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G protein‐dependent activation of a phosphoinositide‐specific phospholipase C in UMR‐106 osteosarcoma cell membranes
Author(s) -
Babich Michael,
King Kathleen L.,
Nissenson Robert A.
Publication year - 1989
Publication title -
journal of bone and mineral research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.882
H-Index - 241
eISSN - 1523-4681
pISSN - 0884-0431
DOI - 10.1002/jbmr.5650040414
Subject(s) - gtp' , g protein , guanosine , phospholipase c , inositol , inositol trisphosphate , biology , medicine , chemistry , endocrinology , biochemistry , microbiology and biotechnology , receptor , enzyme
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 PIP 2 hydrolysis. Addition of GTP (10 −4 M) or guanosine 5′‐0‐(3‐thiotriphosphate, GTP γ S, 10 −5 M) to membranes prepared from UMR‐106 cells labeled with [ 3 H]myo‐inositol increased both [ 3 H]inositol trisphosphate (IP 3 ) and [ 3 H]inositol bisphosphate (IP 2 ) formation. The increases in [ 3 H]IP 2 and [ 3 H]IP 3 produced by GTP were 8.6‐ and 4.3‐fold, respectively. GTP γ S produced a 17.6‐ and 11.9‐fold increase in [ 3 H]IP 2 and [ 3 H]IP 3 , respectively. The stimulatory effects of GTP and GTP γ S were dose dependent (GTP ED 50 = 3.9 × 10 −6 M; GTP γ S ED 50 = 2.5 × 10 −7 M) and progressive over 10 minutes and required the presence of Mg 2+ . GTP (10 −4 M) and GTP γ S (10 −5 M) decreased membrane [ 3 H]phosphoinositides concomitantly with increased [ 3 H]IP 2 and [ 3 H]IP 3 . The GDP analog guanosine 5′‐ O ‐(2‐thiodiphosphate, GDPβ) alone did not alter [ 3 H]IP 2 or [ 3 H]IP 3 production but at 10 −4 M blocks the stimulatory effects of GTP and GTP γ S. NaF (3 × 10 −2 M) produced a 2.8‐ and 2.0‐fold stimulation of [ 3 H]IP 2 and [ 3 H]IP 3 , respectively. In the presence of 10 −4 M GTP, bPTH‐(1–34) (1 μg/ml) produced an increase in [ 3 H]IP 3 and [ 3 H]IP 2 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 [ 3 H]IP 3 and [ 3 H]IP 2 . 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.