The work described here illustrates direct, concentration-dependent stimulation of purified PLC-η2 in phospholipid vesicles reconstituted with purified Gβ1γ2. Gβγ-dependent activation of PLC-η2 also is observed in intact cells with multiple forms of the heterotrimeric subunit. Thus, we have identified a novel effector of Gβγ and have implicated this PLC isozyme in signaling responses downstream of heterotrimeric G protein-coupled receptors.
The involvement of multiple G proteins in receptor-promoted regulation of inositol lipid signaling was first realized in studies of the PLC-β isozymes. Gα subunits of the Gq
family initially were shown to mediate guanine nucleotide-dependent activation (12
), but subsequent studies revealed that PLC-β2 and PLC-β3 also are activated by Gβγ subunits of heterotrimeric G proteins (16
). The apparent complexity of G protein-dependent regulation of inositol lipid signaling was further increased by realization that the small GTPase Rac binds and activates PLC-β2 (19
). Moreover, the most divergent member of the PLC isozyme family, PLC-ε, was initially discovered as a Ras-binding protein in Caenorhabditis elegans
, and the mammalian isoform is dually activated by Ras (22
) and Rho (26
) through direct binding of these GTPases to two different regions of the isozyme. Although PLC-γ isozymes are activated by phosphorylation by receptor and other tyrosine kinases, PLC-γ2 is additionally activated by Rac (11
). Transient expression studies in intact COS-7 cells implicated activation of PLC-η2 by Gβγ subunits, and the results presented here illustrate that this novel PLC isozyme is a direct effector of Gβγ.
We previously screened all of the heterotrimeric Gα subunits and many Ras superfamily GTPases for stimulation of PLC-η2 after coexpression in COS-7 cells, and only Gβγ was identified as a potential activator of the isozyme (31
). Additional comprehensive analyses are needed, but within the limitations of our screens to date, PLC-η2 is the only PLC isozyme that is solely regulated by Gβγ. Thus, our data are consistent with the idea that G protein-coupled receptors activate PLC-η2 by release of Gβγ from heterotrimeric G proteins. The broad expression of PLC-η2 in the brain together with the high levels of neuronal Gαo
and other members of the Gi
subfamily suggests that this PLC isozyme may in part function downstream of Gi
-coupled receptors in the neuronal signaling pathways. For example, PLC-η2 is strongly expressed in hippocampus, cerebral cortex, and olfactory bulb in mouse brain (30
). Expression of PLC-η2 was detected in early developmental stages in mouse brain and was proposed to be important in formation and/or maintenance of the neuronal networks in postnatal brain (30
). The exact roles of G protein-mediated regulation of PLC-η2 in brain development await further exploration.
Gβ1γ2 was the only G protein dimer tested as an activator of purified PLC-η2. However, the relative effects of Gβγ subunits of various subunit composition on PLC-η2-versus PLC-β2-promoted activity were very similar in transfection studies, and therefore, it is unlikely that remarkable differences exist in the selectivity of regulation of these two PLC isozymes by certain Gβγ subunits. Given its similarity in sequence with PLC-η2, we also tested whether Gβ1γ2 activates PLC-η1. Multiple coexpression experiments with COS-7 cells carried out under a variety of conditions failed to reveal any Gβγ-dependent activation of PLC-η1. Thus, within the limitations of these transfection experiments, we conclude that Gβγ differentially regulates the two PLC-η isozymes. Whether PLC-η1 is regulated by small GTPases or by other heterotrimeric G protein subunits remains unreported.
The last three exons of the PLC-η2 gene are alternatively spliced in both mice and humans to produce at least four splice variants containing markedly variable C-terminal domains beyond the C2 domain (31
). The physiological relevance of these different forms of PLC-η2 is not yet known, but as illustrated here, removal of the 545 C-terminal amino acids does not demonstrably affect its regulation by Gβ1
. Previous studies have suggested that Gβγ interacts with multiple sites in PLC-β2, and both the catalytic and PH domains have been implicated in Gβγ-dependent activation of the isozyme (19
). A construct of PLC-η2 that encompassed the PH through C2 domain retained activation by Gβ1
both in vivo and in vitro, but observation of similar levels of activation of a fragment of PLC-η2 lacking the PH domain indicates that in contrast to PLC-β2, the PH domain of PLC-η2 appears to be relatively unimportant in Gβ1
-mediated regulation. However, Nakahara and co-workers (30
) illustrated that PLC-η2 is predominantly associated with the plasma membrane in mouse brain and reported that the PH domain is necessary for membrane localization.
The mechanism by which Gβγ activates PLC-β isozymes, and now PLC-η2, remains to be defined. Activation could follow from simple Gβγ-dependent recruitment to phosphoinositide substrate-containing membranes, or activation may involve G protein-promoted conformational changes in the catalytic core of the enzyme. Mechanistic insight into the activation of PLC isozymes has accrued from our recent structural and biochemical studies illustrating that PLC-β2 and, ostensibly, PLC-δ and PLC-ε are autoinhibited by the unstructured X–Y linker region of the isozymes (ref 21
and unpublished observations of S. Hicks et al., 2007). These studies strongly suggest that the active site-occluding X–Y linker moves as a consequence of dense negative charges in the linker encountering negatively charged membrane surfaces; autoinhibition is relieved, and access to the PtdIns(4,5)P2
substrate is promoted. Thus, our structures of PLC-β2 alone and PLC-β2 in a GTP-dependent complex with Rac1 illustrate that Rac-mediated activation of PLC-β2 follows from simple recruitment of the enzyme to membrane surfaces. In contrast, biochemical studies of Gβγ- dependent activation of PLC-β2 suggest a more complex mode of regulation involving the catalytic core of the enzyme, and therefore, the mechanism(s) by which Gβγ activates PLC-β2 and other PLC isozymes remains unclear. The Gβγ-binding interface has not yet been structurally identified in any PLC isozyme, and it will be important to establish unambiguously whether Gβγ binds to PLC-η2 and PLC-β2 through similar or different determinants.
A notable difference between PLC-η2 and PLC-β2 is that the overall charge of PLC-η2 is much more positive than that of PLC-β2, and the X–Y linker of PLC-η2 is essentially devoid of sequence containing dense negative charge and overall is positively charged. Thus, we anticipate that PLC-η2 exhibits less autoinhibition and greater electrostatic force-dependent attraction to phosphoinositide substrate-containing membrane surfaces. Such properties may explain the high basal activity of PLC-η2, and given these differences in PLC-β2 and PLC-η2, it is perhaps not surprising that the fold activation of PLC-β2 by Gβ1
was greater than that observed with PLC-η2. Nomikos and co-workers (38
) recently concluded that a dense positive region in the X–Y linker of PLC-ζ also is important for membrane association and activity of this sperm-specific isozyme.
In conclusion, we have demonstrated that Gβ1γ2 directly activates PLC-η2. Future experiments will delineate the region of Gβ1γ2 interaction and reveal the mechanism of activation. The physiological role of Gβ1γ2-mediated activation of PLC-η2 is under investigation.