Olfactory receptor signaling is regulated by the post-synaptic density 95, Drosophila discs large, zona-occludens (PDZ) scaffold multi-PDZ domain protein Ruth Dooley1,2,*, Sabrina Baumgart2,*, Sebastian Rasche1, Hanns Hatt1 and Eva M Neuhaus3 Molecular Medicine Lab RCSI, Education & Research Centre Smurfit Building, Beaumont Hospital, Dublin, Republic of Ireland Department of Cell Physiology, Ruhr University Bochum, Germany ´ NeuroScience Research Center, Charite, Universitatsmedizin Berlin, Germany ă Keywords MUPP1; olfactory neuron; olfactory receptor; PDZ protein; signal transduction Correspondence E M Neuhaus, NeuroScience Research ´ Center, Charite, Universitatsmedizin Berlin, ¨ 10117 Berlin, Germany Fax: +49 30 450 539 970 Tel: +49 30 450 539 702 E-mail: eva.neuhaus@charite.de *These authors contributed equally to this work (Received September 2009, revised October 2009, accepted 12 October 2009) doi:10.1111/j.1742-4658.2009.07435.x The unique ability of mammals to detect and discriminate between thousands of different odorant molecules is governed by the diverse array of olfactory receptors expressed by olfactory sensory neurons in the nasal epithelium Olfactory receptors consist of seven transmembrane domain G protein-coupled receptors and comprise the largest gene superfamily in the mammalian genome We found that approximately 30% of olfactory receptors possess a classical post-synaptic density 95, Drosophila discs large, zona-occludens (PDZ) domain binding motif in their C-termini PDZ domains have been established as sites for protein–protein interaction and play a central role in organizing diverse cell signaling assemblies In the present study, we show that multi-PDZ domain protein (MUPP1) is expressed in the apical compartment of olfactory sensory neurons Furthermore, on heterologous co-expression with olfactory sensory neurons, MUPP1 was shown to translocate to the plasma membrane We found direct interaction of PDZ domains + of MUPP1 with the C-terminus of olfactory receptors in vitro Moreover, the odorant-elicited calcium response of OR2AG1 showed a prolonged decay in MUPP1 small interfering RNA-treated cells We have therefore elucidated the first building blocks of the putative ‘olfactosome’, brought together by the scaffolding protein MUPP1, a possible central nucleator of the olfactory response Structured digital abstract l MINT-7290305: OR2AG1 (uniprotkb:Q9H205) physically interacts (MI:0915) with MUPP1 (uniprotkb:O75970) by anti tag coimmunoprecipitation (MI:0007) l MINT-7289999, MINT-7290250, MINT-7290063, MINT-7290110: OR2AG1 (uniprotkb:Q9H205) binds (MI:0407) to MUPP1 (uniprotkb:O75970) by peptide array (MI:0081) l MINT-7290162: mOR283-1 (uniprotkb:Q9D3U9) binds (MI:0407) to MUPP1 (uniprotkb:O75970) by peptide array (MI:0081) l MINT-7290128: mOR-EG (uniprotkb:Q920P2) binds (MI:0407) to MUPP1 (uniprotkb:O75970) by peptide array (MI:0081) Abbreviations CamKII, calcium ⁄ calmodulin-dependent protein kinase II; GABAB, c-aminobutyric acid receptor B; GFP, green fluorescent protein; GST, glutathione S-transferase; HRP, horseradish peroxidase; INAD, inactivation no after potential D; MUPP1, multi-PDZ domain protein 1; OMP, olfactory marker protein; OR, olfactory receptor; OSN, olfactory sensory neuron; PDZ, post-synaptic density 95, Drosophila discs large, zona-occludens 1; RNAi, RNA interference; siRNA, small interfering RNA FEBS Journal 276 (2009) 7279–7290 ª 2009 The Authors Journal compilation ª 2009 FEBS 7279 PDZ proteins interact with olfactory receptors l l l l R Dooley et al MINT-7290219: hOR3A1 (uniprotkb:P47881) binds (MI:0407) to MUPP1 (uniprotkb:O75970) by peptide array (MI:0081) MINT-7290191: hOR1D2 (uniprotkb:P34982) binds (MI:0407) to MUPP1 (uniprotkb:O75970) by peptide array (MI:0081) MINT-7289922: AC3 (uniprotkb:Q8VHH7) and MUPP1 (uniprotkb:O75970) colocalize (MI:0403) by fluorescence microscopy (MI:0416) MINT-7289933, MINT-7289954, MINT-7289978: OR2AG1 (uniprotkb:Q9H205) binds (MI:0407) to MUPP1 (uniprotkb:O75970) by pull down (MI:0096) Introduction Detection of an odorant is initiated by activation of a fraction of many hundreds of G protein-coupled odorant receptors (ORs) expressed in olfactory sensory neurons (OSNs) of the mammalian olfactory epithelium [1] Signal transduction begins when an odorant molecule binds to an OR, resulting in the activation of adenylyl cyclase type III [2] via the olfactory G protein Gaolf [3] cAMP then binds to a cyclic nucleotide-gated channel [4–7], allowing it to conduct cations such as Na+ ⁄ Ca2+ The calcium ions then bind to a calcium-gated chloride channel [8], further depolarizing the cell How these diverse signaling molecules find each other in the complex and densely-packed environment of the cell, avoiding cross-talk with other signaling pathways, in order to ensure the rapidity and specificity of signaling, remains an unanswered question The idea of the existence of an ‘olfactosome’, or highly ordered multi-component protein network involving the olfactory signal transducing molecules, has been previously discussed [9–11]; however, until now, no concrete evidence has been provided Scaffolding networks have been investigated in detail in the visual system of Drosophila melanogaster, where inactivation no after potential D (INAD), made up of five postsynaptic density 95, Drosophila discs large, zonaoccludens (PDZ) domains, has the ability to bind to various molecules in the signal transduction cascade, thereby bringing them into close proximity and ensuring a rapid and specific signal transduction [9,12] PDZ domains are modular protein–protein interaction domains, which are amongst the most abundant protein interaction domains in organisms from bacteria to mammals, and have been implicated in various processes, including clustering, targeting and routing of their binding partners [13–15] PDZ target specificity is usually dependent on the extreme carboxyl-terminal amino acid sequence of the interacting protein; however, for some ligands, residues as far back as the )10 position may influence binding energy [16] Peptidebinding preferences of PDZ domains led to their 7280 division into three discrete functional classes [16], which may not be as strict as initially anticipated because predictions of PDZ domain–peptide interactions were recently shown to be evenly distributed throughout selectivity space [17] The multi-PDZ domain protein (MUPP1) is composed of thirteen PDZ domains, each diverse with respect to its amino acid sequence It was first identified through a yeast two-hybrid screening as an interaction partner of the C-terminus of 5-hydroxytryptamine receptor type 2C [18] Subsequently, many diverse interaction partners of MUPP1 have been characterized, including G-protein coupled c-aminobutyric acid receptor B (GABAB) [19] and the calcium ⁄ calmodulin-dependent protein kinase II (CamKII) [20] In the present study, we introduce ORs as novel interaction partners of MUPP1 Results MUPP1 is expressed at the sites of olfactory signal transduction ORs are expressed on the ciliary membranes of OSNs, the first point of contact of the sensory cell with incoming odorant molecules To investigate whether a receptor centered multi-component protein network might exist, we tested for the expression of PDZ scaffolding proteins in the olfactory epithelium using RT-PCR (Fig 1A) We detected robust expression of MUPP1 and weak expression of ZO-1, but could not detect Patj, Erbin or DLG-2 Because MUPP1 mRNA was highly abundant, we examined the expression of the protein by western blotting Upon fractionation of the olfactory epithelium [21], we found MUPP1 to be present to a greater extent in the cilia-enriched fraction compared to the remaining cell fractions (Fig 1B) Using olfactory marker protein (OMP)-green fluorescent protein (GFP) transgenic mice, expressing GFP in every mature olfactory sensory neuron [22], we investigated the cellular localization of MUPP1 in the olfactory epithelium and found MUPP1 to be FEBS Journal 276 (2009) 7279–7290 ª 2009 The Authors Journal compilation ª 2009 FEBS R Dooley et al PDZ proteins interact with olfactory receptors A B C Fig MUPP1 expression in olfactory sensory neurons (A) Expression of mRNA of different PDZ scaffolding proteins in the olfactory epithelium by RT-PCR *Weak band for ZO-1 (B) Fractional preparation of whole olfactory epithelium shows MUPP1, at 220 kDa, enriched in the cilia fraction (1) compared to the remaining cell fractions (2–4); a Coomassie-stained gel is shown as a loading control (C) MUPP1 is co-localized with adenylyl cyclase in the apical layer of the olfactory epithelium Immunohistochemical staining of 14 lm cryosections of OMP-GFP mouse olfactory epithelium using specific antibodies against MUPP1 (green) and adenylyl cyclase (red) Overlay shows mature OSNs in blue White arrow denotes the apical layer Scale bars = 50 lm (D) Higher magnification image of MUPP1 ⁄ adenylyl cyclase stained olfactory epithelium The arrow shows MUPP1 expression in cilia and in dendritic knobs Scale bar = lm D expressed in the apical part of OSNs, mainly in the cilia layer (Fig 1C) Double immunolabeling showed co-localization with adenylyl cyclase 3, a central molecule in the olfactory signal transduction cascade in the cilia of OSNs (Fig 1C, D) Interaction of PDZ domains + of MUPP1 with OR2AG1 in vitro PDZ domain interactions have been well characterized and modes of binding have been grouped into three main classes of PDZ binding motifs, occurring at the C-terminus of the interacting proteins [16] We scanned the human olfactory receptor repertoire for putative binding motifs and discovered them in the extreme C-termini of approximately 30% of human ORs, with examples from each of the three classes being outlined to date (7% Class I, 12% Class II and 10% Class III; Fig 2A) Intriguingly, this suggested that a subset of ORs could have the ability to interact with PDZ domains of MUPP1 We performed co-immunoprecipitation experiments to verify the ability of ORs containing a PDZ interaction motif to bind to MUPP1 Hana3A cells were transfected with HA-OR2AG1 These cells express members of the RTP and REEP family, which comprise molecular chaperones known to promote the expression of ORs [23] Antibodies to the HA tag were used to co-immunoprecipitate MUPP1, as shown by a band of 220 kDa in western blots (Fig 2B, immunoprecipitation: a-HA), whereas no MUPP1 immunoreactivity could be observed in precipitates of nontransfected cells (Fig 2B, control) These results indicated an interaction between OR2AG1 and MUPP1 in the recombinant expression system MUPP1 is made up entirely of thirteen different PDZ domains, each being diverse in sequence We set out to determine which of these PDZ domains were involved in the molecular interaction with ORs We created a glutathione S-transferase (GST) fusion peptide of the C-terminus of OR2AG1 and in vitro translated the PDZ domains of MUPP1, in pairs (1 + 2, FEBS Journal 276 (2009) 7279–7290 ª 2009 The Authors Journal compilation ª 2009 FEBS 7281 PDZ proteins interact with olfactory receptors R Dooley et al + 4, etc.) (Fig 2C) Interaction assays were then carried out by incubating different pairs of PDZ domains as in vitro translation products with OR2AG1 C-terminus GST fusion peptides A specific binding of PDZ domains + was determined via western blotting, whereas, for example, in vitro translated PDZ domains + did not have the ability to bind to the C-terminus of OR2AG1 (Fig 2D) None of the PDZ domains could bind to GST alone We then tested binding of single PDZ domains + 2, and found that both could bind to the OR C-terminus (Fig 2D) Next, we investigated the ability of PDZ domains + to bind to receptor C-termini of 15 amino acids in length, which were spotted on microarrays The arrays were probed four times with PDZ domains + fused to the HA tag for subsequent A analysis of binding by antibody incubation and chemiluminescence detection (Fig 2E) Interactions were analyzed in every experiment with positive (HA tag spotted directly) and negative (FLAG tag spotted directly and A1 ⁄ A2) control spots Interactions that yielded robust interactions of PDZ domains + with the olfactory receptors hOR2AG1 (S-T-L), mOR283-1 (A-T-V) and hOR3A1 (S-L-A), which all contain PDZ domain binding motifs in their C-termini, were scored as array positives (Fig 2E) hOR1D2 and mOR-EG, which are olfactory receptors that not contain classical PDZ interaction motifs in their C-termini, also showed positive interactions but, in the case of hOR1D2, only in two out of four experiments Other olfactory receptors, such as mOR167-4, mOR199-1, M71, M72 and mOR241-1 B C D E Fig The C-terminus of OR2AG1 interacts with MUPP1 in vitro (A) Pie chart illustrating the abundance of classical PDZ motifs in human OR C-termini (B) MUPP1 was immunoprecipitated in HA-OR2AG1 expressing Hana3A cells using a-HA antibodies, detection was performed with a-MUPP1 (*MUPP1) and a control was performed with identical amounts of nontransfected cell lysates from Hana3A cells The blot shown is representative of three independent immunoprecipitation experiments (C) Western blot using HA-specific antibodies showing the in vitro translation products of PDZ domain pairwise constructs Three nonspecific bands appear at 170, 70 and 30 kDa Specific bands at the correct molecular weights are outlined (white asterisk) (D) PDZ domains + both interact with OR2AG1_GST in vitro Interaction assay using in vitro translated PDZ domains + 2, PDZ domains + 4, PDZ domain and PDZ domain with GST alone or C-terminus OR2AG1_GST The blots shown are representative of four independent experiments for each interaction assay described (E) Peptide microarray with C-termini of different receptors incubated with PDZ domains + fused to HA; chemiluminescence detection on film after incubation with a-HA antibodies and HRP-coupled secondary antibodies The array shown is representative of four independent experiments; peptide sequences for spots A1–A12 (row 1), A13–A24 (row 2) and B1 (FLAG tag) and B2 (HA tag, positive control) are listed in Table S1 A1 and A2 serve as negative controls 7282 FEBS Journal 276 (2009) 7279–7290 ª 2009 The Authors Journal compilation ª 2009 FEBS R Dooley et al PDZ proteins interact with olfactory receptors A B C D Fig MUPP1 plasma membrane translocation on co-expression of odorant receptors (A) MUPP1-GFP expressed alone in Hana3A cells exhibits a diffuse cytosolic expression Co-transfection of OR2AG1 with MUPP1-GFP leads to a predominant plasma membrane expression of MUPP1-GFP with clustering apparent Truncation of OR2AG1 from the final eight amino acids leads to a cytosolic expression of MUPP1GFP; at least five independent experiments were performed for each condition (B) Higher magnification of the plasma membrane of the cells shown in (A) (C) In vitro interaction properties of truncated hOR2AG1 C-terminus Western blot showing HA-PDZ1 + probed with 2AG1-GST and trunc8-GST, at 55 kDa, using a-HA antibodies The experiment was repeated three times with similar results being obtained (D) Co-expression of hOR1D2 and hOR3A1 also resulted in translocation of MUPP1-GFP to the plasma membrane; at least five independent experiments were performed for each receptor Scale bars = 20 lm and mGluR2, as well as the olfactory cyclic nucleotide-gated ion channel subunit A2, did not show any interaction with the PDZ domains investigated We furthermore investigated the binding determinants in the C-terminus of hOR2AG1 by spotting peptides that correspond to mutated or shortened receptor C-termini Truncation of the last amino acids abolished binding of the C-terminus of hOR2AG1 to PDZ domains + hOR2AG1 constructs where the last four amino acids H-S-T-L were mutated to H-A-T-A [OR2AG1_deltaPDZ(A)] still bound to PDZ domains + 2, whereas mutation to H-W-T-W [OR2AG1_deltaPDZ(W)] completely abolished binding (Fig 2E) MUPP1 shows plasma membrane localization upon co-expression of ORs MUPP1 is a cytosolic protein, and MUPP1-GFP, similar to endogenous MUPP1, shows a homogenous, predominantly cytosolic distribution when expressed in Hana3A cells (Fig 3A) Interestingly, when co-expressed with hOR2AG1, MUPP1 exhibited a largely plasma membrane expression in a subset of cells, forming clusters at the cell surface (Fig 2A, B) Approximately 5% of transfected cells exhibited this translocation effect of MUPP1-GFP This apparently low proportion of cells reflects the notoriously low expression rate of olfactory receptors in heterologous systems and the relatively high amount of cells expressing MUPP1-GFP However, the results obtained in the present study correlate with various studies showing a similar proportion of transiently transfected cells responding to odorant in ratiometric calcium imaging experiments [24–26] This alteration in the subcellular distribution of MUPP1 upon co-expression with ORs supported the finding of a physical association between MUPP1 and ORs in the heterologous expression system We hypothesized that, by deleting the components of the PDZ binding motif, we could disrupt MUPP1 translocation Truncation of the receptor from the final eight amino acids (amino acids 309–316) did result in a clear abrogation of the association, as outlined by the predominantly cytosolic localization of MUPP1GFP (Fig 3A) We then investigated the in vitro binding properties of the truncated C-terminal mutant of OR2AG1 by creating a GST fusion construct The FEBS Journal 276 (2009) 7279–7290 ª 2009 The Authors Journal compilation ª 2009 FEBS 7283 PDZ proteins interact with olfactory receptors R Dooley et al A B C D E F G H I Fig Functional role of MUPP1 in OR signaling (A) Western blot showing MUPP1 expression in Hana3A cells (control) compared to 48 and 72 h after siRNA (exon5) transfection (B) Representative ratiometric calcium imaging responses of transiently transfected Hana3A cells [siRNA(1)]; the arrow represents the beginning of application of amylbutyrate, lasting for 10 s (C) Western blot showing MUPP1 expression in Hana3A cells (control) compared to scrambled siRNA, siRNA against exon of MUPP1 and siRNA against exon 45 of MUPP1, 72 h after transfection (D) Bar chart showing the rise time (10–90%) of Hana3A cells responding to amylbutyrate, transfected with OR2AG1 (ctrl) (n = 15) or siRNA(exon5)-treated Hana3A cells transfected with OR2AG1 (RNAi) (n = 15) (E) Time of decay from 90% of peak amplitude to 10% of average baseline (n = 15) for each condition and the percentage of cell responses decaying to basal levels within the time-frames outlined Cell responses not decaying within the time-frame of experiment were included in the > 20 s section; n = 15 for control, n = 27 for RNAi(exon5) (F) Bar chart showing the rise time (10–90%) for siRNA(exon45) transfected Hana3A cells; n = 12 for control, n = 12 for RNAi(exon45) (G) Bar chart showing the time of decay (90–10%) and percentages of cell responses for siRNA(exon45) transfected Hana3A cells; n = 12 for control, n = 12 for RNAi(exon45) (H) Bar chart showing the rise time (10–90%) for Hana3A cells transfected with scrambled siRNA (I) Bar chart showing the time of decay (90–10%) and percentages of cell responses for scrambled siRNA transfected Hana3A cells Error bars show the SEM **P < 0.01, ***P < 0.001 C-terminal mutant peptide was incubated with PDZ domains + of MUPP1 and failed to interact with truncated mutant (Fig 3C) 7284 Consistent with this observation is the fact that other olfactory receptors showing interactions with PDZ domains + (hOR1D2, hOR3A1) also caused FEBS Journal 276 (2009) 7279–7290 ª 2009 The Authors Journal compilation ª 2009 FEBS R Dooley et al PDZ proteins interact with olfactory receptors A B C D E F Fig Interaction of MUPP1 with OR2AG1 is important for controlled signal decay (A) Immunocytochemistry (a-HA antibody) showing stable expression of MUPP1-PDZ1 + 2-HA in Hana3A cells Scale bar = 20 lm (B) Representative ratiometric calcium imaging traces for Hana3A cells (control) and MUPP1-PDZ1 + 2-HA cells (1 + 2) transiently transfected with OR2AG1 Arrows denote amylbutyrate application (C) Bar chart showing the rise time (10–90%) for Hana3A cells stably expressing MUPP1-PDZ1 + 2-HA, transiently transfected with OR2AG1; n = 13 for control, n = 24 for PDZ domains + (D) Decay of response (90–10%) (n = 13 for control, n = 24 for PDZ domains + 2) and the percentage of responses to amylbutyrate decaying to basal levels within the given time-frames (E) Transient expression of a truncated version of OR2AG1 missing the last eight amino acids (trunc8) Bar chart showing the rise time (10–90%) for Hana3A cells expressing the truncated receptor (n = 12 for control, n = 17 for OR2AG1-trunc8) (F) Decay of response (90–10%) (n = 12 for control, n = 17 for OR2AG1-trunc8) and the percentage of responses to amylbutyrate decaying to basal levels within the given time-frames Error bars show the SEM **P < 0.01, ***P < 0.001 MUPP1-GFP translocation to the plasma membrane in the Hana3A cells (Fig 3D) MUPP1 controls the duration of Ca2+ signaling mediated by recombinant ORs To determine whether MUPP1 has the ability to regulate OR function, we investigated the role of this scaffolding protein in hOR2AG1-mediated Ca2+ mobilization by performing ratiometric Ca2+ imaging in Hana3A cells These cells express MUPP1 endogenously and, by reducing the amount of MUPP1 by RNA interference, we aimed to investigate the functionality of the interaction between MUPP1 and hOR2AG1 Transfection of small interfering RNA (siRNA) against Mupp1 led to an almost complete knockdown of the MUPP1 protein, as shown by western blotting (Fig 4A) Next, we monitored the response of transiently transfected OR2AG1 to its specific odorant ligand, amylbutyrate [24] via ratiometric calcium imaging in siRNA-treated cells (Fig 4B) When MUPP1 was largely absent, the OR-elicited response exhibited a similar rise time to that of the control cells, 5.54 ± 0.87 s for RNA interference (RNAi) compared to 4.27 ± 0.65 s for the control (Fig 4B, C) However, the response failed to decay within the normal average time-frame in siRNA-treated cells (19.3 ± 2.9 s) compared to the control cells (7.2 ± 2.1 s) (Fig 4B, D) Using another siRNA directed against an alternative exon of Mupp1, similar results were obtained The rise time of the OR-elicited response was similar to that of the control cells FEBS Journal 276 (2009) 7279–7290 ª 2009 The Authors Journal compilation ª 2009 FEBS 7285 PDZ proteins interact with olfactory receptors R Dooley et al (Fig 4C), although the decay was prolonged significantly (Fig 4D) Cells transfected with a scrambled version of the siRNA did not show any significant differences in the kinetics of the OR-elicited Ca2+ response compared to nontransfected cells (Fig 4E, F) Interaction with MUPP1 is important for controlling OR-mediated Ca2+ signaling Because PDZ domains + interact with MUPP1 (Fig 2), we generated a cell line over-expressing these two PDZ domains (Fig 5A) Interestingly, the prolonged signal decay observed in the siRNA experiments was mirrored in the OR-dependent responses of Hana3A cells stably over-expressing PDZ domains + of MUPP1 (Fig 5B) When monitoring the response of transiently transfected OR2AG1 to amylbutyrate, we found that the OR-elicited response did not decay within the normal average time-frame in cells over-expressing PDZ domains + (26.2 ± 1.8 s) compared to control cells (10.2 ± 1.9 s) (Fig 5D) As in siRNA-treated cells, the rise time after odorant stimulation was almost indistinguishable in cells over-expressing PDZ domains + (4.87 ± 0.2 s) compared to control cells (4.71 ± 0.69 s) (Fig 5C) In summary, when the interaction between MUPP1 and OR2AG1 is inhibited by over-expressing the interacting PDZ domains, the resulting response of OR2AG1 to odorant is modified in that the rapid decay of signal is impaired We finally examined the effect of deletion of the components of the PDZ binding motif on the signaling properties of the receptor in Ca2+ imaging experiments Truncation of the receptor from the final eight amino acids (amino acids 309–316) resulted in a prolonged signal decay, similar to that observed in the siRNA experiments and in the cells over-expressing PDZ domains + 2, whereas the rise time of the signals was again not affected (Fig 5E, F) Discussion Until now, the involvement of PDZ domain scaffolding proteins in olfactory signal transduction has gone unstudied It has previously been suggested that such scaffolding networks or ‘olfactosomes’ may exist [9,10] but, to date, no evidence for this phenomenon has been outlined In the present study, we have uncovered a PDZ protein as a novel interaction partner of olfactory receptors and have elucidated the molecular details of this interaction 7286 The primary source of olfactory signaling and the sites of expression of ORs are the ciliary structures of the OSNs Interestingly, we found MUPP1 to be predominantly expressed in the apical compartment of OSNs and enriched in the cilia fraction of a preparation of whole murine olfactory epithelium We hypothesize that MUPP1, through its multivalent capabilities, could play a key role as a central nucleator of olfactory signal transduction With its thirteen PDZ domains, each diverse in its amino acid sequence, MUPP1 holds great potential for organizing signal transduction molecules into defined protein networks and thereby regulating signaling events MUPP1 has previously been found to interact with a diverse array of molecules, including G protein-coupled receptors such as the 5-hydroxytryptamine receptor type 2C [18,27] and the GABAB receptor [19] We postulated that MUPP1 could directly interact with the olfactory receptor itself We scanned the entire human olfactory receptor repertoire and discovered that up to 30% of receptors contain putative PDZ binding motifs in their C-termini, following the previously outlined rules of binding [16] PDZ domains + of MUPP1 indeed showed direct interaction with OR C-terminal petides Moreover, upon co-expression of ORs containing classical PDZ binding motifs in their C-termini, MUPP1-GFP exhibited a translocation from the cytosol to the plasma membrane in the heterologous expression system, suggesting a physical association between both proteins within the cell We found this translocation to be dependent on the final amino acids of the receptor protein Similar to the other PDZ domain interactions that have been shown to be abolished by mutating amino acids at position and )2 from the C-terminus [16], we found that binding of the hOR2AG1 Cterminus to PDZ domains + did not occur when positions and )2 were mutated to tryptophans, which are not present in the PDZ binding motifs of other membrane proteins [17] We also found interaction of PDZ domains + with ORs showing no classical PDZ binding motifs, indicating that the understanding of the exact molecular rules of OR PDZ interaction will require further analysis Previous work with other proteins has already indicated that it is highly likely that a large number of PDZ domain interactions will not fit into the confined class definitions and that PDZ domains may have been optimized across the proteome in order to minimize cross-reactivity [17] We observed that a reduction of MUPP1 resulted in a significant increase in the duration of Ca2+ responses evoked by the activation of recombinantly expressed FEBS Journal 276 (2009) 7279–7290 ª 2009 The Authors Journal compilation ª 2009 FEBS R Dooley et al hOR2AG1 Similarly, over-expression of the OR-interacting PDZ domains + of MUPP1 also resulted in odorant-evoked Ca2+ responses that persisted longer than those in control cells When over-expressed, PDZ domains + may bind to the C-terminus of hOR2AG1, thus having a blocking effect on the binding of the less abundant endogenous MUPP1 A truncated receptor no longer containing a PDZ motif in the C-terminus showed the same effect of prolonged signal duration Thus, all of the experiments revealed that the association of hOR2AG1 with MUPP1 regulates signal duration To a certain extent, the impaired signal desensitization resembles the effect of the absence of the multi-PDZ domain protein INAD in the Drosophila visual signal transduction cascade Flies lacking INAD exhibit a profound reduction of the light response [28] Interestingly, INAD is also required for normal deactivation of visual signaling by positioning eye protein kinase C in close proximity to TRP to facilitate its phosphorylation, ultimately resulting in deactivation of the channel [12,29,30] On the other hand, in contrast to the findings of the present study, MUPP1 was shown to prolong the duration of GABAB receptor signaling and increase the stability of the receptor [19] However, we must note that the situation in the recombinant expression system is different from that in the olfactory neurons, where alternative binding partners of MUPP1 presumably exist It is therefore possible that MUPP1 could exhibit alternative effects on the dynamics of calcium responses induced by ORs in the neurons compared to those induced by heterologously expressed ORs The observed effects can therefore only be taken as proof of the functional significance of the observed interaction Further studies are necessary to shed light on the function of this interaction in the in vivo situation By influencing the duration of the Ca2+ signal of ORs in the cilia of the sensory neurons, MUPP1 could have a strong influence on the olfactory signaling pathway An interesting interaction partner of MUPP1 outlined to date is CamKII, which is known to play an important role in olfactory adaptation [20] By phosphorylation of adenylyl cyclase in OSNs, CamKII provides an important mechanism for the attenuation of odorant-stimulated cAMP increases [31] Alternatively, because different pathways, such as those involving phosphoinositide 3-kinase [32], are ultimately engaged after OR stimulation, MUPP1 may control OR activity by acting as a scaffold to link different signaling pathways In conclusion, we have outlined a novel aspect of the olfactory signal transduction cascade by uncovering a previously unknown interaction partner of olfactory PDZ proteins interact with olfactory receptors receptors and a putative regulator of signaling processes in OSNs It is tempting to speculate that a so-called ‘olfactosome’ exists in the cilia of olfactory sensory neurons, organizing the vast array of signaling molecules and ensuring the specificity of signaling How exactly MUPP1 could carry out such an important task remains to be elucidated, although the answer may lie in the remaining and as yet unidentified interaction partners of MUPP1 in the olfactory sensory cell Experimental procedures DNA constructs and primers pCDNA3_MUPP1-GFP and in vitro translation tandem PDZ domain constructs in vector pBAT were provided by H Lubbert (Ruhr-University, Bochum, Germany) ă pCDNA3_OR2AG1 was cloned as described previously [33] C-terminal mutant constructs of OR2AG1 [^PDZ_ pCDNA3 (S314A, L316A), trunc4_pCDNA3 (amino acids 313–316) and trunc8_pCDNA3 (amino acids 309–316)], were obtained by PCR using varying 3¢ primers and pCDNA3_OR2AG1 as a template GST fusion constructs of the C-terminus of OR2AG1, and mutant thereof (trunc8) were created by cloning the region between amino acids 293 and 316 from the receptor into pGEX-3X vector (Amersham Pharmacia Biotech, Piscataway, NJ, USA), using varying reverse primers PDZ domains + were cloned using pBAT_1 + 2_HA as a template The stable cell line construct, pCMV ⁄ Bsd_PDZ1 + 2_HA, was cloned using pCDNA3_MUPP1-GFP as a template All constructs were verified by sequencing For RT-PCR, mRNA was extracted from adult mouse olfactory epithelium, and the primers were used were: 5¢-CAAAACGCTCTACAGGC TCC-3¢, 5¢-GAAGAGCTGGACAGAGGTGG-3¢ (ZO-1), 5¢-TTATGGGCCACCGGATATTA-3¢, 5¢-GGAGAGTCA CTGAAGGCTGG-3¢ (DLG-2), 5¢-AAGCTAAGAGGCA CGGAACA-3¢, 5¢-TCCTTATTGCCAGCGAGACT-3¢ (Patj), 5¢-TTGCAGACGGAAGAGGTTCT-3¢, 5¢-GGCCACTT TCAGCATCAAAT-3¢ (Erbin), 5¢-GCGGATCCGCAT GTTGGAAACCATAGAC-3¢ and 5¢-GCGAATTCGA CATTTTTAGTGAGTTCCAC-3¢ (MUPP1) Antibodies Primary antibodies used were: anti-adenylyl cyclase 3, rabbit polyclonal (Santa Cruz Biotechnology, Santa Cruz, CA, USA), directly labeled using DyLightÔ549 Microscale Antibody Labeling Kit (Pierce, Rockford, IL, USA); antiMUPP1, rabbit polyclonal (provided by H Lubbert, Ruhră University); anti-GFP, rabbit polyclonal (#ab290-50; Abcam, Cambridge, MA, USA); a-HA antibody, mouse monoclonal (#H9658; Sigma, St Louis, MO, USA) Secondary antibodies used were goat anti-rabbit Alexa546nm FEBS Journal 276 (2009) 7279–7290 ª 2009 The Authors Journal compilation ª 2009 FEBS 7287 PDZ proteins interact with olfactory receptors R Dooley et al (Molecular Probes, Carlsbad, CA, USA) and horseradish peroxidase (HRP) coupled goat anti-mouse and goat antirabbit IgGs (Bio-Rad, Hercules, CA, USA) standard cages at room temperature Each cage was surrounded by a Perspex chamber with ventilation suction to maintain a constant air-flow Cell culture and transfection GST fusion peptides and in vitro interaction assays All tissue culture media and related reagents were purchased from Invitrogen (Carlsbad, CA, USA) Hana3A cells [23] (provided by H Matsunami, Duke University Medical Center, Durham, NC, USA), were maintained in DMEM plus 10% fetal bovine serum and 1% penicillin ⁄ streptomycin, at 37 °C and 5% CO2, and transfections were carried out using a standard calcium phosphate precipitation technique MUPP1-GFP and OR plasmid DNAs were transfected in a ratio of : 10, with approximately lg of total DNA per dish All images were acquired using a Zeiss LSM 510 Meta confocal microscope (Carl Zeiss, Oberkochen, Germany) Hana3A cells were stably transfected with pCMV ⁄ Bsd plasmid (Invitrogen) containing tandem PDZ domains + along with an HA tag Positive clones were selected for using blasticidin (10 lgỈmL)1) and stable transfection was confirmed by immunocytochemistry The C-terminal region of OR2AG1 was found to lie between amino acids 293 and 316, as predicted by tmhmm, a transmembrane helices prediction program based on a hidden Markov model [34] OR2AG1 C-terminus GST fusion proteins and mutant construct thereof (trunc8-GST) were produced in Escherichia coli XL1 blue and purified on glutathione sepharose beads (Becton-Dickinson Biosciences, Franklin Lakes, NJ, USA) PDZ domains were in vitro translated using the TNTÒT3 Coupled Reticulocyte Lysate System (Promega, Madison, WI, USA) Interaction assays were carried out by incubating 10 lL of in vitro translation product with 50 lL of GST fusion peptide bead slurry for h at °C with gentle shaking After a series of washing steps using Buffer S (20 mm Hepes, 100 mm KCl, 0.5 mm EDTA, mm dithiothreitol, pH 7.9), specific interactions were assessed via immunoblotting GST alone was used as a negative control Cell membrane preparation and western blotting The olfactory epithelium of CD1 mice was fractionated by mechanical agitation as described previously [21] Equal amounts of protein from each fraction were loaded on an SDS gel and subjected to immunoblotting on poly(vinylidene difluoride) membrane (Millipore, Billerica, MA, USA) and Coomassie staining Detection was performed using the ECL western blotting detection system (GE Healthcare, Milwaukee, WI, USA) For co-immunoprecipitation, Hana3A cells were transfected with OR2AG1-HA and MUPP1_pCDNA3 or untransfected The nucleus-free cell lysates were incubated with biotinylated a-HA antibody and precipitated protein collected using Dynabeads (Invitrogen) and DynaMag (Invitrogen) Any interaction was detected using MUPP1 antibodies Immunohistochemistry Mice were raised and maintained according to governmental and institutional care instructions Immunohistochemistry was carried out on 14 lm horizontal sections, and fluorescence images were obtained with a confocal microscope (Zeiss LSM 510 Meta) with ·40 objective Control experiments in the absence of any primary antibody revealed a very low level of background staining For odorant exposure experiments, OMP-GFP mice were exposed to a mixture of 100 different odorant molecules (Henkel KGaA, Dusseldorf, Germany) for specied amounts of ă time Control mice were housed in a separate room free from artificial odorant stimulation All mice were held in 7288 Peptide microarray CelluSpotsÔ Peptide Arrays (Intavis AG, Cologne, Germany) were blocked for h at room temperature with 5% skimmed milk in NaCl ⁄ Tris ⁄ Tween The arrays were incubated with a PDZ1 + 2_HA fusion protein (produced as described in E coli) overnight at °C CelluSpotsÔ were incubated with the a-HA antibody for h at room temperature Detection was performed with HRP coupled secondary antibody and using ECL western blotting detection reagent (GE Healthcare) Mupp1 siRNA Pre-synthesized and tested Mupp1 siRNA (identification #107246 and #216971) and a custom designed scrambled version of Mupp1 (CUGACUGUGUAUCGAACGGtt) were purchased from Ambion (Austin, TX, USA) siRNA was transfected 72 h prior to calcium imaging using Lipofectamine 2000 (Invitrogen), in serum-free medium (Opti-Mem; Invitrogen) Forty-eight hours prior to calcium imaging, lg of OR2AG1 plasmid DNA were transfected per dish using ExGen 500 transfection reagent (Fermentas, Glen Burnie, MD, USA) Medium was exchanged for fresh DMEM 24 h post-transfection Ratiometric Ca2+ imaging in heterologous cells Stably ⁄ transiently transfected Hana3A cells were incubated with 7.5 lm FURA-2 AM (Invitrogen) Ratiometric FEBS Journal 276 (2009) 7279–7290 ª 2009 The Authors Journal compilation ª 2009 FEBS R Dooley et al calcium imaging was performed as described previously [25] using a Zeiss inverted microscope equipped for ratiometric imaging Cells were exposed to 100 lm amylbutyrate (Henkel GmbH), which is a typical odorant concentration used for heterologously expressed ORs [23,35,36], employing a specialized microcapillary application system The rise time was calculated as the time in seconds from 10% of peak response, starting from the average baseline value, to 90% of peak amplitude The response decay duration was calculated as the time in seconds between 90% and 10% of the maximum amplitude Acknowledgements We thank H Bartel and J Gerkrath for their excellent technical assistance; H Matsunami (Duke University Medical Center, Durham, NC, USA) for the donation of Hana3A cells; P Mombaerts (MPI Biophysics, Frankfurt, Germany) for the donation of OMP-GFP transgenic mice; and H Lubbert ⁄ X Zhu (Ruhr Uniă versity, Bochum, Germany) for the donation of MUPP1 antibodies and constructs This work was supported by the International Max-Planck Research School in Chemical Biology (IMPRS-CB), the Studienstiftung 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mapping the odorant-binding site J Neurosci 25, 1806– 1815 36 Spehr M, Schwane K, Riffell JA, Barbour J, Zimmer RK, Neuhaus EM & Hatt H (2004) Particulate adenylate cyclase plays a key role in human sperm olfactory receptor-mediated chemotaxis J Biol Chem 279, 40194– 40203 Supporting information The following supplementary material is available: Table S1 Peptide microarray This supplementary material can be found in the online version of this article Please note: As a service to our authors and readers, this journal provides supporting information supplied by the authors Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset Technical support issues arising from supporting information (other than missing files) should be addressed to the authors FEBS Journal 276 (2009) 7279–7290 ª 2009 The Authors Journal compilation ª 2009 FEBS ... in the case of hOR1D2, only in two out of four experiments Other olfactory receptors, such as mOR167-4, mOR199 -1, M 71, M72 and mOR2 41- 1 B C D E Fig The C-terminus of OR2AG1 interacts with MUPP1... (19 99) The organization of INAD -signaling complexes by a multivalent PDZ domain protein in Drosophila photoreceptor cells ensures sensitivity and speed of signaling Cell Calcium 26, 16 5? ?17 1 13 ... 16 5? ?17 1 13 Harris BZ & Lim WA (20 01) Mechanism and role of PDZ domains in signaling complex assembly J Cell Sci 11 4, 3 219 –32 31 14 Nourry C, Grant SG & Borg JP (2003) PDZ domain proteins: plug