Gβγ engages PLCβ3 at multiple sites to reorient and facilitate its activation.

Phospholipase C β (PLCβ) enzymes are activated by heterotrimeric G protein subunits, increasing hydrolysis of phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) at the plasma membrane. All four human PLCβ isoforms (PLCβ1-4) are activated by Gαq, whereas PLCβ1-3 are activated to varying extents by Gβγ. The binding sites for Gαq on PLCβ are well established, and much has been learned about its mechanism of activation, but comparatively little is known about Gβγ-dependent activation. In this work, we used cryo-electron microscopy single-particle analysis, functional assays, and bioluminescence resonance energy transfer to investigate how Gβγ interacts with PLCβ3 in concert with activated Gαq to regulate phospholipase activity. Gβγ heterodimers bind multiple surfaces of PLCβ3 to promote activation, but alone do not recruit the enzyme to the plasma membrane. Instead, Gβγ facilitates activation by Gαq, most likely by reorienting the phospholipase catalytic site at the membrane to maximize PI(4,5)P2 hydrolysis and downstream Ca2+ release. Cell-based functional assays demonstrate that Gβγ is required for maximal PLCβ3 activation, even when Gq heterotrimers are the sole source of Gβγ. Together, these findings demonstrate that Gβγ acts as a critical positive allosteric modulator that regularly acts in concert with Gαq to activate PLCβ3 at the plasma membrane.