title: A rapid and efficient immunoenzymatic assay to detect receptor protein interactions: G protein-coupled receptors.
abstract: G protein-coupled receptors (GPCRs) represent one of the largest families of cell surface receptors, and are the target of at least one-third of the current therapeutic drugs on the market. Along their life cycle, GPCRs are accompanied by a range of specialized GPCR-interacting proteins (GIPs), which take part in receptor proper folding, targeting to the appropriate subcellular compartments and in receptor signaling tasks, and also in receptor regulation processes, such as desensitization and internalization. The direction of protein-protein interactions and multi-protein complexes formation is crucial in understanding protein function and their implication in pathological events. Although several methods have been already developed to assay protein complexes, some of them are quite laborious, expensive, and, more important, they do not generate fully quantitative results. Herein, we show a rapid immunoenzymatic assay to quantify GPCR interactionswith its signaling proteins. The recently de-orphanized GPCR, GPR17, was chosen as a GPCR prototype to optimize the assay. In a GPR17 transfected cell line and primary oligodendrocyte precursor cells, GPR17 interaction with proteins involved in the typical GPCR regulation, such as desensitization and internalization machinery, was investigated. The obtained results were validated by co-immunoprecipitation experiments, confirming this new method as a rapid and quantitative assay to study protein-protein interactions.
authors.affiliation: Department of Pharmacy, University of Pisa, 56126 Pisa, Italy. elisa.zappelli@for.unipi.it.
authors.forename: Elisa
authors.initials: E
authors.lastname: Zappelli
authors.affiliation: Department of Pharmacy, University of Pisa, 56126 Pisa, Italy. simona.daniele@for.unipi.it.
authors.forename: Simona
authors.initials: S
authors.lastname: Daniele
authors.affiliation: Department of Pharmacological and Biomolecular Sciences, University of Milan, 20133 Milan, Italy. mariapia.abbracchio@unimi.it.
authors.forename: Maria P
authors.initials: MP
authors.lastname: Abbracchio
authors.affiliation: Department of Pharmacy, University of Pisa, 56126 Pisa, Italy. claudia.martini@unipi.it.
authors.forename: Claudia
authors.initials: C
authors.lastname: Martini
authors.affiliation: Department of Pharmacy, University of Pisa, 56126 Pisa, Italy. maria.trincavelli@farm.unipi.it.
authors.forename: Maria Letizia
authors.initials: ML
authors.lastname: Trincavelli
mesh_terms: D000818:Animals; D019390:Arrestins / Q000378:metabolism; D002478:Cells, Cultured; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D047468:Immunoprecipitation; D060066:Protein Interaction Maps; D051381:Rats; D017207:Rats, Sprague-Dawley; D043562:Receptors, G-Protein-Coupled / Q000032:analysis* / Q000276:immunology / Q000378:metabolism; D015398:Signal Transduction; D000071557:beta-Arrestins
chemical_list: D019390:Arrestins; C121670:GPR17 protein, human; D043562:Receptors, G-Protein-Coupled; D000071557:beta-Arrestins
references.citation: Nature. 2002 May 23;417(6887):399-403
references.pmid: 12000970
references.citation: Pharmacol Rev. 2001 Mar;53(1):1-24
references.pmid: 11171937
references.citation: Trends Endocrinol Metab. 2006 May-Jun;17(4):159-65
references.pmid: 16595179
references.citation: EMBO J. 2006 Oct 4;25(19):4615-27
references.pmid: 16990797
references.citation: Nat Biotechnol. 2004 Oct;22(10):1317-21
references.pmid: 15470473
references.citation: Purinergic Signal. 2011 Dec;7(4):463-8
references.pmid: 21773766
references.citation: Pharmacol Ther. 2012 Jan;133(1):40-69
references.pmid: 21903131
references.citation: Biochem Soc Trans. 2004 Nov;32(Pt 5):851-5
references.pmid: 15494032
references.citation: Curr Opin Pharmacol. 2003 Apr;3(2):121-6
references.pmid: 12681232
references.citation: J Biol Chem. 2013 Feb 15;288(7):5241-56
references.pmid: 23288840
references.citation: J Biol Chem. 2011 Mar 25;286(12):10593-604
references.pmid: 21209081
references.citation: Annu Rev Physiol. 2007;69:511-34
references.pmid: 17305472
references.citation: Annu Rev Physiol. 2007;69:483-510
references.pmid: 17305471
references.citation: Bioanalysis. 2013 May;5(10):1299-313
references.pmid: 23721450
references.citation: Genome Res. 2003 Jun;13(6A):1146-54
references.pmid: 12799350
references.citation: J Pharmacol Exp Ther. 2011 Aug;338(2):559-67
references.pmid: 21531793
references.citation: Mol Pharmacol. 2003 Jun;63(6):1256-72
references.pmid: 12761335
references.citation: Cell Signal. 2014 Jun;26(6):1310-25
references.pmid: 24613411
references.citation: Biochem J. 2005 Feb 1;385(Pt 3):625-37
references.pmid: 15504107
references.citation: Br J Pharmacol. 2010 Mar;159(5):1092-105
references.pmid: 20148890
references.citation: PLoS One. 2008;3(10):e3579
references.pmid: 18974869