2016
DOI: 10.1002/cpph.15
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Detection of Receptor Heteromerization Using In Situ Proximity Ligation Assay

Abstract: Although G protein-coupled receptor (GPCR) heteromerization has been extensively demonstrated in vitro using heterologous cells that overexpress epitope-tagged receptors, their presence in endogenous systems is less well established. This is because a criterion to identify receptor heteromerization is the demonstration that the two interacting receptors are present not only in the same cell but also in the same subcellular compartment in close enough proximity to allow for direct receptor-receptor interaction.… Show more

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Cited by 54 publications
(44 citation statements)
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“…The PLA can be carried out in situ in a cell membrane environment, which provides evidence for the functional relevance of the SBA results. While PLA has been used to detect GPCR heteromers (39), to our knowledge, there have been no reports on its use to detect GPCR-RAMP complexes. Cells cotransfected with CALCRL and RAMP2 and treated with both primary Abs showed a significant PLA signal against all negative controls, indicating that the assay is robust.…”
Section: Discussionmentioning
confidence: 99%
“…The PLA can be carried out in situ in a cell membrane environment, which provides evidence for the functional relevance of the SBA results. While PLA has been used to detect GPCR heteromers (39), to our knowledge, there have been no reports on its use to detect GPCR-RAMP complexes. Cells cotransfected with CALCRL and RAMP2 and treated with both primary Abs showed a significant PLA signal against all negative controls, indicating that the assay is robust.…”
Section: Discussionmentioning
confidence: 99%
“…The PLA can be carried out in situ in a cell membrane environment, which provides evidence for the functional relevance of the SBA results. While PLA has been used to detect GPCR heteromers (38) , to our knowledge there have been no reports on its use to detect GPCR-RAMP complexes. Cells co-transfected with CALCRL+RAMP2 and treated with both primary Abs showed a significant (P<0.0001) PLA signal against all the negative controls, indicating that the assay is robust.…”
Section: Discussionmentioning
confidence: 99%
“…The potential applications of this method are huge, since it can be used in principle with any pair of antibodies, allowing co-detection of any endogenous antigen, including posttranslational modifications, such as specific phosphorylated sites. This flexibility explains its increasing popularity, in fields as diverse as cell biology, pharmacology, immunology, virology, proteomics, biomarkers for cancer, pathogen diagnostic, or even astrobiology [5,7,8,[10][11][12][13][14][15][16][17].…”
Section: Introductionmentioning
confidence: 99%