Functional Interactions of Recombinant α2 Adrenergic Receptor Subtypes and G Proteins in Reconstituted Phospholipid Vesicles

Hitoshi Kurose, John W Regan, Marc G. Caron, Robert J. Lefkowitz

Research output: Contribution to journalArticle

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Abstract

The functional interaction of the recombinant α2 adrenergic receptor subtypes, α2-C10 (the human platelet α2 receptor, equivalent to the α2A subtype) and α2-C4 (an α2 receptor subtype cloned from a human kidney cDNA library), with G proteins was characterized in an in vitro reconstitution system. These receptor subtypes were overexpressed in COS-7 cells and were purified to a specific activity of 1.1-3.3 nmol/mg of protein. The G proteins consisted of Gs (adenylyl cyclase stimulatory) and members of the inhibitory family, including Gi1 Gi2, and Gi3, and G0. The cloned α subunits of these G proteins were overexpressed in Escherichia coli and were purified to homogeneity. Prior to use, G holoproteins were prepared by mixing the α subunits with βγ subunits that had been purified from bovine brain. Following reconstitution into phospholipid vesicles, both α2 receptor subtypes could couple to the inhibitory G proteins but not to Gs, as assessed by agonist stimulation of GTPase activity. The pharmacological specificity of this interaction was preserved with respect to the two receptor subtypes. Between the different inhibitory G proteins, the α2-C10 adrenergic receptor subtype showed the following preference: Gi3 > Gi1 > Gi2 > G0. The stimulation of GTPase activity (turnover number) ranged from 6.4-fold (Gi3) to 1.5-fold (G0). The preference of G-protein interaction for the α2-C4 receptor subtype was the same as that observed for the α2-C10, but the extent of activation was slightly lower. The results show that in vitro each of the α2 adrenergic receptor subtypes can activate multiple G proteins but that clear preferences exist with respect to the individual inhibitory G-protein subtypes. Additionally, it appears that α2-C10 is coupled more efficiently to G-protein activation than is α2-C4.

Original languageEnglish (US)
Pages (from-to)3335-3341
Number of pages7
JournalBiochemistry
Volume30
Issue number13
DOIs
StatePublished - Apr 1 1991

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GTP-Binding Proteins
Adrenergic Receptors
Phospholipids
GTP Phosphohydrolases
Chemical activation
COS Cells
Platelets
Gene Library
Adenylyl Cyclases
Escherichia coli
Brain
Blood Platelets
Pharmacology
Kidney

ASJC Scopus subject areas

  • Biochemistry

Cite this

Functional Interactions of Recombinant α2 Adrenergic Receptor Subtypes and G Proteins in Reconstituted Phospholipid Vesicles. / Kurose, Hitoshi; Regan, John W; Caron, Marc G.; Lefkowitz, Robert J.

In: Biochemistry, Vol. 30, No. 13, 01.04.1991, p. 3335-3341.

Research output: Contribution to journalArticle

Kurose, Hitoshi ; Regan, John W ; Caron, Marc G. ; Lefkowitz, Robert J. / Functional Interactions of Recombinant α2 Adrenergic Receptor Subtypes and G Proteins in Reconstituted Phospholipid Vesicles. In: Biochemistry. 1991 ; Vol. 30, No. 13. pp. 3335-3341.
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