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Vol. 56, Issue 4, 684-692, October 1999
Subunit of Gi1: Differences in
Receptor-Constitutive Activity Imparted by Single Amino Acid
Substitutions in Gi1
Molecular Pharmacology Group, Division of Biochemistry and
Molecular Biology, Institute of Biomedical and Life Sciences,
University of Glasgow, Glasgow, Scotland, United Kingdom
Fusion proteins were generated between the human
5-hydroxytryptamine (5-HT)1A receptor and both
wild-type (Cys351) and pertussis toxin-resistant
(Gly351 and Ile351) forms of Gi1.
These were expressed stably. Pertussis toxin treatment substantially
reduced basal high-affinity GTPase activity in clones expressing the
5-HT1A receptor wild-type Gi1
construct but
not in clones expressing 5-HT1A receptor
(Gly351)Gi1 or
(Ile351)Gi1. Spiperone functioned as an
inverse agonist in membranes expressing the 5-HT1A receptor
wild-type Gi1 fusion protein and in those expressing
5-HT1A receptor (Ile351)Gi1 but
not the 5-HT1A receptor
(Gly351)Gi1 fusion protein. The effect of
spiperone at the 5-HT1A receptor wild-type
Gi1 construct but not the 5-HT1A receptor
(Ile351)Gi1 construct was blocked by
pertussis toxin treatment. By contrast, agonists functioned with equal
effectiveness at the three fusion proteins and were unaffected by
pertussis toxin treatment of the (Ile351)Gi1- and
(Gly351)Gi1-containing constructs. 5-HT
resulted in strong inhibition of forskolin-amplified adenylyl cyclase
in intact cells expressing the isolated 5-HT1A receptor. In
fusion protein-expressing cells, 5-HT-mediated inhibition of adenylyl
cyclase was also observed. Pertussis toxin treatment obliterated
5-HT-mediated inhibition in cells expressing the isolated receptor and
the 5-HT1A receptor wild-type Gi1 fusion
protein but not in those expressing the 5-HT1A receptor
(Ile351) or (Gly351)Gi1 fusion
proteins. These studies demonstrate that alteration of a single amino
acid in Gi1 located at a key contact site between the G
protein and a G protein-coupled receptor can regulate
agonist-independent constitutive activity of the G protein-coupled
receptor and that fusion proteins can directly regulate adenylyl cyclase.
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