Advertisement

Probing the Interactome of Corticotropin-Releasing Factor Receptor Heteromers Using Mass Spectrometry

  • Burcu Hasdemir
  • Juan A. Oses-Prieto
  • Alma Burlingame
  • Aditi BhargavaEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1947)

Abstract

Mass spectrometry is a sensitive technique used in the field of proteomics that allows for simultaneous detection and characterization of several proteins in a sample. It is also a powerful methodology to elucidate protein–protein interactions in a sequence-dependent and unbiased manner. G protein-coupled receptors (GPCRs) seldom function in isolation and characterization of proteins present in the receptor complex (or its interactome) is critical for understanding the vast spectrum of functions these receptors perform in a context-dependent manner. Here, we describe a mass spectrometry-based method to sequence and characterize proteins present in heteromeric complexes formed by corticotropin-releasing factor (CRF) receptors that belong to class B GPCRs. CRF receptor heteromeric complexes were identified in HEK293 cells co-transfected with tagged CRF receptors 1 and 2. CRF receptors were immunoprecipitated using antibodies against the tags from transfected HEK293 cells and proteins in their interactome were identified using liquid chromatography mass spectrometry method (LC-MS/MS). Both CRF receptors were identified in this interactome. A few of the proteins identified in the CRF receptor interactome using MS were confirmed to be true interactions using traditional co-immunoprecipitation and Western blotting methods.

Key words

Antibody, Computational analysis, Co-immunoprecipitation, GPCRs, Proteome, Tagged receptors, Western blot analysis 

Notes

Acknowledgments

This work was supported by NIH grants DK080787 to A. Bhargava and GM8P41GM103481 to A. Burlingame. B.H. was supported by T32 AT003997.

References

  1. 1.
    Hasdemir B, Mahajan S, Oses-Prieto J, Chand S, Woolley M, Burlingame A, Grammatopoulos DK, Bhargava A (2017) Actin cytoskeleton-dependent regulation of corticotropin-releasing factor receptor heteromers. Mol Biol Cell 28(18):2386–2399.  https://doi.org/10.1091/mbc.E16-11-0778CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Milligan G (2009) G protein-coupled receptor hetero-dimerization: contribution to pharmacology and function. Br J Pharmacol 158(1):5–14.  https://doi.org/10.1111/j.1476-5381.2009.00169.xCrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Vischer HF, Watts AO, Nijmeijer S, Leurs R (2011) G protein-coupled receptors: walking hand-in-hand, talking hand-in-hand? Br J Pharmacol 163(2):246–260.  https://doi.org/10.1111/j.1476-5381.2011.01229.xCrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Shrestha D, Jenei A, Nagy P, Vereb G, Szollosi J (2015) Understanding FRET as a research tool for cellular studies. Int J Mol Sci 16(4):6718–6756.  https://doi.org/10.3390/ijms16046718CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Michel MC, Wieland T, Tsujimoto G (2009) How reliable are G-protein-coupled receptor antibodies? Naunyn Schmiedeberg’s Arch Pharmacol 379(4):385–388.  https://doi.org/10.1007/s00210-009-0395-yCrossRefGoogle Scholar
  6. 6.
    Gomes I, Gupta A, Bushlin I, Devi LA (2014) Antibodies to probe endogenous G protein-coupled receptor heteromer expression, regulation, and function. Front Pharmacol 5:268.  https://doi.org/10.3389/fphar.2014.00268CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Chung KY, Day PW, Velez-Ruiz G, Sunahara RK, Kobilka BK (2013) Identification of GPCR-interacting cytosolic proteins using HDL particles and mass spectrometry-based proteomic approach. PLoS One 8(1):e54942.  https://doi.org/10.1371/journal.pone.0054942CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Ciruela F, Burgueno J, Casado V, Canals M, Marcellino D, Goldberg SR, Bader M, Fuxe K, Agnati LF, Lluis C, Franco R, Ferre S, Woods AS (2004) Combining mass spectrometry and pull-down techniques for the study of receptor heteromerization. Direct epitope-epitope electrostatic interactions between adenosine A2A and dopamine D2 receptors. Anal Chem 76(18):5354–5363.  https://doi.org/10.1021/ac049295fCrossRefPubMedGoogle Scholar
  9. 9.
    Matsubara S, Shiraishi A, Sakai T, Okuda T, Satake H (2017) Heterodimerization of the prostaglandin E2 receptor EP2 and the calcitonin receptor CTR. PLoS One 12(11):e0187711.  https://doi.org/10.1371/journal.pone.0187711CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Hasdemir B, Mahajan S, Bunnett NW, Liao M, Bhargava A (2012) Endothelin-converting enzyme-1 actions determine differential trafficking and signaling of corticotropin-releasing factor receptor 1 at high agonist concentrations. Mol Endocrinol 26(4):681–695.  https://doi.org/10.1210/me.2011-1361CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Guan S, Price JC, Prusiner SB, Ghaemmaghami S, Burlingame AL (2011) A data processing pipeline for mammalian proteome dynamics studies using stable isotope metabolic labeling. Mol Cell Proteomics 10(12):M111 010728.  https://doi.org/10.1074/mcp.M111.010728CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Rosenfeld J, Capdevielle J, Guillemot JC, Ferrara P (1992) In-gel digestion of proteins for internal sequence analysis after one- or two-dimensional gel electrophoresis. Anal Biochem 203(1):173–179CrossRefGoogle Scholar
  13. 13.
    UCSF in-gel digestion protocol. http://msf.ucsf.edu/protocols.html. Accessed 24 May 2018
  14. 14.
    LI-COR (Biosciences) protein electrotransfer methods. https://www.licor.com/documents/t932473b94z25454q3kqazh3t830xfoy. Accessed 24 May 2018
  15. 15.
    Sambrook J (2000) Molecular cloning, a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NYGoogle Scholar
  16. 16.
    Hasdemir B, Bunnett NW, Cottrell GS (2007) Hepatocyte growth factor-regulated tyrosine kinase substrate (HRS) mediates post-endocytic trafficking of protease-activated receptor 2 and calcitonin receptor-like receptor. J Biol Chem 282(40):29646–29657.  https://doi.org/10.1074/jbc.M702974200CrossRefPubMedGoogle Scholar
  17. 17.
    Klockenbusch C, Kast J (2010) Optimization of formaldehyde cross-linking for protein interaction analysis of non-tagged integrin beta1. J Biomed Biotechnol 2010:927585.  https://doi.org/10.1155/2010/927585CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Clauser KR, Baker P, Burlingame AL (1999) Role of accurate mass measurement (+/− 10 ppm) in protein identification strategies employing MS or MS/MS and database searching. Anal Chem 71(14):2871–2882CrossRefGoogle Scholar
  19. 19.
    LI-COR (BIOSCIENCES) Odyssey classic application protocols. https://www.licor.com/documents/o2lbjk5lhkb0ehh5e89x. Accessed 24 May 2018
  20. 20.
    LI-COR (BIOSCIENCES) Introduction to chemiluminescence western detection. https://www.licor.com/bio/applications/chemiluminescent_western_blot/index.html. Accessed 24 May 2018

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Burcu Hasdemir
    • 1
    • 2
  • Juan A. Oses-Prieto
    • 3
  • Alma Burlingame
    • 4
  • Aditi Bhargava
    • 1
    • 2
    Email author
  1. 1.The Osher Center for Integrative MedicineUniversity of California, San FranciscoSan FranciscoUSA
  2. 2.Department of Ob-GynUniversity of California, San FranciscoSan FranciscoUSA
  3. 3.Pharmaceutical ChemistryUniversity of California San FranciscoSan FranciscoUSA
  4. 4.Department of Pharmaceutical ChemistryUniversity of California, San FranciscoSan FranciscoUSA

Personalised recommendations