SUMMARY Phospholipase C β (PLCβ) enzymes are dramatically activated by heterotrimeric G proteins. Central to this response is the robust autoinhibition of PLCβ by an X–Y linker region within its catalytic core and by the Hα2′ helix in the C-terminal extension of the enzyme. The molecular mechanism of each and their mutual dependence are poorly understood. Herein it is shown that distinct regions within the X–Y linker have specific roles in regulating activity. Most importantly, an acidic stretch within the linker stabilizes a lid that occludes the active site, consistent with crystal structures of variants lacking this region. Inhibition by the Hα2′ helix is independent of the X–Y linker and likely regulates activity by limiting membrane interaction of the catalytic core. Full activation of PLCβ thus requires multiple independent molecular events induced by membrane association of the catalytic core and by the binding of regulatory proteins.

Phospholipase C‐β (PLCβ) enzymes hydrolyze the inner membrane lipid PIP2 to produce the second messengers inositol‐1,4,5‐triphosphate and diacylglycerol. PLCβ enzymes are characterized by low basal activity, due in part to a linker region within catalytic core that physically occludes the active site. We previously identified a highly conserved autoinhibitory helix in the PLCβ C‐terminal regulatory region that makes extensive interactions with the PLCβ catalytic core. Mutations within this interface lead to dramatic increases in basal activity and decreased thermal stability. Furthermore, this autoinhibitory helix docks in close proximity to the PLCβ active site and the regulatory linker. However, the molecular basis for how this inhibitory helix regulates basal activity, and whether or how it works in conjunction with the catalytic core regulatory linker remains unclear. We have used mass spectrometry, site‐directed mutagenesis, and biochemical assays to begin to elucidate the molecular basis of autoinhibition of lipid hydrolysis by these various regulatory elements. A better understanding of the mechanisms governing PLCβ activity are essential for developing novel therapeutic approaches for the treatment of cardiac hypertrophy and heart failure. Grant Funding Source: NIH grants HL086865 and HL071818 (J.J.G.T.) and AHA Fellowship 13POST1637009 (A.M.L.).

Phospholipase C β (PLCβ) enzymes hydrolyze phosphatidylinositol‐4,5‐bisphosphate to produce the second messengers inositol‐1,4,5‐triphosphate and diacylglycerol. PLCβ enzymes are autoinhibited by an X—Y linker region within catalytic core that occludes the active site and a helix from within the C‐terminal extension. Protease protection assays, site‐directed mutagenesis, biochemical assays, and X‐ray crystallography were used to investigate the molecular basis of autoinhibition by these regulatory elements and define their respective roles. Distinct regions within the X—Y linker were shown to have specific roles in regulating access to the PLCβ active site, with the conserved acidic stretch within the linker being the most autoinhibitory element. Crystal structures of PLCβ3 variants lacking the acidic stretch have disordered X—Y linkers and bind IP3. The C‐terminal inhibitory helix acts independently of the linker and prevents full activation of the enzyme in the absence of Gαq. Our results demonstrate that PLCβ activation is a multi‐step process requiring independent molecular events induced by membrane association and the binding of activating proteins.