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Quantitative analysis of a cysteine 351 glycine mutation in the G protein G i1 α: effect on α 2A ‐adrenoceptor‐G i1 α fusion protein activation
Author(s) -
Carr I.Craig,
Burt Andrew R,
Jackson Vicky N,
Wright Jason,
Wise Alan,
Rees Stephen,
Milligan Graeme
Publication year - 1998
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(98)00476-1
Subject(s) - fusion protein , g protein , mutant , lipid bilayer fusion , stimulation , chemistry , receptor , wild type , pertussis toxin , gtpase , biochemistry , microbiology and biotechnology , biology , endocrinology , membrane , recombinant dna , gene
Fusion proteins were constructed between the porcine α 2A ‐adrenoceptor and either wild‐type (Cys 351 ) or a pertussis toxin‐resistant (Gly 351 ) form of the G protein G i1 α. Addition of adrenaline to membranes expressing the fusion proteins resulted in concentration‐dependent stimulation of their high affinity GTPase activity. The α 2A ‐adrenoceptor‐wild type G i1 α fusion protein produced substantially higher maximal stimulation of GTPase activity in response to adrenaline than that containing Gly 351 G i1 α. Treatment of the fusion proteins as agonist‐regulated enzymes allowed measurement of V max and turnover number for adrenaline‐stimulation of the GTPase activity of each fusion construct. The turnover number of the α 2A ‐adrenoceptor‐Cys 351 Gly G i1 α fusion protein was only 44% of that for the α 2A ‐adrenoceptor‐wild type G i1 α 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.