G protein-dependent activation of a phosphoinositide-specific phospholipase C in UMR-106 osteosarcoma cell membranes

Recent evidence suggests that guanyl nucleotide binding (G) proteins are involved in receptor-mediated bone resorption and in osteoblastic function, but the nature of the G protein coupled to effectors that are involved in these skeletal effects is unknown. The purposes of this study were to determine (1) whether a G protein mediates activation of phosphoinositide-specific phospholipase C in UMR-106 rat osteosarcoma cells, and (2) whether parathyroid hormone (PTH) and a PTH-like protein (PLP) associated with humoral hypercalcemia of malignancy promote GTP-dependent PIP₂ hydrolysis. Addition of GTP (10^-4 M) or guanosine 5'-0-(3-thiotriphosphate, GTPγS, 10^-5 M) to membranes prepared from UMR-106 cells labeled with [³H]myo-inositol increased both [³H]inositol trisphosphate (IP₃) and [³H]inositol bisphosphate (IP₂) formation. The increases in [³H]IP₂ and [³H]IP₃ produced by GTP were 8.6- and 4.3-fold, respectively. GTPγS produced a 17.6- and 11.9-fold increase in [³H]IP₂ and [³H[IP₃, respectively. The stimulatory effects of GTP and GTPγS were dose dependent (GTP ED₅₀ = 3.9 x 10^-6 M; GTPγS ED₅₀ = 2.5 x 10^-7 M) and progressive over 10 minutes and required the presence of Mg²⁺. GTP (10^-4 M) and GTPγS (10^-5 M) decreased membrane [³H]phosphoinositides concomitantly with increased [³H]IP₂ and [³H]IP₃. The GDP analog guanosine 5'-O-(2-thiodiphosphate, GDPβS) alone did not alter [³H]IP₂ or [³H]IP₃ production but at 10^-4 M blocks the stimulatory effects of GTP and GTPγS. NaF (3 x 10^-2 M) produced a 2.8- and 2.0-fold stimulation of [³H]IP₂ and [³H]IP₃, respectively. In the presence of 10^-4 M GTP, bPTH-(1-34) (1 μg/ml) produced an increase in [³H]IP₃ and [³H[IP₂ of 23.5 ± 3.0% (p < 0.001) and 14.1 ± 2.5% (p < 0.01) within 2 minutes. hPLP-(1-34)amide (1 μg/ml) produced a 19.8 ± 5.3% (p < 0.05) and 13.2 ± 4.8% (p < 0.05) increase in [³H]IP₃ and [³H]IP₂. We conclude that UMR-106 membranes possess a G protein-sensitive phosphoinositide-specific phospholipase C. Conceivably, this signal transduction pathway contributes to the skeletal actions of PTH and PLP.