Kinetic Scaffolding Mediated by a Phospholipase C- and Gq Signaling Complex

Department of Pharmacology, University of North Carolina School of Medicine, Chapel Hill, NC 27599, USA.
Science (Impact Factor: 33.61). 11/2010; 330(6006):974-980. DOI: 10.1126/science.1193438


Transmembrane signals initiated by a broad range of extracellular stimuli converge on nodes that regulate phospholipase C
(PLC)–dependent inositol lipid hydrolysis for signal propagation. We describe how heterotrimeric guanine nucleotide–binding
proteins (G proteins) activate PLC-βs and in turn are deactivated by these downstream effectors. The 2.7-angstrom structure
of PLC-β3 bound to activated Gαq reveals a conserved module found within PLC-βs and other effectors optimized for rapid engagement of activated G proteins.
The active site of PLC-β3 in the complex is occluded by an intramolecular plug that is likely removed upon G protein–dependent
anchoring and orientation of the lipase at membrane surfaces. A second domain of PLC-β3 subsequently accelerates guanosine
triphosphate hydrolysis by Gαq, causing the complex to dissociate and terminate signal propagation. Mutations within this domain dramatically delay signal
termination in vitro and in vivo. Consequently, this work suggests a dynamic catch-and-release mechanism used to sharpen spatiotemporal
signals mediated by diverse sensory inputs.

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    • "The differential ability of Gα16QL and GαzQL to interact with Fhit (Figure 2A) permits identification of Fhit-interacting regions on Gα16 through gain of function analyses. Since the effector interacting domain is likely to reside in the carboxyl half of the Gα subunit [36,37], we have selected chimeras composed of Gαz backbones with their C-terminal regions increasingly replaced by Gα16 sequences all the way up to the β2 domain (Figure 4A); mirror images of selected chimeras were also included. Among the various chimeras examined, constitutively active N188QL and N210QL (N-terminal 188 or 210 amino acids from Gαz, respectively) were more efficiently pulled down by the anti-Flag affinity gel than their corresponding wild-types; both chimeras were as effective as, if not better than, Gα16QL (Figure 4B). "
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    ABSTRACT: The FHIT tumor suppressor gene is arguably the most commonly altered gene in cancer since it is inactivated in about 60% of human tumors. The Fhit protein is a member of the ubiquitous histidine triad proteins which hydrolyze dinucleoside polyphosphates such as Ap3A. Despite the fact that Fhit functions as a tumor suppressor, the pathway through which Fhit inhibits growth of cancer cells remains largely unknown. Phosphorylation by Src tyrosine kinases provides a linkage between Fhit and growth factor signaling. Since many G proteins can regulate cell proliferation through multiple signaling components including Src, we explored the relationship between Galpha subunits and Fhit. Several members of the Galphaq subfamily (Galpha16, Galpha14, and Galphaq) were found to co-immunoprecipitate with Fhit in their GTP-bound active state in HEK293 cells. The binding of activated Galphaq members to Fhit appeared to be direct and was detectable in native DLD-1 colon carcinoma cells. The use of Galpha16/z chimeras further enabled the mapping of the Fhit-interacting domain to the alpha2-beta4 region of Galpha16. However, Galphaq/Fhit did not affect either Ap3A binding and hydrolysis by Fhit, or the ability of Galphaq/16 to regulate downstream effectors including phospholipase Cbeta, Ras, ERK, STAT3, and IKK. Functional mutants of Fhit including the H96D, Y114F, L25W and L25W/I10W showed comparable abilities to associate with Galphaq. Despite the lack of functional regulation of Gq signaling by Fhit, stimulation of Gq-coupled receptors in HEK293 and H1299 cells stably overexpressing Fhit led to reduced cell proliferation, as opposed to an enhanced cell proliferation typically seen with parental cells. Activated Galphaq members interact with Fhit through their alpha2-beta4 region which may result in enhancement of the growth inhibitory effect of Fhit, thus providing a possible avenue for G protein-coupled receptors to modulate tumor suppression.
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    • "Some Gα subunit effectors also enhance GTPase activity; particularly, phospholipase-Cβ serves as a GAP for Gαq, and the Gα12/13 subfamily RGS-RhoGEF effectors possess a GTPase-accelerating domain (the rgRGS domain) with distant homology to RGS proteins.40, 43 An additional class of Gα regulators is the GoLoco motif protein family, members of which serve as guanine nucleotide dissociation inhibitors (GDIs) by binding directly to Gα·GDP and preventing nucleotide release.44 "
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    • "PLC-ε is regulated directly by small GTPases of the Ras and Rho families, as well as subunits of G proteins (Bunney et al., 2006; Harden et al., 2009; Harden and Sondek, 2006). Crystal structures of human PLC-b3 (enzyme core or full-length) in complex with Gaq were recently solved, revealing novel structural insights into the function of each domain involved in Gaq-induced activation of PLC-b3 (Lyon et al., 2013, Waldo et al., 2010). Structure and regulation of PLC by GPCRs and other means have been recently reviewed (Gresset et al., 2012; Kadamur and Ross, 2013). "

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