Visualizing G Protein‐Coupled Receptor‐Receptor Interactions in Brain Using Proximity Ligation In Situ Assay

Jaume Taura1, Víctor Fernández‐Dueñas1, Francisco Ciruela2

1 Unitat de Farmacologia, Departament Patologia i Terapèutica Experimental, Facultat de Medicina, IDIBELL‐Universitat de Barcelona, Barcelona, 2 Department of Physiology, Faculty of Sciences, University of Ghent, Ghent
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 17.17
DOI:  10.1002/0471143030.cb1717s67
Online Posting Date:  June, 2015
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library


G protein‐coupled receptors (GPCRs) constitute the largest family of plasma membrane receptors, thus representing the more investigated drug targets in the design of new therapeutic strategies. The existence of receptor‐receptor interactions has revolutionized the field, since GPCR oligomerization might confer new intervention opportunities in pharmacotherapy. However, demonstrating the existence of such receptor‐receptor interactions in native tissue has been a bottleneck in GPCR pharmacology. Here, we discuss an experimental approach, the proximity ligation in situ assay (P‐LISA), which provides enough sensitivity to evaluate a receptor's close proximity within a named GPCR oligomer. Indeed, we provide a detailed step‐by‐step protocol for P‐LISA experiments visualizing receptor‐receptor interactions in brain slices. Additionally, we provide instructions for slide observation, data acquisition and quantification. Finally, we also discuss these critical aspects determining the success of the technique, namely the fixation process and the validation of the primary antibodies used. Overall, the P‐LISA is a powerful and straightforward technique to visualize receptor‐receptor interactions when performed under optimal conditions. © 2015 by John Wiley & Sons, Inc.

Keywords: GPCR; proximity ligation assay; brain; oligomerization

PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Visualization of GPCR Oligomers by Proximity Ligation In Situ Assay
  • Support Protocol 1: Slide Observation, Image Acquisition, and Quantifcation
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
PDF or HTML at Wiley Online Library


Basic Protocol 1: Visualization of GPCR Oligomers by Proximity Ligation In Situ Assay

  • CD1 mouse (in‐house colony)
  • Ketamine (50 mg/ml; Ketalar, Panpharma)
  • Xylazine (20 mg/ml; Rompun, Bayer)
  • 70% ethanol
  • 4% formaldehyde solution (see recipe)
  • 1× PBS (see recipe)
  • Walter's antifreezing solution (see recipe)
  • Blocking solution:
    • 10% normal donkey serum (NDS; Jackson ImmunoResearch Laboratories)
    • and 0.05% Triton X‐100 (Sigma‐Aldrich) in 1× PBS
  • Normal donkey serum (NDS; Jackson ImmunoResearch Laboratories)
  • Duolink in situ PLA probe anti‐goat PLUS (Sigma‐Aldrich)
  • Duolink in situ PLA probe anti‐rabbit MINUS (Sigma‐Aldrich)
  • 1× ligation solution (see recipe)
  • T4 DNA ligase (1 U/μl; Promega)
  • P‐LISA wash buffer A (see recipe)
  • 1× rolling‐circle amplification (RCA) solution
  • phi29 DNA polymerase (Life Technologies)
  • P‐LISA wash buffer B (see recipe)
  • Duolink in situ mounting medium containing 4′,6‐diamidino‐2‐phenylindole (DAPI; Sigma‐Aldrich)
  • Transparent nail polish
  • Surgical scissors
  • Forceps
  • 27‐gauge needle
  • Rack (for dissection)
  • Tape or pins
  • 1‐cc syringe
  • 25‐gauge, 25‐mm perfusion needle
  • Vibratome (VT1200, Leica Lasertechnik GmbH)
  • Round brush, sizes 10/0 to 2/10
  • Microscope slides and coverslips

Support Protocol 1: Slide Observation, Image Acquisition, and Quantifcation

  • Laser scanning confocal microscope (LSCM; TCS 4D, Leica Lasertechnik GmbH)
  • Fiji program (Fiji Is Just ImageJ;
PDF or HTML at Wiley Online Library



Literature Cited

Literature Cited
  Agnati, L.F., Fuxe, K., Zini, I., Lenzi, P., and Hokfelt, T. 1980. Aspects on receptor regulation and isoreceptor identification. Med. Biol. 58:182‐187.
  Albizu, L., Cottet, M., Kralikova, M., Stoev, S., Seyer, R., Brabet, I., Roux, T., Bazin, H., Bourrier, E., Lamarque, L., Breton, C., Rives, M.L., Newman, A., Javitch, J., Trinquet, E., Manning, M., Pin, J.P., Mouillac, B., and Durroux, T. 2010. Time‐resolved FRET between GPCR ligands reveals oligomers in native tissues. Nat. Chem. Biol. 6:587‐594.
  Besmer, P., Miller, R.C. Jr., Caruthers, M.H., Kumar, A., Minamoto, K., Van de Sande, J.H., Sidarova, N., and Khorana, H.G. 1972. Studies on polynucleotides. CXVII. Hybridization of polydeoxynucleotides with tyrosine transfer RNA sequences to the r‐strand of phi80psu + 3 DNA. J. Mol. Biol. 72:503‐522.
  Clark, J.D., Gebhart, G.F., Gonder, J.C., Keeling, M.E., and Kohn, D.F. 1997. Special report: The 1996 guide for the care and use of laboratory animals. ILAR J. 38:41‐48.
  Fernández‐Dueñas, V., Taura, J.J., Cottet, M., Gómez‐Soler, M., López‐Cano, M., Ledent, C., Watanabe, M., Trinquet, E., Pin, J.‐P., Luján, R., Durroux, T., and Ciruela, F. 2015. Untangling dopamine‐adenosine receptor‐receptor assembly in experimental parkinsonism in rats. Dis. Model. Mech. 8:57‐63.
  Flecknell, P.A. 1993. Anaesthesia of animals for biomedical research. Br. J. Anaesth. 71:885‐894.
  Fredriksson, S., Gullberg, M., Jarvius, J., Olsson, C., Pietras, K., Gústafsdóttir, S.M., Ostman, A., and Landegren, U. 2002. Protein detection using proximity‐dependent DNA ligation assays. Nat. Biotechnol. 20:473‐477.
  Fuxe, K., Agnati, L.F., Benfenati, F., Celani, M., Zini, I., Zoli, M., and Mutt, V. 1983. Evidence for the existence of receptor‐receptor interactions in the central nervous system. Studies on the regulation of monoamine receptors by neuropeptides. J. Neural Transm. 18:165‐179.
  Jarvius, M., Paulsson, J., Weibrecht, I., Leuchowius, K.‐J., Andersson, A.‐C., Wählby, C., Gullberg, M., Botling, J., Sjöblom, T., Markova, B., Ostman, A., Landegren, U., and Söderberg, O. 2007. In situ detection of phosphorylated platelet‐derived growth factor receptor β using a generalized proximity ligation method. Mol. Cell Proteomics 6:1500‐1509.
  Matamales, M., Bertran‐Gonzalez, J., Salomon, L., Degos, B., Deniau, J., Valjent, E., Hervé, D., and Girault, J. 2009. Striatal medium‐sized spiny neurons: Identification by nuclear staining and study of neuronal subpopulations in BAC transgenic mice. PLoS One 4:e4770.
  Miller, J. and Stagljar, I. 2004. Using the yeast two‐hybrid system to identify interacting proteins. Methods Mol. Biol. 261:247‐262.
  Palczewski, K. 2006. G protein‐coupled receptor rhodopsin. Annu. Rev. Biochem. 75:743‐767.
  Ramos‐Vara, J.A. and Miller, M.A. 2014. When tissue antigens and antibodies get along: Revisiting the technical aspects of immunohistochemistry—the red, brown, and blue technique. Vet. Pathol. 51:42‐87.
  Ritter, S.L. and Hall, R.A. 2009. Fine‐tuning of GPCR activity by receptor‐interacting proteins. Nat. Rev. Mol. Cell Biol. 10:819‐830.
  Schindelin, J., Arganda‐Carreras, I., Frise, E., Kaynig, V., Longair, M., Pietzsch, T., Preibisch, S., Rueden, C., Saalfeld, S., Schmid, B., Tinevez, J.Y., White, D.J., Hartenstein, V., Eliceiri, K., Tomancak, P., and Cardona, A. 2012. Fiji: An open‐source platform for biological‐image analysis. Nat. Methods 9:676‐982.
  Selbach, M. and Mann, M. 2006. Protein interaction screening by quantitative immunoprecipitation combined with knockdown (QUICK). Nat. Methods 3:981‐983.
  Smith, C.L. 2011. Basic confocal microscopy. Curr. Protoc. Neurosci. 56:2.2.1‐2.2.18.
  Söderberg, O., Leuchowius, K.‐J., Gullberg, M., Jarvius, M., Weibrecht, I., Larsson, L.‐G., and Landegren, U. 2008. Characterizing proteins and their interactions in cells and tissues using the in situ proximity ligation assay. Methods 45:227‐232.
  Söderberg, O., Gullberg, M., Jarvius, M., Ridderstråle, K., Leuchowius, K.‐J., Jarvius, J., Wester, K., Hydbring, P., Bahram, F., Larsson, L.‐G., and Landegren, U. 2006. Direct observation of individual endogenous protein complexes in situ by proximity ligation. Nat. Methods 3:995‐1000.
  Trifilieff, P., Rives, M.‐L., Urizar, E., Piskorowski, R.A., Vishwasrao, H.D., Castrillon, J., Schmauss, C., Slättman, M., Gullberg, M., and Javitch, J.A. 2011. Detection of antigen interactions ex vivo by proximity ligation assay: Endogenous dopamine D2‐adenosine A2A receptor complexes in the striatum. Biotechniques 51:111‐118.
PDF or HTML at Wiley Online Library