G(i2) mediates α2-adrenergic inhibition of adenylyl cyclase in platelet membranes: In situ identification with G(α) C-terminal antibodies

W. F. Simonds, P. K. Goldsmith, J. Codina, C. G. Unson, Allen M. Spiegel

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Abstract

A panel of antibodies to synthetic decapeptides corresponding to the C termini of guanine nucleotide-binding regulatory protein (G protein) α subunits has been generated in rabbits. The specificity of each antibody was assessed by ELISA for peptide binding and by immunoblotting for binding to defined, recombinant G(α) subunits expressed in Escherichia coli. Immunoblotting of human platelet membranes with these antibodies identified a variety of endogenous G proteins including G(s) (stimulatory), G(i2) (inhibitory), G(i3), and G(x(z)) (unknown function). Pretreatment of platelet membranes with C-terminal antibodies reactive with G(i2), but not with antibodies to G(i3) or G(x(z)), blocked α2-adrenergic inhibition of adenylyl cyclase. This identifies G(i2) as the dominant mediator of cyclase inhibition in this pathway. This approach may provide a general means of identifying relevant functional interactions of G proteins with receptors an effectors in situ.

Original languageEnglish (US)
Pages (from-to)7809-7813
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume86
Issue number20
StatePublished - 1989
Externally publishedYes

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GTP-Binding Proteins
Adenylyl Cyclases
Adrenergic Agents
Blood Platelets
Membranes
Antibodies
Immunoblotting
Antibody Specificity
Protein Subunits
Carrier Proteins
Enzyme-Linked Immunosorbent Assay
Escherichia coli
Rabbits
Peptides

ASJC Scopus subject areas

  • General
  • Genetics

Cite this

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title = "G(i2) mediates α2-adrenergic inhibition of adenylyl cyclase in platelet membranes: In situ identification with G(α) C-terminal antibodies",
abstract = "A panel of antibodies to synthetic decapeptides corresponding to the C termini of guanine nucleotide-binding regulatory protein (G protein) α subunits has been generated in rabbits. The specificity of each antibody was assessed by ELISA for peptide binding and by immunoblotting for binding to defined, recombinant G(α) subunits expressed in Escherichia coli. Immunoblotting of human platelet membranes with these antibodies identified a variety of endogenous G proteins including G(s) (stimulatory), G(i2) (inhibitory), G(i3), and G(x(z)) (unknown function). Pretreatment of platelet membranes with C-terminal antibodies reactive with G(i2), but not with antibodies to G(i3) or G(x(z)), blocked α2-adrenergic inhibition of adenylyl cyclase. This identifies G(i2) as the dominant mediator of cyclase inhibition in this pathway. This approach may provide a general means of identifying relevant functional interactions of G proteins with receptors an effectors in situ.",
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T1 - G(i2) mediates α2-adrenergic inhibition of adenylyl cyclase in platelet membranes

T2 - In situ identification with G(α) C-terminal antibodies

AU - Simonds, W. F.

AU - Goldsmith, P. K.

AU - Codina, J.

AU - Unson, C. G.

AU - Spiegel, Allen M.

PY - 1989

Y1 - 1989

N2 - A panel of antibodies to synthetic decapeptides corresponding to the C termini of guanine nucleotide-binding regulatory protein (G protein) α subunits has been generated in rabbits. The specificity of each antibody was assessed by ELISA for peptide binding and by immunoblotting for binding to defined, recombinant G(α) subunits expressed in Escherichia coli. Immunoblotting of human platelet membranes with these antibodies identified a variety of endogenous G proteins including G(s) (stimulatory), G(i2) (inhibitory), G(i3), and G(x(z)) (unknown function). Pretreatment of platelet membranes with C-terminal antibodies reactive with G(i2), but not with antibodies to G(i3) or G(x(z)), blocked α2-adrenergic inhibition of adenylyl cyclase. This identifies G(i2) as the dominant mediator of cyclase inhibition in this pathway. This approach may provide a general means of identifying relevant functional interactions of G proteins with receptors an effectors in situ.

AB - A panel of antibodies to synthetic decapeptides corresponding to the C termini of guanine nucleotide-binding regulatory protein (G protein) α subunits has been generated in rabbits. The specificity of each antibody was assessed by ELISA for peptide binding and by immunoblotting for binding to defined, recombinant G(α) subunits expressed in Escherichia coli. Immunoblotting of human platelet membranes with these antibodies identified a variety of endogenous G proteins including G(s) (stimulatory), G(i2) (inhibitory), G(i3), and G(x(z)) (unknown function). Pretreatment of platelet membranes with C-terminal antibodies reactive with G(i2), but not with antibodies to G(i3) or G(x(z)), blocked α2-adrenergic inhibition of adenylyl cyclase. This identifies G(i2) as the dominant mediator of cyclase inhibition in this pathway. This approach may provide a general means of identifying relevant functional interactions of G proteins with receptors an effectors in situ.

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