Measurement of agonist efficacy using an alpha2A-adrenoceptor-Gi1alpha fusion protein
| Autor: | Graeme Milligan, Alan Wise, I. Craig Carr, D. Alex Groarke |
|---|---|
| Rok vydání: | 1998 |
| Předmět: |
Agonist
Efficacy medicine.drug_class G protein Swine GTP-Binding Protein alpha Subunits Recombinant Fusion Proteins Biophysics GTPgammaS Biology GTP-Binding Protein alpha Subunits Gi-Go Pertussis toxin Ligands Biochemistry GTP Phosphohydrolases Adrenaline chemistry.chemical_compound Structural Biology Receptors Adrenergic alpha-2 Genetics medicine Animals Virulence Factors Bordetella Molecular Biology G protein-coupled receptor G alpha subunit Cell Biology Fusion protein Rats chemistry Pertussis Toxin Guanosine 5'-O-(3-Thiotriphosphate) COS Cells Adrenergic alpha-Agonists Receptor |
| Zdroj: | FEBS letters. 419(1) |
| ISSN: | 0014-5793 |
| Popis: | A fusion protein was constructed between the porcine alpha2A-adrenoceptor and a pertussis toxin-insensitive (Cys351Gly) form of the alpha subunit of the G protein Gi1. Addition of agonist ligands to membranes of COS-7 cells transiently transfected to express this construct, and treated with pertussis toxin prior to cell harvest, resulted in stimulation of both high affinity GTPase activity and enhanced binding of [35S]GTPgammaS. By considering the fusion protein as an agonist-activated enzyme and measuring Vmax of GTP hydrolysis a range of agonist ligands displayed varying efficacy in their capacity to activate the receptor-associated G protein with adrenaline = noradrenaline = alpha-methylnoradrenaline > UK14304 > BHT933 > or = xylazine = clonidine. A similar rank order was observed following independent co-expression of the alpha2A-adrenoceptor and Cys351Gly-Gi1alpha. These data demonstrate the utility and applicability of using a receptor-G protein fusion protein approach, in which the stoichiometry of receptor and G protein is fixed at 1:1, to measure and further understand the nature of agonist efficacy. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|
Plný text