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, GTP gamma S, 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. GTP gamma S produced a 17.6- and 11.9-fold increase in [3H]IP2 and [3H]IP3, respectively. The stimulatory effects of GTP and GTP gamma S were dose dependent (GTP ED50 = 3.9 x 10(-6) M; GTP gamma S ED50 = 2.5 x 10(-7) M) and progressive over 10 minutes and required the presence of Mg2+.GTP (10(-4) M) and GTP gamma S (10(-5) M) decreased membrane [3H]phosphoinositides concomitantly with increased [3H]IP2 and [3H]IP3. The GDP analog guanosine 5'-O-(2-thiodiphosphate, GDP beta S) alone did not alter [3H]IP2 or [3H]IP3 production but at 10(-4) M blocks the stimulatory effects of GTP and GTP gamma S. NaF (3 x 10(-2)M) produced a 2.8- and 2.0-fold stimulation of [3H]IP2 and [3H]IP3, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
Babich
M
M
Endocrine Section, Veterans Administration Medical Center, San Francisco, CA.
King
K L
KL
Nissenson
R A
RA
D002462:Cell Membrane / Q000201:enzymology*; D019204:GTP-Binding Proteins / Q000494:pharmacology*; D006160:Guanosine Triphosphate / Q000378:metabolism; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D009363:Neoplasm Proteins / Q000494:pharmacology; D012516:Osteosarcoma / Q000201:enzymology*; D010281:Parathyroid Hormone / Q000494:pharmacology; D044162:Parathyroid Hormone-Related Protein; D010716:Phosphatidylinositols / Q000378:metabolism*; D015398:Signal Transduction; D014407:Tumor Cells, Cultured; D010738:Type C Phospholipases / Q000378:metabolism*
D009363:Neoplasm Proteins; D010281:Parathyroid Hormone; D044162:Parathyroid Hormone-Related Protein; D010716:Phosphatidylinositols; D006160:Guanosine Triphosphate; D010738:Type C Phospholipases; D019204:GTP-Binding Proteins; D008274:Magnesium