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.
|Original language||English (US)|
|Number of pages||8|
|Journal||Journal of Bone and Mineral Research|
|State||Published - 1989|
ASJC Scopus subject areas