune reactivity. To combine the data the band intensity was compared to background staining (i.e. a region equal in shape and size as that covered by the ERK-band). According to the summary in Fig. 4B the differences in the time course failed to reach statistical significance as they were apparently due to inter-assay variability. Thus, the DVELL-to-RVRAA mutation in the
A2A receptor c-tail did not change extent and time course of agonist-stimulated ERK- phosphorylation.
44
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.
4.7 Mapping the USP4 recognition site on the A2A receptor c-tail
USP4 has been established as a regulator of A2A receptor surface expression; if co- expressed with the A2A receptor it increases the level of functional receptors by de- ubiquitinating the receptor protein. The deubiquitinated receptor is thought to be redirected from the degradation pathway to the cell surface (Milojevic, et al., 2006).
45
The interaction with the receptor is due to the DUSP subdomain that recognizes a specific though undefined structural feature in the receptor c-tail (the DUSP subdomain has been isolated from a human brain cDNA library as cognate partner of the receptor c-tail). Therefore, the c-tail constructs were fused to the pB42-activation domain (see section 1) to narrow down the DUSP binding site in the receptor c-tail.
The EGY48 yeast strain was double transformed with each of the c-tail constructs and the USP4-DUSP subdomain fused to LexA. Figure 5A shows that yeast growth was induced by DUSP-binding to each of the c-tail constructs, i.e. the full-length and truncated c-tail
(A2AR 391, 380, 370 and 360). There was no meaningful difference between any of the truncated c-tail constructs compared to the full-length c-tail. Although growth appeared somewhat less robust with 360 it was clearly set off from growth in the negative control (empty pB42-AD vector).
The interaction test in Fig. 5A was performed with the human c-tail orthologue. In order to confirm the specificity of the interaction it was prepared analogous c-tail constructs using mouse cDNA (the human and mouse A2A receptors have substantial sequence homology but are not identical).
Figures 5B and 5C show two yeast interaction plates using the mouse A2AR-sequence. In both yeast growth was less with constructs 385, 360 and 345 relative to the full-length construct. Thus, there appeared to be a distinct difference between mouse and human. While a shorter human c-tail (360) still supported growth the orthologous mouse construct did much less so. Because of this ambiguity, it was obvious to study the interaction of receptor and USP4 in mammalian cells.
46
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.
4.8 Time course of the effect of USP4 on the expression of functional
A2A receptors HEK293 cells were transiently transfected with plasmids encoding the following sets of proteins: CFP-tagged A2A receptor, GFP-tagged USP4 and as control a GFP-vector with no insert. Receptor expression was quantified in membranes of cells that had been co- transfected with A2A receptor plus either the control vector or the vector encoding USP4 (the amount of DNA had to be kept constant in the transfection experiment). Cells were harvested at the indicated time points after transfection. Membranes were prepared and
47 used for radioligand binding with [3H]ZM241385; the radioligand was added at a single saturating concentration; unspecific binding was measured in the presence of 10µM XAC.
Figure 6 shows the summary of 4-8 experiments for each time point; data are given as fmol per 10 cm-culture dish ( standard error). It appears that the synthesis of ligand-binding receptors reached steady state at about 24 hours after transfection; at later time points the receptor turn-over was in equilibrium of synthesis and degradation. The time course differed between cells with or without over-expressed USP4. In the presence of USP4 the receptor number was increased at 19 hours; an USP4-dependent increase was also found at 23 and 30 hours but the increment was less pronounced.
Thus USP4 acted to enhance receptor expression which is in accordance with the previous report (Milojevic, et al., 2006). To observe the difference it was necessary to measure within a distinct time window (19- 23 hours after transfection). The size of the USP4 effect was rather modest; therefore, the number of co-transfected cells receiving only one type of DNA instead of both was higher than expected.
5000
4000
3000
2000 wild type ZM241385 bound (fmol/dish) ZM241385 1000 wild type+USP4
15 20 25 30 time after transfection
Figure 6 - Radioligand binding to membranes from HEK293 cells transfected with the A2A adenosine receptors in the presence and absence of USP4.
48 4.9 FACS analysis of A2A receptor surface expression It was exploited an epitope-tag fused to the N-terminus of the receptor to analyze the receptor level with an antibody. In order to identify cells that were double-transfected a GFP-tagged version of USP4 was used for co-transfection; a plasmid driving the expression of unfused GFP was used as control. By means of CaPO4-precipitation HEK293 cells (10 cm dish) were transiently transfected as in Figure 6. After 24 hours cells were washed and harvested with EDTA in Tris.NaOH solution. The suspended cells were incubated with an anti-FLAG antibody (dilution) and an anti-mouse IgG labeled with the fluorophore PE-Cy7 (dilution). After incubation at 4°C an aliquot of about 100,000 cells (corresponding to about 1/5 of the total cell population) was subjected to FACS.
Figure 7 - A2A receptor surface expression in cells co-transfected with USP4
Figure 7 demonstrates the principle of FACS and shows that the method is more accurate than determining the level of radioligand binding. The y-axis represents GFP emission (that closely corresponds to the emission spectrum of FITC-A), the x-axis represents the PE-Cy7 emission emanating from the secondary anti-mouse antibody. Receptor-positive cells were recorded in quadrants Q2 and Q4, whereas cells positive for GFP showed up in Q1 and Q2. Evidently most of the receptor expressing cells had been effectively co- transfected because they were allocated to Q2.
The difference between controls (“GFP”, left diagram) and USP4-transfected cells (right diagram) can be gleaned from the distribution of scoring events in Q1 and Q2. It is evident that the ratio of dots in Q2 over Q1 was greater with USP4-transfected cells than with the
49 controls. The difference in shape of the cloud of dots (y-axis scale) was likely due to the fact that unfused GFP is brighter because of less restricted expression than USP4-fused GFP. The mean intensity of PE-Cy7 fluorescence increased due to USP4 from 1733 to 2911. The increase appeared to be of comparable size but more pronounced than the increase detected by radioligand binding (Fig. 6). Thus, the more sensitive method in determining receptor regulation is by FACS when the results depend on co-transfection and should be appropriate for elucidating the preference of USP4 for receptor c-tail variants.
50 5 Discussion and Conclusions
The A2A adenosine receptor has an extended cytoplasmic carboxyl terminal tail that contains putative docking sites for accessory cellular proteins. The binding of two candidate interaction partners to the receptor c-tail were investigated; these were (i) the MAGUK (membrane-associated guanylate kinase-like domain) protein SAP102 (synapse associated protein of 102 kDa) and (ii) the ubiquitin-specific protease USP4.
5.1 Mapping the binding-site for SAP102 on the A2A adenosine receptor A yeast-two-hybrid interaction test was employed to verify the interaction with SAP102. The subdomains of SAP102 were isolated and expressed as fusion constructs with the B42- AD; in the interaction test only the GUK-subdomain supported yeast growth. On the other hand, a truncated c-tail lacking the distal 21 amino acid residues was sufficient for interaction with the GUK domain whereas a shorter c-tail construct lacking another 11 residues (c-tail 380) did not. Mutation of a DVELL sequence that is inherent to these 11 residues to RVRAA in the full-length c-tail reduced the binding affinity; as opposed to the wild-type sequence, the mutant c-tail construct failed to promote yeast growth in the presence of the LexA-GUK fusion on the appropriate synthetic drop-out medium.
The DVELL sequence conforms to a DxxLL sorting signal present in transmembrane proteins that cycle between the trans-Golgi network and endosomes (Bonifacino and Traub, 2003). In these proteins (such as LDL-related receptor proteins LRP 3 and 10) the motif is preceded by a serine, surrounded by several other acidic residues and located near the extreme end of a carboxyl terminus. In the A2A receptor the preceding serine is conserved (however there are no acidic residues in the immediate neighbourhood nor is the DVELL sequence close to the extreme c-terminal end). Since the neighbouring serine is part of a casein-kinase substrate sequence ([S/T]xxD) the interaction could in theory be modulated through protein phosphorylation.
A cognate partner for proteins with the DXXLL motif is the mammalian GGAs, ADP- ribosylation factor dependent clathrin adaptors, localized to the Golgi network and endosomes. They link membrane proteins to the endocytosis machinery. The A2A receptor however does not internalize even upon continuous activation during an extended time
51 period; in addition its expression level is not altered by inhibitors of endosomal degradation. Thus, it is unlikely that the receptor be sorted to endosomes or retained in the
Golgi cisternae. The DVELL sequence in the A2A receptor hence must be recognized even when the receptor resides in the plasmalemmal membrane at the cell surface. According to our data the motif provides for a direct interaction with SAP102.
In order to assess the importance of the sequence motif for the principal signaling properties, a receptor variant with the DVELL-to-RVRAA mutation was evaluated in mammalian cells. The results indicated that the mutation did not impair receptor-dependent activation of the cognate G-protein, GS. GS stimulates adenylyl cyclase leading to the formation of the second messenger cAMP. In HEK293 cells, cAMP causes rapid phosphorylation of the MAP-kinase, ERK1/2; the receptor-dependent delayed phase of ERK1/2 phosphorylation however is via a G-protein independent signaling pathway. The potency of the receptor agonist and the extent of receptor-dependent cAMP-formation were similar with the wild-type and mutant A2A receptor. Similarly, time course and the extent of ERK1/2 phosphorylation – in the initial and the delayed phase - were indistinguishable.
Of note, the receptor-effect on cAMP was much greater in a stable clone than in transiently transfected cells. The increase was observed for potency and efficacy; the EC50 was ~50 nM in transient transfectants, only ~5 nM in the stable lines and even less in one clone expressing mutant receptor; the maximum increased correspondingly. Since the receptor levels were quite comparable between the cells (1 to 4 pmol/mg) the difference unlikely reflected a receptor reserve (i.e. enhanced potency with higher receptor number). More likely, it was due to a selection process that yielded cells with high sensitivity for activated receptor or activated GS.
The data obtained in HEK293 model cell (the mammalian fibroblast cell type was used) however may not faithfully predict the situation in a polarized neuronal cell with its specific protein equipment. Thus, while in HEK293 cells the mutant receptor is indistinguishable from wild-type this may not be the case in a nerve cell. A neuronal cell gathers SAP102 and other members of the PSD-protein family in the postsynaptic density where they are thought to mediate local clustering of receptors and ion channels. There is evidence that the A2A receptor resides at or in close vicinity to the postsynaptic density.
52 Possibly the receptor undergoes a regulatory control by SAP102. While reported interactions with membrane proteins (such as the NMDA-type glutamate receptors) have been via the PDZ subdomains, our yeast data is the first evidence that the GUK-subdomain (which is a degenerate inactive kinase domain) can also be involved in the recognition of binding partners.
5.2 The binding site of USP4 on the A2A adenosine receptor The gained interaction data in the yeast-two-hybrid system with USP4 were however less explicit - in regard to the USP4-docking site - than those with SAP102. The original observation published by Milojevic and coworkers was that USP4 binds to the c-terminus and splits ubiquitin from the receptor. This weakens the quality control for misfolded A2A receptors and leads to an increase of their surface expression levels (Milojevic, et al., 2006). The interaction between the USP4-DUSP domain and the receptor c-tail could have been reproduced. However, using progressively truncated c-tail constructs the USP4- DUSP recognition site could not be mapped to a specific peptide segment. In the mouse species orthologue the interaction was diminished after truncation by 26 amino acids (but not 20); in contrast in yeast transformed with the human orthologue the interaction decreased gradually with each distal peptide segment removed. The discrepancy between mouse and human was surprising given the high degree of sequence similarity. Thus, the yeast-two-hybrid hunt was an inappropriate model system to study the binding of USP4 to the receptor and therefore proceeded with co-expression experiments in mammalian cells.
In HEK293 cells transiently co-transfected with USP4 and the human A2A receptor the apparent effect was an increase in the level of receptor on the cell surface. Two approaches have been used: first, radioligand binding to membranes of disrupted cells; second, antibody staining of the receptor on the surface of intact cells. The results from both approaches were compatible but FACS analysis of the antibody-labelled cells found a larger increment – due to co-expression with USP4 - than that indicated by radioligand binding. The difference is likely due to the advantage of encompassing co-transfected cells as FACS selected for cells positive in USP4; in the radioligand binding assay double- and single-transfected (negative in USP4) were not distinguished. Nevertheless the experiments uniformly suggested that the USP4-dependent receptor increase was due to an accelerated export to the cell surface but that the effect size was modest.
53 5.3 Conclusion
In summary, the experimental work presented here confirms the hypothesis that the A2A adenosine receptor c-tail serves as binding site for regulatory proteins. The PSD-protein SAP102 binds to the receptor by virtue of a distinct peptide motif (“DVELL”) that has been hitherto appreciated as an intracellular sorting signal. Disruption of the DVELL sequence reduced binding of the SAP102 GUK-subdomain to the receptor c-tail. Nevertheless, signaling by a mutant receptor (where DVELL had been substituted by RVRAA) was normal in a fibroblast cell line. The effect of the mutation on receptor function therefore needs to be re-evaluated in a nerve cell where SAP102 may be present to control localization of or signaling by the receptor. Thus, the data underscore the usefulness of the yeast-two-hybrid approach in determining the coordinates of protein- protein interaction. However, the data with USP4 raise a cautionary note. May be due to the interference by yeast-endogenous protein components an apparently direct interaction could in fact be only indirect. To assess the interaction of USP4 and receptor c-tail an in vitro approach should be preferred where the binding of purified proteins can be tested with no interference by proteins from a foreign host organism.
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59 v. Appendix
60 v.1 Zusammenfassung
Der A2A Rezeptor hat einen abstehenden cytoplasmatischen carboxyl-terminalen Schwanz (c- tail). Dieser beinhaltet potentielle Andockstellen für bestimmte zelluläre Proteine. Die Bindung von zwei Interaktionspartnern an diesen c-tail wurde untersucht. Zu diesen zählen (i) das MAGUK (Membran Assoziierte Guanylase Kinase-ähnliche Domain) SAP102 (Synapse Assoziiertes Protein von 102kDa) und (ii) die Ubiquitin-spezifische Protease USP4. Mittels Yeast-two-Hybrid Test wurde die Interaktion zwischen diesen zwei Proteinen und verschiedenen Varianten des c-tail, mit originaler Länge und vom distalen Ende an trunkierte Varianten, untersucht. Vom Multidomain Protein SAP102 wurden zusätzlich die verschiedenen Subdomains (PDZ, SH und GUK) isoliert und getrennt getestet, sowie von USP4 die Substraterkennungsdomain (DUSP).
Im Falle von SAP102 konnte in mehreren Interaktionstests gezeigt werden, dass der c-tail des
A2A Rezeptors mittels 5 Aminosäuren zwischen Position 382-386 an die GUK Domain bindet. Die Signaltransduktion wurde auch in einer bindungs-unfähigen Mutante, in welcher anstatt der originalen Sequenz (DVELL) eine alternative Sequenz (RVRAA) verwendet wurde, untersucht. In HEK293 Zellen führte weder die Expression des Rezeptors, noch die rezeptor- abhängige cAMP-Produktion oder die Aktivierung der MAP-Kinase ERK1/2 zu unterschiedlichen Ergebnissen im Vergleich zwischen mutierten und Wildtyp Rezeptor.
Im Unterschied zu SAP102, konnte für USP4 keine eindeutige Bindungsstelle am c-tail des
A2A Rezeptors entdeckt werden. Durch progressive Trunkierung des c-tails konnte eine potentielle Interaktions-Sequenz ermittelt werden. Diese konnte jedoch in analogen
Experimenten mit den homologen A2A Rezeptor c-tail der Maus nicht bestätigt werden. Aus diesen Ergebnissen kann daher geschlossen werden, dass (i) der Yeast-two-Hybrid Methode in diesem Fall an bestimmte Grenzen stößt, und diese Ergebnisse durch andere unabhängige
Methoden reevaluiert werden sollten, und dass (ii) die SAP102-Interaktionsstelle im A2A Rezeptor, sowohl als Wildtyp als auch als Mutante, nachfolgend in Nervenzellen untersucht werden sollte.
61