Project Details


DESCRIPTION (Verbatim from the Applicant's Abstract): Hormones and
neurotransmitters can activate intracellular signal transduction by binding
receptors linked to heterotrimeric guanine nucleotide-binding or "G" proteins.
G protein a subunits cycle between active, GTP-bound and inactive, GDP-bound
states, and thus signal duration is controlled by their intrinsic GTPase
activity. "Regulator of G-protein signaling" (RGS) proteins are considered key
desensitizers of G protein-coupled signaling given the ability of their
hallmark "RGS-box" domains to accelerate Ga GTPase activity. However, as RGS
proteins have only recently been identified, their physiological functions in
the overall dynamics of signal onset, integration, and termination are poorly
defined. Recent identification of Ras-family GTPase- and Ga-interaction domains
("RFL" and "GoLoco" domains) within RGS 12 and RGS 14 presents the opportunity
to define the molecular mechanisms these two RGS proteins use to transact
higher-order functions in G protein signaling modulation. This proposal is
focused on determining the binding specificities and structural determinants of
RGS 12/14 GoLoco and RFL domains, as well as the effects of domain interactions
both on the nucleotide cycle of the bound G protein and on RGS-box
GTPase-accelerating activity. Studies in Aim 1 test the hypothesis that the
GoLoco region binds to GDP-bound Gi-class Ga subunits and acts as a
receptor-independent guanine-nucleotide exchange factor. Studies in Aim 2 test
the hypothesis that the RFL domains bind GTP-bound Ras-family G proteins and
inhibit nucleotide dissociation. Studies in Aim 3 will define the Ga
selectivites of RGS12/14 ROS-boxes as well as test the hypothesis that
GoLoco/Gax and/or RFL/Ras-family protein interactions modify RGS box function.
In all three Aims, binding specificity and affinity will be determined by a
combination of yeast two-hybrid analyses, coprecipitation studies, biosensor
measurements, and cell co-immunoprecipitation assays; effects of these
interactions on nucleotide binding/hydrolysis will be analysed by in vitro
nucleotide binding assays, single-turnover and steady state GTP hydrolysis
measurements, and cellular readouts of receptor signaling outcomes. As
perturbation of G protein-coupled signal transduction can cause human disease,
yet forms the basis of many drug actions, defining the mechanisms by which
RGS12 and RGS14 assemble and regulate specific heterotrimeric and Ras-family G
proteins should ultimately lead to novel drug discovery targets with exquisite
Effective start/end date1/02/0131/01/02