THESIS
2019
2 unnumbered pages, xviii, 167 pages : illustrations (some color) ; 30 cm
Abstract
The heterotrimeric G proteins act as key molecular switches in eukaryotic cells to
transmit signals from hundreds of receptors to multiple effectors. The conventional
theory of G protein activation entails the physical dissociation of Gβγ from the GTP-bound
Gα subunit, thereby producing two separate signals for effector regulation. The
concept of subunit dissociation is primarily based on the Gβγ-binding characteristics of
Gα subunits in solution, but there is little or no in vivo evidence in support of this
mechanism. Here, a difference in subunit dissociation was observed among G protein
families by co-immunoprecipitation assays. Gα subunits of G
q and G
s family remained
bound to Gβγ even when they were activated by AlF
4- or GTPγS, whereas Gα subunits
of G
i family became sepa...[
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The heterotrimeric G proteins act as key molecular switches in eukaryotic cells to
transmit signals from hundreds of receptors to multiple effectors. The conventional
theory of G protein activation entails the physical dissociation of Gβγ from the GTP-bound
Gα subunit, thereby producing two separate signals for effector regulation. The
concept of subunit dissociation is primarily based on the Gβγ-binding characteristics of
Gα subunits in solution, but there is little or no in vivo evidence in support of this
mechanism. Here, a difference in subunit dissociation was observed among G protein
families by co-immunoprecipitation assays. Gα subunits of G
q and G
s family remained
bound to Gβγ even when they were activated by AlF
4- or GTPγS, whereas Gα subunits
of G
i family became separated from the Gβγ dimers under the same condition. To
determine whether subunit dissociations of various G protein families are required for the activation of their effectors, a series of Gβ-Gα fusion proteins were created. Fusion
proteins as well as their constitutively active mutants were examined for effector
activations. Fusion proteins of G
s and G
q families remain functional even when the Gα and Gβ subunits were tightly fused with a single residue linker, while the G
i fusion
proteins with the same linker region failed regulate their effector. In addition to the
major effector, the Gβ
1-Gα
q fusion proteins remained capable of activating other G
q
effectors including GRK2, p63RhoGEF and TPR1. Moreover, the G
q fusion proteins
could also cooperate with “free” Gβγ dimers released from G
i activation to
synergistically activate PLCβ3. These results suggest that only the activation of G
i
family heterotrimer requires subunit dissociation but not for G
q and G
s families. The in
cellulo crosslinking of the G protein subunits also confirmed this family-wise difference.
Such difference in subunit dissociation explains some long-lasting confusions such as
Gβγ signal specificity.
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