Simultaneous coupling of α2-adrenergic receptors to two G-proteins with opposing effects

Subtype-selective coupling of α2C10, α2C4, and α2C2 adrenergic receptors to G(i) and G(s)

M. G. Eason, Hitoshi Kurose, B. D. Holt, J. R. Raymond, S. B. Liggett

Research output: Contribution to journalArticle

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Abstract

Coupling of the three α2-adrenergic receptor (α2AR) subtypes to G(i) and G(s) was studied in membranes from transfected CHO cells. We observed that in the presence of low concentrations of the α2AR agonist UK-14304, α2C10 mediated inhibition of adenylyl cyclase activity, whereas at high concentrations of agonist, α2C10 mediated stimulation of adenylyl cyclase activity. We considered that this biphasic response was due to the coupling of α2C10 to both G(i) and G(s). To isolate functional G(s) and G(i) coupling, cells were treated with pertussis toxin or cholera toxin in doses sufficient to fully ADP-ribosylate the respective G-proteins. Following treatment with cholera toxin, agonists elicited only α2C10-mediated inhibition (~50%) of adenylyl cyclase while after pertussis toxin treatment, agonists elicited only α2C10-mediated stimulation (~60%) of adenylyl cyclase. Incubation of membranes with antisera directed against the carboxyl- terminal portion of G(5α) blocked this functional α2AR·G(s) coupling to the same extent as that found for β2AR·G(s) coupling. In addition to functional G(s) coupling, we also verified direct, agonist-dependent, physical coupling of α2AR to G(sα). In agonist-treated membranes, an agonist-receptor-G(sα) complex was immunoprecipitated with a specific α2C10 antibody, and the G(s) component identified by both western blots using G(sα) antibody, and cholera toxin mediated ADP-ribosylation. Due to the differences in primary amino acid structure in a number of regions of the α2AR subtypes, we investigated whether G-protein coupling was subtype- selective, using UK-14304 and cells with the same α2AR expression levels (~5 pmol/mg). Coupling to G(i) was equivalent for α2C10, α2C4, and α2C2: 53.4 ± 8.8% versus 54.9 ± 1.0% versus 47.6 ± 3.5% inhibition of adenylyl cyclase, respectively. In marked contrast, distinct differences in coupling to G(s) were found between the three α2AR subtypes: stimulation of adenylyl cyclase was 57.9 ± 6.3% versus 30.7 ± 1.1% versus 21.8 ± 1.7% for α2C10, α2C4, and α2C2, respectively. Thus, α2AR have the potential to couple physically and functionally to both G(i) and G(s); for G(i) coupling we found a rank order of α2C10 = α2C4 = α2C2, while for G(s) coupling, α2C10 > α2C4 > α2C2.

Original languageEnglish
Pages (from-to)15795-15801
Number of pages7
JournalJournal of Biological Chemistry
Volume267
Issue number22
Publication statusPublished - Jan 1 1992
Externally publishedYes

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GTP-Binding Proteins
Adenylyl Cyclases
Adrenergic Receptors
Cholera Toxin
Pertussis Toxin
Membranes
Adenosine Diphosphate
Adrenergic Agonists
CHO Cells
Antibodies
Immune Sera
Western Blotting
Amino Acids

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Simultaneous coupling of α2-adrenergic receptors to two G-proteins with opposing effects : Subtype-selective coupling of α2C10, α2C4, and α2C2 adrenergic receptors to G(i) and G(s). / Eason, M. G.; Kurose, Hitoshi; Holt, B. D.; Raymond, J. R.; Liggett, S. B.

In: Journal of Biological Chemistry, Vol. 267, No. 22, 01.01.1992, p. 15795-15801.

Research output: Contribution to journalArticle

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abstract = "Coupling of the three α2-adrenergic receptor (α2AR) subtypes to G(i) and G(s) was studied in membranes from transfected CHO cells. We observed that in the presence of low concentrations of the α2AR agonist UK-14304, α2C10 mediated inhibition of adenylyl cyclase activity, whereas at high concentrations of agonist, α2C10 mediated stimulation of adenylyl cyclase activity. We considered that this biphasic response was due to the coupling of α2C10 to both G(i) and G(s). To isolate functional G(s) and G(i) coupling, cells were treated with pertussis toxin or cholera toxin in doses sufficient to fully ADP-ribosylate the respective G-proteins. Following treatment with cholera toxin, agonists elicited only α2C10-mediated inhibition (~50{\%}) of adenylyl cyclase while after pertussis toxin treatment, agonists elicited only α2C10-mediated stimulation (~60{\%}) of adenylyl cyclase. Incubation of membranes with antisera directed against the carboxyl- terminal portion of G(5α) blocked this functional α2AR·G(s) coupling to the same extent as that found for β2AR·G(s) coupling. In addition to functional G(s) coupling, we also verified direct, agonist-dependent, physical coupling of α2AR to G(sα). In agonist-treated membranes, an agonist-receptor-G(sα) complex was immunoprecipitated with a specific α2C10 antibody, and the G(s) component identified by both western blots using G(sα) antibody, and cholera toxin mediated ADP-ribosylation. Due to the differences in primary amino acid structure in a number of regions of the α2AR subtypes, we investigated whether G-protein coupling was subtype- selective, using UK-14304 and cells with the same α2AR expression levels (~5 pmol/mg). Coupling to G(i) was equivalent for α2C10, α2C4, and α2C2: 53.4 ± 8.8{\%} versus 54.9 ± 1.0{\%} versus 47.6 ± 3.5{\%} inhibition of adenylyl cyclase, respectively. In marked contrast, distinct differences in coupling to G(s) were found between the three α2AR subtypes: stimulation of adenylyl cyclase was 57.9 ± 6.3{\%} versus 30.7 ± 1.1{\%} versus 21.8 ± 1.7{\%} for α2C10, α2C4, and α2C2, respectively. Thus, α2AR have the potential to couple physically and functionally to both G(i) and G(s); for G(i) coupling we found a rank order of α2C10 = α2C4 = α2C2, while for G(s) coupling, α2C10 > α2C4 > α2C2.",
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T1 - Simultaneous coupling of α2-adrenergic receptors to two G-proteins with opposing effects

T2 - Subtype-selective coupling of α2C10, α2C4, and α2C2 adrenergic receptors to G(i) and G(s)

AU - Eason, M. G.

AU - Kurose, Hitoshi

AU - Holt, B. D.

AU - Raymond, J. R.

AU - Liggett, S. B.

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N2 - Coupling of the three α2-adrenergic receptor (α2AR) subtypes to G(i) and G(s) was studied in membranes from transfected CHO cells. We observed that in the presence of low concentrations of the α2AR agonist UK-14304, α2C10 mediated inhibition of adenylyl cyclase activity, whereas at high concentrations of agonist, α2C10 mediated stimulation of adenylyl cyclase activity. We considered that this biphasic response was due to the coupling of α2C10 to both G(i) and G(s). To isolate functional G(s) and G(i) coupling, cells were treated with pertussis toxin or cholera toxin in doses sufficient to fully ADP-ribosylate the respective G-proteins. Following treatment with cholera toxin, agonists elicited only α2C10-mediated inhibition (~50%) of adenylyl cyclase while after pertussis toxin treatment, agonists elicited only α2C10-mediated stimulation (~60%) of adenylyl cyclase. Incubation of membranes with antisera directed against the carboxyl- terminal portion of G(5α) blocked this functional α2AR·G(s) coupling to the same extent as that found for β2AR·G(s) coupling. In addition to functional G(s) coupling, we also verified direct, agonist-dependent, physical coupling of α2AR to G(sα). In agonist-treated membranes, an agonist-receptor-G(sα) complex was immunoprecipitated with a specific α2C10 antibody, and the G(s) component identified by both western blots using G(sα) antibody, and cholera toxin mediated ADP-ribosylation. Due to the differences in primary amino acid structure in a number of regions of the α2AR subtypes, we investigated whether G-protein coupling was subtype- selective, using UK-14304 and cells with the same α2AR expression levels (~5 pmol/mg). Coupling to G(i) was equivalent for α2C10, α2C4, and α2C2: 53.4 ± 8.8% versus 54.9 ± 1.0% versus 47.6 ± 3.5% inhibition of adenylyl cyclase, respectively. In marked contrast, distinct differences in coupling to G(s) were found between the three α2AR subtypes: stimulation of adenylyl cyclase was 57.9 ± 6.3% versus 30.7 ± 1.1% versus 21.8 ± 1.7% for α2C10, α2C4, and α2C2, respectively. Thus, α2AR have the potential to couple physically and functionally to both G(i) and G(s); for G(i) coupling we found a rank order of α2C10 = α2C4 = α2C2, while for G(s) coupling, α2C10 > α2C4 > α2C2.

AB - Coupling of the three α2-adrenergic receptor (α2AR) subtypes to G(i) and G(s) was studied in membranes from transfected CHO cells. We observed that in the presence of low concentrations of the α2AR agonist UK-14304, α2C10 mediated inhibition of adenylyl cyclase activity, whereas at high concentrations of agonist, α2C10 mediated stimulation of adenylyl cyclase activity. We considered that this biphasic response was due to the coupling of α2C10 to both G(i) and G(s). To isolate functional G(s) and G(i) coupling, cells were treated with pertussis toxin or cholera toxin in doses sufficient to fully ADP-ribosylate the respective G-proteins. Following treatment with cholera toxin, agonists elicited only α2C10-mediated inhibition (~50%) of adenylyl cyclase while after pertussis toxin treatment, agonists elicited only α2C10-mediated stimulation (~60%) of adenylyl cyclase. Incubation of membranes with antisera directed against the carboxyl- terminal portion of G(5α) blocked this functional α2AR·G(s) coupling to the same extent as that found for β2AR·G(s) coupling. In addition to functional G(s) coupling, we also verified direct, agonist-dependent, physical coupling of α2AR to G(sα). In agonist-treated membranes, an agonist-receptor-G(sα) complex was immunoprecipitated with a specific α2C10 antibody, and the G(s) component identified by both western blots using G(sα) antibody, and cholera toxin mediated ADP-ribosylation. Due to the differences in primary amino acid structure in a number of regions of the α2AR subtypes, we investigated whether G-protein coupling was subtype- selective, using UK-14304 and cells with the same α2AR expression levels (~5 pmol/mg). Coupling to G(i) was equivalent for α2C10, α2C4, and α2C2: 53.4 ± 8.8% versus 54.9 ± 1.0% versus 47.6 ± 3.5% inhibition of adenylyl cyclase, respectively. In marked contrast, distinct differences in coupling to G(s) were found between the three α2AR subtypes: stimulation of adenylyl cyclase was 57.9 ± 6.3% versus 30.7 ± 1.1% versus 21.8 ± 1.7% for α2C10, α2C4, and α2C2, respectively. Thus, α2AR have the potential to couple physically and functionally to both G(i) and G(s); for G(i) coupling we found a rank order of α2C10 = α2C4 = α2C2, while for G(s) coupling, α2C10 > α2C4 > α2C2.

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