Transducin and the inhibitory nucleotide regulatory protein inhibit the stimulatory nucleotide regulatory protein mediated stimulation of adenylate cyclase in phospholipid vesicle systems

Richard A. Cerione, Juan Codina, Brian F. Kilpatrick, Claudia Staniszewski, Peter Gierschik, Robert L. Somers, Allen M. Spiegel, Allen M. Spiegel, Marc G. Caron, Robert J. Lefkowitz

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Abstract

The adenylate cyclase coupled inhibitory nucleotide regulatory protein (Ni) and the bovine retinal nucleotide regulatory protein transducin (T) appear to share some common functional properties since their GTPase activity is stimulated to similar extents by the retinal photoreceptor rhodopsin. In the present work, we sought to assess whether these functional similarities might extend to their interaction with adenylate cyclase. This necessitated the development of reconstitution systems in which guanine nucleotide regulatory protein mediated inhibition of adenylate cyclase activity could be demonstrated and characterized in a lipid milieu. In the absence of the pure human erythrocyte stimulatory nucleotide regulatory protein (Ns), the insertion into phospholipid vesicles of either pure Ni from human erythrocytes or pure bovine T with the resolved catalytic moiety of bovine caudate adenylate cyclase (C) does not establish GppNHp inhibition of either Mg2+- or forskolin-stimulated adenylate cyclase. However, the coinsertion into lipid vesicles of either Ni or T with Ns and resolved C results in an inhibition of Ns(GppNHp) stimulatable C activity. As is the case in intact membranes, the reconstituted inhibition of the Ns-stimulated C activity extends into the steady-state phase of time courses of activity. This inhibition is highly sensitive to the MgCl2 concentration. At 2 mM MgCl2, the inhibition is greater than 80% while at 50 mM MgCl2 it is only ∼20%. Overall these results suggest (1) that the inhibition of adenylate cyclase activity by Ni is due to an interference with the Ns stimulation of the C activity rather than a direct effect on the intrinsic activity of the catalytic moiety itself and (2) that the retinal nucleotide regulatory protein, transducin, substitutes for Ni in causing inhibition of Ns-stimulated C activity.

Original languageEnglish (US)
Pages (from-to)4499-4503
Number of pages5
JournalBiochemistry
Volume24
Issue number17
StatePublished - 1985
Externally publishedYes

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Transducin
Adenylyl Cyclases
Phospholipids
Nucleotides
Magnesium Chloride
Proteins
Erythrocytes
Gs GTP-Binding Protein alpha Subunits
Gi-Go GTP-Binding Protein alpha Subunits
Lipids
Vertebrate Photoreceptor Cells
Rhodopsin
GTP Phosphohydrolases
Colforsin
GTP-Binding Proteins
Membranes

ASJC Scopus subject areas

  • Biochemistry

Cite this

Cerione, R. A., Codina, J., Kilpatrick, B. F., Staniszewski, C., Gierschik, P., Somers, R. L., ... Lefkowitz, R. J. (1985). Transducin and the inhibitory nucleotide regulatory protein inhibit the stimulatory nucleotide regulatory protein mediated stimulation of adenylate cyclase in phospholipid vesicle systems. Biochemistry, 24(17), 4499-4503.

Transducin and the inhibitory nucleotide regulatory protein inhibit the stimulatory nucleotide regulatory protein mediated stimulation of adenylate cyclase in phospholipid vesicle systems. / Cerione, Richard A.; Codina, Juan; Kilpatrick, Brian F.; Staniszewski, Claudia; Gierschik, Peter; Somers, Robert L.; Spiegel, Allen M.; Spiegel, Allen M.; Caron, Marc G.; Lefkowitz, Robert J.

In: Biochemistry, Vol. 24, No. 17, 1985, p. 4499-4503.

Research output: Contribution to journalArticle

Cerione, RA, Codina, J, Kilpatrick, BF, Staniszewski, C, Gierschik, P, Somers, RL, Spiegel, AM, Spiegel, AM, Caron, MG & Lefkowitz, RJ 1985, 'Transducin and the inhibitory nucleotide regulatory protein inhibit the stimulatory nucleotide regulatory protein mediated stimulation of adenylate cyclase in phospholipid vesicle systems', Biochemistry, vol. 24, no. 17, pp. 4499-4503.
Cerione, Richard A. ; Codina, Juan ; Kilpatrick, Brian F. ; Staniszewski, Claudia ; Gierschik, Peter ; Somers, Robert L. ; Spiegel, Allen M. ; Spiegel, Allen M. ; Caron, Marc G. ; Lefkowitz, Robert J. / Transducin and the inhibitory nucleotide regulatory protein inhibit the stimulatory nucleotide regulatory protein mediated stimulation of adenylate cyclase in phospholipid vesicle systems. In: Biochemistry. 1985 ; Vol. 24, No. 17. pp. 4499-4503.
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abstract = "The adenylate cyclase coupled inhibitory nucleotide regulatory protein (Ni) and the bovine retinal nucleotide regulatory protein transducin (T) appear to share some common functional properties since their GTPase activity is stimulated to similar extents by the retinal photoreceptor rhodopsin. In the present work, we sought to assess whether these functional similarities might extend to their interaction with adenylate cyclase. This necessitated the development of reconstitution systems in which guanine nucleotide regulatory protein mediated inhibition of adenylate cyclase activity could be demonstrated and characterized in a lipid milieu. In the absence of the pure human erythrocyte stimulatory nucleotide regulatory protein (Ns), the insertion into phospholipid vesicles of either pure Ni from human erythrocytes or pure bovine T with the resolved catalytic moiety of bovine caudate adenylate cyclase (C) does not establish GppNHp inhibition of either Mg2+- or forskolin-stimulated adenylate cyclase. However, the coinsertion into lipid vesicles of either Ni or T with Ns and resolved C results in an inhibition of Ns(GppNHp) stimulatable C activity. As is the case in intact membranes, the reconstituted inhibition of the Ns-stimulated C activity extends into the steady-state phase of time courses of activity. This inhibition is highly sensitive to the MgCl2 concentration. At 2 mM MgCl2, the inhibition is greater than 80{\%} while at 50 mM MgCl2 it is only ∼20{\%}. Overall these results suggest (1) that the inhibition of adenylate cyclase activity by Ni is due to an interference with the Ns stimulation of the C activity rather than a direct effect on the intrinsic activity of the catalytic moiety itself and (2) that the retinal nucleotide regulatory protein, transducin, substitutes for Ni in causing inhibition of Ns-stimulated C activity.",
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AU - Codina, Juan

AU - Kilpatrick, Brian F.

AU - Staniszewski, Claudia

AU - Gierschik, Peter

AU - Somers, Robert L.

AU - Spiegel, Allen M.

AU - Spiegel, Allen M.

AU - Caron, Marc G.

AU - Lefkowitz, Robert J.

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