In: Biology
Cell Biology Short Answer Question: Review the steps for ligand-induced activation of effector protein mediated by trimeric G proteins. Suppose you have isolated a mutant Gα that has increased GTPase activity, what effect would this mutation have on the G protein and the effector protein?
The heterotrimeric G protein G(s) couples hormone receptors (as
well as other receptors) to the effector enzyme adenylyl cyclase
and is therefore required for hormone-stimulated intracellular cAMP
generation. Receptors activate G(s) by promoting exchange of GTP
for GDP on the G(s) alpha-subunit (G(s)alpha) while an intrinsic
GTPase activity of G(s)alpha that hydrolyzes bound GTP to GDP leads
to deactivation.
Substitution of Arg258, a residue within the GTPase domain of the
heterotrimeric guanine nucleotide binding protein (G protein)
-subunit (s), to alanine (s-R258A) results in decreased activation
by receptor or aluminum fluoride (AlF4) and increased basal GDP
release. Arg258 interacts with Gln170 in the helical domain, and,
presumably, loss of this interaction between the GTPase and helical
domain leads to more rapid GDP release, resulting in decreased
activation by AlF4 and increased thermolability. In this study, we
mutate Gln170 to alanine (s-Q170A) and demonstrate that this
mutant, like s-R258A, has decreased activation by AlF4, increased
thermolability (both reversed in the presence of excess guanine
nucleotide), and an increased rate of GDP release. However, unlike
s-R258A, s-Q170A does not have impaired receptor-mediated
activation.
Therefore, this interdomain interaction is critical to maintain
normal guanine nucleotide binding (and hence normal activation by
AlF4) but is not important for receptor-mediated activation. In
single turnover GTPase assays, the catalytic rate for GTP
hydrolysis of s-R258A was 14-fold higher than normal whereas that
of s-Q170A was unaffected. Examination of the s crystal structure
suggests that Arg258, through interactions with Glu50, might
constrain the position of Arg201, a residue critical for catalyzing
the GTPase reaction. This is an example of a mutation in a
heterotrimeric G protein that results in an increased intrinsic
GTPase activity and provides another mechanism by which G protein
mutations can impair signal transduction.
Impaired Receptor-Mediated Activation of s-R258A is Associated with Increased Intrinsic GTPase Activity. The Arg258-Gln170 interdomain interaction is critical for maintaining the GDP-bound basal state because disruption of this interaction by substituting either residue increases GDP release, which leads to decreased activation by AlF4 and increased thermolability. However, the fact that activation by isoproterenol plus GTP is unaffected by the Gln170 substitution suggests that this interdomain interaction is not important for receptor-mediated activation and that substitution of Arg258 impairs receptor-mediated activation by another mechanism.
It is possible that the s-R258A mutant has minor defects in receptor-catalyzed guanine nucleotide exchange the receptor-mediated activation defect of s-R258A is caused primarily by a markedly increased GTPase activity. Evidence that increased GTPase rates can lead to decreased receptor-mediated activation is provided by the effects of regulators of G protein signaling proteins (which increase the GTPase rate of -subunits) on receptor signaling.
This is an example in which receptor signaling is impaired by a mutation that activates the intrinsic GTPase activity of a heterotrimeric G protein; mutations that lead to increased GTPase activity have been identified in the small guanine nucleotide binding proteins EF-Tu and ras. This underscores the need to examine the GTPase activity when assessing the function of mutant G proteins with impaired receptor-mediated activation. Finally, these studies demonstrate that identification and analysis of naturally occurring G protein mutations in patients can lead to significant advances in our understanding of how G proteins function.