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Department of Pharmacology and Therapeutics, McGill University,
Montreal, Quebec, Canada (P.M.C.L., M.H.G., P.R.A.), and
Department of
Medicine, Neuroscience Research Institute, University of Ottawa,
Ottawa, Ontario, Canada K1H 8M5 (S.J.M., P.R.A.)
Productive interaction between receptors and G proteins involves
multiple intracellular receptor domains, but the role of individual
receptor amino acids in directing the selection of specific signaling
pathways has not yet been identified. Sequence alignment of several G
protein-coupled receptors identified a highly conserved threonine
residue in the i2 loop of the 5-hydroxytryptamine 1A
(5-HT1A) receptor that is a putative protein kinase C
phosphorylation consensus site and is located in a predicted
amphipathic 
-helical domain. To examine the role of this conserved
threonine residue in 5-HT1A receptor coupling to
Gi/Go proteins, this residue was mutated to
alanine (T149A mutant). Wild-type and mutant 5-HT1A receptors were stably transfected into both Ltk
and GH4C1
cells to investigate receptor coupling to multiple signaling pathways.
In both cell lines, the T149A mutant displayed similar agonist
affinities as the wild-type receptor. In Ltk cells, the
T149A 5-HT1A receptor inhibited cAMP accumulation by 30%
compared with wild-type (83%). A 2.6-fold increase in intracellular calcium (due to phospholipase C-mediated calcium mobilization) was
observed for the wild-type receptor upon the addition of 100 nM 5-HT; whereas the T149A 5-HT1A receptor
failed to mediate a calcium mobilization response at equivalent
receptor levels to wild-type. When transfected in GH4C1 cells, the
T149A receptor mutant fully inhibited basal cAMP and partially
inhibited Gs-stimulated cAMP accumulation compared with
wild-type receptor (57 ± 14% versus 86 ± 2%). In
contrast, the T149A 5-HT1A receptor mutant failed to block
the influx of calcium induced by calcium channel agonist (±)-Bay
K8644, whereas the wild-type 5-HT1A receptor inhibited the
calcium influx by 40%. Thus, the Thr149 residue is directly involved
in G protein coupling to calcium mobilization (mediated by 
subunits of Gi2) and to inhibition of calcium channel
activation (mediated by subunits of Go) but plays a
minor role in coupling to i-mediated inhibition of cAMP
accumulation. The conserved i2 loop threonine may serve as a G protein
contact site to direct the signaling specificity of multiple receptors.
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