Affiliation:
1. Laboratory of Molecular Neurobiology and Biophysics, The Rockefeller University, New York, NY 10065
2. HHMI, The Rockefeller University, New York, NY 10065
Abstract
PLCβ
(
Phospholipase Cβ
) enzymes cleave phosphatidylinositol 4,5-bisphosphate (
PIP2)
producing
IP3
and
DAG
(diacylglycerol).
PIP2
modulates the function of many ion channels, while
IP3
and
DAG
regulate intracellular Ca
2+
levels and protein phosphorylation by protein kinase C, respectively.
PLCβ
enzymes are under the control of G protein coupled receptor signaling through direct interactions with G proteins
Gβγ
and
Gα
q
and have been shown to be coincidence detectors for dual stimulation of
Gα
q
and
Gα
i
-coupled receptors.
PLCβs
are aqueous-soluble cytoplasmic enzymes but partition onto the membrane surface to access their lipid substrate, complicating their functional and structural characterization. Using newly developed methods, we recently showed that
Gβγ
activates
PLCβ3
by recruiting it to the membrane. Using these same methods, here we show that
Gα
q
increases the catalytic rate constant,
k
cat
, of
PLCβ3
. Since stimulation of
PLCβ3
by
Gα
q
depends on an autoinhibitory element (the X-Y linker), we propose that
Gα
q
produces partial relief of the X-Y linker autoinhibition through an allosteric mechanism. We also determined membrane-bound structures of the
PLCβ3·Gα
q
and
PLCβ3·Gβγ
(2)
·Gα
q
complexes, which show that these G proteins can bind simultaneously and independently of each other to regulate
PLCβ3
activity. The structures rationalize a finding in the enzyme assay, that costimulation by both G proteins follows a product rule of each independent stimulus. We conclude that baseline activity of
PLCβ3
is strongly suppressed, but the effect of G proteins, especially acting together, provides a robust stimulus upon G protein stimulation.
Funder
Howard Hughes Medical Institute
HHS | NIH | National Institute of General Medical Sciences
Publisher
Proceedings of the National Academy of Sciences