Functional reconstitution of the α2-adrenergic receptor with guanine nucleotide regulatory proteins in phospholipid vesicles

R. A. Cerione, J. W. Regan, H. Nakata, J. Codina, J. L. Benovic, P. Gierschik, R. L. Somers, Allen M. Spiegel, L. Birnbaumer, R. J. Lefkowitz

Research output: Contribution to journalArticle

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Abstract

We describe the successful reconstitution of functional interactions between an inhibitory adenylate cyclase-coupled receptor and various nucleotide-binding regulatory proteins in phospholipid vesicles. The receptor is the α2-adrenergic receptor (α2AR) whcih has been partially purified (~500-5000-fold) from human platelet membranes. The nucleotide-binding regulatory proteins include purified preparations of human erythrocyte N(i) and N(s), bovine retinal transducin and the recently discovered bovine brain N(o). Addition of the physiologic ligand, epinephrine, to vesicles containing the α2AR and N(i) results in stimulation of the GTPase activity in N(i). This stimulation of GTPase activity by epinephrine is prevented in the presence of the α-adrenergic antagonist, phentolamine, which indicates that a functional reconstitution of the α2AR and N(i) has been established. The maximum turnover number for the α2AR-mediated epinephrine-stimulated GTPase activity in N(i) is similar to the maximal turnover numbers obtained for the β-adrenergic receptor-mediated isoproterenol-stimulated GTPase activity in N(s) and the rhodopsin-mediated light-stimulated GTPase activity in transducin (0.5-1.5 mol of P(i) released per min per mol of nucleotide regulatory protein). Functional similarities between the α2AR and rhodopsin are observed in their interactions with the various nucleotide-binding regulatory proteins. Thus, both of these receptor proteins are capable of promoting the maximal activation of N(i) and N(o) while being much less effective in promoting the activation of N(s). However, there are differences between the α2AR and rhodopsin in their interactions with transducin. Specifically, while rhodopsin will maximally activate transducin, the α2AR is much less effective in promoting this activation (i.e ~20% as effective as rhodopsin). Overall, these results suggest the following specificities of interaction: for rhodopsin, transducin ≃ N(i) ≃ N(o) >> N(s); while for α2AR, N(i) ≃ N(o) > transducin ≥ N(s).

Original languageEnglish (US)
Pages (from-to)3901-3909
Number of pages9
JournalJournal of Biological Chemistry
Volume261
Issue number8
StatePublished - 1986
Externally publishedYes

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GTP-Binding Proteins
Transducin
Adrenergic Receptors
Phospholipids
Rhodopsin
GTP Phosphohydrolases
Nucleotides
Epinephrine
Carrier Proteins
Chemical activation
Proteins
Adrenergic Antagonists
Phentolamine
Platelets
Isoproterenol
Adenylyl Cyclases
Brain
Blood Platelets
Erythrocytes
Ligands

ASJC Scopus subject areas

  • Biochemistry

Cite this

Cerione, R. A., Regan, J. W., Nakata, H., Codina, J., Benovic, J. L., Gierschik, P., ... Lefkowitz, R. J. (1986). Functional reconstitution of the α2-adrenergic receptor with guanine nucleotide regulatory proteins in phospholipid vesicles. Journal of Biological Chemistry, 261(8), 3901-3909.

Functional reconstitution of the α2-adrenergic receptor with guanine nucleotide regulatory proteins in phospholipid vesicles. / Cerione, R. A.; Regan, J. W.; Nakata, H.; Codina, J.; Benovic, J. L.; Gierschik, P.; Somers, R. L.; Spiegel, Allen M.; Birnbaumer, L.; Lefkowitz, R. J.

In: Journal of Biological Chemistry, Vol. 261, No. 8, 1986, p. 3901-3909.

Research output: Contribution to journalArticle

Cerione, RA, Regan, JW, Nakata, H, Codina, J, Benovic, JL, Gierschik, P, Somers, RL, Spiegel, AM, Birnbaumer, L & Lefkowitz, RJ 1986, 'Functional reconstitution of the α2-adrenergic receptor with guanine nucleotide regulatory proteins in phospholipid vesicles', Journal of Biological Chemistry, vol. 261, no. 8, pp. 3901-3909.
Cerione, R. A. ; Regan, J. W. ; Nakata, H. ; Codina, J. ; Benovic, J. L. ; Gierschik, P. ; Somers, R. L. ; Spiegel, Allen M. ; Birnbaumer, L. ; Lefkowitz, R. J. / Functional reconstitution of the α2-adrenergic receptor with guanine nucleotide regulatory proteins in phospholipid vesicles. In: Journal of Biological Chemistry. 1986 ; Vol. 261, No. 8. pp. 3901-3909.
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AU - Benovic, J. L.

AU - Gierschik, P.

AU - Somers, R. L.

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N2 - We describe the successful reconstitution of functional interactions between an inhibitory adenylate cyclase-coupled receptor and various nucleotide-binding regulatory proteins in phospholipid vesicles. The receptor is the α2-adrenergic receptor (α2AR) whcih has been partially purified (~500-5000-fold) from human platelet membranes. The nucleotide-binding regulatory proteins include purified preparations of human erythrocyte N(i) and N(s), bovine retinal transducin and the recently discovered bovine brain N(o). Addition of the physiologic ligand, epinephrine, to vesicles containing the α2AR and N(i) results in stimulation of the GTPase activity in N(i). This stimulation of GTPase activity by epinephrine is prevented in the presence of the α-adrenergic antagonist, phentolamine, which indicates that a functional reconstitution of the α2AR and N(i) has been established. The maximum turnover number for the α2AR-mediated epinephrine-stimulated GTPase activity in N(i) is similar to the maximal turnover numbers obtained for the β-adrenergic receptor-mediated isoproterenol-stimulated GTPase activity in N(s) and the rhodopsin-mediated light-stimulated GTPase activity in transducin (0.5-1.5 mol of P(i) released per min per mol of nucleotide regulatory protein). Functional similarities between the α2AR and rhodopsin are observed in their interactions with the various nucleotide-binding regulatory proteins. Thus, both of these receptor proteins are capable of promoting the maximal activation of N(i) and N(o) while being much less effective in promoting the activation of N(s). However, there are differences between the α2AR and rhodopsin in their interactions with transducin. Specifically, while rhodopsin will maximally activate transducin, the α2AR is much less effective in promoting this activation (i.e ~20% as effective as rhodopsin). Overall, these results suggest the following specificities of interaction: for rhodopsin, transducin ≃ N(i) ≃ N(o) >> N(s); while for α2AR, N(i) ≃ N(o) > transducin ≥ N(s).

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