The GalR1-GalR2 heteroreceptor complexes can be the receptor for galanin fragment 1-15 Dasiel O. Borroto-Escuela*, Feliciano Calvo, Manuel Narváez, Wilber Romero-Fernández, Carmelo Millon, Michael Di Palma, Mileidys Pérez Alea, Mercé Tena, Luigi F. Agnati, Alexander Tarakanov, Pere Garriga, Zaida Díaz-Cabiale and Kjell Fuxe * Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden. Email: [email protected] BACKGROUND Previous work has established homodimerization and internalization of GalR1 in living CHO cells using FRET and time lapse confocal imaging1. Thus, GalR1 can exist as a dimer in the plasma membrane which may undergo desensitization and internalization upon agonist activation with Gal1-29. We have examined the possible existence of GalR-5-HT1A heteromersin HEK-293 cells using a proximity-based FRET assay2. Furthermore, the three cloned galanin receptors show a higher affinity for galaninthan for galaninN-terminal fragments like Gal1-153. A substantial further development was therefore the demonstration of specific Gal1-15 binding sites in the rat CNS indicating a relevant role of Gal fragments in central galanin communication, especially in dorsal hippocampus, neocortex and striatum which show only few high affinity Gal1-29 binding sites4. We have therefore introduced the hypothesis that these Gal1-15 preferring sites may be formed through the formation of GalR1- GalR2 heteroreceptor complexes which leads to conformational changes in their galanin recognition sites converting GalR1 and/or GalR2 into galanin fragment preferring binding sites with reduced affinity for Gal1-295. METHODS & RESULTS We used a dual luciferase reporter assay to indirectly detect variations Figure 1. GalR1-GalR2 heteroreceptor complexes in hipocampal and of cAMP levels, PLC-b activity, or activations of MAPK pathway in median raphe sections of rat detected by In situ PLA. In situ PLA was transiently transfected cell lines treated with different compounds. For performed as described previously6 and using primary antibodies of luciferase assays, cells were co-transfected with plasmids different species directed to GalR1 and GalR2. The detected corresponding to three constructs as follows: 2 g firefly luciferase- heteroreceptor complexes are seen as red clusters indicated by arrows. encoding experimental plasmid (pGL4-CRE-luc2p or pGL4-NFAT- Specific GalR1-GalR2 clusters are visualized within discrete regions of luc2p; 1 g of GalR1 plus GalR2 expression vectors and 0.5 g Rluc- the hippocampus and median raphe. They were almost absent in the encoding internal control plasmid (phRG-B). The luciferase activity of corpus callosum (cc). The relative densities of PLA cluster distributions cell extracts was determined in a POLARstar Optima plate reader using are schematically illustrated by the density of red puncta. Bregma level a 535 nm filter with a 30-nm bandwidth. Firefly luciferase was –1.7 and –7.64. measured as firefly luciferase luminescence over a 15s reaction period. Figure 3. Agonist-induced GalR1 and GaLR2 receptor activation in a DG DRN forskolin-induced CRE-luciferase reporter gene assay. The data represent the mean S.E.M. of five independent experiments performed in quadruplicate. (EC50 ~ 0.92nM in Gal1-15 group and EC50 ~ 35.8nM in the Gal 1-29 group). 120 [GalR1-GalR2 1:1] [GalR2-GalR2 1:1] 120 120 φφφ 80 +++ +++ φφφ 80 80 CREB *** CREB CREB 40 &&& 40 40 (% max response FK, 30 min) 30 FK, response max (% FK, 30response min) (% max (% max response FK, 30 FK,min) 30 response (% max [GalR1-GalR2 1:1] 0 0 0 l M M M M M M -14 -12 -10 -8 -6 -4 -2 µ µ µ µ ro µ µ 1 1 0nM 0nM 1 1 50nM ont 5 5 control 35 c ] 87 871 Log [peptide](M) M 871 1 871 1 M 1-15] M M M [ + [1-15 M + l + + al [1-29] 50nM M + a G 0n G Gal Gal [1-29] 0n ] 5 50nM ] 5 50nM 29 ] ] 50nM Gal [1-15] Gal [1-29] 29 [1- 1-15] [1- [ [1-15 [1-29 Gal Gal [1-15] 50nM + M35 1 Gal Gal Gal Gal +++ [GalR1-GalR2 1:1] [GalR2-GalR2 1:1] 150 *** 200 &&& &&& Figure 4. Agonist-induced *** 150 +++ GalR1 and GaLR2 receptor 100 100 NFAT NFAT ** +++ 50 protomer activation in an 50 (% basal, over 30 min) (% over basal, 30 min) 30 basal, over (% NFAT-luciferase reporter 0 0 M M M M M M µ µ rol nM µ µ nM µ µ ntrol 0nM nt o 5 o 50 50 c gene assay. c ] ] 9 5 2 M871 1 M871 1 M871 1 + [1-29] + M871 1 [1-2 + [1-1 + l M + M35 1 M + M35 1 M a Figure 2. (left) BRET saturation curves for the GalR1-GalR2 n n Gal [1-15] 50nM G nM Gal nM Gal nM 50 50 50n 50 5] -29] [1-1 1 2 al [1-29] al [1-15] G G Gal Gal [ heteromers with increasing expression levels of the GFP tagged GalR2 Gal [1-29] 50 Gal [1-15] 50 receptor. Plotted on the X-axis is the fluorescence value obtained from DISCUSSION & CONCLUSIONS the GFP2, normalized with the luminescence value of GalR1Rluc ! GalR1-GalR2 heteroreceptor complexes exist in cellular models and expression 10 min after coelenterazine incubation. Mean S.E.M.; n = raphe-hippocampal system. 10, in triplicate. The GalR1-GalR2 curve fitted better to a saturation ! Gal1-15 vs Gal1-29 preferentially activates the GalR1 protomer Gi/o curve than to a linear regression as found with mixed pool of cell from mediated signaling cell individually expressing GalR1Rluc + GalR2GFP2 (F test ! Gal1-15 cannot activate the GalR2 (P < 0.01). (right) BRET2 competition experiment for the GalR1- protomer Gq mediated signaling in GalR2 heteromers. the heteromers. GalR1Rluc-GalR2GFP2 ! Gal1-29 strongly increases GalR2 100 BRET = 81.4 3.9 max Gq mediated signaling in GalR1- BRET50 = 1.54 0.13 80 GalR2 heteromers and GalR2 120 homomers. 60 80 ! The dominance of GalR1 vs ratio (mBU) ratio 2 */** 40 signal 2 ** GalR2 induced by Gal1-15 suggests ** pcDNA3.1+ BRET 40 5-HT1A strong depression like actions 20 BRET GalR1 GalR2 [% of max response] 0 0 GalR1-GalR2 BRET pair 1:1 REFERENCES 0 3 6 9 0 2 4 6 8 10 1Wirz SA et al., 2005. Neuropeptides 39,535-546.; 2Borroto-Escuela DO et al., 2010. Biochem Biophys Res 2 2 3 4 (GFP -GFP 0)/Rluc (AU) cDNA (µµµg) Commun 393,767-772.; Branchek T et al., 1998. Ann N Y Acad Sci 863, 94-107.; Hedlund PB et al., 1992. Eur J Pharmacol 224, 203-205.; 5Fuxe K et al., 2008. Brain Res Rev 58, 415-452.; 6Borroto-Escuela DO et al., 2013. GalR1-Rluc/GalR2-GFP 2 control (-) Methods Enzymol 521, 281-294. Copyright © 2014 Dasiel O. Borroto-Escuela, Mobil: + 46 706177287, email: [email protected] Bio-CV Dasiel O. Borroto-Escuela , PhD, MBA Name: Dasiel Oscar Borroto Escuela Birth data: 14/11/1979, Yaguajay-Cuba. Address: Department of Neuroscience. Karolinska Institutet. Retzius väg 8. 17177 Stockholm, Sweden. +46 706 177 287; [email protected] During my doctoral studies (Ph.D. in Polymers and Biopolymers, UPC, Barcelona, Spain), I was focused into the understanding of muscarinic acetylcholine receptor family interacting proteins and their potential roles in receptor function. The conducted research work earned me 5 relevant publication in the field and the UPC Excellence Thesis Award. In the last years, I have mainly focused on understanding the molecular integration of signals in the brain via receptor-receptor interaction and its functional effects. The existence of receptor heteromers with allosteric receptor-receptor interactions markedly increases the diversity of GPCR recognition, trafficking and signalling; a current scientific issue of high relevance to brain function and integration. The starting point of my research projects (Postdoc, Neuro-KI) has been to figure out whether alterations in specific heteromers and their receptor-receptor interactions are associated or considered to play a role in pathogenic mechanisms contributing to brain disease development, inter alia Parkinson's disease, schizophrenia, addiction and depression. To understand the role of receptor heteromers in some of these diseases I have primarily used as models D2likeR, serotonin 5-HT receptor and galanin receptor heteromers as well as GPCR-RTK interaction (5HT1A/FGFR1) through a multidisciplinary approach. I have focused on the interactions between 5-HT1A and FGFR1 receptors and their relevance for neuroplasticity and depression (Borroto- Escuela et al., Biological psychiatry 71, 84-91 (2012) and Borroto-Escuela et al., BBRC. 441(2):387-92 (2013)). Also, on developing and applying both FRET and BRET techniques to demonstrate a direct molecular receptor-receptor interaction and more recently to develop in situ Proximity Ligation Assay to demonstrate the existence of heteroreceptor complexes in the brain (Borroto-Escuela et al., Methods in enzymology 521, 281-294 (2013)). I have been specially interested in the study of serotonin 5-HT1A receptor containing heteroreceptor complexes and their relevance for neurological disorders (Borroto- Escuela et al., BBRC 393(4):767-72 (2010) and Borroto-Escuela et al., BBRC 441(2):387-92. (2013)). My work on the characterization of the GPCR heteromer interface interaction has been pioneering in the field. I have described the interface interaction of the D2R-A2AR heterodimer and later on the interface interactions of the M5-M3, 5-HT1A-FGFR1 and the GalR1-GalR2 heteroreceptor complexes, demonstrating their potential use as drug targets by means of the use of small interfering peptides (Borroto-Escuela et al., BBRC.; 394(1):222-7 (2010), Borroto-Escuela et al., Biochim Biophys Acta.;1803(7):813-25. (2010) and Borroto-Escuela et al., BBRC.;402(4):801-7.(2010)). The most important impact of my research has been on the discovery of the existence of GPCR-GPCR and GPCR-RTK receptor-receptor interactions in the CNS and the characterization of their interface interactions (Borroto-Escuela et al., IUBMB Life; 63(7):463-72 (2011)).