Assay of RGS protein activity in vitro using purified components

Andrejs M. Krumins, Alfred G. Gilman

Research output: Contribution to journalArticle

22 Citations (Scopus)

Abstract

Single-turnover and steady-state GTPase assays are an effective means to identify and characterize interactions between RGS and Gα proteins in vitro. The advantage of the single turnover GTPase assay is that it permits simple and rapid assessment of RGS protein activity toward a putative Gα-GTP substrate. Moreover, once an interaction between an RGS protein and a Gα-GTP subunit has been identified, the single-turnover assay can be used to determine Michaelis-Menten constants and/or KI values for other competing Gα substrates. A disadvantage of the single-turnover assay is that a negative result does not preclude the possibility of an interaction between given RGS and Gα proteins in vivo. Inappropriate reaction conditions or the presence (or absence) of appropriate posttranslational modifications may result in small or undetectable increases in RGS protein-dependent GTPase activity. In these cases it may be tempting to examine RGS protein activity using steady-state GTPase assays in phospholipid vesicles reconstituted with receptors and heterotrimetric G proteins. The advantage to monitoring steady-state GTPase activity in reconstituted proteoliposomes is that ligand-dependent activation of the receptor facilitates GDP dissociation, such that effects of RGS proteins can be observed; multiple cycles of GTP binding and hydrolysis then amplify the GTPase signal. Additionally, the presence of the phospholipid membrane can increase the local RGS protein concentration ∼104-fold, permitting observation of interactions that are weak in solution. The primary disadvantage of the reconstituted system is the requirement for receptor purification, a technically demanding undertaking in comparison to the purification of Gα, Gβγ, and most RGS proteins.

Original languageEnglish (US)
Pages (from-to)673-685
Number of pages13
JournalMethods in Enzymology
Volume344
DOIs
StatePublished - 2001

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RGS Proteins
Assays
GTP Phosphohydrolases
Guanosine Triphosphate
GTP-Binding Proteins
Purification
Phospholipids
In Vitro Techniques
Post Translational Protein Processing
Hydrolysis
Chemical activation
Observation
Ligands

ASJC Scopus subject areas

  • Biochemistry

Cite this

Assay of RGS protein activity in vitro using purified components. / Krumins, Andrejs M.; Gilman, Alfred G.

In: Methods in Enzymology, Vol. 344, 2001, p. 673-685.

Research output: Contribution to journalArticle

Krumins, Andrejs M. ; Gilman, Alfred G. / Assay of RGS protein activity in vitro using purified components. In: Methods in Enzymology. 2001 ; Vol. 344. pp. 673-685.
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