Selectivity of the β-adrenergic receptor among Gs, Gi's, and Go: Assay using recombinant α subunits in reconstituted phospholipid vesicles

Ronald C. Rubenstein, Maurine E. Linder, Elliott M. Ross

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Abstract

The selective regulation of Gs (long and short forms), Gi's (1,2, and 3), and Go by the β-adrenergic receptor was assessed quantitatively after coreconstitution of purified receptor, purified G-protein βγ subunits, and individual recombinant G-protein α subunits that were expressed in and purified from Escherichia coli. Receptor and βγ subunits were incorporated into phospholipid vesicles, and the α subunits bound to the vesicles stoichiometrically with respect to βγ. Efficient regulation of α subunit by receptor required the presence of βγ. Regulation of G proteins was measured according to the stimulation of the initial rate of GTPγS binding, steady-state GTPase activity, and equilibrium GDP/GDP exchange. The assays yielded qualitatively similar results. GDP/GDP exchange was a first-order reaction for each subunit. The rate constant increased linearly with the concentration of agonist-liganded receptor, and the dependence of the rate constant on receptor concentration was a reproducible measurement of the efficiency with which receptor regulated each G protein. Reconstituted αs (long or short form) was stimulated by receptor to approximately the extent described previously for natural Gs. Both αi,1 and αi,3 were regulated with 25-33% of that efficiency. Stimulation of αo and αi,2 was weak, and stimulation of αo was barely detectable over its high basal exchange rate. Reduction of the receptor with dithiothreitol increased the exchange rates for all G proteins but did not alter the relative selectivity of the receptor.

Original languageEnglish (US)
Pages (from-to)10769-10777
Number of pages9
JournalBiochemistry
Volume30
Issue number44
StatePublished - 1991

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GTP-Binding Proteins
Adrenergic Receptors
Assays
Phospholipids
Protein Subunits
Rate constants
Ion exchange
Dithiothreitol
GTP Phosphohydrolases
Recombinant Proteins
Escherichia coli

ASJC Scopus subject areas

  • Biochemistry

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Selectivity of the β-adrenergic receptor among Gs, Gi's, and Go : Assay using recombinant α subunits in reconstituted phospholipid vesicles. / Rubenstein, Ronald C.; Linder, Maurine E.; Ross, Elliott M.

In: Biochemistry, Vol. 30, No. 44, 1991, p. 10769-10777.

Research output: Contribution to journalArticle

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