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G Protein-Coupled Receptor Signaling pp 269–285Cite as

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

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Part of the book series: Methods in Molecular Biology ((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.

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References

  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.E16-11-0778

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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.x

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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.x

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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/ijms16046718

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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-y

    Article  CAS  Google Scholar 

  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.2014.00268

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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.0054942

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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/ac049295f

    Article  CAS  PubMed  Google Scholar 

  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.0187711

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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.2011-1361

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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.M111.010728

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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):173–179

    Article  CAS  PubMed  Google Scholar 

  13. UCSF in-gel digestion protocol. http://msf.ucsf.edu/protocols.html. Accessed 24 May 2018

  14. LI-COR (Biosciences) protein electrotransfer methods. https://www.licor.com/documents/t932473b94z25454q3kqazh3t830xfoy. Accessed 24 May 2018

  15. Sambrook J (2000) Molecular cloning, a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY

    Google Scholar 

  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.M702974200

    Article  CAS  PubMed  Google Scholar 

  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/10.1155/2010/927585

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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):2871–2882

    Article  CAS  PubMed  Google Scholar 

  19. LI-COR (BIOSCIENCES) Odyssey classic application protocols. https://www.licor.com/documents/o2lbjk5lhkb0ehh5e89x. Accessed 24 May 2018

  20. LI-COR (BIOSCIENCES) Introduction to chemiluminescence western detection. https://www.licor.com/bio/applications/chemiluminescent_western_blot/index.html. Accessed 24 May 2018

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Acknowledgments

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

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Authors and Affiliations

  1. The Osher Center for Integrative Medicine, University of California, San Francisco, San Francisco, CA, USA

    Burcu Hasdemir & Aditi Bhargava

  2. Department of Ob-Gyn, University of California, San Francisco, San Francisco, CA, USA

    Burcu Hasdemir & Aditi Bhargava

  3. Pharmaceutical Chemistry, University of California San Francisco, San Francisco, CA, USA

    Juan A. Oses-Prieto

  4. Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, CA, USA

    Alma Burlingame

Authors
  1. Burcu Hasdemir
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  2. Juan A. Oses-Prieto
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  3. Alma Burlingame
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  4. Aditi Bhargava
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Corresponding author

Correspondence to Aditi Bhargava .

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Editors and Affiliations

  1. Ottawa Hospital Research Institute (Neurosciences), Ottawa, ON, Canada

    Mario Tiberi

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Hasdemir, B., Oses-Prieto, J.A., Burlingame, A., Bhargava, A. (2019). Probing the Interactome of Corticotropin-Releasing Factor Receptor Heteromers Using Mass Spectrometry. In: Tiberi, M. (eds) G Protein-Coupled Receptor Signaling. Methods in Molecular Biology, vol 1947. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9121-1_15

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  • DOI: https://doi.org/10.1007/978-1-4939-9121-1_15

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-9120-4

  • Online ISBN: 978-1-4939-9121-1

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