Tubby protein binds with high affinity and selectivity to phosphatidylinositol 4,5-bisphosphate (PIP₂), and GFP-Tubby has been shown to enrich to the plasma membrane and translocate to the cytosol on activation of Gα_q-coupled GPCRs (Santagata et al., 2001), making it a good candidate for a PIP₂-selective biosensor. Here, we have investigated the relative contributions of inositol 1,4,5-trisphosphate (IP₃) production and PIP₂ depletion to the translocation of GFP-Tubby and eGFP-PH-PLCδ1 in response to Gα_q-coupled GPCR activation. Both eGFP-PH-PLCδ1 and GFP-Tubby are predominantly localized to the plasma membrane when transiently expressed in SH-SY5Y neuroblastoma cells. Activation of the endogenous M₃ mACh receptor population (with methacholine (MCh)) caused both probes to translocate from plasma membrane to cytosol in a reversible (on agonist washout) and concentration-dependent manner (pEC₅₀ values: 5.19 ± 0.11 (n = 9) for eGFP-PH-PLCδ1 and 4.53 ± 0.21 (n = 5) for GFP-Tubby). For both eGFP-PH-PLCδ1 and GFP-Tubby, cytosolic fluorescence returned to baseline within 4 min of agonist washout in control cells while, in the presence of a high concentration of wortmannin (10 μM), both eGFP-PH-PLCδ1 and GFP-Tubby responses to MCh (1 mM) were prolonged (responses maintained at significantly elevated plateau levels of 44 ± 8% (n=12) and 79 ± 6% (n=10), respectively (p<0.001 in each case)). In the presence of a lower concentration of wortmannin (1 μM), responses were not significantly different from controls, indicating that the wortmannin effect is due to inhibition of phosphatidylinositol 4-kinase, not phosphoinositide 3-kinase activity. Co-expression of a dsRed2-tagged IP₃ 3-kinase construct significantly attenuated MCh-mediated translocation of eGFP-PH-PLCδ1 (27 ± 8% in cells co-expressing 3-kinase, versus 129 ± 17% in controls (n≥10; p<0.001)). In contrast, GFP-Tubby translocation in response to a similar stimulus was unaffected by co-expression with the IP₃ 3-kinase construct (53 ± 11% in cells co-expressing 3-kinase, versus 47 ± 13% in controls (n≥12)). Therefore, unlike eGFP-PH-PLCδ1, agonist-induced translocation of GFP-Tubby does not report changes in cytosolic IP₃. These data indicate that eGFP-PH-PLCδ1 translocation in response to Gα_q-PCR activation is primarily driven by changes in cytosolic IP₃, in agreement with our previous findings (Nash et al., 2002, 2004), while GFP-Tubby translocation reflects dynamic changes in plasma membrane PIP₂ levels and is independent of changes in cytosolic IP₃. GFP-Tubby therefore represents a selective fluorescent biosensor for investigating the dynamic regulation of cellular [PIP₂].