Quantitative analysis of a cysteine351glycine mutation in the G protein Gi1alpha: effect on alpha2A-adrenoceptor-Gi1alpha fusion protein activation.
Fusion proteins were constructed between the porcine alpha2A-adrenoceptor and either wild-type (Cys351) or a pertussis toxin-resistant (Gly351) form of the G protein Gi1alpha. Addition of adrenaline to membranes expressing the fusion proteins resulted in concentration-dependent stimulation of their high affinity GTPase activity. The alpha2A-adrenoceptor-wild type Gi1alpha fusion protein produced substantially higher maximal stimulation of GTPase activity in response to adrenaline than that containing Gly351 Gi1alpha. Treatment of the fusion proteins as agonist-regulated enzymes allowed measurement of Vmax and turnover number for adrenaline-stimulation of the GTPase activity of each fusion construct. The turnover number of the alpha2A-adrenoceptor-Cys351 Gly Gi1alpha fusion protein was only 44'S, of that for the alpha2A-adrenoceptor-wild type Gi1alpha fusion protein. These data provide the first direct quantitative evaluation of the effects of a mutation of a G protein on the capacity of an agonist-occupied receptor to activate the mutant.
Carr
I C
IC
Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Scotland, UK.
Burt
A R
AR
Jackson
V N
VN
Wright
J
J
Wise
A
A
Rees
S
S
Milligan
G
G
D000818:Animals; D019556:COS Cells; D003545:Cysteine / Q000235:genetics / Q000378:metabolism*; D004837:Epinephrine / Q000494:pharmacology; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000235:genetics / Q000378:metabolism*; D005998:Glycine / Q000235:genetics / Q000378:metabolism*; D037342:Pertussis Toxin; D017354:Point Mutation; D018341:Receptors, Adrenergic, alpha-2 / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D018341:Receptors, Adrenergic, alpha-2; D011993:Recombinant Fusion Proteins; D010566:Virulence Factors, Bordetella; D037342:Pertussis Toxin; D020558:GTP Phosphohydrolases; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D003545:Cysteine; D005998:Glycine; D004837:Epinephrine