Specificity of the functional interactions of the β-adrenergic receptor and rhodopsin with guanine nucleotide regulatory proteins reconstituted in phospholipid vesicles

R. A. Cerione, C. Staniszewski, J. L. Benovic, R. J. Lefkowitz, M. G. Caron, P. Gierschik, R. Somers, Allen M. Spiegel, J. Codina, L. Birnbaumer

Research output: Contribution to journalArticle

129 Citations (Scopus)

Abstract

We have assessed the functional interactions of two pure receptor proteins with three different pure guanine nucleotide regulatory proteins in phosphatidylcholine vesicles. The receptor proteins are the guinea pig lung β-adrenergic receptor (βAR) and the retinal photon receptor rhodopsin. The guanine nucleotide regulatory proteins were the stimulatory (N(s)) and inhibitory (N(i)) proteins of the adenylate cyclase system and transducin (T), the regulatory protein from the light-activated cyclic GMP phosphodiesterase system in retinal rod outer segments. The insertion of N(s) with βAR in lipid vesicles increases the extent of binding of [35S]GTPγS to N(s) and in parallel, the total GTPase activity. However, there is little change in the actual rate of catalytic turnover of GTPase activity (defined as mol of P(i) released/min/mol of N(s)-guanine nucleotide complexes). Enhancement of this turnover rate requires the β-agonist isoproterenol and is accounted for by an isoproterenol-promoted increase in the rate and extent of [35S]GTPγS binding to N(s). The co-insertion of the βAR with N(i) or transducin results in markedly lower stimulation by isoproterenol of both the GTPase activity and [35S]GTPγS binding to these nucleotide regulatory proteins indicating that their preferred order of interaction with βAR is N(s) >> N(i) > T. This contrasts with the preferred order of interaction of these different nucleotide regulatory proteins with light-activated rhodopsin which we find to be T ≃ N (i) >> N (s). Nonetheless the fold stimulation of GTPase activity and [35S]GTPγS binding in T, induced by light-activated rhodopsin, is significantly greater than the 'fold' stimulation of these activities in N(i). This reflects the greater intrinsic ability of N(i) to hydrolyze GTP and bind guanine nucleotides (at 10 mM MgCl2, 100-200 nM GTP or [35S]GTPγS) compared to T. The maximum turnover numbers for the rhodopsin-stimulated GTPase in both N(i) and T are similar to those obtained for isoproterenol-stimulated activity in N(s). This suggests that the different nucleotide regulatory proteins are capable of a common upper limit of catalytic efficiency which can best be attained when coupled to the appropriate receptor.

Original languageEnglish (US)
Pages (from-to)1493-1500
Number of pages8
JournalJournal of Biological Chemistry
Volume260
Issue number3
StatePublished - 1985
Externally publishedYes

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Rhodopsin
GTP-Binding Proteins
Adrenergic Receptors
Phospholipids
GTP Phosphohydrolases
Isoproterenol
Proteins
Transducin
Guanine Nucleotides
Nucleotides
Guanosine Triphosphate
Light
Retinal Photoreceptor Cell Outer Segment
Rod Cell Outer Segment
Retinal Rod Photoreceptor Cells
Magnesium Chloride
Cyclic GMP
Phosphoric Diester Hydrolases
Phosphatidylcholines
Photons

ASJC Scopus subject areas

  • Biochemistry

Cite this

Cerione, R. A., Staniszewski, C., Benovic, J. L., Lefkowitz, R. J., Caron, M. G., Gierschik, P., ... Birnbaumer, L. (1985). Specificity of the functional interactions of the β-adrenergic receptor and rhodopsin with guanine nucleotide regulatory proteins reconstituted in phospholipid vesicles. Journal of Biological Chemistry, 260(3), 1493-1500.

Specificity of the functional interactions of the β-adrenergic receptor and rhodopsin with guanine nucleotide regulatory proteins reconstituted in phospholipid vesicles. / Cerione, R. A.; Staniszewski, C.; Benovic, J. L.; Lefkowitz, R. J.; Caron, M. G.; Gierschik, P.; Somers, R.; Spiegel, Allen M.; Codina, J.; Birnbaumer, L.

In: Journal of Biological Chemistry, Vol. 260, No. 3, 1985, p. 1493-1500.

Research output: Contribution to journalArticle

Cerione, RA, Staniszewski, C, Benovic, JL, Lefkowitz, RJ, Caron, MG, Gierschik, P, Somers, R, Spiegel, AM, Codina, J & Birnbaumer, L 1985, 'Specificity of the functional interactions of the β-adrenergic receptor and rhodopsin with guanine nucleotide regulatory proteins reconstituted in phospholipid vesicles', Journal of Biological Chemistry, vol. 260, no. 3, pp. 1493-1500.
Cerione, R. A. ; Staniszewski, C. ; Benovic, J. L. ; Lefkowitz, R. J. ; Caron, M. G. ; Gierschik, P. ; Somers, R. ; Spiegel, Allen M. ; Codina, J. ; Birnbaumer, L. / Specificity of the functional interactions of the β-adrenergic receptor and rhodopsin with guanine nucleotide regulatory proteins reconstituted in phospholipid vesicles. In: Journal of Biological Chemistry. 1985 ; Vol. 260, No. 3. pp. 1493-1500.
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AU - Caron, M. G.

AU - Gierschik, P.

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N2 - We have assessed the functional interactions of two pure receptor proteins with three different pure guanine nucleotide regulatory proteins in phosphatidylcholine vesicles. The receptor proteins are the guinea pig lung β-adrenergic receptor (βAR) and the retinal photon receptor rhodopsin. The guanine nucleotide regulatory proteins were the stimulatory (N(s)) and inhibitory (N(i)) proteins of the adenylate cyclase system and transducin (T), the regulatory protein from the light-activated cyclic GMP phosphodiesterase system in retinal rod outer segments. The insertion of N(s) with βAR in lipid vesicles increases the extent of binding of [35S]GTPγS to N(s) and in parallel, the total GTPase activity. However, there is little change in the actual rate of catalytic turnover of GTPase activity (defined as mol of P(i) released/min/mol of N(s)-guanine nucleotide complexes). Enhancement of this turnover rate requires the β-agonist isoproterenol and is accounted for by an isoproterenol-promoted increase in the rate and extent of [35S]GTPγS binding to N(s). The co-insertion of the βAR with N(i) or transducin results in markedly lower stimulation by isoproterenol of both the GTPase activity and [35S]GTPγS binding to these nucleotide regulatory proteins indicating that their preferred order of interaction with βAR is N(s) >> N(i) > T. This contrasts with the preferred order of interaction of these different nucleotide regulatory proteins with light-activated rhodopsin which we find to be T ≃ N (i) >> N (s). Nonetheless the fold stimulation of GTPase activity and [35S]GTPγS binding in T, induced by light-activated rhodopsin, is significantly greater than the 'fold' stimulation of these activities in N(i). This reflects the greater intrinsic ability of N(i) to hydrolyze GTP and bind guanine nucleotides (at 10 mM MgCl2, 100-200 nM GTP or [35S]GTPγS) compared to T. The maximum turnover numbers for the rhodopsin-stimulated GTPase in both N(i) and T are similar to those obtained for isoproterenol-stimulated activity in N(s). This suggests that the different nucleotide regulatory proteins are capable of a common upper limit of catalytic efficiency which can best be attained when coupled to the appropriate receptor.

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