Investigating receptor-protein interactions in vitro is a complicated task. Receptor integrity must be maintained in order to achieve meaningful proteomics results. Membrane extraction methods can degrade or alter structural conformation, which changes receptor binding sites, disrupting the interactome so that binding partners may be absent during analysis.
Roy et al. validated two gel-free extraction methods for examining G-protein coupled receptors in cell culture.1 Using the human β2-adrenergic receptor (β2-AR) and the prostaglandin D2 receptor (DP) as examples, the researchers demonstrated that receptor-protein interactions in the endoplasmic reticulum (ER) were maintained using their methods. Furthermore, the extraction procedure resulted in material suitable for valid analysis by liquid chromatography–tandem mass spectrometry (LC-MS/MS). Their results revealed new maturation and degradation pathways for the receptors.
Human embryonic kidney cells were transfected with FLAG- or TAP-tagged β2-AR constructs. After lysis, samples were divided into either crude membrane preparations or further fractionated by ultra-centrifugation. Following immunopurification, the prepared samples were analyzed by LC-MS/MS using an LTQ XL linear ion trap mass spectrometer (Thermo Scientific).
The researchers investigated different detergent protocols for solubilizing receptors from cell membranes, validating methods for each of the tagging constructs used in transfection. Because detergents have the potential to alter receptor conformation, these methods were optimized to retain maximal interactome stability in the receptors harvested. The researchers found that N-dodecyl-β-D-maltoside (DDM), a mild non-ionic detergent, preserved β2-AR structural integrity following TAP-tagged transfection. A different protocol, using octyglucoside, was validated for FLAG-β2-AR constructs.
After receptor identity was confirmed using traditional gel electrophoresis and immunoblotting, binding ability was confirmed with radioligand binding assays. Maintenance of this binding ability throughout the purification procedures was shown using the Gβ subunit, a protein known to associate with the β2-AR in vivo. Immunoblots for this subunit confirmed that cellular receptor interactions were maintained.
Once the extraction methods were validated, the researchers scaled up the procedure to investigate protein interactions between β2-AR and potential chaperones on the ER. Samples were prepared for LC-MS/MS analysis using a filter-aided sample preparation technique.
LC-MS/MS data showed 97 proteins interacting with the receptor using the TAP constructs, and 177 using the FLAG method. This screen included previously reported protein partners of β2-AR, thus confirming protocol validity and the importance of multiple screening approaches.
With validation of their research protocols, the researchers were able to show interaction between the G-protein coupled receptors β2-AR and DP, and the proteins RNF5 and JAMP. These proteins are involved in the ER quality-control system, ER-associated degradation (ERAD), which detects and targets mis-folded proteins for proteasomal destruction. These results were possible because the receptor interactome was preserved due to the novel gel-free extraction method established in this study.
1. Roy, S.J., et al. (2013) “Novel, Gel-free Proteomics Approach Identifies RNF5 and JAMP as Modulators of GPCR Stability,” Molecular Endocrinology, 27(8) (pp. 1245–66).
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